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Role of intercellular adhesion molecule 2(ICAM-2) in the murine immune system Carpenito, Carmine 1997

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ROLE OF INTERCELLULAR ADHESION MOLECULE 2 (ICAM-2) IN THE MURINE IMMUNE SYSTEM by CARMINE CARPENITO B . S c , The University of British Columbia, 1987 M . S c , The University of British Columbia, 1990  A T H E S I S S U B M I T T E D IN PARTIAL F U L F I L L M E N T O F THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Department of Medical Genetics, Genetics Program)  We accept this thesis as conforming to the required standard  T H E UNIVERSITY O F BRITISH C O L U M B I A June 1997 © Carmine Carpenito, 1997  In  presenting  degree at the  this  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be granted her  for  It  is  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department  of  /%P?fpL-  &faJGTtCi  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  TYj<0£  2^)h 7  1  (^^J,r%  f^OG/^  ABSTRACT  Intercellular adhesion molecule-2 (ICAM-2; CD102) is one of three ligands for the p leukocyte  integrin  LFA-1 (CD11a/CD18).  Although  ICAM-2 expression  is  limited  2  to  lymphocytes, monocytes, granulocytes, and endothelium, the biological role of ICAM-2 has remained unknown. In this thesis, the murine ICAM-2 cDNA was cloned in order to assist the functional investigation. Sequence analysis of both the cDNA and the genomic clone revealed that ICAM-2 is a member of the immunoglobulin superfamily.  The cDNA and antibody were  used to examine the role of ICAM-2 in T cell activation and leukocyte  transendothelial  migration. In order to examine the role of ICAM-2 in antigen presentation to T cells, murine fibroblastic L cells expressing allogeneic class II M H C (l-E ) were transfected with the ICAM-2 d  c D N A and tested for the ability to stimulate splenic T cells. significantly  The expression of ICAM-2  increased the stimulation of T cells in an LFA-1-dependent manner.  The  increased T cell response was also observed when the class II M H C and ICAM-2 were expressed in separate cells combined together.  This indicated that ICAM-2 is actually  transmitting a costimulatory signal rather than merely enhancing T cell adhesion to the antigen presenting cell. T cells stimulated with ICAM-2-transfected L cells expressing the class II M H C were able to respond to an allogeneic secondary stimulation.  In contrast, T cells stimulated  with L cells expressing only the class II M H C were not able to respond to allogeneic stimulation in the secondary response.  These results indicated that ICAM-2 may provide a necessary  costimulatory signal to the T cell that is required for the aversion of an anergic state. Endothelial cells transfected with the ICAM-2 cDNA were examined for the ability to assist leukocyte migration. A system was set up in which transendothelial migration could be  easily quantitated. It was found that a lymphocytic cell line and bone marrow neutrophils were able to utilize ICAM-2 for the migratory process without destroying the endothelial monolayer. These results demonstrate that ICAM-2 is able to play a role in two physiological processes that are of central importance to the normal function of the immune system.  iii  TABLE OF CONTENTS Abstract List of tables List of figures List of abbreviations Acknowledgments  ii v vi viii x  Chapter 1: Introduction 1:1 Cellular adhesion 1:1.1 Discovery of adhesion molecules 1:1.2 Adhesion molecules in the immune system 1:2 Leukocyte integrins 1:2.1 Nomenclature 1:2.2 Tissue distribution 1:2.3 Structure and biosynthesis 1:2.4 Chromosomal location of leukocyte integrin genes 1:3 Leukocyte adhesion deficiency (LAD) 1:4 Identification of LFA-1 counter-receptors 1:4.1 Intercellular adhesion molecule 1 (ICAM-1) 1:4.2 Intercellular adhesion molecule 2 (ICAM-2) 1:4.3 Intercellular adhesion molecule 3 (ICAM-3) 1:5 Thesis objectives 1:6 References  1 4 4 5 6 7 9 11 11 12 13 23 27 29 30  Chapter 2: Cloning 2:1 2:2 2:3 2:4 2:5  46 49 67 86 96  and characterization of murine ICAM-2 Introduction Materials and Methods Results Discussion References  Chapter 3: Costimulatory role of ICAM-2 in T cell response to allogeneic class II M H C 3:1 Introduction 104 3:2 Materials and Methods 109 3:3 Results 112 3:4 Discussion 128 3:5 References 134 a Chapter 4: Role of ICAM-2 in leukocyte transendothelial migration 4:1 Introduction 4:2 Materials and Methods 4:3 Results 4:4 Discussion 4:5 References  140 143 149 163 170  Chapter 5: Summary and Discussion 5:1 Conclusion and future directions 5:2 References  176 184  iv  LIST OF TABLES  T A B L E 1 Various members of the immunoglobulin, integrin, and selectin families involved in leukocyte function TABLE 2  Red bead fluorescence does not interfere with yellow cell fluorescence  v  3 162  LIST OF FIGURES  Figure 1  Comparison of human and murine ICAM sequences  68  Figure 2  Alignment of protein sequences from the P C R clone with human ICAM-2  70  Figure 3  Nucleic acid and protein sequence of the murine ICAM-2 c D N A (G3-1.1)  72  Figure 4  Comparison of amino acid sequence between cDNA-encoded (G3-1.1) protein and human ICAM-2  73  Figure 5  Expression of murine ICAM-2 by Northern blot analysis  75  Figure 6  Genomic Southern blot analysis of murine ICAM-2  76  Figure 7  Nucleotide sequence of the murine ICAM-2 genomic clone  78  Figure 8  Southern blot analysis of murine ICAM-2 genomic clone (BM 1-1.1)  79  Figure 9  Restriction map of the murine ICAM-2 genomic ICAM-2 clone  80  Figure 10  Immunopurification of ICAM-2  81  Figure 11  Adhesion of murine splenic T cells to purified ICAM-1 and ICAM-2  83  Figure 12  Adhesion of splenic T cells to L cells transfected with ICAM-2  84  Figure 13  Flow cytometric analysis of RT10.3 cells  113  Figure 14  Adhesion of splenic T cells to RT10.3 cells expressing ICAM-1 and ICAM-2 116  Figure 15  Schematic representation of T cell stimulation by allogeneic M H C class II and ICAM-2 Dose response of splenic T cells to RT10.3 cells expressing ICAM-1 or ICAM-2  Figure 16  Figure 17  117 119  Kinetics of the allogeneic T cell response to ICAM-1- or ICAM-2-transfected RT10.3 cells  120  Figure 18  Effects of antibodies on allogeneic T cell response to RT10.3 cells  121  Figure 19  Schematic representation of T cell response to mixed stimulators  123  Figure 20  Stimulation of T cells with RT10.3 cells and ICAM-2-transfected L cells  124  Figure 21  Schematic representation of secondary stimulation of T cells  126  vi  Figure 22  Secondary responses of T cells  127  Figure 23  Flow cytometric analysis of S V E C 4 . 1 0 cells  150  Figure 24  Immunoprecipitation of ICAM-1 and ICAM-2 from transfected S V E C 4 . 1 0 cells  151  Figure 25  Kinetics of bead diffusion across S V E C monolayer  153  Figure 26  Ability of TIL1 cells to utilize ICAM-1 and ICAM-2 for migration across the S V E C 4 . 1 0 monolayer S V E C 4 . 1 0 cells expressing ICAM-1 or ICAM-2 are able to mediate TIL1  154  transendothelial migration in an LFA-1-dependent manner  156  Figure 28  Expression of adhesion molecules on mouse bone marrow neutrophils  157  Figure 29  Neutrophil binding to S V E C 4 . 1 0 monolayers  158  Figure 30  Neutrophil migration across S V E C 4 . 1 0 monolayers  160  Figure 31  Antibody blocking of neutrophil migration across S V E C 4 . 1 0 monolayers  161  Figure 32  Simultaneous assessment of neutrophil migration and S V E C 4 . 1 0 monolayer permeability  164  Figure 27  vii  LIST OF ABBREVIATIONS  Ag APC bp BSA CAM CD cDNA CMV cpm CTL DMEM DNA dNTP DTT ECM EDTA Fab EtBr FACS FCS FITC fMLP HBSS HEV hr HSA HUVEC ICAM IFN ig IL IPTG kb kD LAD  LFA-1 LPS mAb  Mac-1 Mad MALA-2 2-ME MHC min MLR MOPS  anitgen antigen-presenting cell base pair bovine serum albumin cell adhesion molecule cluster of differentiation complementary DNA cytomegalovirus counts per minute cytolytic T lymphocyte Dulbecco's modified minimum essential medium deoxyribonucleic acid deoxynucleotidetriphosphate dithiothreitol extracellular matrix ethylenediamine tetraacetic acid antigen-binding fragment ethidium bromide fluorescence-activated cell sorter fetal calf serum fluorescein isothiocyanate f-Met-Leu-Phe Hank's balanced salt solution high endothelial venule hour heat stable antigen human umbilical vein endothelial cell intercellular adhesion molecule interferon immunoglobulin interleukin isopropylthio-p-D-galactoside kilobase kilodalton leukocyte adhesion deficiency leukocyte function-associated antigen-1 lipopolysaccharide monoclonal antibody macrophage antigen-1 mucosal addressin murine activation lymphocyte antigen-2 2-mercaptoethanol major histocompatibility complex minute mixed lymphocyte reaction 3-[N-Morpholino]-propane-sulfonic acid viii  M mRNA NCAM NK NP-40 ORF OVA PAGE PBL PBS PCR PEG PHA PMA PMSF PSGL-1 RBC RGD RNA SDS SEM SSC SSPE TAE T TcR [ H]-TdR T TE TNE Tris TWEEN-20 UTR UV VCAM VLA X-gal r  c  3  h  molecular mass messenger R N A neural cell adhesion molecule natural killer cell nonidet p-40 open reading frame ovalbumin polyacrylamide gel electrophoresis peripheral blood lymphocytes phosphate buffered saline polymerase chain reaction polyethylene glycol phytohaemagglutinin phorbol 12-myristate 13-acetate phenylmethylsulfonyl fluoride P-selectin glycoprotein ligand-1 red blood cell Arg-Gly-Asp ribonucleic acid sodium dodecyl sulphate standard error of the mean saline sodium citrate buffer saline sodium phosphate EDTA buffer Tris acetate E D T A buffer cytolytic T lymphocyte T cell receptor [methyl- H]-thymidine deoxyribose T helper lymphocyte Tris E D T A buffer Tris sodium chloride E D T A buffer tris(hydroxymethyl)aminomethane polyoxyethylene-sorbitan monolaurate untranslated region ultraviolet vascular adhesion molecule very late antigen 5-bromo-4-chloro-3-indolyl-p-D-galactoside 3  ix  ACKNOWLEDGMENTS  I would like to acknowledge the input of numerous people who have contributed to this thesis. Starting with my supervisor, Dr. Fumio Takei, for his guidance and support in leading this sometimes difficult project through the dark moments. I also thank my advisory committee, Dr. Graeme Dougherty, Dr. Keith Humphries, Dr. Dixie Mager, and Dr. Ann Rose, for critical comments and discussion. In addition, I would like to express my gratitude to Andrew Pyszniak, Mike Ohh, and Dean Thacker for their support and friendship, to Blythe Miyagawa for the work in cloning the genomic ICAM-2, and every member of the T F L who has ever answered any of my endless and sometimes thoughtless questions.  x  CHAPTER 1 INTRODUCTION  1:1  Cellular A d h e s i o n  T h e ability of cells to a d h e r e both to o n e another a n d to extracellular matrix c o m p o n e n t s is a n e s s e n t i a l requirement for maintaining organ structure a n d integrity a s well a s function.  C e l l surface m o l e c u l e s mediate t h e s e a d h e s i v e interactions a n d  a s s u c h play critical roles in d e v e l o p m e n t , tissue organization, cell migration  and  function in multicellular o r g a n i s m s ( H y n e s , 1987; E d e l m a n , 1989; Turner, 1992).  The  i m m u n e s y s t e m is c o m p r i s e d of a multiplicity of cells s p e c i a l i z e d in the recognition a n d presentation of non-self antigens.  In contrast to the rather p e r m a n e n t a d h e s i o n  o b s e r v e d in solid o r g a n s , the cells of the i m m u n e s y s t e m (leukocytes) only temporarily a d h e r e to their targets (Dustin a n d Springer, 1989; v a n K o o y k et al., 1989).  This  transient  The  a d h e s i o n is regulated by the activation state of the  expression  profile  of  cell a d h e s i o n m o l e c u l e s ( C A M s )  on  leukocytes.  leukocytes can  also  determine w h i c h counter-receptors on o p p o s i n g cells are utilized, thus affecting the extent  to  which  the  appropriate  immune  response  is  delivered.  Molecular  characterization of C A M s e x p r e s s e d by leukocytes a s well a s n o n - h e m a t o p o i e t i c cells h a s greatly a d v a n c e d the understanding of the i m m u n e r e s p o n s e . A s the list of C A M s participating in various a s p e c t s of the i m m u n e r e s p o n s e e x p a n d s , it is a p p a r e n t that t h e s e m o l e c u l e s s h a r e structural similarity.  T h e s e structurally a n d functionally related  a d h e s i o n receptors of the i m m u n e s y s t e m are g r o u p e d into three families ( T A B L E 1).  1  Collectively, t h e s e m o l e c u l e s allow cells of the immune s y s t e m to function in leukocyte recirculation, inflammation, a n d antigen-specific removal of p a t h o g e n s by mediating a d h e s i o n b e t w e e n leukocytes a n d the appropriate targets (Dustin a n d Springer, 1 9 9 1 ; B e v i l a c q u a , 1993; C a r l o s a n d H a r l a n , 1994). T h i s thesis is c o n c e r n e d with intercellular a d h e s i o n m o l e c u l e - 2 ( I C A M - 2 ) , a m e m b e r of o n e of t h e s e protein families, a n d its potential role in antigen presentation a s well a s leukocyte transendothelial migration.  2  TABLE 1  V a r i o u s m e m b e r s of the immunoglobulin, intergih, a n d selectin families involved in leukocyte function  Immunoglobulin (Ig) Superfamily: common feature is varying numbers of an Ig-like domain, which consists of 100-110 amino acids arranged in two anti-parallel p-sheets stabilized together by an intradomain disulphide bond. Mediates leukocyte adhesion to hematopoietic and non-hematopoietic cells. Important in antigen recognition and leukocyte recirculation. Expression lymphoid cells, myeloid cells, fibroblasts, endothelium, keratinocytes endothelium, lymphoid cells, myeloid cells, platelets lymphoid cells, myeloid cells  ICAM subfamily ICAM-1 ICAM-2 ICAM-3  Counter-receptors and ligands LFA-1, Mac-1, CD43, fibrinogen LFA-1 LFA-1, a p d  2  Leukocyte Integrin (p ) Family: common feature is a heterodimeric structure with one of four a-chains non-covalently associated with pychain. Expression is restricted to leukocytes. Binding capacity controlled by activation state of leukocyte playing critical role in inflammation and antigen recognition. 2  LFA-1 Mac-1 p150/95 a p d  2  Expression lymphoid cells, myeloid lymphoid cells, myeloid lymphoid cells, myeloid lymphoid cells, myeloid  cells cells cells cells  Counter-receptors and ligands ICAM-1,-2,-3 ICAM-1, fibrinogen fibrinogen ICAM-3  Selectin Family: common features include N-terminal lectin-like domain, an epidermal growth factor repeat, followed by a variable number of modules similar to complement binding proteins. Function primarily in leukocyte trafficking and inflammation.  E-selectin L-selectin P-selectin  Expression endothelium lymphoid cells, myeloid cells endothelium, platelets  Counter-receptors and ligands sialyl Lewis* , E-selectin sialyl Lewis*' , GlyCAM-1, CD34, MadCAM-1 sialyl Lewis" , PSGL-1 3  3  3  1:1.1 Discovery of adhesion molecules T h e e x i s t e n c e of C A M s w a s postulated before their actual d i s c o v e r y b a s e d on the r e a s o n i n g that a m e c h a n i s m must exist to fix cells in position giving rise to organ structure a n d thus s p e c i a l i z e d function ( E d e l m a n , 1976; E d e l m a n , 1977).  T h e initial  a p p r o a c h e m p l o y e d to identify cell a d h e s i o n m o l e c u l e s involved attempting to disrupt cellular a d h e s i o n to adjacent cells with antibodies (Muller and G e r i s c h , 1978).  It w a s  o b s e r v e d that slime mold and neural cell a g g r e g a t e s could be disrupted with F a b fragments of IgG purified from polyclonal antisera raised against the respective cell type.  T h i s disruption could then be reversed with the addition of various fractions of  solubilized m e m b r a n e proteins that allowed cellular a d h e s i o n to recur. key  evidence  that  cell  adhesion  was  mediated  by  specific  T h i s provided  membrane-bound  m o l e c u l e s . T h e next step w a s to e x a m i n e what other cell types a l s o e x p r e s s e d C A M s a n d to identify the individual C A M s responsible for the a d h e s i o n a s well a s their p o s s i b l e function.  In the c a s e of the i m m u n e s y s t e m , C A M s h a v e b e e n identified by  m o n o c l o n a l antibodies (mAb) recognizing the specific m o l e c u l e s (Kohler a n d Milstein, 1975).  1:1.2 Adhesion molecules in the immune system T h e ability of leukocytes to r e c o g n i z e and eliminate foreign invaders is crucial to the function of the i m m u n e s y s t e m .  W h e n a T lymphocyte e n c o u n t e r s a n antigen  presenting cell ( A P C ) or a target cell, there is a n initial w e a k a d h e s i o n b e t w e e n the two cells (Shortman a n d G o l d s t e i n , 1979).  If the T cell receptor (TcR) r e c o g n i z e s the  4  appropriate antigen, then the T cell will elicit its helper or cytolytic function. If the T cell d o e s not r e c o g n i z e the appropriate antigen, it will d e t a c h a n d continue circulating throughout the body.  In the i m m u n e s y s t e m , s e v e r a l lines of e v i d e n c e s u p p o r t e d the  notion that C A M s w e r e involved in immune function. antigen:MHC  S i n c e T c R recognition of  w a s thought to provide very w e a k a d h e s i o n , other m o l e c u l e s w e r e  postulated to mediate the actual a d h e s i o n (Martz, 1975;  Shortman and Goldstein,  1979; Martz, 1980). A n t i g e n - i n d e p e n d e n t a d h e s i o n of cytolytic T l y m p h o c y t e s ( C T L ) to target cells provided the first experimental e v i d e n c e that s u g g e s t e d i m m u n e cell a d h e s i o n involved a c c e s s o r y m o l e c u l e s (Martz,  1975; Z a g u r y et al., 1975). T h i s n o n -  specificity of conjugate formation b e t w e e n C T L s a n d target cells lacking t h e c o g n a t e antigen lead r e s e a r c h e r s to attempt to identify the m o l e c u l e s involved in the a d h e s i o n .  1:2 Leukocyte integrins T h e first a d h e s i o n m o l e c u l e s d i s c o v e r e d to play a role in the i m m u n e s y s t e m w e r e m e m b e r s of the integrin superfamily.  T h e s e m o l e c u l e s a r e heterodimeric cell  s u r f a c e m o l e c u l e s , e a c h mediating specific interactions with other cells or extracellular matrix c o m p o n e n t s ( H y n e s ,  1987). T h e integrin superfamily is divided into s u b f a m i l i e s  b a s e d o n o n e of three c o m m o n l y s h a r e d p subunits (p^ p , p ); although the p subunits 2  are distinct, they a r e n o n e t h e l e s s structurally similar. restricted e x p r e s s i o n of the  3  B e c a u s e of t h e leukocyte-  p leukocyte integrins (Larson a n d Springer, 1990), this 2  subfamily is involved solely in leukocyte function. M e m b e r s of the p integrins s h a r e a 2  common  p c h a i n which a s s o c i a t e s noncovalently with unique a c h a i n s ( H o g g , 1989).  5  The p  2  c h a i n ( C D 1 8 ) c a n a s s o c i a t e with either t h e oc chain ( C D 1 1 a ) , a L  (CD11b), a  x  M  chain  c h a i n ( C D 1 1 c ) , or a forming L F A - 1 , M a c - 1 , p 1 5 0 / 9 5 , o r a p , respectively d  d  2  (Corbi etal., 1987; C o r b i etal., 1 9 8 8 a ; L a r s o n etal., 1989; D a n i l e n k o e r a / . , 1 9 9 5 ; V a n der V i e r e n et al., 1995).  T h e p leukocyte integrins a r e the most extensively studied 2  family of a d h e s i o n m o l e c u l e s functioning in the immune s y s t e m . A l t h o u g h Mac-1 w a s the first p integrin to b e r e c o g n i z e d (Springer et al., 1979), 2  it w a s u s e d primarily a s a marker for myeloid cells. T h e first p leukocyte integrin to b e 2  c h a r a c t e r i z e d functionally w a s the lymphocyte function-associated antigen-1 CD11a/CD18).  (LFA-1,  It w a s first defined by a m A b s c r e e n e d for the ability to interfere with  C T L killing of target cells a n d T cell proliferation (Davignon et al., 1 9 8 1 ; K u r z i n g e r et al., 1 9 8 1 ; P i e r r e s et al., 1982).  M o r e intense examination indicated that t h e A b  b l o c k a g e o c c u r r e d at the stage of conjugate formation (between the T cell a n d t h e target cell) rather than the actual function elicited ( K r e n s k y et al., 1984; S p i t s et al., 1986).  In contrast, antibodies against the T c R a r e able to block t h e function of  cytolytic T cells but not the initial a d h e r e n c e to target cells ( K a u f m a n a n d B e r k e , 1 9 8 3 ; Martz, 1987).  T h i s provided further e v i d e n c e that there is a n initial a d h e s i o n event  w h i c h p r e c e d e s the functional activity of the leukocyte a n d b l o c k a g e of this preliminary step a b r o g a t e s leukocyte function.  1:2.1 Nomenclature T h e leukocyte integrins s h a r e both structural a n d functional similarities ( H o g g , 1989; L a r s o n a n d Springer, 1990).  T h e c o m m o n l y s h a r e d p subunit h a s b e e n 2  6  a s s i g n e d to the cluster of differentiation ( C D ) 1 8 a n d the a c h a i n s of L F A - 1 , M a c - 1 , a n d p150/95  have  been  assigned  the designations  CD11a,  CD11b,  and  CD11c,  respectively. T h e recently d i s c o v e r e d a chain of a (3 (Danilenko et al., 1995) h a s yet d  2  to b e a s s i g n e d a cluster of differentiation.  1:2.2 Tissue Distribution T h e distribution of L F A - 1 is restricted to cells of hematopoietic origin.  L F A - 1 is  e x p r e s s e d by most leukocytes (Kurzinger et al., 1981; K r e n s k y et al., 1983), with the e x c e p t i o n of s o m e murine tissue m a c r o p h a g e s ( S t r a s s m a n et al., 1985). e x p r e s s e d o n ~ 5 0 % of b o n e marrow cells.  L F A - 1 is  It is a l s o present o n the majority of  t h y m o c y t e s , neutrophils, m a c r o p h a g e s , m o n o c y t e s , a n d peripheral l y m p h o c y t e s . L F A 1 e x p r e s s i o n is a b s e n t or low on myeloid a n d erythroid precursor cells (Miller et al., 1 9 8 5 ; C a m p a n a et al., 1986).  In B cell a n d myeloid l i n e a g e s , L F A - 1 first a p p e a r s in  the p r e - B cell a n d late myeloblast s t a g e s , respectively.  H o w e v e r , L F A - 1 m a y be lost  from B cells during terminal differentiation to p l a s m a cells ( M i e d e m a et al., 1985). S i n c e most leukocytes e x p r e s s L F A - 1 , it is not surprising that antibodies a g a i n s t L F A - 1 h a v e b e e n s h o w n to inhibit a multitude of immune functions by blocking a d h e s i o n to counter-receptors o n o p p o s i n g cells (Springer et al., 1987; L a r s o n a n d Springer, 1 9 9 0 ; S p r i n g e r , 1990).  Essentially every leukocyte function requiring a d h e s i v e interactions  c a n in s o m e w a y be abrogated with L F A - 1 A b s . T h e distribution of Mac-1 is more restricted than that of L F A - 1 . Mac-1  is  confined  to  monocytes,  macrophages,  7  granulocytes,  E x p r e s s i o n of large  granular  l y m p h o c y t e s , a n d immature B cells (Flotte et al., 1983; S p r i n g e r a n d U n k l e s s , 1984; d e la H e r a et al.,  1988).  Mac-1 is a l s o e x p r e s s e d on C D 5  activated killer cell precursors. ligands.  B cells a n d  lymphokine  receptor that c a n bind  Initially, a n anti-Mac-1 A b w a s s h o w n to block m o n o c y t e a n d  binding to erythrocytes 1982).  It is a multifunctional  +  multiple  granulocyte  coated with C 3 b i , a c o m p l e m e n t c o m p o n e n t (Beller et  al.,  Eventually, Mac-1 w a s demonstrated to bind any cell type c o a t e d with C 3 b i  (Rothlein a n d Springer, 1985; R a m o s et al., 1988).  U n d e r certain conditions, Mac-1  c a n a l s o bind a n L F A - 1 ligand (Diamond et al., 1991).  Mac-1 h a s a l s o b e e n reported  to bind the s o l u b l e ligand fibrinogen (Wright et al., 1988). T h e s e a d h e s i v e interactions are  important  aggregation  of  in  myeloid  cell function.  neutrophils,  granulocyte  Anti-Mac-1  Abs  transendothelial  can  block  migration,  as  homotypic well  as  m o n o c y t e a n d neutrophil c h e m o t a x i s and a d h e r e n c e ( A n d e r s o n et al., 1986; D a n a et al., 1986; W a l l i s etal., 1986). T h e p 1 5 0 / 9 5 protein is similar to Mac-1 in distribution. activated l y m p h o c y t e s .  Like M a c - 1 , p 1 5 0 / 9 5 c a n also act a s a C 3 b i receptor (Micklem  a n d S i m , 1 9 8 5 ; Malhorta et al. , 1986). molecule.  It is a l s o found on s o m e  It a l s o probably functions a s a cell a d h e s i o n  A n t i b o d i e s against p 1 5 0 / 9 5 are able to inhibit neutrophil a n d  peripheral  blood m o n o c y t e binding to endothelium, phagocytosis, and c h e m o t a x i s ( A n d e r s o n al.,  1986; K e i z e r et  al.,  1987a).  In s o m e c a s e s , the p 1 5 0 / 9 5  protein  et  can also  contribute a d h e s i v e strength in C T L conjugate formation with target cells d e p e n d i n g on its e x p r e s s i o n levels on the activated T cells and lymphoid cell lines ( K e i z e r et 1987b).  8  al.,  The a p d  2  m o l e c u l e h a s b e e n detected o n peripheral blood l e u k o c y t e s .  Human  l y m p h o c y t e s e x p r e s s low levels of this molecule ( V a n der V i e r e n et al., 1995), w h e r e a s only a s m a l l s u b s e t of c a n i n e C D 8 Human granulocytes also express  T cells e x p r e s s a p  +  d  which c a n b e upregulated from a n intracellular  a p , d  2  pool by f - M L P treatment of the granulocytes. s p l e e n h a s r e v e a l e d that a p d  2  (Danilenko et al., 1995).  2  Immunohistological staining of h u m a n  e x p r e s s i o n is localized to the red pulp c o r d s a n d s i n u s e s  on s m a l l a n d large m o n o n u c l e a r cells a n d granulocytes. white pulp is limited to scattered dendritic cells. blocking h a s yet to b e d o n e .  E x p r e s s i o n of a p d  in the  2  Functional inhibition by antibody  H o w e v e r , the predominant e x p r e s s i o n of a p d  s p e c i a l i z e d cells in t i s s u e s s u g g e s t s that the major functions of a p d  2  2  on  m a y b e restricted  to s p e c i f i c microenvironments.  1:2.3 Structure and biosynthesis T h e two c o m p l e m e n t a r y D N A s ( c D N A ) e n c o d i n g the p a n d the a 2  L  subunits  h a v e b e e n isolated a n d characterized in both the h u m a n (Kishimoto et al., 1 9 8 7 a ; L a w et al., 1987; L a r s o n etal., 1989) a n d murine s y s t e m s (Wilson et al., 1989; K a u f m a n n et T h e s e two c D N A s e n c o d e s e p a r a t e subunit precursors of 7 2 k D ( p ) a n d  al., 1991).  2  130 k D ( a ) . L  two  bare  High m a n n o s e N-glycoside carbohydrate groups are then a d d e d to t h e s e  polypeptide  b a c k b o n e s (Sastre et al., 1986a).  Heterogeneity  in the  glycosylation pattern b e t w e e n cells h a s b e e n o b s e r v e d ; N-linked c a r b o h y d r a t e s a r e sulfated only o n T cells ( D a h m s a n d Hart, 1985). In addition, sialylation patterns differ b e t w e e n B a n d T cells ( T a k e d a , 1987).  9  In order to further p r o c e s s t h e g l y c o s y l a t e d  p r e c u r s o r s to the c o m p l e x type N-linked carbohydrate structures, the p  a r | 2  d the a  subunits must first a s s o c i a t e intracellular^ (Ho a n d Springer, 1983; S p r i n g e r et  L  al.,  1984). T h i s o l i g o s a c c h a r i d e modification o c c u r s in the G o l g i a p p a r a t u s from w h i c h the mature L F A - 1 m o l e c u l e s are transported to the cell surface. T h e d e d u c e d a m i n o acid s e q u e n c e s b a s e d on the c D N A s e q u e n c e s indicates that the a a n d the p subunits h a v e the c l a s s i c a l features of integral type I m e m b r a n e L  2  proteins (Kishimoto et al., 1 9 8 7 a ; L a w et al., 1987; L a r s o n et al., 1989). large  extracellular  domains  with  multiple  potential The p  transmembrane and cytoplasmic domains.  2  N-glycosylation  Both h a v e sites,  subunit has a n unusually  short high  c y s t e i n e content (7.4%) concentrated over a four-fold repeat region (20%) s p a n n i n g 186 a m i n o acid residues near the C-terminus (Kishimoto et al., 1990). that this g i v e s the p subunit a rigid tertiary globular structure. 2  striking h o m o l o g y with the oc a n d a M  x  It is believed  T h e oc subunit s h a r e s L  subunits (44% in human). A significant feature of  a subunits is the three t a n d e m repeats w h i c h contain putative divalent cation binding sites (Kishimoto et al., 1990). T h e s e d o m a i n s are similar to the C a loop" sites in c a l m o d u l i n , troponin C , a n d parvalbumin.  + +  binding " E F - h a n d  T h e s e m a y be the  putative  metal-binding sites responsible for the M g - d e p e n d e n t a d h e s i o n d i s p l a y e d by the ++  leukocyte integrins (Rothlein a n d Springer, 1986).  A n o t h e r key feature of the ot  L  subunit is the p r e s e n c e of an I d o m a i n which h a s b e e n s p e c u l a t e d to be directly involved in binding to counter structures on o p p o s i n g cells ( R a n d i a n d H o g g , 1994; H u a n g a n d Springer, 1995).  10  1:2.4 Chromosomal location of leukocyte integrin genes T h e c h r o m o s o m a l localization of the g e n e s e n c o d i n g the h u m a n integrins h a s provided s o m e interesting results.  leukocyte  T h e a subunits a , a , a n d a L  M  x  were  localized to the s a m e band on c h r o m o s o m e 16 (16p11.1-p13) (Corbi et al., 1988b); the location of the a  d  subunit has not b e e n m a p p e d .  This defined a cluster of g e n e s  belonging to the s a m e family of proteins involved in leukocyte a d h e s i o n .  Isolation of  the g e n o m i c c l o n e s of the a subunits h a s revealed that t h e s e g e n e s h a v e a n a l o g o u s intron/exon organization (Sastre et al., 1986b; C o r b i et al., 1990; W o n g et al., T h e protein homology, exon/intron boundary similarity, a n d proximal  1996).  chromosomal  location s u g g e s t s that the leukocyte integrins a r o s e by g e n e duplication of a n a n c e s t r a l a subunit. T h e h u m a n p g e n e is located on c h r o m o s o m e 21 a n d not found n e a r other 2  p g e n e s (Corbi etal., 1988b).  1:3 Leukocyte Adhesion Deficiency (LAD) Patients with a n inherited leukocyte integrin-dependent i m m u n o d e f i c i e n c y h a v e b e e n identified ( A n d e r s o n a n d Springer, 1987).  T h e s e patients consistently exhibit a  defect in a d h e r e n c e - d e p e n d e n t leukocyte functions a n d suffer from recurrent threatening  bacterial  and  fungal  infections  (Fischer  et  al.,  1988).  Symptoms  a s s o c i a t e d with this d i s e a s e include recurrent gingivitis, defective neutrophil and  phagocytosis,  lack  of  pus  formation,  and  absence  of  life-  mobility  lymphocytes  and  g r a n u l o c y t e s in infected lesions despite chronic leukocytosis. T h e defect in leukocyte function w a s found to be s e c o n d a r y to an abnormality in a d h e s i o n ( C r o w l e y et  11  al.,  1980).  L e u k o c y t e s from t h e s e patients w e r e found to lack e x p r e s s i o n of  leukocyte integrins ( L F A - 1 , M a c - 1 , p150/95) (Springer et al., 1984; Beatty et al., 1984).  three  1984; A r n a o u t et  al.,  However, levels of cell surface e x p r e s s i o n vary b e t w e e n  patients a n d correlates with the severity of the d i s e a s e .  M o d e r a t e l y deficient patients  m a y survive to adulthood if their recurrent infections are a d e q u a t e l y treated while the s e v e r e l y deficient patients require a b o n e marrow transplant to extend life e x p e c t a n c y past early childhood ( F i s c h e r et al.,  1990).  T h e deficiency in L F A - 1 , M a c - 1 ,  and  p 1 5 0 / 9 5 e x p r e s s i o n (a (3 not e x a m i n e d ) w a s traced to a b n o r m a l e x p r e s s i o n of the p d  2  2  subunit (Marlin et al., 1986; Kishimoto e r a / . , 1987b). Normal a m o u n t s of t h e s e three a c h a i n p r e c u r s o r s are detected in patient cells ( L i s o w s k a - G r o s p i e r r e et  al.,  1986).  H o w e v e r , a b s e n c e of a functional p subunit prevents proper intracellular a s s o c i a t i o n 2  of the heterodimers, w h i c h is required for c o m p l e t e p r o c e s s i n g a n d transport to the cell surface.  E x p r e s s i o n of t h e s e three integrins c a n be r e s c u e d by introduction of the  h u m a n or murine p c D N A e n c o d i n g a protein w h i c h c a n a s s o c i a t e with any of the 2  h u m a n a c h a i n precursors (Hibbs et al., 1990).  T h e r e s c u e d m o l e c u l e s function in  exactly the s a m e m a n n e r that they do on normal leukocytes.  1:4 Identification of LFA-1 counter-receptors Stimulated lymphoid cells form homotypic a g g r e g a t e s that are L F A - 1 - d e p e n d e n t (Rothlein a n d Springer, 1986).  L y m p h o i d cells from L A D patients d o not form t h e s e  a g g r e g a t e s s i n c e t h e s e cells d o not e x p r e s s L F A - 1 . (LFA-1") are c o m b i n e d with normal L F A - 1  12  +  However, when L A D lymphocytes  lymphoid cells, c o a g g r e g a t e s form in a n  L F A - 1 - d e p e n d e n t manner.  T h i s lead investigators to s e a r c h for L F A - 1  counter-  receptors o n L A D cells w h i c h eventually lead to the d i s c o v e r y of a family of L F A - 1 receptors o n various cell types.  1:4.1 Intercellular Adhesion Molecule-1 (ICAM-1) The  observation  that  stimulated  lymphocytes  were  able  to  adhere  to  l y m p h o c y t e s from L A D patients in an L F A - 1 - s p e c i f i c m a n n e r s u g g e s t e d that there exists a structure on L A D cells which could interact with L F A - 1 (Rothlein a n d Springer, 1986).  In order to identify the L F A - 1 counter-receptor, m A b s raised against L A D cells  w e r e s c r e e n e d for their ability to block the L F A - 1 - d e p e n d e n t aggregation of stimulated lymphoid cells (Rothlein et al., 1986).  T h e first m A b obtained from this p r o c e d u r e  r e c o g n i z e d a heavily glycosylated protein of ~ 8 6 - 1 1 4 k D that w a s s u b s e q u e n t l y termed intercellular a d h e s i o n m o l e c u l e 1 ( I C A M - 1 , C D 5 4 ) .  Murine I C A M - 1 w a s  similarly  identified using a m A b that inhibited an in vitro mixed lymphocyte reaction ( M L R ) (Takei, 1985).  Unlike L F A - 1 , I C A M - 1 e x p r e s s i o n is more widely distributed.  d e t e c t e d on cells of both hematopoietic a n d nonhematopoietic origin (Dustin et 1986).  It is al.,  A l t h o u g h e x p r e s s i o n m a y be very low on nonhematopoietic cells a n d resting  lymphoid cells, I C A M - 1 c a n be greatly upregulated on t h e s e cells by various cytokines (Dustin  et  al.,  1986; Dustin et  al.,  1988a).  This i n c r e a s e d I C A M - 1  expression  correlates with i n c r e a s e d L F A - 1 - d e p e n d e n t a d h e s i o n of l y m p h o c y t e s to i n d u c e d cells. I C A M - 1 e x p r e s s i o n is a l s o closely coordinated with the p r o g r e s s i o n of the  13  immune  r e s p o n s e indicating that the L F A - 1 : I C A M - 1 interaction plays very important roles in all a s p e c t s of the i m m u n e s y s t e m .  1:4.1 a) ICAM-1 cDNA cloning T h e g e n e s coding for both the h u m a n ( S i m m o n s et al., 1988; S t a u n t o n et  al.,  1988) a n d murine I C A M - 1 (Horley et al., 1989; S i u et al., 1989) h a v e b e e n c l o n e d a n d sequenced. kD.  I C A M - 1 is a t r a n s m e m b r a n e protein with a polypeptide b a c k b o n e of 5 5  Both the h u m a n a n d m o u s e s p e c i e s h a v e multiple potential glycosylation sites  w h i c h are extensively utilized. T h e d e g r e e of glycosylation c a n vary d e p e n d i n g on the cell  type  as  evidenced  by  the  size  variation  (85-115  kD)  seen  immunoprecipitated product (Dustin et al., 1986; M a k g o b a et al., 1988).  in  the  The amino  acid s e q u e n c e of I C A M - 1 s h a r e s sufficient homology with other protein s e q u e n c e s a n d harbors  a common  repeated  structural  motif to  place it in the  s u p e r g e n e family (Williams, 1987; Williams a n d Barclay, 1988).  immunoglobulin  M e m b e r s h i p within  this superfamily is b a s e d on the p r e s e n c e of a c o n s e r v e d tertiary protein structure, a n Ig-like structural motif. T h e Ig-like d o m a i n consists of approximately 110 a m i n o a c i d s w h i c h fold in two anti-parallel p-sheets.  Five Ig-like d o m a i n s are present in the  extracellular portion of I C A M - 1 followed by short t r a n s m e m b r a n e a n d c y t o p l a s m i c regions ( S i m m o n s et al., 1988; Staunton et al., 1988; Horley et al., 1989; S i u et  al.,  1989) . In addition to t h e s e d o m a i n s , I C A M - 1 a l s o displays overall structural h o m o l o g y with two  unique C A M s ,  m o l e c u l e (Dustin et al.,  myelin a s s o c i a t e d glycoprotein 1988b), bearing multiple  14  a n d neural cell a d h e s i o n  Ig-like d o m a i n s .  The  homology  b e t w e e n h u m a n a n d murine ICAM-1 is s o m e w h a t limited ( 6 5 % nucleotide homology, 5 0 % a m i n o acid homology).  G e n o m i c cloning has revealed that t h e five Ig-like  d o m a i n s of I C A M - 1 are e n c o d e d by s e p a r a t e e x o n s (Voraberger et al., 1 9 9 1 ; Degitz et al., 1991). T h e h u m a n I C A M - 1 g e n e has b e e n m a p p e d to c h r o m o s o m e 19 (Katz et al., 1985) a n d the murine I C A M - 1 h a s b e e n m a p p e d to c h r o m o s o m e 9 (Ballantyne et al., 1991).  Identification of I C A M - 1 a s a ligand for L F A - 1 w a s the first e x a m p l e of a n  interaction occuring b e t w e e n superfamilies functioning in the i m m u n e s y s t e m (Marlin a n d Springer, 1987).  1:4.1  b) T i s s u e d i s t r i b u t i o n a n d c y t o k i n e i n d u c t i o n  In contrast to its counter-receptor, L F A - 1 , I C A M - 1 e x p r e s s i o n is not restricted to cell of the hematopoietic lineage.  I C A M - 1 has b e e n detected at low levels o n cells  including resting leukocytes, follicular dendritic cells, fibroblasts, v a s c u l a r e n d o t h e l i u m , keratinocytes, synovial cells, a n d certain epithelial cells (Dustin et al., 1986; P o b e r et al., 1986; te V e l d e et al., 1987; Dustin et al., 1 9 8 8 a ; M e n t z e r et al., 1988; Rothlein et al., 1988; Dustin a n d Springer, 1988a).  H o w e v e r , I C A M - 1 levels c a n be dramatically  i n c r e a s e d by various cytokines (Dustin et al., 1986; P o b e r et al., 1986; Rothlein et al., 1988).  T h i s is most prominent on endothelial cells, fibroblasts, m e s e n c h y m a l cells,  a n d epithelial cells (Dustin etal., 1986; W a n t z i n etal., 1989; M u n r o etal., 1989; Dustin a n d Springer, 1991).  C y t o k i n e s s u c h a s 1 ( 3 , T N F - a , IFN-y, a n d L P S a r e pro-  inflammatory a n d c a u s e the upregulation of I C A M - 1 on cells a b l e to receive the signal m e d i a t e d by t h e s e m o l e c u l e s (Dustin et al., 1986; Rothlein etal., 1988). It is of interest  15  to note that the cytokine receptors which c a u s e I C A M - 1 upregulation a l s o c a u s e the e x p r e s s i o n of other g e n e s . O n e e x a m p l e of this is IFN-y induction of both I C A M - 1 a n d M H C c l a s s II ( O ' C o n n e l l a n d E d i d i n , 1990; Farrar a n d S c h r e i b e r , 1993).  T h i s allows  application of T helper (T ) function to appropriate target cells s i n c e both a d h e s i o n h  b e t w e e n the two cells a n d efficient antigen presentation are required for T (Springer,  1990; Dustin a n d Springer, 1991).  h  function  E x p r e s s i o n of I C A M - 1 a l s o plays a  significant role in inflammation a n d migration of leukocytes out of the b l o o d s t r e a m a n d into the sites of infection ( d i s c u s s e d in chapter 4) ( B e v i l a c q u a , 1 9 9 3 ; C a r l o s a n d H a r l a n , 1994).  T h i s upregulation usually requires de novo s y n t h e s i s (Rothlein et  al.,  1988; J o h n s o n et al., 1989) but in certain instances rapid mobilization of I C A M - 1 from intracellular stores h a s b e e n o b s e r v e d (Dougherty et al., 1988). a l s o b e e n o b s e r v e d on lymphocytes.  I C A M - 1 induction h a s  R e s t i n g B a n d T cells lack d e t e c t a b l e I C A M - 1  e x p r e s s i o n (Dustin et al., 1986; Clark et al., 1986; W a w r y k et al., e x p r e s s i o n is i n c r e a s e d during efficient interaction with L F A - 1  +  activation  of the l y m p h o c y t e s  1989).  thus  However,  allowing  more  cells a n d coordination of the i m m u n e r e s p o n s e s i n c e  l y m p h o c y t e s must c o m m u n i c a t e with e a c h other to e n s u r e a r e s p o n s e .  1:4.1 c) Physiological significance of LFA-1 MCAM-1 interaction T h e formal proof that I C A M - 1 w a s a counter-receptor for L F A - 1 c a m e from s t u d i e s using artificial m e m b r a n e s bearing purified h u m a n I C A M - 1 protein (Marlin a n d Springer, 1987). bind L F A - 1  +  I C A M - 1 protein incorporated into planar lipid bilayers w a s a b l e to  cells. T h e a d h e s i o n w a s specifically inhibited by pretreating the cells with  16  anti-LFA-1 mAb or anti-ICAM-1 mAb treatment of the membranes. Such adhesion was found to require divalent cations such as M g  ++  or C a  + +  since the chelating agent EDTA  would block the adhesion. This is in agreement with the sequence data of LFA-1 which indicates the presence of a divalent cation binding domain within the extracellular region of the a subunit (Larson et al., 1989). L  Energy and functional  microfilaments are also required for binding as demonstrated by sodium azide together with 2-deoxy-D-glucose which block energy production and T lymphoblastoid binding to ICAM-1-bearing membranes (Marlin and Springer, 1987).  Cell binding is  temperature dependent, being maximal at 37°C, reduced at 14°C, and completely inhibited at 4°C. The adhesion is also inhibited by cytochalasin B, a microfilament inhibitor.  It has been found that LFA-1 is indeed attached to several cytoskeletal  proteins (Burn et al., 1988; Kupfer et al., 1990; Pavalko and LaRoche, 1993). Reciprocal studies using purified LFA-1 incorporated into membranes do not mimic the same binding requirements (Dustin and Springer, 1989; Kishimoto et al., 1990). This indicates that the active, dynamic process is occurring on the LFA-1-bearing cell side of the interaction. Several integrins which act as receptors for extracellular matrix components bind  their  ligands  through  Arg-Gly-Asp (RGD)  sequences  (Ruoslahti  and  Pierschbacher, 1986; Hynes, 1987). Oligopeptides containing the RGD sequence can block the interaction between these integrins and their ligands. The human ICAM-1 does not contain an RGD sequence, but does contain several RGD-like sequences (Staunton et al., 1988).  However, the RGD and RGD-like oligopeptides could not  17  inhibit binding of L F A - 1 cells to I C A M - 1 m e m b r a n e s (Marlin a n d Springer, 1987). This +  s u g g e s t s that leukocyte integrin binding specificity h a s diverged from that of other integrins. L e u k o c y t e s bearing L F A - 1 circulate throughout the body a s single unattached cells e v e n though they continuously e n c o u n t e r I C A M - 1 later o b s e r v e d that in order for L F A - 1  +  cells.  It w a s s u s p e c t e d a n d  cells to a d h e r e to I C A M - 1  +  +  cells, the L F A - 1  +  cells n e e d to be in an activated state (Dustin a n d Springer, 1989; v a n K o o y k et al., 1989).  L y m p h o c y t e activation by P M A , or antibody triggering through C D 3 or T c R  crosslinking results in a strongly i n c r e a s e d L F A - 1 - d e p e n d e n t a d h e s i v e n e s s .  The  i n c r e a s e d a d h e s i o n following C D 3 or T c R antibody crosslinking a p p e a r e d almost immediately after activation a n d returned to normal after 30 min. speculated  that  this  relatively  quick  increased  adhesion  may  It h a s b e e n be  due  to  a  conformational c h a n g e in L F A - 1 e x p o s i n g a binding site. E v i d e n c e for this hypothesis c o m e s from a m A b directed against a n L F A - 1 epitope w h i c h is only detected w h e n the l y m p h o c y t e is activated (Dransfield a n d H o g g , 1989). disappears when M g  + +  E x p r e s s i o n of this epitope  is r e m o v e d , temperature is lowered, or metabolic inhibitors are  present. R e l e a s e of a low m o l e c u l a r weight lipid called integrin modulating factor (IMF) h a s b e e n reported to a l s o induce i n c r e a s e d a d h e s i o n ( H e r m a n o s k i - V o s a t k a et 1992) a n d p o s s i b l e conformational c h a n g e . controlled by the redistribution  al.,  T h e i n c r e a s e d a d h e s i o n m a y a l s o be  of L F A - 1 or I C A M - 1 on the cell s u r f a c e .  LFA-1  localization h a s b e e n detected at regions of focal contact with I C A M - 1 (Kupfer a n d S i n g e r , 1 9 8 9 a ; Kupfer a n d Singer, 1989b).  18  In another study, L F A - 1 e x p r e s s i o n w a s  found to b e uniformly distributed o n the cell surface, however, avid L F A - 1 (able to bind I C A M - 1 - c o a t e d b e a d s ) w a s only detected at localized regions ( P y s z n i a k et al., 1994). A s well, I C A M - 1 e x p r e s s i o n localized to uropods has a l s o b e e n o b s e r v e d (Dougherty etal., 1988). T h e L F A - 1 :ICAM-1 attachment is a highly regulated interaction a n d plays a major role in a d h e s i o n - d e p e n d e n t immunological r e s p o n s e s . A n t i b o d i e s against L F A - 1 or I C A M - 1 h a v e b e e n s h o w n to interfere with a multitude of i m m u n e r e s p o n s e s (Martz, 1987; S p r i n g e r et al., 1987; Dustin a n d Springer, 1991).  In addition to blocking C T L -  m e d i a t e d killing of target cells (Davignon et al., 1 9 8 1 ; P i e r r e s et al., 1982; D i a l y n a s et al., 1982), antibodies against L F A - 1 a n d I C A M - 1 a r e a l s o able to block natural killer (NK)  cell-mediated  cytotoxicity  a n d antibody-dependent  cytotoxicity  m e d i a t e d by  g r a n u l o c y t e s a n d peripheral blood m o n o n u c l e a r cells (Kohl et al., 1984; M i e d e m a et al., 1984; S c h m i d t et al., 1985).  T h e s e two antibodies together h a v e b e e n s h o w n to  inhibit virtually every a d h e s i o n - d e p e n d e n t immune r e s p o n s e . killer  ( L A K ) cell  Staphylococcus antibodies.  mediated  cytolysis  by neutrophils  (Nishimura  ( R o s s et  et  al.,  Lymphokine-activated  1985) a n d ingestion  al., 1985) a r e a l s o b l o c k e d  of  by t h e s e  T h e L F A - 1 :ICAM-1 interaction is a l s o involved in T lymphocyte functions.  T h e s e antibodies  block T cell proliferation  in r e s p o n s e to v i r u s e s ,  alloantigens,  x e n o a n t i g e n s , a n d mitogens (Davignon et al., 1 9 8 1 ; Pierres et al., 1982; K r e n s k y et al., 1 9 8 3 ; Hildreth a n d A u g u s t , 1985; Dougherty a n d H o g g , 1987).  T cell-dependent  antibody r e s p o n s e s by B cells are a l s o inhibited by anti-LFA-1 m A b s a s is interleukin-2 (IL-2) production by T cell hybrids stimulated by a l l o g e n e i c A P C s ( D a v i g n o n et al.,  19  1 9 8 1 ; K a u f m a n a n d B e r k e , 1983; G o l d e et al., 1985; H o w a r d et al., 1986; F i s c h e r et al.,  1986).  T h e inhibition experiments are consistent with the notion that L F A - 1 is  involved in the strengthening of cellular a d h e s i o n which facilitates the e x e c u t i o n of the i m m u n e r e s p o n s e . In the c a s e of lymphocyte proliferation or cytolysis, the a n t i - L F A - 1 m A b s c a n c a u s e dissociation of preformed conjugates a s long a s the antibodies are a d d e d no later than two hours after the initial contacts are m a d e ( K r e n s k y et al., 1984; Spits et al., 1986).  After s u c h a time, the antibodies cannot inhibit t h e s e functions,  further supporting the idea that L F A - 1 : I C A M - 1 interactions are involved in the induction of proliferation or cytotoxicity rather than the p r o c e s s itself. In addition to the i m m u n e r e s p o n s e s mentioned a b o v e , L F A - 1 a n d I C A M - 1 play significant roles in other functions. Transendothelial migration of l e u k o c y t e s h a s b e e n s h o w n to h a v e a n L F A - 1 - a n d I C A M - 1 - d e p e n d e n t c o m p o n e n t ( O p p e n h e i m e r - M a r k s et al., 1 9 9 1 ; K a v a n a u g h et al., 1 9 9 1 ; Furie et al., 1 9 9 1 ; B e v i l a c q u a , 1993; C a r l o s a n d H a r l a n , 1994). A n t i b o d i e s blocking t h e s e interactions inhibit leukocyte binding to a n d s u b s e q u e n t migration a c r o s s endothelial cells. Administration of blocking antibodies to rabbits h a s a l s o interfered with leukocyte migration (Barton et al., 1989; D o e r s c h u k et al., 1990; L o etal., 1992). T h e L F A - 1 a n d I C A M - 1 antibody blocking studies are in a g r e e m e n t with the results obtained from genetically deficient  mice.  I C A M - 1 deficient  m i c e display  a b n o r m a l inflammatory r e s p o n s e s a n d i n c r e a s e d n u m b e r s of circulating a n d l y m p h o c y t e s (Sligh et al., 1993; X u et al., 1994). neutrophil  migration  T h e s e mice exhibit impaired  in r e s p o n s e to c h e m i c a l peritonitis  20  neutrophils  a s well a s d e l a y e d type  hypersensitivity.  T h e mutant cells from t h e s e animals failed to stimulate a l l o g e n e i c  l y m p h o c y t e s in a M L R , although they proliferated normally a s r e s p o n d e r cells.  ICAM-1  deficient m i c e w e r e a l s o resistant to septic s h o c k , by reducing either T cell activation or neutrophil infiltration.  M i c e deficient for the (3 subunit display similar impairment in the 2  inflammatory r e s p o n s e to c h e m i c a l peritonitis (Wilson et al., 1 9 9 3 ; Bullard et al., 1996). T h e s e m i c e a l s o display d e l a y s in transplantation rejection.  L F A - 1 - d e f i c i e n t mice a l s o  exhibited defective leukocyte function (Schmits et al., 1996). L y m p h o c y t e s d i s p l a y e d a d e c r e a s e d r e s p o n s e in a n M L R . was also reduced.  T h e inflammatory r e s p o n s e to c h e m i c a l  T h e s e mice w e r e a l s o not able to reject grafted  peritonitis  immunogenic  tumors. Perturbation  of  normal  adhesion  between  LFA-1  and  ICAM-1  has  been  d e m o n s t r a t e d to c o i n c i d e with the metastatic capacity of certain cells. T h i s a d h e s i v e interaction allows adjacent cells to remain attached.  H o w e v e r , if the L F A - 1 :ICAM-1  binding is not present, the total a d h e s i o n is l e s s e n e d and the likelihood that the cells c a n d i s s o c i a t e is i n c r e a s e d . In B-lymphoid tumors, I C A M - 1 e x p r e s s i o n correlates with m e t a s t a s i s ( B o y d et al., 1989; W a w r y k et al., 1989).  L a r g e B cell tumors w h i c h form  bulky, solitary m a s s e s consistently exhibit intermediate to strong I C A M - 1 e x p r e s s i o n . C o n v e r s e l y , l y m p h o m a cells which s h o w a diffuse, infiltrative, n o n - a d h e s i v e pattern of m e t a s t a s i s exhibit little or no I C A M - 1 e x p r e s s i o n . In contrast to B cell tumors, I C A M - 1 e x p r e s s i o n s e e m s to be a n indicator of metastatic progression of m e l a n o m a cells ( J o h n s o n et  al.,  1988; J o h n s o n et al.,  1989).  I C A M - 1 is not normally found  on  q u i e s c e n t m e l a n o c y t e s or benign melanocytic lesions. H o w e v e r , a n i n c r e a s e in tumor  21  s i z e is a c c o m p a n i e d by a n upregulation  in I C A M - 1 and results  in a n  increased  probability of metastasis. This i n c r e a s e d I C A M - 1 e x p r e s s i o n m a y be a s i d e effect of cytokines  released  from  lesion-infiltrating  m e l a n o m a cells m a y interact with L F A - 1  +  lymphocytes.  As  well,  the  ICAM-1  +  recruited leukocytes c a u s i n g a reduction in  homotypic aggregation between m e l a n o m a cells. T h e d i s s o c i a t e d m e l a n o m a cells c a n attach to the L F A - 1 m o l e c u l e s on the mobile leukocytes present in the lesion. E x p r e s s i o n of I C A M - 1 on l y m p h o m a s may not only affect dissociation from the primary tumor, but a l s o e s c a p e from the i m m u n e surveillance of circulating leukocytes. Burkitt's l y m p h o m a cells downregulate L F A - 1 and I C A M - 1 ( C l a y b e r g e r et al.,  1987;  G r e g o r y et al., 1988). It is thought that lack of t h e s e a d h e s i o n m o l e c u l e s on the tumor cells m a y contribute to their inability to initiate an effective i m m u n e r e s p o n s e thus leading to e s c a p e from immunosurveillance.  This is supported by the o b s e r v a t i o n that  L F A - 1 a n d I C A M - 1 r e a p p e a r on t h e s e tumor cells after s e v e r a l p a s s a g e s in culture a n d b e c o m e sensitive to lysis by E p s t e i n - B a r r virus ( E B V ) - s p e c i f i c C T L s . In addition to its role in mediating cellular contact in the i m m u n e s y s t e m , I C A M - 1 h a s a l s o b e e n s h o w n to function in other capacities. It h a s b e e n s h o w n , for e x a m p l e , to s e r v e a s a receptor for the major serotype of rhinoviruses ( G r e v e et al., Staunton  et  al.,  1 9 8 9 a ; T o m a s s i n i et  al.,  1989).  R h i n o v i r u s e s are the  c a u s a t i v e agent of the c o m m o n cold (Sperber and H a y d e n , 1988). infect  cells  expressing  transmembrane  ICAM-1.  A  soluble  and c y t o p l a s m i c d o m a i n s  infections v i a I C A M - 1 (Marlin et al., 1990).  22  version  of  1989; primary  Viroid particles  ICAM-1  lacking  is able to specifically block  the  rhinovirus  In addition to the c o m m o n c o l d , I C A M - 1  a l s o acts a s a receptor for Plasmodium falciparum infected erythrocytes (Berendt et al., 1989). T h e initial event in the p a t h o g e n e s i s of malaria is the a d h e r e n c e of infected erythrocytes to endothelium in the liver.  In malaria, elevated levels of cytokines h a v e  b e e n d e t e c t e d , a n d t h e s e cytokines a r e a l s o responsible for the induction of I C A M - 1 e x p r e s s i o n o n resting endothelium. Similar to rhinovirus infection, a n i m m u n o a d h e s i n v e r s i o n of I C A M - 1 is a b l e to inhibit P. falciparum-\nfected  erythrocyte a d h e s i o n to  I C A M - 1 s u r f a c e s (Staunton etal., 1992).  1:4.2 Intercellular Adhesion Molecule-2 (ICAM-2) 1:4.2 a) ICAM-2 cDNA cloning A l t h o u g h I C A M - 1 interaction with L F A - 1 plays a major role in the overall function of the i m m u n e s y s t e m , it is not the only L F A - 1 - d e p e n d e n t interaction.  Initially, it w a s  o b s e r v e d that the L F A - 1 - d e p e n d e n t binding of T cells to endothelial cells contains both a n I C A M - 1 - d e p e n d e n t a n d a n I C A M - 1 - i n d e p e n d e n t c o m p o n e n t (Dustin a n d Springer, 1988a).  T h e ICAM-1-dependent  i n d e p e n d e n t pathway is constitutive.  pathway  is inducible  whereas  the  ICAM-1-  In addition, purified L F A - 1 in planar m e m b r a n e s  is a b l e to strongly bind a n I C A M - 1 " T cell line (Dustin a n d Springer, 1989). predicted t h e e x i s t e n c e of at least o n e alternate L F A - 1 counter-receptor.  This  A human  c D N A e n c o d i n g this m o l e c u l e w a s c l o n e d by s c r e e n i n g C O S cells transfected with a p l a s m i d - b a s e d e x p r e s s i o n library (Staunton et al., 1989b).  T h e C O S cells w e r e then  s e l e c t e d for their ability to a d h e r e to purified h u m a n L F A - 1 protein in the p r e s e n c e of anti-ICAM-1 antibody.  T h i s n e w L F A - 1 ligand w a s d e s i g n a t e d I C A M - 2 ( C D 102).  23  I C A M - 2 is a n integral m e m b r a n e protein with two extracellular Ig-like d o m a i n s . T h e s e two d o m a i n s s h a r e highest s e q u e n c e identity with the two N-terminus Ig-like d o m a i n s of h u m a n I C A M - 1 (34%) s u g g e s t i n g that the crucial interactions involved in a d h e s i o n to L F A - 1 are mediated by t h e s e two d o m a i n s .  T h e L F A - 1 binding region of I C A M - 1  h a s b e e n m a p p e d to d o m a i n 1 a n d e x t e n d s partly into d o m a i n 2 indicating that the similarity is both structural a s well a s functional (Staunton et al., 1990). S i n c e M a c - 1 is a l s o a b l e to bind I C A M - 1 through the third Ig-like d o m a i n ( D i a m o n d et al.,  1990;  D i a m o n d et al., 1991), it s u g g e s t s that I C A M - 2 is not able to act a s a counter-receptor for  Mac-1.  The  inability  of  I C A M - 2 to  mediate  a d h e s i o n to  Mac-1  has  been  d e m o n s t r a t e d (de F o u g e r o l l e s et al., 1995). D e s p i t e the structural a n d functional similarity b e t w e e n I C A M - 1 a n d I C A M - 2 , there is a n apparent difference in affinity for L F A - 1 . a d h e s i o n more effectively than I C A M - 2 .  I C A M - 1 is able to mediate L F A - 1  Initial e v i d e n c e to support this  differential  binding capability of L F A - 1 c a m e from s e v e r a l a d h e s i o n a n d d e t a c h m e n t studies (Kishimoto et al., 1990).  I C A M - 1 - t r a n s f e c t e d C O S cells a d h e r e more avidly to L F A - 1 -  c o a t e d plastic petri plates than do I C A M - 2 - t r a n s f e c t e d cells ( I C A M - 2 C O S cells w a s h off more readily).  A n t i b o d i e s against I C A M - 1 are a b l e to effectively inhibit cellular  binding to L F A - 1 - c o a t e d plastic w h e n the L F A - 1 is c o a t e d at a low density.  However,  w h e n the L F A - 1 is c o a t e d at a high density, the anti-ICAM-1 m A b s are not a b l e to completely block the binding.  T h i s implies that w h e n a limited n u m b e r of L F A - 1  m o l e c u l e s are available, the higher affinity ligand, I C A M - 1 , will o u t c o m p e t e the lower affinity ligand, I C A M - 2 .  T h i s differential binding capacity is specific to the tertiary  24  structure of the binding sites.  E x t e n s i o n of the I C A M - 2 binding site by the addition of  five extra Ig-like d o m a i n s d o e s not e n h a n c e L F A - 1 a d h e s i o n (Damle et al., 1992a). A s well, r e m o v a l of Ig-like d o m a i n s 3-5 from I C A M - 1 d o e s not diminish binding.  These  results are consistent with the idea that the strength of the L F A - 1 : I C A M binding is d e t e r m i n e d by the structure of the binding site and not by the d i s t a n c e that the binding d o m a i n e x t e n d s from the cell surface. T h e g e n e e n c o d i n g I C A M - 2 is a single c o p y g e n e located on c h r o m o s o m e 17 in h u m a n s (Hogg et al., 1991) and c h r o m o s o m e 11 in the murine s y s t e m (Kuramoto al., 1994).  et  C l o n i n g the h u m a n c D N A has revealed that it c o n s i s t s of a core protein of  2 9 k D with six potential N-glycosylation sites yielding a mature glycoprotein of 50-60 kD (Staunton etal., 1989b; d e F o u g e r o l l e s etal., 1991). A l t h o u g h e x p r e s s i o n patterns of I C A M - 1 a n d I C A M - 2 are different, there is s o m e overlap.  I C A M - 1 is e x p r e s s e d at  low levels on a subpopulation of lymphocytes, m o n o c y t e s , and endothelial cells (Dustin et al., 1986; P o b e r et al., 1986; te V e l d e et al., 1987; Dustin et al., 1 9 8 8 a ; M e n t z e r et al., 1988; Rothlein et al., 1988; Dustin and Springer, 1988a), but is strongly induced on t h e s e cells a n d fibroblasts and epithelial cells by cytokines a n d inflammatory (Dustin et al., 1986; P o b e r etal., 1986; Rothlein et al., 1988). expression  is restricted  to lymphocytes,  monocytes,  agents  In c o m p a r i s o n , I C A M - 2  a n d vascular  endothelium  (Staunton etal., 1989b; de F o u g e r o l l e s etal., 1991; Nortamo etal., 1991). In addition, I C A M - 2 e x p r e s s i o n is not affected by cytokines. platelets a n d granulocytes (Diacovo et al., 1994).  25  I C A M - 2 h a s a l s o b e e n found o n  1:4.2  b) P h y s i o l o g i c a l s i g n i f i c a n c e of L F A - 1 : I C A M - 2 i n t e r a c t i o n  Relatively little investigation has b e e n d o n e on the physiological function of the L F A - 1 : I C A M - 2 interaction.  B a s e d on its e x p r e s s i o n on endothelium a n d its ability to  m e d i a t e L F A - 1 - d e p e n d e n t a d h e s i o n of lymphocytes, it is s u s p e c t e d that I C A M - 2 is involved in lymphocyte recirculation (Dustin a n d Springer, 1 9 9 1 ; C a r l o s a n d H a r l a n , 1994).  In o n e study, a 22 residue peptide c o r r e s p o n d i n g to a region in d o m a i n 1 of  h u m a n I C A M - 2 w a s s h o w n to inhibit a B cell line from binding to endothelial cells (Li et al., 1993a). T h i s s a m e peptide is a l s o able to activate N K cells a n d T cells (Li et al., 1 9 9 3 b ; S o m e r s a l o et al., 1995; Li et al., 1995).  T h e peptide-activated N K cells h a v e  i n c r e a s e d cytotoxicity a n d are more readily able to migrate through a B o y d e n c h a m b e r . It is s u s p e c t e d that this peptide m a y transmit a signal to the cell through L F A - 1 s i n c e binding of the  peptide c o i n c i d e s with i n c r e a s e d phosphorylation of two  proteins  ( S o m e r s a l o et al., 1995). T h e activated state c a n dictate the i n c r e a s e d function of the cell.  T h e ability of the I C A M - 2 protein to transmit a signal is a l s o o b s e r v e d in  lymphocyte activation ( d i s c u s s e d in chapter 3).  A l t h o u g h tested only in a n artificial  s y s t e m , the I C A M - 2 protein is able to a u g m e n t the primary activation s i g n a l delivered through the T c R / C D 3 c o m p l e x (Damle et al., studies h a v e implicated  1 9 9 2 a ; D a m l e et al.,  I C A M - 2 e x p r e s s i o n in m a l i g n a n c y .  1992b).  Endothelial I C A M - 2  e x p r e s s i o n is significantly higher in malignant lymph n o d e s than in n o d e s ( R e n k o n e n et al., 1992).  Other  nonmalignant  High e x p r e s s i o n of I C A M - 2 w a s a l s o d e t e c t e d on  H o d g k i n ' s d i s e a s e - d e r i v e d cell lines (Ellis et al., 1992).  A s well, Burkitt's l y m p h o m a  (BL) cells are lysed by E B V - s p e c i f i c C T L s w h e n I C A M - 2 is present on the B L cells  26  e v e n if other C A M s s u c h a s I C A M - 1 are a b s e n t ( K h a n n a et al., 1993).  However,  susceptibility to lysis is i n c r e a s e d if I C A M - 1 is a l s o present s u g g e s t i n g that I C A M - 2 c a n play a key role in the i m m u n e s y s t e m w h e n I C A M - 1 is absent. Natural killer (NK) cells are a l s o a b l e to utilize the L F A - 1 : I C A M - 2 pathway for the lysis of target cells ( J a c k s o n etal., 1992; K a t s a n a s etal., 1994; H e l a n d e r et al., 1996).  1:4.3 I n t e r c e l l u l a r A d h e s i o n M o l e c u l e - 3 (ICAM-3) In similar fashion to the w a y that I C A M - 2 w a s identified, a third L F A - 1 counterreceptor w a s a l s o identified.  T h e L F A - 1 - d e p e n d e n t a d h e s i o n of o n e h u m a n lymphoid  cell line to purified L F A - 1 w a s greater than the total I C A M - 1 - a n d I C A M - 2 - d e p e n d e n t a d h e s i o n (de F o u g e r o l l e s et al., 1 9 9 1 ; d e F o u g e r o l l e s a n d Springer, 1992).  A third  m o l e c u l e , I C A M - 3 ( C D 5 0 ) , w a s identified by s e v e r a l g r o u p s . O n e group c h a r a c t e r i z e d I C A M - 3 b a s e d on available s e q u e n c e information from s e v e r a l C A M s ( V a z e u x et al., 1992).  Another  group  generated  antibodies  against  ICAM-3 and subsequently  e x p r e s s i o n c l o n e d the c D N A (Fawcett et al., 1992). A third group utilized a m i n o acid s e q u e n c e d a t a from the purified protein to construct oligonucleotides w h i c h w e r e u s e d to s c r e e n a c D N A library (de F o u g e r o l l e s et al., 1993)  I C A M - 3 is a l s o a typical  t r a n s m e m b r a n e glycoprotein with a core polypeptide of 56 k D containing 15 potential N-glycosylation sites. T h e d e g r e e of glycosylation is evident in the s i z e of the mature protein (124 kD).  I C A M - 3 contains five Ig-like d o m a i n s , e a c h with varying similarities  to the c o r r e s p o n d i n g d o m a i n s of the other m e m b e r s of the I C A M subfamily.  Based on  s e q u e n c e c o m p a r i s o n a n d the predicted n u m b e r of d o m a i n s , I C A M - 3 is most similar to  27  I C A M - 1 with 5 2 % overall amino acid identity and 7 7 % identity in the s e c o n d d o m a i n . T h e first two d o m a i n s of I C A M - 3 are 3 7 % identical to the two Ig-like d o m a i n s of I C A M 2. E v e n though the m e m b e r s of the I C A M subfamily s h a r e structural a n d functional similarities, functions.  their  distribution  is  unique  possibly  reflecting  distinct  physiological  I C A M - 3 is detected on resting lymphocytes, m o n o c y t e s , neutrophils, a n d  e p i d e r m a l L a n g e r h a n s cells (de F o u g e r o l l e s and Springer, 1992; A c e v e d o et al., 1993; S t a q u e t et al., 1995).  It is not inducible by cytokines or other inflammatory  agents.  R e s t i n g l y m p h o c y t e s bind L F A - 1 primarily through I C A M - 3 ; this fact c o u p l e d with the observed  higher  c o m p a r e d to  expression  of  I C A M - 3 on  resting  lymphocytes  and  I C A M - 1 or I C A M - 2 s u g g e s t s that L F A - 1 : I C A M - 3 m a y  monocytes  be critical  in  initiating a n i m m u n e r e s p o n s e (de F o u g e r o l l e s and Springer, 1992; d e F o u g e r o l l e s et al., 1994).  Similar to I C A M - 1 and I C A M - 2 , purified I C A M - 3 is a l s o able to provide a  costimulatory signal which e n h a n c e s the primary signal delivered through the T c R / C D 3 c o m p l e x (de F o u g e r o l l e s et al., 1994).  A n t i - I C A M - 3 m A b c a n inhibit peripheral blood  lymphocyte proliferation in r e s p o n s e to phytohemagglutinin, a s well a s a l l o g e n e i c and anti-specific proliferation (de F o u g e r o l l e s ef al., 1994). T h e observation that a cocktail of a n t i - I C A M - 1 , - 2 , and -3 m A b s c a n effectively inhibit homotypic aggregation to the s a m e extent that anti-LFA-1 antibodies c a n s u g g e s t s that there m a y not be a n y more m e m b e r s in the I C A M subfamily. 3 c a n a l s o a d h e r e to  a (3 d  2  In addition, o n e group h a s d e m o n s t r a t e d that I C A M -  (Van der V i e r e n ef al., 1996).  28  1:5 T h e s i s o b j e c t i v e T h e interaction a n d functional significance of L F A - 1 : I C A M - 1 a d h e s i o n h a s b e e n extensively  studied.  It is involved  in virtually  every  leukocyte function  tested.  H o w e v e r , the physiological significance of the L F A - 1 : I C A M - 2 interaction h a s not b e e n e x a m i n e d a s thoroughly.  A l t h o u g h preliminary studies are indicative of p o s s i b l e  functions, they are still not definitive.  B a s e d on distribution s t u d i e s a n d preliminary T  cell activation d a t a , it is s p e c u l a t e d that I C A M - 2 m a y play a role in delivering a n e c e s s a r y costimulatory signal to lymphocytes in order for them to proliferate. a l s o s u s p e c t e d that I C A M - 2 m a y play a role in leukocyte recirculation.  It is  T h e overall  objective of this thesis w a s to e x a m i n e the p o s s i b l e roles of murine I C A M - 2 in the i m m u n e a n d inflammatory  responses.  U n d e r s t a n d i n g of I C A M - 2 functions in the  murine s y s t e m will provide a better b a s i s for the construction of a n a n i m a l m o d e l in the future.  W h e n this project w a s initiated, murine I C A M - 2 w a s not yet c h a r a c t e r i z e d .  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W o n g D A , D a v i s E M , L e B e a u M , a n d Springer T A (1996) C l o n i n g a n d c h r o m o s o m a l localization of a novel g e n e e n c o d i n g a h u m a n (3 -integrin a subunit. Gene 1 7 1 : 2 9 1 . 2  X u H , G o n z a l o J A , St. Pierre Y , Williams IR, K u p p e r T S , C o t r a n R S , S p r i n g e r T A , a n d G u t i e r r e z - R a m o s J C (1994) L e u k o c y t o s i s a n d resistance to septic s h o c k in intercellular a d h e s i o n molecule 1-deficient mice. J. Exp. Med. 180:95. Z a g o u r y D, B e r n a r d J , T h i e r n e s s N, F e l d m a n M , a n d B e r k e G (1975) Isolation a n d characterization of individual functionally reactive cytotoxic T lymphocytes: conjugation, killing a n d recycling at the single cell level. Eur. J. Immunol. 5:818.  45  Chapter 2 C l o n i n g a n d c h a r a c t e r i z a t i o n of m u r i n e I C A M - 2  T h e work p r e s e n t e d in this chapter a p p e a r s in the following publications: i) O h h M , S m i t h C A , C a r p e n i t o C , a n d T a k e i F (1994) R e g u l a t i o n of intercellular a d h e s i o n molecule-1 g e n e e x p r e s s i o n involves multiple mRNA stabilization m e c h a n i s m s : effects of interferon-y a n d phorbol myristate acetate. Blood 8 4 : 2 6 3 2 ii) C a r p e n i t o C , O h h M , a n d T a k e i F (1995) C l o n i n g a n d characterization of murine intercellular a d h e s i o n m o l e c u l e - 2 ( I C A M - 2 ) : a functional a n d m o l e c u l a r a n a l y s i s .  Allerg. Immunol. (Life Sci. Adv.) 14:43 iii) X u H, Bickford J K , Luther E , C a r p e n i t o C , T a k e i F, a n d S p r i n g e r T A (1996) C h a r a c t e r i z a t i o n of murine intercellular a d h e s i o n m o l e c u l e - 2 . J. Immunol. 1 5 6 : 4 9 0 9  2:1  Introduction  T h e i m m u n e s y s t e m c o n s i s t s of a network of cells that maintain b a s i c d e f e n s e s a g a i n s t m i c r o o r g a n i s m s , parasites, a n d c a n c e r cells. L e u k o c y t e s travel throughout the multicellular o r g a n i s m surveying for the p r e s e n c e of t h e s e d i s e a s e - c a u s i n g p a t h o g e n s . A s d i s c u s s e d in the previous chapter, m a n y different cell s u r f a c e proteins e x p r e s s e d by l e u k o c y t e s h a v e b e e n s h o w n to play a role in determining their functional activity. A m o n g t h e most important a r e m e m b e r s of the immunoglobulin (Ig) s u p e r g e n e family (Williams a n d B a r c l a y , 1988; Springer, 1990).  These molecules share sequence and  structural similarities with the variable a n d constant d o m a i n s of Igs a n d contain at least o n e region of a c o n s e r v e d Ig-like tertiary protein structure, called a h o m o l o g y unit (Iglike d o m a i n ) . T h e h o m o l o g y unit c o n s i s t s of approximately 1 0 0 - 1 1 0 a m i n o a c i d s w h i c h  46  fold into a s a n d w i c h of two anti-parallel (3-sheets (Amit et al., 1986; W i l l i a m s , 1987; A l z a r i et al.,  1988; W i l l i a m s a n d Barclay, 1988).  This structure is stabilized by a  c o n s e r v e d disulphide link between the two s h e e t s . s e v e r a l potential glycosylation sites. soluble  proteins,  transmembrane  m e m b r a n e proteins.  T h e d o m a i n m a y a l s o harbor  M o l e c u l e s with t h e s e Ig-like d o m a i n s m a y be  proteins,  or  glycophospholipid  (GPI)  attached  T h e g e n o m i c structure of most of t h e s e m o l e c u l e s s h o w s that  e a c h Ig-like d o m a i n is often e n c o d e d by o n e e x o n (Hunkapiller a n d H o o d , 1989). Introns separating the e x o n s are all in p h a s e .  T h i s supports the notion that a  primordial g e n e , w h i c h e n c o d e d a n Ig-like structure, duplicated, t r a n s p o s e d throughout the g e n o m e , a n d diverged from its a n c e s t e r a l g e n e giving rise to the m a n y different Iglike structures e n c o d e d in the g e n o m e (Williams a n d Barclay, 1988; Hunkapiller a n d H o o d , 1989). A l t h o u g h the m e m b e r s of the Ig superfamily perform a variety of functions, the central f o c u s of their role in the i m m u n e s y s t e m is cell s u r f a c e recognition.  Various  m e m b e r s are k n o w n to play a role specifically in leukocyte function. A small s u b s e t of t h e s e m o l e c u l e s display not only structural but a l s o functional similarity. T h e m e m b e r s of the I C A M subfamily are t r a n s m e m b r a n e glycoproteins with varying n u m b e r s of Iglike d o m a i n s ( S i m m o n s et al., 1988; Staunton et al., 1988; Horley et al., 1989; S i u et al., 1989; S t a u n t o n et al., 1989; X u et al., 1992; Fawcett et al., 1992; V a z e u x et  al.,  1992; d e F o u g e r o l l e s et al., 1993). A s d e s c r i b e d in the previous chapter, three I C A M s ( I C A M - 1 , - 2 , -3) h a v e b e e n identified.  T h e key functional a s p e c t w h i c h they s h a r e is  the ability to bind a c o m m o n counter-receptor e x p r e s s e d on leukocytes, L F A - 1 .  47  T h e three known L F A - 1 counter-receptors ( I C A M - 1 , - 2 , a n d -3), are s h o w n to be typical type  I m e m b r a n e glycoproteins.  T h r e e I C A M s h a v e b e e n identified  and  c h a r a c t e r i z e d in the h u m a n s y s t e m . I C A M - 1 a n d I C A M - 2 h a v e b e e n c h a r a c t e r i z e d in the murine s y s t e m . T h e murine v e r s i o n s of the I C A M s are a s s i g n e d their designation b a s e d on structural homology, s i z e similarity, s e q u e n c e identity, a n d similar distribution patterns. Although  all three  I C A M s h a v e b e e n s h o w n to bind  L F A - 1 , their  relative  contributions to L F A - 1 - d e p e n d e n t i m m u n e r e s p o n s e s , particularly I C A M - 2 a n d - 3 , are still unclear. T h e physiological significance of the L F A - 1 :ICAM-1 interaction h a s b e e n e x a m i n e d thoroughly  (Springer, 1990; Dustin a n d Springer, 1991).  previoulsy in chapter 1, studies on the L F A - 1 : I C A M - 2 interaction definitive.  Functional examination  of the  As  mentioned  h a v e not  L F A - 1 : I C A M - 2 interaction  has  been  involved  purified I C A M - 2 a n d anti-TcR m A b crosslinking or antibody b l o c k a d e of a n i m m u n e r e s p o n s e ( D a m l e et al., 1992; de F o u g e r o l l e s et al., 1994) or I C A M - 2 peptide studies on leukocyte activation a n d transendothelial migration (Li et al., 1 9 9 3 a ; Li et al., 1993b; S o m e r s a l o et al., 1995; Li et al., 1995).  This project t a k e s a different a p p r o a c h in  e x a m i n i n g the significance of the L F A - 1 : I C A M - 2 interaction.  S y s t e m s to e x a m i n e the  role of I C A M - 2 in T cell stimulation a n d transendothelial migration w e r e e s t a b l i s h e d . T h e effect on e a c h of t h e s e a s p e c t s of the i m m u n e s y s t e m w e r e e x a m i n e d w h e n the I C A M - 2 c D N A w a s introduced into t h e s e s y s t e m s . W h e n this project w a s intiated, I C A M - 2 in the murine s y s t e m had not yet b e e n identified.  T h e first step towards examining the role of I C A M - 2 in the i m m u n e s y s t e m  48  w a s to c l o n e the c D N A e n c o d i n g the murine I C A M - 2 .  In this chapter, cloning a n d  functional characterization of murine I C A M - 2 is d e s c r i b e d . T h e results in this chapter d e m o n s t r a t e the high d e g r e e of similarity between h u m a n a n d murine I C A M - 2 in their s e q u e n c e s a n d function. T h e binding properties of the murine I C A M - 2 (purified protein a n d c D N A - e n c o d e d ) to murine L F A - 1 are a l s o e x a m i n e d .  2.2 Materials and Methods 2:2.1 Animals B A L B / c m i c e w e r e p u r c h a s e d from C h a r l e s R i v e r C a n a d a , Q u e b e c , C a n a d a a n d maintained in the Joint A n i m a l Facility of the B . C . C a n c e r R e s e a r c h C e n t r e .  2:2.2 Cell lines and antibodies T h e m o u s e fibroblast L cell line (Sanford et al., 1948) w a s maintained in D u l b e c c o ' s modified minimum e s s e n t i a l m e d i a ( D M E M ) containing  1 0 % fetal  calf  s e r u m ( F C S ) a n d antibiotics (50 U/ml penicillin a n d 5 0 u.g/ml streptomycin). N S - 1 (Kohler et al., 1976), a B A L B / c p l a s m a c y t o m a cell line, a n d B W 5 1 4 7 ( R a l p h a n d Nakoinz,  1973), a n A K R thymic  l e u k e m i a cell line, w e r e  maintained  in D M E M  containing 5 % F C S a n d antibiotics. T h e T l y m p h o m a line E L - 4 (Old et al., 1965) w a s maintained in R P M I 1640 m e d i u m containing 1 0 % F C S . A l l m o n o c l o n a l antibodies (mAb) w e r e u s e d a s purified immunoglobulins. T h e rat m A b Y N 1 / 1 . 7 . 4 ( l g G ) r e c o g n i z e s the murine I C A M - 1 a n d h a s b e e n d e s c r i b e d 2a  (Takei, 1 9 8 5 ; Horley et al., 1989).  T h e rat anti-mouse I C A M - 2 ( 3 C 4 , l g G ) antibody 2 a  49  u s e d for functional and inhibition studies w a s p u r c h a s e d from P h a r m i n g e n ( S a n D i e g o , C A ) a n d the antibody u s e d for the purification of I C A M - 2 w a s a g e n e r o u s gift from Dr. T. S p r i n g e r (Centre for B l o o d R e s e a r c h , Harvard M e d i c a l S c h o o l ) , a n d h a s b e e n c h a r a c t e r i z e d (Xu et al., 1996). T h e rat hybridoma cell lines F D 4 4 1 . 8 (Dialynas et al., 1982) w h i c h p r o d u c e s anti-mouse L F A - 1 ( C D 1 1 a , A T C C TIB 2 1 3 , l g G ) a n d R 1 - 2 2 b  ( H o l z m a n n a n d W e i s s m a n , 1989; H o l z m a n n et al., 1989) w h i c h p r o d u c e s rat antimouse VLA-4 (CD49d, A T C C  H B 2 2 7 , l g G ) w e r e obtained from A m e r i c a n 2 b  Type  Culture C o l l e c t i o n (Rockville, M D ) .  2:2.3 PCR cloning a murine specific probe for ICAM-2 Total R N A from E L - 4 cells w a s isolated using the a c i d - p h e n o l extraction method ( C h o m c z y n s k i a n d S a c c h i , 1987). buffered  C e l l s (5 x 10 ) w e r e w a s h e d twice with p h o s p h a t e 6  s a l i n e ( P B S ) a n d d i s s o l v e d in 0.5 ml of solution  D (4 M  guanidinium  thiocyanate, 2 5 m M s o d i u m citrate p H 7.0, 0 . 5 % s a r k o s y l , 0.1 M 2-mercaptoethanol). Following this, 50 uJ of 2 M s o d i u m acetate p H 4.0, 0.5 ml water-saturated-phenol, and 0.1 ml chloroform:isoamyl alcohol (49:1) w e r e a d d e d . T h i s solution w a s centrifuged at 14,000 g for 15 min at 4°C. T h e a q u e o u s layer, which w a s free of g e n o m i c D N A , w a s mixed with 0.5 ml isopropanol and put at -70°C for 1 hr. T h e solution w a s centrifuged at 14,000 g for 20 min at 4°C and the pellet w a s then s u s p e n d e d in 0.3 ml solution D followed by 0.3 ml of ethanol.  T h e solution w a s kept at -70°C for another hour and  then centrifuged for 20 min at 4°C. T h e pellet w a s then w a s h e d twice with 1 ml 7 0 % ethanol a n d finally s u s p e n d e d in 20 uJ of diethylpyrocarbonate ( D E P C ) - t r e a t e d water.  50  Total R N A (5 |ug) from E L - 4 cells w a s c o m b i n e d with 1.5 u.g oligo-dT primers (12-18  mer  ) in a final v o l u m e of 30 ul containing 50 m M Tris p H 8.3, 60 m M K C I , 3 m M  M g C I , 10 m M D T T , 500 u M d N T P s ( P h a r m a c i a ; B a i e d'Urfe, P Q ) , 4 ug acetylated 2  B S A ( P r o m e g a ; M a d i s o n , W l ) , 4 0 units of R N a s i n ( P r o m e g a ) a n d 6 0 0 units of M o l o n e y murine l e u k e m i a virus reverse transcriptase ( C a n a d i a n Life T e c h n o l o g i e s ; Burlington, ON).  T h i s mixture w a s incubated at 42°C for 1 hr to g e n e r a t e s i n g l e - s t r a n d e d c D N A  ( S a m b r o o k et al., 1989).  In a final v o l u m e of 50 ul, 1/5 of the a b o v e c D N A reaction  w a s c o m b i n e d with the d e g e n e r a t e oligonucleotides 5 ' - G T C A A C T G ( C / T ) A G ( C / T ) ( A / T ) C C ( A / T ) C ( A / C ) T G - 3 ' a n d 5 ' - C A C A G ( C / T ) ( C / G ) ( A / C ) G ( A / G ) C A G G A G A A ( A / G ) T T - 3 ' (1 u.M concentration e a c h ) in a mixture containing 10 m M Tris p H 8.3, 50 m M K C I , 1.5 mM  MgCI , 2  0.1%  Technologies).  gelatin,  and  1.5  units  of  Taq  polymerase  (Canadian  Life  T h i s cocktail mix w a s subjected to 30 rounds of p o l y m e r a s e c h a i n  amplification ( P C R ) (94°C, 30 s e c ; 50°C, 60 s e c ; 72°C, 6 0 s e c ) . T h e amplified D N A fragment (450 bp) w a s then purified by a g a r o s e gel electrophoresis. It w a s then blunt e n d e d by incubating the fragment in 50 m M Tris p H 7.5, 10 m M M g C I , 5 m M D T T , 5 0 0 2  U.M d N T P s , a n d 5 units of T  4  D N A p o l y m e r a s e ( C a n a d i a n Life T e c h n o l o g i e s ) at 37°C  for 5 min. T h e e n d s of the fragment w e r e then phosphorylated with 5 units  T  4  polynucleotide k i n a s e ( C a n a d i a n Life T e c h n o l o g i e s ) in 60 m M Tris p H 7.6, 10 m M M g C I , 2.5 m M D T T , 1 m M A T P at 37°C for 1 hr. 2  Finally, the P C R fragment w a s  ligated into a Smal cut Bluescipt vector ( p B S T ) ( P h a r m a c i a ) with 2 units of T  4  DNA  ligase ( C a n a d i a n Life T e c h n o l o g i e s ) in 50 m M Tris p H 7.6, 10 m M M g C I , 1 m M A T P , 1 2  mM  DTT, 5%  PEG-8000  at 4°C for  8-12  51  hrs.  The  ligation  mixture  was  then  transformed  into competent E. coli D H 5 a cells ( C a n a d i a n Life T e c h n o l o g i e s ) a n d  plated o n L B a g a r containing ampicillin (50 u.g/ml) a n d colour selection with 0 . 0 2 % IPTG and 0.04% X-gal .  Individual colonies were s u b s e q u e n t l y picked for plasmid  mini-preps a n d s e q u e n c e d by the S a n g e r dideoxynucleotide chain termination  method  ( S a n g e r et al., 1977) using the T s e q u e n c i n g kit (Pharmacia), [ a - P ] - d C T P (3000 32  7  C i / m m o l ) , T and T primers. 3  7  2:2.4 DNA isolation and Southern blot analysis High molecular weight D N A w a s isolated from B A L B / c s p l e e n cells ( G r o s s Bellard et al., 1977). T h e cells (1 x 10 ) were w a s h e d 3 X with P B S a n d s u s p e n d e d in 8  2 ml of T N E buffer (10 m M Tris p H 8.0, 0.15 M N a C l , 10 m M E D T A ) a n d gently mixed. T o this s u s p e n s i o n , 2 0 pi of 2 0 % S D S and 100 u.g of proteinase K ( S i g m a C h e m i c a l C o m p a n y ; S t . Louis, M O ) were a d d e d a n d incubated at 37°C for 12 hrs. was  then  extracted  (phenolxhloroform)  3X  TNE-saturated-phenol,  3X  with  T h e lysate  TNE-saturated-  (1:1), and finally 3 X with chloroform:isoamyl alcohol (24:1). T h e  a q u e o u s p h a s e w a s dialyzed against two c h a n g e s of 4 I of T E buffer (10 m M Tris p H 8.0,  1 m M EDTA)  each  spectrophotometrically ( A  2 6 0  for 16 hrs at 4 ° C .  The D N A was  quantitated  = 1.00 for 5 0 ucj/ml) and purity w a s determined ( A o / A 8 o 26  2  ~ 2.0) o n a n L K B U L T R O S P E C 4 0 5 0 (Cambridge, England). P l a s m i d mini-preps were prepared by alkaline lysis (Brinboim a n d Dolby, 1979). Bacterial cultures w e r e grown in 2 ml of L B broth overnight at 37°C a n d pelleted. T h e pellet w a s s u s p e n d e d in 0.2 ml G T E buffer (50 m M g l u c o s e , 2 5 m M Tris p H 8 . 0 , 10 m M  52  E D T A ) containing 4 mg/ml of l y s o z y m e a n d incubated for 5 min at room temperature. T o this mixture, 0.4 ml of 1% S D S , 0.2 M N a O H w a s a d d e d a n d incubated on ice for 5 min.  Finally, 0.3 ml of 3 M p o t a s s i u m acetate, 2 M acetic acid w a s a d d e d a n d the  mixture w a s centrifuged (14,000 g) for 20 min at 4°C.  T h e clear supernatant w a s  r e c o v e r e d a n d extracted with 0.6 ml a phenol:chloroform:isoamyl a l c o h o l solution (25:24:1), a n d mixed with 1 ml of isopropanol.  Nucleic acids were allowed  to  precipitate by incubating on ice for 10 min followed by spinning in a microfuge for 15 min at 4 ° C . T h e pellet w a s then d i s s o l v e d in 0.1 ml of T E buffer containing 2 0 0 Lig/ml of R N a s e A ( S i g m a C h e m i c a l C o . ) , 20 U/ml R N a s e Tj (Boehringer M a n n h e i m ; L a v a l , P Q ) a n d incubating first at 37°C for 30 min a n d then at 50°C for 5 min.  T o the  a q u e o u s p h a s e , 60 jal of 7.5 M a m m o n i u m acetate p H 7.0 w a s a d d e d a n d incubated o n ice for 5 min. T h e solution w a s centrifuged for 5 min a n d the supernatant r e c o v e r e d w a s mixed with 160 uJ of ethanol.  This w a s then centrifuged for 5 min at room  temperature a n d the pellet w a s w a s h e d with 1 ml of 7 0 % ethanol. T h e D N A w a s then d i s s o l v e d in 50 uJ of T E buffer a n d ready for either s e q u e n c i n g or restriction e n z y m e analysis. Large scale  plasmid purification  was done  in a slightly  different f a s h i o n .  Overnight bacterial cultures grown in 500 ml of L B broth w e r e pelleted a n d s u s p e n d e d in similar f a s h i o n a s the minipreps.  T h e y w e r e lysed a n d neutralized a s before.  H o w e v e r , the isopropanol-precipitated nucleic a c i d s w e r e then purified by the W i z a r d M a x i p r e p kit ( P r o m e g a ) . T h e isolated D N A w a s then ethanol precipitated a n d w a s h e d with 7 0 % e t h a n o l before being d i s s o l v e d in sterile T E buffer.  53  T h e restriction e n z y m e s u s e d for restriction digest a n a l y s i s w e r e p u r c h a s e d from either C a n a d i a n Life T e c h n o l o g i e s , P h a r m a c i a , or B o e h r i n g e r M a n n h e i m . T h e conditions a n d temperature manufacturer.  Approximately  for the digestions w e r e t h o s e  recommended  by the  1 u.g of plasmid D N A w a s digested with 5 units of  e n z y m e for 2 - 3 hrs. High molecular weight g e n o m i c D N A (12 u.g) w a s d i g e s t e d with a large e x c e s s of e n z y m e ( s ) (10 units/ucj of g e n o m i c D N A ) for 6-8 hrs. Both types of s a m p l e s w e r e then precipitated by adding 1/10 v o l u m e of 2.5 M s o d i u m acetate p H 4 . 5 a n d 2 v o l u m e s of cold ethanol, a n d incubated on dry ice for 2 0 min. T h e solution w a s microfuged (14,000 g) for 2 0 min at 4°C a n d the pellet w a s h e d with 1 ml of 7 0 % ethanol.  T h e air dried pellet w a s then redissolved in 1 0 - 3 0 uJ of T A E buffer (40 m M  Tris acetate p H 7.2, 2 0 m M s o d i u m acetate, 1 m M E D T A ) .  T o this, 1/10 v o l u m e D N A  loading buffer ( 0 . 2 5 % xylene c y a n o l , 0 . 2 5 % b r o m o p h e n o l blue, 5 0 % glycerol in T A E ) w a s a d d e d a n d loaded onto a n 0 . 8 % T A E a g a r o s e g e l .  P l a s m i d digests  were  e l e c t r o p h o r e s e d at 5 0 volts for 3-4 hrs. Digested g e n o m i c D N A w a s e l e c t r o p h o r e s e d at 2 5 volts for 16-18 hrs. Ethidium bromide ( C a n a d i a n Life T e c h n o l o g i e s ) w a s present in both the a g a r o s e g e l a n d the T A E running buffer at a concentration of 5 0 0 ng/ml. M o l e c u l a r weights w e r e determined from molecular weight s t a n d a r d s s u c h a s Hindlll d i g e s t e d X D N A a n d X (Hindlll + EcoRI) fragments.  T h e gels were  photographed  under U V light with a Polaroid c a m e r a . T h e S o u t h e r n blot hybridization w a s d o n e a s d e s c r i b e d (Southern, 1975). T h e g e l w a s first treated in 0.1 M H C I for 15 min followed by treatment in 0.5 M N a O H , 1.5 M N a C l for 3 0 min, a n d finally neutralized in 1.0 Tris p H 7.0, 2.5 M N a C l for a n additional 3 0 min ( S a m b r o o k et al., 1989)).  54  T h e gel w a s  then layered on top of a strip of 3 M M W h a t m a n filter p a p e r ( S c h l e i c h e r a n d S c h u e l l ; K e e n e , N H ) s o a k e d in 2 0 X S S C (3 M N a C l , 0.15 M s o d i u m citrate p H 7.0). T h e e n d s of the filter p a p e r are s o a k e d in a pool of 2 0 X S S C buffer. A p i e c e of Z e t a P r o b e G T m e m b r a n e ( B i o R a d Laboratories; M i s s i s s a u g a , O N ) cut to the s i z e of the gel w a s first s o a k e d in double-distilled water for 10 min and then layered on top of the g e l . wetted  p i e c e s of W h a t m a n  p a p e r were then  layered on top  of the  Four  ZetaProbe  m e m b r a n e , followed by a stack of paper towels (~10 cm) cut to the s a m e s i z e a s the gel a n d a g l a s s plate.  T h e D N A w a s covalently fixed to the m e m b r a n e by U V c r o s s -  linking in a U V Stratalinker 1800 (Stratagene; L a Jolla, C A ) . T h e m e m b r a n e w a s prehybridized in a solution of 6 X S S C , 1 0 % d e i o n i z e d f o r m a m i d e ( C a n a d i a n Life T e c h n o l o g i e s ) , 1% S D S , 1% Blotto (Carnation instant s k i m milk), 2 m M E D T A , 0.5 mg/ml heat denatured s a l m o n s p e r m D N A ( P h a r m a c i a ) at 60°C for 4 hrs.  D N A p r o b e s were r a d i o l a b e l e d by the procedure d e v e l o p e d by F e i n b e r g  and Vogelstein (Pharmacia).  (Feinberg DNA  (20  and V o g e l s t e i n , 1983) ng)  was  mixed  with  using the 100  ng  of  T  7  oligolabelling  randomly  kit  generated  h e x a n u c l e o t i d e s a n d boiled for 3 min and immediately c o o l e d on ice. In addition to the cocktail buffer, a nucleotide mix ( d A T P , d G T P , d T T P , [a- P]-dCTP) w a s a l s o a d d e d 32  with T  7  p o l y m e r a s e (final v o l u m e of 10 ul) and allowed to p r o c e e d a c c o r d i n g to  manufacturer's protocol. T h e reaction w a s stopped by the addition of 2 u.l 10 M N a O H . T h e oligolabelled-probe w a s then a d d e d to the hybridization solution containing 6 X SSC,  1 0 % d e i o n i z e d formamide,  1% S D S , 1% Blotto, 2 m M E D T A ,  0.5  d e n a t u r e d s a l m o n s p e r m D N A , and 1 0 % dextran sulfate (Turhan et al., 1988).  55  mg/ml The  filter w a s incubated in this solution at 60°C for 16 hrs a n d then w a s h e d 3 X for 3 0 min e a c h at 65°C in 0 . 3 X S S C , 0 . 1 % S D S , 0 . 1 % s o d i u m p y r o p h o s p h a t e .  T h e filter w a s  then e x p o s e d to K o d a k X A R film at -70°C for 16-36 hrs.  2:2.5 Isolation of a cDNA clone and sequence analysis T h e P C R fragment (described in 2:2.3) w a s u s e d to s c r e e n a ( B 6 x C B A ) F . , m o u s e lung c D N A library in the XZAP II vector (Stratagene; Short et al., 1988). T h e library w a s titrated a c c o r d i n g to manufacturer's protocol.  Briefly, X L 1 - B l u e bacterial  cells w e r e s t r e a k e d o n L B a g a r to obtain a single colony w h i c h w a s u s e d to inoculate 4 0 ml of L B m e d i a s u p p l e m e n t e d with 0 . 2 % maltose a n d 10 m M M g S 0 . 4  T h e s e cells  w e r e grown overnight a n d r e s u s p e n d e d in 2 0 ml of 10 m M M g S 0 . A dilution from the 4  p h a g e library (~10  5  p h a g e particles) w a s incubated with 5 ml of the host cells at 37°C  for 15 min, mixed with 5 0 ml of soft a g a r ( N Z Y C M + 0 . 8 % agar) a n d plated o n N Z Y C M + 1.5% agar. T h e s e plates w e r e incubated overnight at 37°C. T h e plates w e r e chilled at 4 ° C for 2 hrs before being lifted onto duplicate nitrocellulose s h e e t s .  The sheets  w e r e first wetted in water a n d then in 1 M N a C l before being laid o n the plates for 4 min.  E a c h plate w a s lifted in duplicate a n d the orientation w a s m a r k e d with india ink.  T h e filters w e r e then p l a c e d , D N A side up, o n W h a t m a n p a p e r saturated with 1.5 M N a C l , 0.5 M N a O H for 4 min then transferred to another W h a t m a n p a p e r saturated with 1 M Tris p H 7.0, 3 M N a C l for 4 min ( S a m b r o o k et al., 1989). T h e filters w e r e then b a k e d at 80°C for 2 hrs a n d incubated in a p r e w a s h step (50 m M Tris p H 8.0, 1 M N a C l , 1 m M E D T A , 0 . 1 % S D S for 1 hr at 42°C) to r e m o v e bacterial debris. T h e filters  56  w e r e then incubated in a prehydrization solution of 6 X S S C , 0 . 5 % S D S , 1% Blotto, 0.5 mg/ml d e n a t u r e d s a l m o n s p e r m D N A for 2 hrs at 50°C. transferred  into a hybridization  solution  r a d i o l a b e l e d probe) for 14 hrs at 50°C.  (same  T h e filters w e r e  composition  as above  then  plus t h e  T h r e e w a s h e s of 20 min e a c h at 50°C w e r e  then d o n e : a) 5 X S S C , 0 . 1 % , b) 2 X S S C , 0 . 1 % S D S , c) 0 . 5 X S S C , 0 . 1 % S D S . T h e filters w e r e then e x p o s e d to K o d a k X A R film.  T h e autoradiographs w e r e aligned with  their respective duplicates a n d only spots w h i c h a p p e a r e d in both lifts of the s a m e plate w e r e c h o s e n for the next round of s c r e e n i n g w h i c h w a s continued until all p l a q u e s o n the plates hybridized with the probe. T h e X Z A P II vector h a s t h e unique ability to allow in vivo e x c i s i o n a n d recircularization of any c l o n e d insert within the X vector to form a p h a g e m i d containing the insert in p B S T ( S K ) (Short et al., 1988).  Inside E. coli, proteins provided from a  helper p h a g e r e c o g n i z e D N A s y n t h e s i s initiation a n d termination s e q u e n c e s in t h e X ZAP  II. T h e e n d result is to d i s p l a c e the single-stranded D N A b e t w e e n the two sites  r e c o g n i z e d by t h e p h a g e proteins.  T h e d i s p l a c e d single-stranded  D N A is then  circularized a n d p a c k a g e d a s a p h a g e m i d w h i c h c a n infect bacterial host cells. T h e p h a g e m i d c a n propagate a s double-stranded p B S T with t h e c l o n e d insert inside t h e cell a n d will a p p e a r a s a bacterial colony. described  above  and used  for s e q u e n c i n g  T h e plasmid D N A c a n b e isolated a s a n d restriction  enzyme  S e q u e n c e d a t a w a s g e n e r a t e d from s e q u e n c i n g both strands of the insert.  57  digestion.  2:2.6 RNA isolation and Northern blot analysis Total R N A w a s isolated from the various lymphoid a n d non-lymphoid o r g a n s (Davis et al., 1986). S p l e e n , thymus, a n d b o n e marrow w e r e t e a s e d with t w e e z e r s a n d p a s s e d through a syringe to generate single cell s u s p e n s i o n s .  Heart, lung, kidney,  a n d liver w e r e cut into small p i e c e s a n d i m m e r s e d into liquid nitrogen.  T h e frozen  p i e c e s w e r e then m i n c e d with a mortar a n d pestle until a fine p o w d e r w a s g e n e r a t e d a n d a l l o w e d to thaw before being lysed in a guanidinium isothiocyanate solution (4 M guanidinium  isothiocyanate,  mercaptoethanol).  25  m M sodium  acetate  pH  6.0, 0.12 M  2-  T h e single cell s u s p e n s i o n s of s p l e e n , thymus, a n d b o n e marrow  w e r e a l s o lysed in this s a m e solution.  T h e lysate w a s then layered o n a c a e s i u m  chloride c u s h i o n (5.7 M C s C I , 2 5 m M s o d i u m acetate p H 6.0) a n d centrifuged in a B e c k m a n L 8 - 6 0 M ultracentrifuge (174,000 g in a n S W 4 1 rotor for 2 0 hr at 20°C). T h e d e n s e r R N A is s e p a r a t e d from the D N A , proteins, a n d lipids a n d isolated at the bottom of t h e c u s h i o n .  T h i s pellet w a s d i s s o l v e d in 2 0 u.l of D E P C - t r e a t e d doubly-distilled  w a t e r a n d a n aliquot (10 u.g R N A ) w a s c o m b i n e d with R N A loading buffer s u c h that the final solution contained 5 0 % d e i o n i z e d formamide, 36 m M 3-[N-Morpholino]-propanesulfonic acid ( 1 X M O P S ) p H 7, 7 % formaldehyde, 6 % glycerol, 0 . 5 % b r o m o p h e n o l blue. T h e s a m p l e s w e r e heated to 95°C for 2 min a n d then l o a d e d into t h e wells of a 1% a g a r o s e g e l in 1 X M O P S buffer with 2 % f o r m a l d e h y d e a n d 1 X M O P S a s the running buffer.  T h e g e l w a s run at 120 volts for 3-4 hrs.  Molecular weights were  d e t e r m i n e d from the R N A ladder ( C a n a d i a n Life T e c h n o l o g i e s ) . After e l e c t r o p h o r e s i s , the lane with the R N A ladder w a s cut from the rest of the gel a n d w a s h e d twice in 500  58  ml of 1 0 X S S C for 15 min e a c h . containing 1  Lig/ml  for 1 hr e a c h .  T h e g e l slab w a s then stained in 2 0 0 ml 1 X S S C  Ethidium bromide a n d d e s t a i n e d in 2 c h a n g e s of 500 ml of 1 X S S C  T h e slab w a s then photographed under U V light.  T h e g e l with the  isolated R N A s a m p l e s w a s rinsed for 2 0 min e a c h in two c h a n g e s of 5 0 0 ml of 1 0 X S S C in order to r e m o v e the formaldehyde. R N A in the gel w a s then transferred onto a Z e t a P r o b e m e m b r a n e by capillary action for 16 hrs using 1 0 X S S C . T h e m e m b r a n e w a s then rinsed in 2 0 X S S C a n d U V fixed with a U V Stratalinker 1800. After this, the m e m b r a n e w a s incubated in a prehybridization solution containing 1.5X S S P E ( 2 0 X S S P E : 3.6 M N a C l , 0.2 M N a H P 0 2  4  p H 7.4, 2 0 m M E D T A ) , 1% S D S , 0 . 5 % Blotto, 0.5  mg/ml heat d e n a t u r e d s a l m o n s p e r m D N A for 2 hrs at 60°C. T h e m e m b r a n e w a s then transferred to a hybridization solution containing 1.5X S S P E , 1% S D S , 0 . 5 % Blotto, 0.5 mg/ml heat denatured s a l m o n s p e r m D N A , a n d oligolabelled probe for 16 hrs at 60°C. T h e filter w a s then w a s h e d in a) 2 X S S C , 0 . 1 % S D S at 20°C for 15 min, b) 0 . 5 X S S C , 0 . 1 % S D S at 20°C for 15 min, c) 0 . 3 X S S C , 0 . 1 % S D S 65°C for 3 0 min. T h e filter w a s then e x p o s e d to K o d a k X A R film.  T h e filter w a s then stripped of the  r a d i o l a b e l e d probe by boiling in three c h a n g e s of 500 ml e a c h of 0.1 X S S C , 1% S D S for 2 0 min. T h e filter w a s then subjected to another prehybridization a n d probing with another oligolabelled probe.  2:2.7 Genomic cloning of the murine ICAM-2 High m o l e c u l a r weight D N A from B A L B / c s p l e e n cells w a s d i g e s t e d with EcoRI. T h e D N A w a s e l e c t r o p h o r e s e d o n a 0 . 8 % a g a r o s e T A E gel a n d three s i z e - s e l e c t e d  59  (4.0-5.5 kb, 5.5-7.0 kb, 7.0-9.0 kb) s e g m e n t s w e r e isolated by electro-elution.  Aliquots  of e a c h fraction w e r e electrophoresed o n a 0 . 8 % T A E a g a r o s e g e l at 3 5 volts for 5 hrs a n d S o u t h e r n blotted a s d e s c r i b e d a b o v e . T h e filter w a s then probed with the isolated I C A M - 2 c D N A G 3 - 1 . 1 . T h e fraction (5.5-7.0 kb) with the most intense b a n d (~ 6.5 kb) w a s ligated into EcoRI cut A.gt10 a r m s (750 ng g e n o m i c D N A with 5 u.g X arms) a n d p a c k a g e d a s p h a g e particles using the X P a c k a g i n g Extract (Invitrogen; S a n D i e g o , CA).  T h e library w a s s c r e e n e d a s d e s c r i b e d a b o v e for the c D N A cloning, e x c e p t that  the host bacterial strain w a s E. coli C 6 0 0 H f l . cloned c D N A ,  G3-1.1.  T h e probe w a s g e n e r a t e d from the  Several genomic clones were  s c r e e n i n g ) a n d s u b c l o n e d into EcoRI cut p B S T .  isolated (three rounds of  T h e c l o n e s w e r e found to h a v e t h e  s a m e restriction e n z y m e pattern a n d thus only o n e , B M 1 - 1 . 1 , w a s a n a l y z e d further by partial s e q u e n c i n g a n d restriction e n z y m e m a p p i n g .  2:2.8 Purification of cell surface ICAM-2 T h e purification of murine I C A M - 2 from B W 5 1 4 7 cells required two preliminary s t e p s , biotinylation of small amount of B W 5 1 4 7 a n d the coupling of t h e anti-mouse I C A M - 2 (3C4) antibody to Affi-gel 10 b e a d s . In the coupling p r o c e s s , 5 m g of purified 3 C 4 antibody in 0.1 M s o d i u m bicarbonate buffer p H 8.0, 0 . 8 5 % N a C l w a s incubated with 2 . 5 ml of Affi-gel  10 b e a d s ( B i o R a d ) p r e w a s h e d with 10 ml with i c e cold  i s o p r o p a n o l a n d then 2 0 ml of ice cold double-distilled water. incubated  with t h e antibody  overnight  with gentle  agitation  T h e b e a d s w e r e then at 4 ° C .  Residual  s u c c i n i m i d e esters w e r e inactivated with the addition of 0.1 M Tris p H 7.5. In the other  60  step, B W 5 1 4 7 cells w e r e w a s h e d twice with biotin labeling buffer p h o s p h a t e s , plus 10 m M N a H C 0  3  p H 7.4) at 4°C.  (HBSS  minus  Immediately before u s e , a stock  solution of sulfo-N-hydroxy s u c c i n i m i d e biotin (sNHS-biotin) ( P i e r c e C h e m i c a l C o . ; R o c k f o r d , IL) w a s prepared at 10 mg/ml in labelling buffer.  B W 5 1 4 7 cells (1 X 10 ) 7  w e r e s u s p e n d e d in 1 ml of labelling buffer a n d 4 0 u.1 of biotin stock solution w a s a d d e d a n d gently vortexed (von B o x b e r g et al., 1990). T h e cells w e r e then incubated on ice for 2 0 min with o c c a s i o n a l s h a k i n g followed by five w a s h e s of 30 ml of D M E M + 2 % F C S . After labeling, the cells w e r e d i s s o l v e d in lysis buffer containing 1.0% nonidet P 40  (NP-40),  120  mM  NaCl,  4  mM  MgCI , 2  20  mM  Tris  pH  7.5,  4  ug/ml  phenylmethylsulfonyl fluoride ( P M S F , S i g m a C h e m i c a l C o . ) , a n d 50 m M L-lys a n d i n c u b a t e d on ice for 1 hr with o c c a s s i o n a l vortexing. T h e solution w a s then microfuged at 14,000 g for 10 min to pellet nuclei a n d other debris. T h e resulting supernatant (1 ml) containing biotinyalted proteins w a s ready to be u s e d a s a tracer in the s u b s e q u e n t purification p r o c e s s . T h e large s c a l e purification of murine I C A M - 2 is similar to that d e s c r i b e d for the murine I C A M - 1 (Horley et al., 1989).  B W 5 1 4 7 cells w e r e grown in s p i n n e r bottles  containing D M E M + 5 % F C S equilibrated with 5 % C 0 . 2  w h e n the cell density r e a c h e d 2-3 X 1 0 per ml. 6  T h e cells w e r e h a r v e s t e d  H a r v e s t e d B W 5 1 4 7 cells (2 X 10 ) 9  w e r e w a s h e d twice with 50 ml of P B S a n d s u s p e n d e d in 4 0 ml 10 m M Tris p H 7.5. T h e cells w e r e then p a s s e d through a 26 g a u g e n e e d l e three times.  Nuclei were  r e m o v e d by low s p e e d centrifugation (1,100 R P M in a B e c k m a n T J - 6 centrifuge) for 15 min at 4°C. After spinning at 19,000 R P M (oakridge t u b e s in a J A - 2 0 rotor) for 50 min  61  at 4°C, the pellet w a s r e s u s p e n d e d in 50 ml of ice cold 10 m M Tris p H 7.5, 1 m M E D T A , 0 . 8 5 % N a C l a n d h o m o g e n i z e d on ice for 5 min with a h o m o g e n i z e r . A n e q u a l v o l u m e of 10 m M Tris p H 7.5, 1 m M E D T A , 0 . 8 5 % N a C l , 2 % Triton X - 1 0 0 containing 2 m M P M S F , 10 Lig/ml leupeptin, a n d 2 u.g/ml aprotinin w a s a d d e d a n d c o m b i n e d with lysate from cell surface biotinylated B W 5 1 4 7 cells (tracer).  T h e solubilized m e m b r a n e  fraction w a s then mixed with a magnetic stir bar for 30 min on ice a n d centrifuged at 10,000 R P M for 30 min. T h e remaining supernatant w a s then c o m b i n e d with the 3 C 4 c o u p l e d Affi-gel 10 b e a d s for 8 hrs under constant agitation at 4°C.  The beads were  then w a s h e d with 4 0 0 ml of 1% Triton X - 1 0 0 , 10 m M Tris p H 7.5, 1 m M E D T A , 0 . 8 5 % N a C l followed by 100 ml of 0 . 1 % Triton X - 1 0 0 , 10 m M Tris p H 7.5, 1 m M E D T A , 0 . 8 5 % N a C l , both at 4°C. B o u n d protein w a s eluted with 0.1 M glycine p H 2.9, 0 . 1 % Triton X 100, 0 . 8 5 % N a C l a n d e a c h 1 ml fraction collected w a s neutralized with 0.5 M N a C 0 . 2  A  10  aliquot  LII  from  e a c h fraction  was  subjected  to  S D S - p o l y a c r y l a m i d e gel  e l e c t r o p h o r e s i s ( S D S - P A G E ) a s outlined previously ( S a m b r o o k et al., fractions  were  analyzed  on  a  10%  polyacrylamide  gel  3  and  1989).  The  electrophoretically  transferred onto a n Immobilon-P m e m b r a n e (Millipore, B e d f o r d , M A ) by electroblotting at 3 5 0 m A for 2 hrs using a Tris-glycine-methanol transfer s y s t e m (0.192 M glycine, 2 5 m M Tris p H 8.3, 2 0 % methanol) (Liu et al., 1994). T h e m e m b r a n e w a s then p l a c e d in blocking buffer ( 2 % B S A , 0 . 0 5 % T w e e n - 2 0 in P B S ) for 2 hrs at room temperature a n d w a s h e d three times in w a s h buffer ( 0 . 1 % B S A , 0 . 0 5 % T w e e n - 2 0 in P B S ) . T h e filter w a s then incubated in w a s h buffer containing streptavidin conjugated to h o r s e radish peroxidase  (1:10,000  dilution)  (Cedarlane;  62  Hornby,  ON)  for  30  min  at  room  temperature.  After three w a s h e s of five min e a c h in w a s h buffer, the m e m b r a n e w a s  w a s h e d in P B S for five min a n d p l a c e in a n e n h a n c e d c h e m i l u m i n e s c e n c e solution ( A m e r s h a m ; Oakville, O N ) for 1 m i n . T h e m e m b r a n e w a s e x p o s e d to X - r a y film ( K o d a k X A R ) for 2-15 min a n d all fractions which d i s p l a y e d a 5 0 - 5 5 k D b a n d w e r e p o o l e d a n d concentrated in a Centricon C - 3 0 ( A m i c o n ; Beverly, M A ) . T h e purity a n d yield w a s a s s e s s e d by S D S - P A G E a n d silver staining of the gel ( O h s a w a a n d E b a t a , 1983). T h e purified protein w a s quantitated by c o m p a r i s o n of b a n d intensity with that of k n o w n a m o u n t s of B S A .  2:2.9 Isolation and activation of murine splenic T lymphocytes M u r i n e s p l e n i c T cells w e r e isolated from B A L B / c mice, 8-12 w e e k s old, using a nylon w o o l ( P o l y s c i e n c e s , Warrington, P A ) c o l u m n a s d e s c r i b e d (Julius et al., 1973). Briefly, 2 g m of nylon w o o l w a s sterilized by autoclave a n d incubated overnight in a p a c k e d c o l u m n containing R P M I 1640 + 5 % F C S at 37°C in a 5 % C 0 atmosphere.  2  humidified  S i n g l e cell s u s p e n s i o n of s p l e e n cells w e r e then incubated in t h e nylon  w o o l c o l u m n for 1 hr. N o n - a d h e r e n t T cells w e r e then eluted with R P M I 1640 + 5 % F C S at a flow rate of ~ 1 ml/min a n d the contaminating red blood cells w e r e lysed with a T r i s - a m m o n i u m chloride solution (17 m M Tris, 140 m M N H C I , p H 7.2; Hunt, 1979). 4  T h e cells w e r e then incubated in R P M I 1640 + 5 % F C S containing 5 0 ng/ml phorbol 12-myristate 13-acetate ( P M A ) for 20 min at 37°C at a concentration of 1 x 1 0 cells/ml 6  ( P y s z n i a k et al., 1994).  Following this, the T cells w e r e w a s h e d three times with  H a n k ' s B a l a n c e d Salt Solution ( H B S S ) a n d labeled with the fluorescent d y e C a l c e i n -  63  A M ( M o l e c u l a r P r o b e s , E u g e n e . O R ) a c c o r d i n g to the manufacturer's protocol. T h e s e labeled T cells w e r e then ready to be u s e d in s u b s e q u e n t a d h e s i o n a s s a y s .  2:2.10 Binding of splenic T cells to purified ICAM-2 Purified murine I C A M - 2 w a s covalently c o u p l e d to microwell plates ( F a l c o n 3 0 7 2 , B e c t o n D i c k i n s o n , Lincoln Park, N J ) a s d e s c r i b e d previously (Horley et al., 1989). Purified recombinant soluble I C A M - 1 , provided by A n d r e w P y s z n i a k (Terry F o x Laboratory, B C C a n c e r R e s e a r c h Centre), (Welder et al., 1993) w a s a l s o c o u p l e d to microwells a n d u s e d a s a positive control.  W e l l s of 96-well microplates w e r e treated  with 100 u.l 0 . 2 % glutaraldehyde, 0.1 M s o d i u m c a r b o n a t e - H C I p H 9.0 for 1 hr at room temperature.  T h e wells w e r e then w a s h e d three times with 0.1 M s o d i u m c a r b o n a t e -  HCI p H 9.0 buffer a n d 5 0 u.l of poly-L-lysine (50 u.g/ml) in 0.05 M s o d i u m c a r b o n a t e p H 9.0 buffer. T h e plates w e r e then incubated for 2 hr at room temperature a n d w a s h e d three times with 0.05 M s o d i u m carbonate p H 9.0 buffer. T h e wells then r e c e i v e d 50 ul of 0 . 2 % glutaraldehyde  in 0 . 0 5 M s o d i u m c a r b o n a t e p H 9.0 for 1 hr at room  temperature a n d w a s h e d three times with 0.05 M s o d i u m c a r b o n a t e p H 9.0 buffer. E a c h well then received 2 5 ul of either  I C A M - 1 (100 ng), I C A M - 2 (100 ng), or 1%  o v a l b u m i n in 0.1 M s o d i u m c a r b o n a t e buffer p H 9.0. After a 2 hr incubation at room temperature, the wells w e r e w a s h e d and received 150 ul of 1% B S A in 0.1 M s o d i u m c a r b o n a t e buffer in order to neutralize the free glutaraldehyde g r o u p s .  The PMA-  activated C a l c e i n - l a b e l e d splenic T cells s u s p e n d e d in H B S S / 5 % F C S a n d d i s p e n s e d into the wells ( 1 0 cells/well) in a final v o l u m e of 100 u l All conditions w e r e tested in 5  64  triplicate.  U n l e s s otherwise stated, anti- I C A M - 1 A b or a n t i - I C A M - 2 A b w e r e a d d e d to  the wells 15 min prior to the addition of the cells at a concentration of 2 Lig/ml or 4 L i g / m l , respectively. A n t i - C D 1 1 a antibody (2 Lig/ml) w a s incubated with s p l e n i c T cells for 15 min at 37°C prior to addition to the wells. T h e plates w e r e centrifuged at 3 0 0 g for 1 m i n , incubated for 8 min at 37°C, a n d w a s h e d five times with H B S S + 5 % F C S (37°C).  F l u o r e s c e n c e of the remaining bound cells w a s m e a s u r e d by a C y t o F l u o r  2 3 0 0 (Millipore) a n d c o m p a r e d with a standard curve.  2:2.11 Cloning of ICAM-2 cDNA into expression vector T h e murine I C A M - 1 (Horley et al., 1989) a n d I C A M - 2 c D N A s w e r e c l o n e d into the m a m m a l i a n e x p r e s s i o n vector p B C M G S ( K a r a s u y a m a et al., 1990).  The cDNAs  w e r e oriented in p B S T ( S K ) s u c h that the 5' e n d of the c D N A w a s adjacent to the Xhol site a n d t h e 3' e n d w a s adjacent to the Notl site. T h e insert w a s liberated from p B S T by digestion with Notl a n d Xhol a n d w a s c l o n e d into p B C M G S digested with Notl a n d Xhol, with t h e Xhol site being c l o s e s t to the promoter.  T h e s e l e c t a b l e m a r k e r s for  p B C M G S a r e ampicillin resistance in bacteria (plated o n L B a g a r with 5 0 Lig/ml of ampicillin) a n d n e o m y c i n resistance in eukaryotic cells (selected in D M E M + 5 % F C S + 0.5 mg/ml G 4 1 8 ) .  p B C M G S is a n e p i s o m a l e x p r e s s i o n vector w h e r e replication is  b a s e d o n a bovine papilloma virus replication s y s t e m .  It maintains a relatively high  c o p y n u m b e r (20-100 copies/cell) a n d the C M V - b a s e d p r o m o t e r / e n h a n c e r s y s t e m aid in high e x p r e s s i o n of the insert.  65  <  2:2.12 Binding of splenic T cells to L cell transfectants expressing I CAMs L cells w e r e transfected with either murine I C A M - 1 (Horley et al., 1989) or murine  ICAM-2 cDNA  in the e x p r e s s i o n vector p B C M G S  ( S i g m a C h e m i c a l C o . ) method (Dong et al., 1993).  by the poly-L-ornithine  Transfectants w e r e then s e l e c t e d  in D M E M + 5 % F C S containing 0.5 mg/ml G 4 1 8 for 5-8 d a y s .  G 4 1 8 - r e s i s t a n t cells  w e r e then a n a l y z e d for e x p r e s s i o n of I C A M - 1 and I C A M - 2 by flow cytometry.  The L  cell transfectants w e r e grown for 2 d a y s in D M E M + 5 % F C S containing 0.5 mg/ml G418.  T h e cells w e r e harvested with P B S + 2 . 5 m M E D T A and incubated with anti-  I C A M - 1 o r a n t i - I C A M - 2 antibodies (3 X 1 0 cells and 4 |^g/ml antibody in 100 uJ H B S S 5  + 2 % F C S + 0 . 1 % N a N ) o n ice for 20 min. T h e s e c o n d a r y stain w a s goat ( F a b ) - a n t i 3  2  rat IgG conjugated to fluorescein isothiocyanate ( G a R l g G - F I T C ) ( C o o p e r B i o m e d i c a l , West Chester, PA).  D e a d cells w e r e stained with 2 u.g/ml propidium iodide. T h e  staining w a s a n a l y z e d on a F A C S t a r ( B e c t o n - D i c k s o n ) . T h e L cells e x p r e s s i n g I C A M - 1 or I C A M - 2 w e r e plated in 96-well flat-bottom microwell plates ( F a l c o n 3 0 7 2 , B e c t o n Dickinson) in 2 0 0 ul of D M E M + 5 % F C S a n d grown for 2 d a y s to a subconfluent monolayer.  A d h e s i o n of P M A - a c t i v a t e d s p l e n i c T  cells to t h e s e m o n o l a y e r s w a s e x a m i n e d in the s a m e w a y a s d e s c r i b e d a b o v e for a d h e s i o n to purified I C A M - 2 .  66  2:3 R e s u l t s 2:3.1 PCR sequence  analysis  T h e nucleotide identity between the h u m a n a n d murine I C A M - 1 is low (50%) (Horley et al., 1989; S i u et al., 1989) a n d using the h u m a n c D N A a s a probe to clone the murine c D N A presented s o m e difficulties.  In order to avert s u c h p r o b l e m s in  cloning the murine I C A M - 2 c D N A , a murine specific probe w a s preferred.  This was  a c h e i v e d by exploiting the c o n s e r v e d s e q u e n c e s a m o n g the known m e m b e r s of the I C A M subfamily. A m i n o acid s e q u e n c e s of h u m a n I C A M - 1 , I C A M - 2 , a n d murine I C A M - 1 (only o n e s available w h e n project began) d i s p l a y e d a high d e g r e e of s e q u e n c e identity a r o u n d the cysteine residues that form disulphide linkages b e t w e e n t h e t w o p-sheets of their Ig-like d o m a i n s .  T h e s e covalent b o n d s a r e thought to stabilize t h e Ig-like  structure a n d a r e a n excellent point to begin a n a l y s i s of c o n s e r v a t i o n of s e q u e n c e s a m o n g m e m b e r s of the I C A M subfamily. A s s e e n in Figure 1, there is a strong d e g r e e of c o n s e r v a t i o n around the cysteine residues in d o m a i n s 1 a n d 2 at both the a m i n o acid a n d nucleotide levels.  W h e n the nucleotide s e q u e n c e in t h e s e two regions is  e x a m i n e d closely, it is apparent that there is a c o n s e n s u s s e q u e n c e with very little d e g e n e r a c y in t h e s e two regions c o n s e r v e d a m o n g all three I C A M m e m b e r s .  In order  to c l o n e a murine-specific probe for I C A M - 2 , t h e s e d e g e n e r a t e oligonucleotides w e r e u s e d to P C R amplify I C A M - l i k e D N A fragments. E L - 4 cells w e r e a g o o d s o u r c e for this amplification s i n c e they d o not e x p r e s s detectable levels of I C A M - 1 a n d thus a n y I C A M - l i k e D N A fragments detected would b e either I C A M - 2 , I C A M - 3 , or s o m e novel  67  Regionl  a)  mICAM-1 hICAM-1 hICAM-2  DAQVSIHPREAFLPQGGSVQ|VNCSSSC|KEDLSLGLETQWLKDE- LESGPNWKLF 53 QTSVSPSKVILPRGGSVLVTCSTSC 3QPKLLGIETPLPKKELLLPGNNRKVY 52 SDEKVFEVHVRPKKLAVEPKGSLEVNCSTTC ^IQPEVGGLETSL - NKI LLDEQAQWKHY 57 P GS V CS C G ET L K  mICAM-1 hICAM-1 hICAM-2  ELSEIGEDSSPLCFENCGTVQSSASATITVYSFPESVELRPLPAWQQVGKDLTLRCHV 111 ELSNVQEDSQPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPSWQPVGKNLTLRCQV 110 LVSNISHDTVLQCHFTCSGKQESMNSNVSVYQPPRQVILTLQPTLVAVGKSFTIECRV 115 S D C C Q V Y P V L P V G K T C V  mICAM-1 hICAM-1 hICAM-2  DGGAPRTQLSAVLLRGEEILSRQPVGGHPKDPKEITFTVLASRGDH--G-ANFSCRTE|166 EGGAPRANLTVVLLRGEKELKREPAVG — EPAEVTTTVLV-RRDHH-G-ANFSCRTE PTVEPLDSLTLFLFRGNETLHYETFGKAAPAPQEATATFNS-TADREDGHRJNFSCLAV P L L R G L P E T T D G Region 2  mICAM-1 hICAM-1 hICAM-2  LDLRPQGLALFSNVSEARSLRTFDLPATIP LDLRPQGLELFENTSAPXQLQTFVLPATPP LDLMSRGGNIFHKHSAPKMLEIYEPVSDSQ LDL G F S L  196 192 202  b) Region 2  Regionl  mICAM-1  GTG AAC TGT TCT TCC TCA TGC Val Asn Cys S e r S e r S e r Cys  mICAM-1  AAT TTC TCA TGC CGC ACA GAA Asn Phe S e r C y s A r g T h r G l u  hICAM-1  GTG ACA TGC AGC ACC TCC TGT Val Thr Cys S e r T h r S e r Cys  hICAM-1  AAT TTC TCG TGC CGC ACT GAA Asn Phe S e r C y s A r g T h r G l u  hICAM-2  GTC AAC TGC AGC ACC ACC TGT Val Asn Cys S e r Thr T h r Cys  hICAM-2  AAC TTC TCC TGC CTG GCT GTG Asn Phe S e r C y s Leu A l a V a l  s e q u e n c e GTC AAA TGC ACC ACC ACA TGC G CC T TGT T T C T  s e q u e n c e AAC TTC TCC TGC CGC ACA GAA T G TG G T TG  oligo  comp. oligo  GTC AAC TGC AGC ACC ACA TG T T T T C  Figure 1  CAC AGC CAG GCA GGA GAA ATT T GC G  Comparison of human and murine ICAM sequences,  s e q u e n c e of m o u s e I C A M - 1 is a l i g n e d with t h e h u m a n ICAM-1  a)  T h e amino  and human  acid  ICAM-2  sequences.  T h e fourth line s h o w s the a m i n o a c i d r e s i d u e s c o m m o n to all three  sequences.  T h e b o x e d regions indicate the a r e a s of c o n s e r v e d h o m o l o g y from w h i c h  the d e g e n e r a t e o l i g o n u c l e o t i d e s w e r e c o n s t r u c t e d , two  regions  including  nucleic  acid  b) S e q u e n c e a n a l y s i s from t h e s e  s e q u e n c e , a c o n s e n s u s s e q u e n c e , a n d the  o l i g o n u c l e o t i d e s e q u e n c e u s e d to P C R amplify a c D N A c o n t a i n e d within t h e s e t w o regions.  D e g e n e r a c y at e a c h position of the o l i g o n u c l e o t i d e is indicated by t h e b a s e s  aligning a b o v e e a c h other.  68  ICAM.  T h e P C R products obtained w e r e characterized by partial s e q u e n c i n g (20  c l o n e s a n a l y z e d ) a n d only two w e r e similar to the h u m a n I C A M - 2 g e n e . T h e other 18 either h a d no long o p e n reading frame or no similarity to any p u b l i s h e d s e q u e n c e s . C o m p a r i s o n of a m i n o acid s e q u e n c e e n c o d e d by the c l o n e d P C R fragment with the p u b l i s h e d h u m a n I C A M - 2 s e q u e n c e revealed 6 3 % amino acid identity in a region in d o m a i n s 1 a n d 2 s p a n n i n g 134 r e s i d u e s (Figure 2). T h e identity w a s e v e n l y distributed throughout the s e q u e n c e with s e v e r a l stretches of continuous identity.  B a s e d o n this,  it w a s c o n c l u d e d that the P C R fragment w a s the partial murine equivalent of the h u m a n I C A M - 2 g e n e . Interestingly, n o n e of the P C R - d e r i v e d c l o n e s w e r e I C A M - 1 . T h e 6 3 % a m i n o acid identity between the 2 P C R c l o n e s a n d the h u m a n I C A M - 2 g e n e strongly indicates that the c l o n e s obtained w e r e derived from the murine h o m o l o g u e of ICAM-2.  O n e of t h e s e c l o n e s w a s u s e d a s a probe to s c r e e n a lung c D N A library  b e c a u s e the e x p r e s s i o n of I C A M - 2 in lung is high (shown in section 2:3.3).  2:3.2 Analysis of mouse ICAM-2 cDNA sequence O f t h e approximately 2 X 1 0 p h a g e from a lung c D N A library that w e r e 5  s c r e e n e d with the c l o n e d P C R fragment, eight p h a g e w e r e isolated w h i c h hybridized to the probe. T h e s e c l o n e s w e r e converted to insert-containing  p B S T plasmid a n d  d i g e s t e d with EcoRI a n d Xhol w h i c h liberated the inserts of three s i z e s (1 of ~0.6 kb, 4 of - 0 . 9 kb, 3 of ~1.2 kb). S i n c e c D N A s of ~ 0.9 kb w e r e found to b e incomplete, t h e 1.2  kb c D N A s w e r e a n a l y z e d further.  T h e s e three  larger c D N A s g a v e similar  restriction e n z y m e digest patterns, thus only o n e (G3-1.1) w a s u s e d for more e x t e n s i v e  69  PCR  clone  hICAM-2  PDMGGLETPTNKIMLEEHPQGKWKQFLVSNVSKDTVFFCHFTCSGKQHSE PEVGGLETSLNKILLDEQAQ--WKHYLVSNISHDTVLQCHFTCSGKQESM P  PCR  clone  hICAM-2  GGLET  clone  hICAM-2  L  E  Q  WK  LVSN  S DTV  CHFTCSGKQ  S  SLNIRVYQPPAQVTLKLQPPRVFVGEDFTIECTVSPVQPLERLTLSLLRG NSNVSVYQPPRQVILTLQPTLVAVGKSFTIECRVPTVEPLDSLTLFLFRG N  PCR  NKI  VYQPP  QV  L  LQP  V VG  FTIEC  V  V  PL  LTL  L  RG  RETLKNQTFGGAETVPQEATATFNSTALKKDGL NETLHYETFGKAAPAPQEATATFNSTADREDGH ETL  TFG  A  PQEATATFNSTA  DG  F i g u r e 2 Alignment of protein sequence from the PCR clone with human ICAM-2. E L 4 c D N A w a s u s e d a s a template for P C R amplification with the d e g e n e r a t e o l i g o n u c l e o t i d e s from R e g i o n 1 a n d R e g i o n 2. T h e c l o n e d fragment w a s s e q u e n c e d a n d the only o p e n reading frame g e n e r a t e d the a m i n o acid s e q u e n c e s h o w n in the top line. It w a s aligned with h u m a n I C A M - 2 s e q u e n c e a n d r e s i d u e s c o m m o n to both w e r e s h o w n o n the third line.  70  sequence analysis.  S m a l l e r fragments of the G3-1.1 insert g e n e r a t e d by cutting the  insert with Stul, Pstl, Asp700l, Aval, Styl, a n d Nhel w e r e s e q u e n c e d o n both strands with the T a n d T primers. T h e G3-1.1 insert is 1124 nucleotides (nt) with t h e largest 3  7  o p e n reading frame ( O R F ) beginning at the A T G initiation c o d o n at position 123 a n d e n d i n g with the stop c o d o n T G A at position 954. T h e 5' untranslated region (5' U T R ) is 122 nt in length (Figure 3). T h e 3' U T R is 170 nt in length with a n 18 nt poly(A) tail 14 nt after the polyadenylation signal ( A A T A C A ) . T h e d e d u c e d a m i n o acid s e q u e n c e of the c D N A - e n c o d e d protein indicates a n O R F of 2 7 7 r e s i d u e s in length with s e q u e n c e characteristic of a t r a n s m e m b r a n e protein.  O f t h e 2 7 7 residues, the first 19 residues m a k e up the h y d r o p h o b i c leader  s e q u e n c e w h i c h is important in directing the translated protein to the cell s u r f a c e . T h e 203  extracellular  residues  a r e followed  by  a  hydrophobic  26  amino  acid  t r a n s m e m b r a n e d o m a i n a n d a hydrophilic 2 9 amino acid c y t o p l a s m i c d o m a i n . T h e extracellular  region  glycosylation sites.  encodes  two Ig-like  domains  containing  five  potential  N-  T h e first d o m a i n has four cysteine r e s i d u e s w h i c h probably form  two disulphide b o n d s to further stabilize the tertiary structure of the d o m a i n .  In  contrast, the s e c o n d d o m a i n h a s only two cysteine r e s i d u e s to form o n e disulphide b o n d . T h e s e q u e n c e identity b e t w e e n murine a n d h u m a n I C A M - 2 is 6 0 % at the a m i n o acid level a n d 7 0 % at the nucleotide level. spread  throughout  the coding  region  A s s h o w n in Figure 4 , the similarity is  with  a stretch  of near  identity  in the  t r a n s m e m b r a n e a n d c y t o p l a s m i c regions. T h i s indicates that the G 3 - 1 . 1 c D N A is the murine h o m o l g u e of I C A M - 2 .  71  CGGGGGAGCGCCAGGCTTCACTCCCCGACCTGTAGCAGACATCTCTC  47  CCTAACCCTCCAGGCAGCCGTCAGCTGTGCCCCTGAAGCCCATAGACTCCACAGACCCCACAGACCCCACCTGAG  122  ATGTCTTCTTTTGCTTGCTGGAGCCTGTCTCTTCTTATCCTGTTCTACAGCCCAGGGTCTGGTGAGAAGGCCTTT MetSerSerPheAlaCysTrpSerLeuSerLeuLeuIleLeuPheTyrSerProGlySerGlyGluLysAlaPhe -19 +1 GAGGTCTACATATGGTCCGAGAAGCAGATAGTAGAAGCCACAGAGTCTTGGAAAATCAACTGCAGCACCAACTGC G l u V a l T y r l l e T r p S e r G l u L y s G l n l l e V a l G l u A l a T h r G l u S e r T r p L y s I l eAsnCysSerThrAsnCys ---CHO--GCAGCCCCAGACATGGGCGGCCTGGAGACGCCCACGAATAAAATAATGTTGGAAGAGCATCCTCAAGGGAAGTGG Al a A l a P r o A s p M e t G l y G l y L e u G l u T h r P r o T h r A s n L y s I l e M e t L e u G l u G l u H i s P r o G l n G l y L y s T r p  197 6 272 31 347 56  AAACAGTTCTTAGTCTCAAACGTCTCCAAAGACACGGTCTTCTTTTGCCATTTCACGTGTTCGGGAAAGCAGCAC LysGlnPheLeuValSerAsnValSerLysAspThrValPhePheCysHi sPheThrCysSerGlyLysGInHi s ---CHO--TCGGAGAGTCTCAACATCAGGGTGTACCAGCCTCCAGCTCAAGTCACACTGAAGCTGCAGCCGCCTCGGGTGTTT SerGluSerLeuAsnlleArgValTyrGlnProProAlaGlnValThrLeuLysLeuGlnProProArgValPhe  422 81  GTGGGTGAAGACTTCACCATTGAGTGCACGGTGTCCCCTGTGCAGCCCCTTGAGAGGCTCACCCTCTCTCTGCTC ValGlyGluAspPheThrlleGluCysThrValSerProValGlnProLeuGl uArgLeuThrLeuSerLeuLeu  572 131  CGTGGCAGAGAGACCCTGAAGAATCAGACCTTTGGGGGAGCAGAAACTGTCCCCCAAGAGGCCACAGCCACGTTC ArgGlyArgGluThrLeuLysAsnGlnThrPheGlyGlyAlaGluThrValProGlnGluAlaThrAlaThrPhe ---CHO--AACAGCACAGCTCTGAAAAAGGACGGTCTCAACTTTTCCTGCCAGGCTGAGCTGGATCTACGGCCCCATGGTGGG AsnSerThrAlaLeuLysLysAspGlyLeuAsnPheSerCysGlnAlaGluLeuAspLeuArgProHisGlyGly ---CHO-----CH0--TATATCATCCGCAGCATCTCGGAGTACCAGATCCTTGAAGTCTATGAGCCGATGCAGGACAACCAAATGGTCATC T y r l l e l l e A r g S e r l l e S e r G I u T y r G l n i l e L e u G l u V a l T y r G l u P r o M e t G l nAspAsnGl n M e t V a l H e  647 156  797 206  ATCATCGTGGTGGTGTCAATACTGCTGTTCTTATTTGTGACATCTGTCCTGCTATGCTTTATCTTTGGCCAGCAC II e l l e V a l V a l V a l S e r l l e L e u L e u P h e L e u P h e V a l T h r S e r V a l L e u L e u C y s P h e l l e P h e G l y G l n H i s  872 231  TGGCACAGAAGACGGACAGGCACCTACGGGGTGCTAGCTGCCTGGAGGAGGCTGCCCCGAGCCTTTCGGGCACGT TrpHi s A r g A r g A r g T h r G l y T h r T y r G l y V a l L e u A l a A l a T r p A r g A r g L e u P r o A r g A l aPheArgAl aArg  947 256  CCCGTGTGAGCCCACGTTGCCAGGCCCCTGGTGGTTACCAGAACTCAACATGGCACCTTCAAGGTGTGGTTCGGC ProVal***  1022 258  ACTGGCTGAAGGACTGTGGCGGCAGCAGCAGATGCGGGGGACATTTCCTCTCCTTTTTAGCCTCAATACAAATAT  1096  CTGGATTTCAAAAAAAAAAAAAAAAAA  497 106  722 181  1124  Figure 3 Nucleic acid and protein sequence of the murine ICAM-2 cDNA (G3-1.1). T h e c o m p l e t e nucleotide s e q u e n c e of the G3-1.1 c l o n e is s h o w n with t h e predicted a m i n o acid residue b e n e a t h its c o r r e s p o n d i n g c o d o n . T h e predicted N-terminal signal peptide is underlined ( ), potential glycosylation sites a r e m a r k e d by — C H O — , c y s t e i n e r e s i d u e s are in bold, the t r a n s m e m b r a n e region is underlined with a thick line {^^^^m), t h e translation termination c o d o n is m a r k e d with *** b e n e a t h it, a n d the polyadenylation signal s e q u e n c e A A T A C A is b o x e d . T h e a m i n o a c i d s e q u e n c e is n u m b e r e d from the predicted c l e a v a g e site of the signal peptide.  72  G3-1.1 hICAM-2  SGEKAFEVYIWSEKQIVEATESWKINCSTNCAAPDMGGLE SDEKVFEVHVRPKKLAVEPKGSLEVNCSTTCNQPEVGGLE S EK FEV K VE S NCST C P GGLE  40 40  G3-1.1 hICAM-2  TPTNKIMLEEHPQGKWKQFLVSNVSKDTVFFCHFTCSGKQ TSLNKILLDEQAQ--WKHYLVSNISHDTVLQCHFTCSGKQ T NKI L E Q WK LVSN S DTV CHFTCSGKQ  80 78  G3-1.1 hICAM-2  HSESLNIRVYQPPAQVTLKLQPPRVFVGEDFTIECTVSPV ESMNSNVSVYQPPRQVILTLQPTLVAVGKS-FTI ECRVPTV S N VYQPP QV L LQP V VG FTIEC V V  120 118  G3-1.1 hICAM-2  QPLERLTLSLLRGRETLKNQTFGGAETVPQEATATFNSTA EPLDSLTLFLFRGNETLHYETFGKAAPAPQEATATFNSTA PL LTL L RG ETL TFG A PQEATATFNSTA  160 158  G3-1.1 hICAM-2  LKKDGL-NFSCQAELDLRPHGGYIIRSISEYQILEVYEPM DREDGHRNFSCLAVLDLMSRGGNIFHKHSAPKMLEIYEPV DG NFSC A LDL GG I S LE YEP  199 198  G3-1.1 hICAM-2  QDNQMVIIIVVVSILLFLFVTSVLLCFIFGQHWHRRRTGT SDSQMVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQRMGT D QMVII VVS LL LFVTSVLLCFIFGQH R GT  239 238  G3-1.1 hICAM-2  YGV LAAWRRLPRAFRARPV YGVRAAWRRLPQAFRP YGV AAWRRLP AFR  258 254  F i g u r e 4 Comparison of amino acid sequence between cDNA-encoded (G3-1.1) protein and human ICAM-2. T h e a m i n o acid s e q u e n c e derived from the c l o n e d G3-1.1 c D N A is aligned with the h u m a n ICAM-2 s e q u e n c e . T h e third line s h o w s t h e residues s h a r e d by the two s e q u e n c e s .  73  2:3.3 Northern blot analysis E x p r e s s i o n of murine I C A M - 2 m R N A in lymphoid a n d non-lymphoid t i s s u e s w a s e x a m i n e d by Northern blot a n a l y s i s (Figure 5). A single s p e c i e s of ~ 1.2 kb w a s d e t e c t e d in t h e various t i s s u e s which e x p r e s s e d I C A M - 2 . L o w levels w e r e d e t e c t e d in b o n e marrow cells, while s p l e e n a n d thymus d i s p l a y e d moderate level of I C A M - 2 expression.  I C A M - 2 w a s not detected in liver tissue.  H o w e v e r , low levels of I C A M - 2  w e r e s e e n in heart a n d kidney t i s s u e s , while very high levels w e r e d e t e c t e d in lung tissue. T h i s e x p r e s s i o n pattern of murine I C A M - 2 is similar to that of h u m a n I C A M - 2 . T h e high level of e x p r e s s i o n in lung is e x p e c t e d b e c a u s e lung tissue is a b u n d a n t in e n d o t h e l i u m , w h i c h constitutively e x p r e s s e s high levels of I C A M - 2 .  2:3.4 Genomic cloning T h e m o u s e I C A M - 2 c D N A w a s then u s e d a s a probe in g e n o m i c S o u t h e r n blot a n a l y s i s of B A L B / c s p l e e n D N A digested with various restriction e n z y m e s (which d o not cut t h e G 3 - 1 . 1 c D N A ) a l o n e a n d in combination (Figure 6). In four c a s e s , a single b a n d w a s d e t e c t e d (BamHI, EcoRI, Hindlll, Xhol).  In the c a s e of Dral, the blot  d i s p l a y e d two b a n d s (possibly d u e to the p r e s e n c e of a Dral site within a n intron). D o u b l e digests a l s o revealed a single banding pattern. It a p p e a r s that murine I C A M - 2 , like its h u m a n h o m o l o g u e , is present a s a single c o p y g e n e in the m o u s e g e n o m e . T h e g e n o m i c organization  of I C A M - 2 could  provide  information  about its  evolution a s well a s its regulation in certain t i s s u e s . In order to isolate a m o u s e I C A M 2 g e n o m i c c l o n e , B A L B / c s p l e e n D N A w a s digested with EcoRI a n d s i z e s e l e c t e d in  74  1 2 3 4  5 67  Actin  F i g u r e 5 Expression of murine ICAM-2 by Northern blot analysis. Total R N A (10 p,g) isolated from various murine t i s s u e s ( B A L B / c ) w a s run o n a f o r m a l d e h y d e g e l a n d blotted onto a Z e t a - p r o b e filter. S o u r c e of R N A in e a c h lane a r e a s follows: 1) heart, 2) lung, 3) liver, 4) kidney, 5) b o n e marrow, 6) s p l e e n , 7) thymus. T h e filter w a s p r o b e d with t h e G3-1.1 c D N A a s well a s a n actin probe.  75  1 2 3 4 5 6 7 8 9  23.1  Figure  6  Genomic Southern blot analysis of murine ICAM-2.  S p l e e n D N A (10 ug) from B A L B / c mice w a s digested with the indicated restriction enzymes, e l e c t r o p h o r e s e d o n a 0 . 8 % a g a r o s e gel, a n d blotted. E n z y m e s in e a c h lane are a s  follows: 1) BamHI, 2) Dral, 3) EcoRI, 4) Hindlll, 5) Xhol, 6) BamHI/EcoRI, 7) EcoRI/Hindlll, 8) EcoRI/Xhol, 9) Hindlll/BamHI. T h e filters w e r e then probed with the G3-1.1 c D N A .  76  the range of 5.5-7.0 kb. A Xgt10 library w a s constructed with the D N A a n d s c r e e n e d with the G 3 - 1 . 1 c D N A .  O n e clone isolated, B M 1 - 1 . 1 , w a s partially s e q u e n c e d (Figure  7) a n d m a p p e d by restriction e n z y m e a n a l y s i s in conjunction with S o u t h e r n  blotting  (Figure 8). It w a s found to b e 6.5 kb in length. T h e coding s e q u e n c e r e s i d e s in a 5 kb region a n d contains a n e x o n coding for the 5' U T R a n d signal peptide, two e x o n s encoding  the two extracellular  Ig-like  domains,  and an  exon  encoding  the  t r a n s m e m b r a n e a n d c y t o p l a s m i c d o m a i n s , a n d the 3' U T R . T h e e x o n s a r e s e p a r a t e d by p h a s e I introns (split e x o n s after the first nucleotide of a codon) a n d the m a p of the g e n o m i c c l o n e is s h o w n in Figure 9. S e q u e n c i n g a l s o g e n e r a t e d 2 3 3 bp of s e q u e n c e 5' to t h e A T G translation initiation start site. Although this is a rather short stretch of s e q u e n c e , there is no apparent T A T A b o x or C A A T - l i k e s e q u e n c e s . T h e r e is h o w e v e r a transcription initiation c o n s e n s u s s e q u e n c e , A T T C T T , at nucleotide at - 2 2 9 (with respect  to t h e translation  start  codon)  suggesting  that there  are  promoter-like  s e q u e n c e s farther upstream.  2:3.5 Purification of ICAM-2 Preliminary binding studies of P M A - a c t i v a t e d s p l e n i c T cells to purified I C A M - 2 c o u l d b e u s e d for e x a m i n i n g parameters of the L F A - 1 : I C A M - 2 a d h e s i o n . T h e murine I C A M - 2 w a s isolated from B W 5 1 4 7 cells, a n A K R thymic l e u k e m i a cell line, b e c a u s e of high e x p r e s s i o n . T h e protein purified by m A b affinity c h r o m a t o g r a p h y w a s a n a l y z e d by S D S - P A G E . T h e silver stained gel (Figure 10) revealed that the isolated protein w a s a single b a n d with a molecular m a s s of 50-55 k D . T h e protein w a s h o m o g e n o u s s i n c e  77  attctt^aggccctaaaggcttgggagctggtctgtgcatattgtttcctgatctcagataattagaggaaatgagctcact  ggcacagaggagattgtggatttcagttgggagcgccaggcttcactccccgacctgtagcagacatctctccctaaccctcca  ggcagccgtcagctgtgcccctgaagcccatagactccacagaccccacagaccccacctgagATGTCTTCTTTT....TTCTA MetSerSerPhe....PheTy CAGCCCAGgtaagccagctcccaggggtttcag.. rSerProG GGGJGTACCgtgagtggctctgctgccgt.. rgValTyrG GJCJAJGgtgaggggaggatccgtaga.. ValTyrG  intronl..cagtggttgattttccagGGJCJGGJGAGAAG. lySerGlyGluLys intron2..cctccttaactccgctgcagAGCCTCCAGCJCAA 1nProProAlaGln  ...ATCA IleA CTTGAA LeuGlu  intron.3..ccacgtcctttgcctcccagAGGCGAJGCAGGAC. . . .GCACGTCC 1uProMetGlnAsp AlaArgPr  CGTGTGAgcccacgttgccaggcccctggtggttaccagaactcaacatggcaccttcaaggtgtggttcggcactggctgaag oVal*** gactgtggcggcagcagcagatgcgggggacatttcctctcctttttagcctcaatacaaatatctggatttc....  Figure 7 Partial nucleotide sequence of the murine ICAM-2 genomic clone.  The partial nucleotide sequence of the ICAM-2 gene determined from the 6.6 kb EcoRI fragment (BM1-1.1) is listed above. Untranslated sequences are in lower case letters, protein coding sequences are in upper case, and intron sequences in bold italics (lower case). Amino acid sequence from the coding region is shown below the corresponding codons of the exons. The consensus transcription start site (ATTCTT) is boxed.  Figure 8  Southern blot analysis of murine ICAM-2 genomic clone (BM1-1.1). T h e 6.6 kb EcoRI insert (750 ng) containing the murine I C A M - 2 g e n o m i c c l o n e w a s digested with various restriction e n z y m e s ( 1 - Xmnl, 2- Aval, 3- BamHI, 4- Kpnl, 5 - Ncol, 6Smal), e l e c t r o p h o r e s e d , a n d blotted onto a filter. T h e filter w a s then probed with the 192 bp fragment g e n e r a t e d by Stul digestion of the G3-1.1 c D N A w h i c h represented the 5' U T R a n d the portion e n c o d i n g the signal peptide. T h e filter w a s a l s o probed with t h e 4 1 1 b p fragment generated by Styl digestion of the G3-1.1 c D N A w h i c h represents the t r a n s m e m b r a n e a n d cytoplasmic portions a s well a s the 3' U T R .  79  Restriction enzymes  B  Aval  A  BamHI  B  Kpnl  K  Ncol  N  Smal  S  XmnI  X  r—W-f K  A  N  lkb Figure 9 Restriction map of the murine ICAM-2 genomic clone. B y combining data from the restriction digestion a n d S o u t h e r n blots of Figure 8 with s e q u e n c e information from the partial s e q u e n c i n g of the g e n o m i c c l o n e a s well a s from the c D N A clone, a m a p of the restriction e n z y m e sites in the BM1-1.1 c l o n e is s h o w n a b o v e . T h e locations of the four e x o n s a r e indicated by filled b o x e s a n d the relative positions of the restriction e n z y m e sites are a l s o s h o w n .  Mr  X  10  F i g u r e 10 Immunopurification of ICAM-2. Murine I C A M - 2 immune-affinity purified from B W 5 1 4 7 p l a s m a m e m b r a n e s w a s subjected to nonreducing S D S - 1 0 % P A G E a n d silver staining.  81  no other b a n d s w e r e detected in the silver stained g e l . A p p r o x i m a t e l y 5 ug of murine I C A M - 2 w a s obtained per litre of cultured B W 5 1 4 7 cells.  2:3.6 Binding to ICAM-2 protein T h e purified murine I C A M - 2 w a s u s e d to e x a m i n e the binding capability of ICAM-2.  P M A - a c t i v a t e d s p l e n i c T cells a d h e r e d to purified  ICAM-1 a n d ICAM-2  c o u p l e d to plastic (Figure 11). This w a s L F A - 1 - s p e c i f i c a s a n t i - C D 1 1 a antibody inhibited the binding to b a c k g r o u n d levels. It w a s a l s o d e p e n d e n t o n divalent cations s u c h a s m a g n e s i u m , s i n c e E D T A a l s o inhibited the binding. A p p r o x i m a t e l y 5 4 % a n d 3 6 % of T cells b o u n d I C A M - 1 a n d I C A M - 2 , respectively. C e l l a d h e s i o n to I C A M - 2 w a s inhibited by t h e I C A M - 2 antibody ( 3 C 4 ) ( 1 1 % of cells bound) but not by the I C A M - 1 antibody.  C e l l a d h e s i o n to I C A M - 1 w a s inhibited by the I C A M - 1 antibody, but w a s  unaffected by the I C A M - 2 antibody.  T h e binding to I C A M - 2 w a s further d e c r e a s e d to  7 % w h e n the I C A M - 2 antibody concentration w a s i n c r e a s e d from 4 u.g/ml to 15 u.g/ml. A s o b s e r v e d previously, resting T cells did not bind to the purified proteins.  2:3.7 Binding to cell surface ICAM-2 In order to e x a m i n e whether the G3-1.1 c D N A e n c o d e s a protein c a p a b l e of binding L F A - 1 , L cells w e r e transfected with the c D N A .  F l o w cytometry r e v e a l e d that  I C A M - 2 w a s detected o n the surface of the transfected cells (Figure 12a).  PMA-  activated s p l e n i c T cells readily a d h e r e d to m o n o l a y e r s of the transfected L cells e x p r e s s i n g I C A M - 1 or I C A M - 2 (Figure 12b). A l t h o u g h the level of I C A M - 2 o n the  82  0 ICAM-2  L + 5 mM EDTAl  B  .'•  ICAM-1  • OVA  + CD11a Ab  binding 0  10  20 30 40 cells bound (%)  Figure 11  50  60  Adhesion of murine splenic T cells to purified ICAM-1 and ICAM-2. I C A M - 2 , I C A M - 1 , a n d ovalbumin w e r e immobilized o n microculture wells. T o e a c h well, 1 0 c a l c e i n A M labeled s p l e n i c T cells ( P M A - a c t i v a t e d or resting) w e r e a d d e d a n d a l l o w e d to p r o c e e d for 8 min at 37°C. Blocking antibodies w e r e a d d e d to P M A activated T cells 15 min prior to the addition of cells to the wells. T h e antibodies w e r e p r e s e n t at a concentration of 2 u.g/ml, except for I C A M - 2 antibody w h i c h w a s present either at 4 u.g/ml or 15 u.g/ml (indicated by *). U n b o u n d T cells w e r e r e m o v e d by w a s h i n g five times with H B S S containing 5 % F C S . R e s u l t s a r e e x p r e s s e d a s a m e a n of triplicate w e l l s ± S E M . 5  83  A)  ICAM-1 Ab  ICAM-2 Ab  L cells  L:ICAM-1  L:ICAM-2  Figure 12 Adhesion of splenic T cells to L cells transfected with ICAM-2. A ) L cells, untransfected or transfected with the I C A M - 1 ( L : I C A M - 1 ) or I C A M - 2 ( L : I C A M - 2 ) c D N A w e r e subjected to flow cytometry using the appropriate antibody a n d F ( a b ' ) goat anti-rat IgG conjugated to F I T C . I C A M - 1 a n d I C A M - 2 e x p r e s s i o n are s h o w n by the solid histogram a n d s e c o n d a r y antibody a l o n e is s h o w n by the outlined histogram. B) (next page) L cells e x p r e s s i n g I C A M - 1 or I C A M - 2 c D N A w e r e grown in 96 well plates. P M A - a c t i v a t e d s p l e n i c T cells labeled with c a l c e i n A M w e r e a d d e d to e a c h well containing a subconfluent m o n o l a y e r of L cells, a n d binding w a s m e a s u r e d a s d e s c r i b e d in F i g u r e 11. 2  84  B)  0  25  50 75 cells bound (%)  (figure legend on previous page)  85  100  transfected L cells w a s higher than that of I C A M - 1 , T cells a d h e r e d to  ICAM-1  transfectants ( 8 6 % of input cells) more efficiently than to I C A M - 2 transfectants (62%). T h e a d h e s i o n of T cells to transfected L cells w a s effectively inhibited by the antiC D 1 1 a m A b a n d the control antibody against C D 4 9 d had no effect on a d h e s i o n . T h e a n t i - I C A M - 2 m A b a l s o inhibited the T cell a d h e s i o n to I C A M - 2 - t r a n s f e c t e d L cells, w h e r e a s the T cell a d h e s i o n to I C A M - 1 - t r a n s f e c t e d L cells w a s effectively inhibited by the m A b to I C A M - 1 .  2:4  Discussion  In the h u m a n s y s t e m , three counter-receptors for L F A - 1 ( I C A M - 1 , I C A M - 2 , a n d I C A M - 3 ) h a v e b e e n identified a n d characterized ( S i m m o n s et al., 1988; S t a u n t o n et al., 1 9 8 8 ; S t a u n t o n et al., 1989; Fawcett et al., 1992; V a z e u x et al., 1992; d e F o u g e r o l l e s et al., 1993). W h e n this project b e g a n , only I C A M - 1 had b e e n identified in the murine s y s t e m (Horley et al., 1989; S i u et al., 1989). In an attempt to isolate additional I C A M like  cDNAs  in the  murine  system, a  PCR-based  strategy  was  explored  using  d e g e n e r a t e oligonucleotides b a s e d on available s e q u e n c e information from the k n o w n ICAMs.  T h e primer s e q u e n c e s w e r e determined on the b a s i s of c o n s e r v e d c y s t e i n e  r e s i d u e s a n d adjacent a m i n o acid s e q u e n c e s from the first two Ig-like d o m a i n s in the h u m a n I C A M - 1 , I C A M - 2 , a n d the murine I C A M - 1 (the only known I C A M s e q u e n c e s at the time).  T h e s e oligoprimers w e r e u s e d to P C R amplify murine I C A M - l i k e D N A  f r a g m e n t s w h i c h w e r e then u s e d a s a probe to s c r e e n for c o m p l e t e c D N A s .  With this  strategy, full length c D N A c l o n e s e n c o d i n g various m o l e c u l e s c a n be isolated.  86  One  group h a d previously c l o n e d the murine I C A M - 1 using the h u m a n I C A M - 1 c D N A a s a probe (Siu et  al.,  1989).  S i n c e the nucleotide homology b e t w e e n the two  DNA  f r a g m e n t s w a s relatively low (50%), the stringency of the s c r e e n w a s a l s o low making the s c r e e n i n g p r o c e s s more difficult.  E L - 4 cells w e r e c h o s e n a s a s o u r c e for P C R  amplification b e c a u s e they exhibit a n L F A - 1 - d e p e n d e n t a d h e s i o n pathway a n d I C A M - 1 e x p r e s s i o n is low (Wuthridge, 1992). This favoured the amplification of murine I C A M like m o l e c u l e s not yet identified. O n e of the P C R c l o n e s generated by the a b o v e a p p r o a c h w a s found to partially e n c o d e a protein with significant similarity to the h u m a n I C A M - 2 . This s u g g e s t e d that the P C R c l o n e w a s derived from the murine equivalent of the h u m a n I C A M - 2 . It a l s o demonstrated  that this strategy  e n c o d i n g related m o l e c u l e s .  h a s the  potential  to  isolate novel c D N A  clones  In the cloning of the h u m a n I C A M - R ( I C A M - 3 ) c D N A , a  similar strategy w a s u s e d ( V a z e u x et al., 1992).  T h e oligonucleotides u s e d a s P C R  primers w e r e derived from s e q u e n c e s of the s e c o n d d o m a i n of I C A M - 1 , I C A M - 2 , N C A M , M A G , P E C A M - 1 , and V C A M - 1 ,  w h e r e a s the primers u s e d in this study s p a n  d o m a i n s o n e a n d two of I C A M - 1 a n d I C A M - 2 . A s well, the template in the I C A M - R P C R cloning w a s g e n o m i c D N A , w h e r e a s in this study it w a s s i n g l e - s t r a n d e d c D N A derived from m R N A of E L - 4 cells. A limitation of using c D N A a s a template for P C R amplification is that the overall effectiveness is determined by e x p r e s s i o n of the m R N A a s well a s relative levels of other similar m R N A s . t h e s e limitations  w e r e circumvented  In the c a s e of the I C A M - R cloning,  by using g e n o m i c D N A a s a template.  A  d r a w b a c k in using g e n o m i c D N A is the p r e s e n c e of introns which m a y be lengthy a n d  87  thus interfere with efficient synthesis of the P C R fragment.  W h i c h e v e r template is  u s e d , the d e g e n e r a t e oligonucleotides m a k e it p o s s i b l e to c l o n e novel I C A M - l i k e s e q u e n c e s without antibodies or specific protein s e q u e n c e information. T h e murine I C A M - 2 c D N A e n c o d e s a type I t r a n s m e m b r a n e protein with two extracellular Ig-like d o m a i n s extending from the cell surface followed by a h y d r o p h o b i c t r a n s m e m b r a n e d o m a i n a n d a hydrophilic cytoplasmic tail. c y s t e i n e r e s i d u e s are c o n s e r v e d .  T h e positions of the  T h e first d o m a i n contains four c y s t e i n e r e s i d u e s  forming two disulphide linkages, while the s e c o n d d o m a i n contains only o n e disulphide b o n d f o r m e d by the two cysteine residues. T h e p r e s e n c e of four c y s t e i n e s in the first d o m a i n of murine I C A M - 2 is also s e e n in h u m a n ( S i m m o n s et al., 1988; S t a u n t o n et al., 1988) a n d murine I C A M - 1 (Horley et al., 1989; S i u et al., 1989), h u m a n I C A M - 2 (Staunton et al., 1989), h u m a n I C A M - 3 (Fawcett et al., 1992; V a z e u x et al., 1992; d e F o u g e r o l l e s et al., 1993), a n d h u m a n (Osborn et al., 1989) a n d murine V C A M - 1 (Araki et al., 1993).  Interestingly, t h e s e are all m e m b e r s of the Ig superfamily w h i c h a d h e r e  to m e m b e r s of the integrin superfamily, L F A - 1 a n d V L A - 4 (Marlin a n d Springer, 1987; E l i c e s et al.,  1990).  Within the I C A M - 2 extracellular region are five potential  glycosylation sites ( A s n - X - S e r / T h r ) .  N-  T h e molecular m a s s of the I C A M - 2 apoprotein  d e d u c e d from the c D N A s e q u e n c e is 28 kD but the mature protein is 55 k D .  This  relatively high i n c r e a s e in molecular m a s s is probably d u e to e x t e n s i v e glycosylation. I C A M - 1 h a s a 55 k D polypeptide b a c k b o n e with the mature protein being ~ 9 0 - 1 0 0 k D a n d the I C A M - 3 polypeptide b a c k b o n e has a molecular m a s s of 57 k D with the mature protein migrating a s a band of 124 kD in S D S - P A G E .  88  I C A M - 1 a n d I C A M - 3 h a v e eight  a n d 15 potential  N-linked glycosylation sites, respectively ( S i m m o n s et  al.,  1988;  S t a u n t o n et al., 1988; Fawcett et al., 1992; V a z e u x et al., 1992; d e F o u g e r o l l e s et al., 1993). T h i s e x t e n s i v e glycosylation in the I C A M s yields a n a v e r a g e i n c r e a s e in m a s s of ~ 4 . 5 k D per glycosylation site.  C o m m o n v a l u e s of N-glycosidic o l i g o s a c c h a r i d e s  are ~ 2-2.5 k D per site ( A s a d a et al., 1991; G a h m b e r g et al., 1 9 9 1 ; N o r t a m o et 1991).  al.,  T h i s higher than a v e r a g e o l i g o s a c c h a r i d e content in I C A M s is c o n s e r v e d not  only a m o n g the m e m b e r s of the subfamily but a l s o b e t w e e n the murine a n d h u m a n s p e c i e s a n d m a y play a role fine tuning the a d h e s i v e property of the  LFA-1:ICAM  interactions. W h e n the h u m a n I C A M - 1 s e q u e n c e is altered s u c h that a glycosylation in the third Ig-like d o m a i n is d e s t r o y e d , it's ability to bind Mac-1 is e n h a n c e d ( D i a m o n d et al.,  1991).  H o w e v e r , it has a l s o b e e n demonstrated that unglycosylated I C A M - 2  purified from a bacterial e x p r e s s i o n s y s t e m is able to bind L F A - 1 ( G a h m b e r g et  al.,  1991). T h e role of the carbohydrate moiety in I C A M - 2 r e m a i n s unclear. A n a l y s i s of the c D N A s e q u e n c e d e m o n s t r a t e s the overall structural identity with the h u m a n I C A M - 2 .  T h e murine I C A M - 2 s h a r e s s e q u e n c e similarity with the h u m a n  I C A M - 2 ( 6 0 % at the a m i n o acid level a n d 7 0 % at the nucleic acid level) with a stretch of n e a r identity s p a n n i n g the t r a n s m e m b r a n e a n d c y t o p l a s m i c regions ( T M a n d Cyto). In the c a s e of I C A M - 2 , there is an 8 5 % a n d a 7 0 % a m i n o acid identity b e t w e e n the murine  and  human  homologue  over  the  TM  and  Cyto  regions,  respectively.  Interestingly, the s e q u e n c e identity b e t w e e n I C A M s of the s a m e s p e c i e s is higher in the  L F A - 1 - b i n d i n g extracellular  domains  than  in  the  TM  and  Cyto  regions.  N o n e t h e l e s s , the conservation in the T M a n d C y t o regions is high b e t w e e n h u m a n a n d  89  their murine equivalents.  T h e s e regions may be important in the function of their  specific I C A M s a n d thus are maintained by s e q u e n c e conservation a c r o s s s p e c i e s . T h e s e regions m a y function in localization on the cell s u r f a c e , interaction with the c y t o s k e l e t o n , a n d signalling.  O n e study has indicated that both h u m a n I C A M - 1 a n d  I C A M - 2 c y t o p l a s m i c tails are able to interact with the a-actinin, a cytoskeletal protein w h i c h c a n a n c h o r actin filaments to the cell m e m b r a n e ( C a r p e n et al., 1992; H e i s k a et al.,  1996).  A l s o , C O S cells transfected with I C A M - 1 exhibited punctated  staining o n the cell surface (Kishimoto et al., 1990; C a r p e n et al., 1992).  ICAM-1 However,  w h e n the c D N A is altered s u c h a s to generate a G P I - l i n k e d version of I C A M - 1 , the staining b e c a m e diffuse.  In addition, I C A M - 2 is able to interact with ezrin ( H e l a n d e r et  al., 1996), a m e m b r a n e - o r g a n i z i n g protein which is thought to act a s a cytoskeletal linker for m e m b r a n e - b o u n d proteins. in a diffuse pattern.  NK-resistant murine target cells e x p r e s s I C A M - 2  H o w e v e r , transfection of ezrin into t h e s e cells c a u s e s the murine  I C A M - 2 to redistribute to uropods and the target cells b e c o m e s e n s i t i z e d to N K - l y s i s . T h e e x p r e s s i o n of murine I C A M - 2 , a s determined by Northern blot a n a l y s i s , is similar to the e x p r e s s i o n pattern of h u m a n I C A M - 2 . I C A M - 2 is constitutively e x p r e s s e d o n l y m p h o c y t e s , granulocytes, platelets, a n d endothelium (de F o u g e r o l l e s et al., 1 9 9 1 ; D i a c o v o et al., 1994).  Detection of I C A M - 2 in lymphoid o r g a n s s u c h a s s p l e e n , b o n e  marrow, a n d t h y m u s may be d u e to the leukocytes present (Xu et al., 1992).  ICAM-2  is a l s o d e t e c t e d in heart, lung, a n d kidney, possibly d u e to leukocytes a n d endothelial cells present in t h e s e o r g a n s . Lung tissue is particularly rich in endothelium a n d this is reflected in the high level of e x p r e s s i o n of I C A M - 2 .  90  T h e s e e x p r e s s i o n patterns h a v e  b e e n confirmed by another group looking at murine I C A M - 2 (Xu et al., d u p l i c a t e s the h u m a n pattern.  1992) a n d  In addition, I C A M - 2 h a s b e e n s h o w n to be n o n -  inducible (de F o u g e r o l l e s et al., 1 9 9 1 ; Nortamo et al., 1 9 9 1 ; O h h et al., 1994).  The  only time I C A M - 2 h a s b e e n found on cells other than the o n e s mentioned a b o v e or at higher levels than normally found in vivo is on n e o p l a s m s ( R o o s , 1 9 9 1 ; Ellis et  al.,  1 9 9 2 ; R e n k o n e n etal., 1992). The  isolation  of  a  murine  genomic  I C A M - 2 D N A fragment  allowed  the  e x a m i n a t i o n of its structure. This is important in understanding its regulation a s well a s the evolutionary origin of m e m b e r s of the I C A M subfamily. T h e g e n o m i c organization of the murine I C A M - 2 g e n e confirms its a s s i g n m e n t a s a m e m b e r of the Ig superfamily (Williams  and  Barclay,  1988;  Hunkapiller  and  Hood,  1989).  The  exon/intron  b o u n d a r i e s of the g e n e are reflected in the structural d o m a i n s of the protein.  The  signal peptide is e n c o d e d by the first e x o n . T h e next two Ig-like d o m a i n s are e n c o d e d by s e p a r a t e e x o n s a n d finally the fourth e x o n c o d e s for the t r a n s m e m b r a n e a n d cytoplasmic domains.  T h e s e e x o n s are s e p a r a t e d by p h a s e I introns in w h i c h the  intron a p p e a r s after the first nucleotide of a c o d o n . P h a s e I introns h a v e b e e n found b e t w e e n n u m e r o u s Ig domain-like e x o n s of other m e m b e r s of the Ig superfamily (Hunkapiller  and  H o o d , 1989).  By maintaining  uniform  intron  phase  in  these  m o l e c u l e s , it allows the possibility of alternate e x o n u s a g e without altering the reading frame.  Uniform intron p h a s e is a l s o important for Ig superfamily evolution, b e c a u s e it  e n s u r e s that m o l e c u l e s with multiple Ig-like d o m a i n s c a n be constructed by e x o n duplication a n d shuffling m e a n w h i l e maintaining the correct reading frame.  91  B a s e d on  s e q u e n c e h o m o l o g y a n d intron/exon boundaries, g e n e duplication a n d  subsequent  d i v e r g e n c e from a primordial d o m a i n are the a c c e p t e d origin of Ig-like m o l e c u l e s ( O w e n s et al., 1987; W i l l i a m s a n d Barclay, 1988; Hunkapiller a n d H o o d , 1989).  The  formation of a distinct subfamily is s o m e w h a t less clear. M e m b e r s of the C D 1 1 family h a v e b e e n found to m a p to a region of o n e c h r o m o s o m e (Corbi et al., evolution  of  this  family  can  be  explained  c h r o m o s o m e s a n d localized to o n e region.  by  unequal  crossing  1988).  over  The  between  H o w e v e r , I C A M - 1 a n d I C A M - 2 in both the  h u m a n a n d murine s y s t e m s are found to be on s e p a r a t e c h r o m o s o m e s (Katz et 1 9 8 5 ; B a l l a n t y n e et al.,  1 9 9 1 ; H o g g et al.,  1 9 9 1 ; K u r a m o t o et al.,  1994).  al.,  Thus,  u n e q u a l c r o s s i n g over cannot explain the generation of the I C A M subfamily in the h u m a n a n d murine s y s t e m s .  It m a y be that the ancestral I C A M - l i k e g e n e duplicated  a n d inserted in various locations in the g e n o m e .  Subsequent divergence may have  o c c u r r e d at e a c h of the insertion sites. A n a l y s i s of the region 5' of the coding region failed to reveal a n y  obvious  regulatory s e q u e n c e s . T h e g e n o m i c c l o n e isolated did h o w e v e r p o s s e s s a c o n s e n s u s transcription initiation s e q u e n c e . A larger portion of the 5' region of the murine I C A M - 2 g e n e h a s b e e n c l o n e d by another group (Xu et al., 1992).  T h e y found that further  u p s t r e a m of the transcription initiation site is a T A T A - l i k e s e q u e n c e a n d a n inverted C A A T box. region.  N o known transcription factor binding sites w e r e identified in the promoter  In contrast, the 5' upstream region of I C A M - 1 h a s NF-KB a n d A P - 1 binding  sites ( V o r a b e r g e r et al., 1991).  I C A M - 2 e x p r e s s i o n is not inducible by cytokines a n d  thus it is not surprising that I C A M - 2 d o e s not p o s s e s s NF-KB a n d A P - 1 binding sites  92  w h i c h h a v e b e e n s h o w n to b e important in transcription of inducible g e n e s s u c h a s I C A M - 1 (Angel etal., 1987; E d b r o o k e etal., 1989). S o m e resting leukocytes e x p r e s s I C A M - 2 in addition to L F A - 1 (de F o u g e r o l l e s et al., 1 9 9 1 ; N o r t a m o et al., 1991).  H o w e v e r , they do not s p o n t a n e o u s l y a g g r e g a t e  b e c a u s e t h e avidity of L F A - 1 d o e s not a p p e a r to b e high e n o u g h (Rothlein a n d Springer,  1986;  Dustin  a n d Springer,  1989; v a n K o o y k  et al., 1989).  e x p e r i m e n t s , s p l e n i c T cells did not a d h e r e to purified murine  In o u r  ICAM-1 or ICAM-2  u n l e s s previously activated with P M A . T h e a d h e s i o n w a s specific a s determined b y inhibition with C D 1 1 a , I C A M - 1 , and I C A M - 2 m A b s . P M A - a c t i v a t e d s p l e n i c T cells a l s o d e m o n s t r a t e d the s a m e specificity towards cell surface I C A M - 1 a n d I C A M - 2 . T cells did not a d h e r e to L cells u n l e s s they w e r e transfected with I C A M - 1 or I C A M - 2 c D N A . Binding of T cells to ICAM-2-transfected L cells w a s consistently lower than to I C A M - 1 transfected L cells, despite the higher level of I C A M - 2 e x p r e s s i o n . T h i s s u g g e s t s that cell a d h e s i o n mediated by L F A - 1 : I C A M - 2 is not a s efficient a s that mediated b y L F A 1:ICAM-1.  Similar results  have b e e n obtained  b y other  groups.  When  LFA-1  e x p r e s s i o n is limiting, I C A M - 1 binding is preferred over I C A M - 2 binding (Dustin et al., 1989; Kishimoto etal., 1990). A s well, I C A M - 1 a d h e s i o n is a l s o stronger than I C A M - 2 . The  comparison  between  I C A M - 2 a n d I C A M - 3 binding  to L F A - 1 h a s not b e e n  e x a m i n e d . N e v e r t h e l e s s , leukocytes e x p r e s s i n g activated L F A - 1 are able to a d h e r e to ICAM-2  +  cells  in t h e a b s e n c e of I C A M - 1  expression.  Certain  cells,  including  unstimulated endothelial cells, e x p r e s s relatively high levels of I C A M - 2 but only low levels of I C A M - 1 (Dustin and Springer, 1988). L e u k o c y t e s e x p r e s s i n g activated L F A - 1  93  m a y b e a b l e to a d h e r e to t h e s e cells by binding to I C A M - 2 a n d thus  facilitate  s u b s e q u e n t functions of the i m m u n e r e s p o n s e . The  binding sites for L F A - 1 a d h e s i o n in I C A M - 1 h a s b e e n m a p p e d by site  directed m u t a g e n e s i s (Staunton et al., 1990). T h e first d o m a i n of I C A M - 1 is critical for binding to L F A - 1 .  B a s e d o n t h e s e q u e n c e conservation of t h e I C A M s a n d their  functional similarity in binding L F A - 1 , it m a y be s p e c u l a t e d that the ligand recognition sites lie in h o m o l o g o u s positions a n d contain k e y c o n s e r v e d r e s i d u e s .  T w o key  r e s i d u e s involved in L F A - 1 binding are glutamic acid at position 3 4 a n d glutamine at position 7 3 . T h e s e are c o n s e r v e d in all five known I C A M s e q u e n c e s . Interestingly, a peptide s p a n n i n g r e s i d u e s 2 1 - 4 2 of the h u m a n I C A M - 2 is able to block binding to L F A 1 indicating the importance of t h e s e r e s i d u e s (Li et al., 1993b). The  binding  of I C A M - 2 to L F A - 1 h a s b e e n well e s t a b l i s h e d in vitro (de  F o u g e r o l l e s et al., 1 9 9 1 ; X u et al., 1992; X u et al., 1996), h o w e v e r t h e functional s i g n i f i c a n c e of this cell a d h e s i o n pathway in vivo remains u n k n o w n .  It is of interest  that inflammatory r e s p o n s e s of ICAM-1-deficient mice are only partially defective (Sligh et al., 1 9 9 3 ; X u et al., 1994).  W h e t h e r I C A M - 2 is c o m p e n s a t i n g for t h e I C A M - 1  deficiency r e m a i n s to be s e e n . It also remains to be determined w h e t h e r the high level of I C A M - 2 e x p r e s s i o n in lung contributes to neutrophil migration in p n e u m o n i a (Burns et al., 1994).  B a s e d o n t h e limited e x p r e s s i o n pattern of I C A M - 2 , l y m p h o c y t e s a n d  e n d o t h e l i u m , it m a y b e p o s s i b l e that I C A M - 2 functions  in T cell activation a n d  leukocyte transendothelial migration. With the c D N A e n c o d i n g murine I C A M - 2 a n d the  94  3 C 4 antibody, the potential functional roles of I C A M - 2 in a n a l l o g e n e i c T cell r e s p o n s e a n d in leukocyte transendothelial migration are e x a m i n e d further in this thesis.  95  2:5 R e f e r e n c e s  A l z a r i P M , L a s c o m b e M , a n d Poljak R J (1988) antibodies. Annu. Rev. Immunol. 6:555.  Three-dimensional  structure  of  A m i t A G , M a r i u z z i R A , Phillips S E V , a n d Poljak R J (1986) Three-dimensional structure of a n antigen-antibody c o m p l e x at 2.8 A resolution. Science 2 3 3 : 7 4 7 . 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X u H, T o n g IL, d e F o u g e r o l l e s A R , a n d Springer T A (1992) Isolation, characterization, a n d e x p r e s s i o n of m o u s e I C A M - 2 c o m p l e m e n t a r y a n d g e n o m i c D N A . J. Immunol. 149:2650.  103  Chapter 3: Costimulatory role of ICAM-2 in T cell response to allogeneic class II MHC  T h e work p r e s e n t e d in this chapter a p p e a r s in the following publication: C a r p e n i t o C , P y s z n i a k A M , a n d T a k e i F (1997) I C A M - 2 provides a costimulatory signal for T cell stimulation by allogeneic c l a s s II M H C . Scand. J. Immunol. 4 5 : 2 4 8  3:1 I n t r o d u c t i o n T l y m p h o c y t e s bearing the a|3 T cell receptor (TcR) r e c o g n i z e p r o c e s s e d antigenic  peptides  presented  by the major  histocompatibility  complex  ( M H C ) of  proteins o n the s u r f a c e of antigen presenting cells ( A P C s ) . T h e interactions b e t w e e n T cells a n d A P C s  a r e c o m p r i s e d of both antigen-specific a n d antigen-nonspecific  c o m p o n e n t s ( R o t h e n b e r g , 1992; G u i n a n et al., 1994). interactions occurring  T h e first s t a g e of t h e s e  involves the r a n d o m , low level a d h e s i o n b e t w e e n A P C s  most  prominently  K i s h i m o t o et al., 1991).  in lymphoid  t i s s u e s (Hemler,  a n d T cells  1 9 9 0 ; Springer, 1 9 9 0 ;  During this w e a k interaction b e t w e e n T cells a n d A P C s , the  a n t i g e n : M H C c o m p l e x , if present in sufficient quantity, is r e c o g n i z e d by the T c R a n d a primary activation signal is delivered within the T cell ( Y a g u e et al., 1985; D e m b i c k et al., 1986; S a i t o et al., 1987; B e r z o f s k y et al., 1988). After ligation of the T c R , the T cell is c o m p e t e n t to r e s p o n d to various s e c o n d a r y or costimulatory s i g n a l s (Geppert et al.,  104  1990; W e a v e r and U n a n u e , 1990). TcR  In contrast to the primary signal delivered by the  w h i c h is both antigen-specific and M H C - r e s t r i c t e d , t h e costimulatory  neither  antigen-specific  nor MHC-restricted.  T h e second  signal  signal is  provides t h e  n e c e s s a r y e n h a n c e m e n t to the primary signal s u c h that cytokine s e c r e t i o n , cellular proliferation, a n d effector function are p o s s i b l e ( C e r d a n et al., 1 9 9 2 a ; C e r d a n et al., 1 9 9 2 b ; B o u s s i o t i s et al., 1993). T h e two signal model p r o p o s e d that a s e c o n d signal is required to amplify the T c R signal (Bretscher and C o h n , 1970; J e n k i n s and S c h w a r t z , 1987; S c h w a r t z , 1990; J e n k i n s , 1992).  If the costimulatory signal is not d e l i v e r e d , the  T cells b e c o m e u n r e s p o n s i v e in an antigen-specific manner. T h e T cells are a n e r g i c to s u b s e q u e n t stimulation, h o w e v e r they are still viable s i n c e they are able to r e s p o n d to e x o g e n o u s l y a d d e d IL-2.  A d h e s i o n m o l e c u l e s at the T cell s u r f a c e are excellent  c a n d i d a t e s for mediating the n e c e s s a r y costimulatory s i g n a l s b e c a u s e the cells are ideally situated to provide a pathway for regulatory  information to b e transmitted.  T h e s e m o l e c u l e s m a k e ideal c a n d i d a t e s for the t r a n s m i s s i o n of this information. T h e most extensively studied pair of a d h e s i o n m o l e c u l e s which h a v e b e e n s h o w n to deliver the required s e c o n d a r y signal is the C D 2 8 : B 7 (Linsley and Ledbetter, 1 9 9 3 ; B o u s s i o t i s et al., 1996). C D 2 8 is a T cell-specific m o l e c u l e that c a n interact with B 7 m o l e c u l e s on APCs.  B 7 c a n a l s o bind a C D 2 8 - l i k e ligand, C T L A 4 .  However, C T L A 4  actually  transmits a negative costimulatory signal ( J a n e w a y and Bottomly, 1994; W a l u n a s et al., 1994; W a t e r h o u s e etal., 1995; Tivol etal., 1996; B l u e s t o n e , 1997) Inhibition of the immune r e s p o n s e is p o s s i b l e b y b l o c k a g e of the a d h e s i v e interactions b e t w e e n A P C s and T cells. A n t i b o d i e s directed against the cell a d h e s i o n  105  m o l e c u l e s mediating the initial interactions or against the T c R inhibits the delivery of the primary signal a n d the immune r e s p o n s e is inhibited (Martz, 1987; Dustin and Springer, 1 9 9 1 ; G u i n a n et al., 1994). conditions, they  If t h e s e T cells are r e m o v e d from the  are able to respond w h e n  rechallenged with the  inhibitory  initial antigen.  H o w e v e r , if the b l o c k a g e o c c u r s at the level of the C D 2 8 : B 7 interaction either with antibodies or with C T L A 4 - l g fusion protein, a soluble high affinity counter receptor for B 7 , the T cells b e c o m e u n r e s p o n s i v e in a n antigen-specific m a n n e r ( J e n k i n s et  al.,  1 9 9 1 ; Harding et al., 1992; T a n et al., 1992). T h e y will not be able to mount a n y kind of a n i m m u n e r e s p o n s e w h e n s u b s e q u e n t l y c h a l l e n g e d with the antigen. the functional prevents  In s u m m a r y ,  o u t c o m e of a b l o c k a d e at the level of a d h e s i o n or T c R  antigen  recognition  resulting  in  immunosuppression.  signaling  B l o c k a d e of  the  C D 2 8 : B 7 interaction results in the induction of T cell anergy. T h e L F A - 1 : I C A M - 1 , -2, -3 pathways have a l s o b e e n e x a m i n e d for their potential roles in the t r a n s m i s s i o n of costimulatory signals to T cells (van S e v e n t e r et al., 1990; v a n S e v e n t e r et al., 1 9 9 1 a ; v a n S e v e n t e r et al., 1991b; D a m l e et al., 1 9 9 2 a ; D a m l e et al., 1 9 9 2 b ; d e F o u g e r o l l e s et al., 1994).  T h e significance of t h e s e interactions h a s  b e e n o b s e r v e d in the mixed lymphocyte reaction ( M L R ) .  S i n c e m A b s against L F A - 1  a n d the three I C A M s c a n either completely or partially inhibit the r e s p o n s e , it w a s plausible  to  s u s p e c t that they  F o u g e r o l l e s et al., 1994).  were  playing  a role  in T cell costimulation  (de  H o w e v e r , A P C s in a n M L R e x p r e s s a large n u m b e r of cell  s u r f a c e m o l e c u l e s w h i c h m a y function not only to e n h a n c e T c e l l : A P C a d h e s i o n , but a l s o in costimulation.  A culture s y s t e m w a s d e v e l o p e d in order to e x a m i n e t h e s e  106  potentially relevant m o l e c u l e s on a n individual basis.  T h e purified m o l e c u l e s w e r e  c o u p l e d to microwells along with a m A b against either the T c R or the C D 3 c o m p l e x . T h e primary s i g n a l c a n be delivered to the T c R by the m A b a n d the  potential  costimulatory signal m a y be transmitted by the purified protein. T h e proliferative T cell r e s p o n s e to the proteins is m e a s u r e d by [ H]-thymidine uptake. All three L F A - 1 : I C A M 3  p a t h w a y s h a v e b e e n s h o w n to play a role in T cell proliferation.  T h e extent of the T  cell stimulation by the three I C A M s is identical to the relative L F A - 1 affinities for e a c h I C A M , with I C A M - 1 inducing the strongest proliferation.  In addition, the T cells w h e n  primed with I C A M - 1 a n d I C A M - 2 costimulation a n d s u b s e q u e n t l y re-stimulated through v a r i o u s receptors, display varying proliferative r e s p o n s e s ( D a m l e et al., 1 9 9 2 a ; D a m l e et al.,  1992b).  It a p p e a r s that I C A M - 1 primed T cells are more r e s p o n s i v e to B 7  costimulation in a s e c o n d a r y r e s p o n s e than they are to B 7 in a primary r e s p o n s e . H o w e v e r , I C A M - 2 primed T cells d o not r e s p o n d to B 7 costimulation in a s e c o n d a r y r e s p o n s e to a n y greater extent than in a primary r e s p o n s e . T h e maturation state of the T cells a l s o a p p e a r s to be important in responding to s u b s e q u e n t re-stimulation. A n t i g e n primed or m e m o r y T cells d o not r e s p o n d well to re-stimulation with I C A M - 1 or ICAM-2.  T h i s h a s b e e n demonstrated in both a proliferative r e s p o n s e a s well a s  r e l e a s e of cytokines ( S e m n a n i et al., 1994). T h e preferred c h o i c e of costimulation for naive T cells is the L F A - 1 :ICAM-1 pathway w h e r e a s m e m o r y T cells a p p e a r to be most r e s p o n s i v e to the C D 2 8 : B 7 pathway (Damle et  al.,  1992b).  A s well, previously  activated T cells are more B 7 r e s p o n s i v e a n d I C A M - 1 a n d I C A M - 2 u n r e s p o n s i v e than naive T cells.  107  Although  purified  proteins  and  a n t i - T c R or a n t i - C D 3 m A b  activation  has  u n c o v e r e d m u c h information about costimulation, the s y s t e m u s e d is highly artificial a n d h a s s e v e r a l potential shortcomings. T h e m A b affinity for the T c R / C D 3 c o m p l e x is m u c h higher than that of the a n t i g e n / M H C (Sagerstrom et al., 1993) a n d therefore m a y not be representative of the actual physiological interactions that o c c u r b e t w e e n T cells and A P C s .  Furthermore, although cell surface I C A M - 2 h a s b e e n s h o w n to m e d i a t e  a d h e s i o n to L F A - 1 cells (Xu et al., 1992; X u et al., 1996), it m a y not be a s effective in +  delivering a costimulatory signal a s the purified protein d u e to g l y c o c a l y x h i n d r a n c e . T h e functional role of cell surface I C A M - 2 in T cell activation is yet to be u n d e r s t o o d . T h e objective of the work presented in this chapter w a s to e x a m i n e the role of I C A M - 2 in T cell activation under more physiological conditions. p a t h w a y w a s functionally  isolated by creating antigen  The LFA-1 :ICAM-2  presenting  cells.  Murine  fibroblast L cells (H-2 ) e x p r e s s i n g c l a s s II l - E m o l e c u l e s w e r e transfected with the k  d  murine I C A M - 2 c D N A a n d e x a m i n e d for the ability to stimulate a l l o g e n e i c s p l e n i c T cells (H-2 ). k  T h e proliferative r e s p o n s e w a s c o m p a r e d to that with untransfected a n d  I C A M - 1 - t r a n s f e c t e d l - E L cells. T h e induction of a n a n e r g i c state w a s a l s o e x a m i n e d d  by rechallenging the stimulated T cells in a s e c o n d a r y r e s p o n s e . A s well, the T cell stimulation w a s repeated with two s e p a r a t e cells e x p r e s s i n g the l - E a n d the I C A M - 2 d  molecules.  A l l the e x p e r i m e n t s w e r e d e s i g n e d to a d d r e s s the i s s u e of w h e t h e r cell  s u r f a c e I C A M - 1 a n d I C A M - 2 merely e n h a n c e T c R recognition of a l l o g e n e i c M H C or w h e t h e r they actually provide a costimulatory signal e s s e n t i a l for the a v o i d a n c e of anergy.  108  3:2 M a t e r i a l s a n d M e t h o d s 3:2.1 Animals C 3 H / H e , B A L B / c , a n d C 5 7 B L / 6 mice u s e d in this study w e r e bred at the Joint A n i m a l Facility of the B . C . C a n c e r R e s e a r c h C e n t r e from the founders p u r c h a s e d from J a c k s o n Laboratories (Bar Harbor, M E ) .  3:2.3 Cell lines and antibodies T h e murine fibroblast L cell line ( H - 2 ; S a n f o r d et al., 1948) w a s maintained in k  D M E M containing 1 0 % F C S . T h e murine fibroblast line R T 1 0 . 3 . B C I (referred to a s R T 1 0 . 3 here after) is a transfected L cell line which e x p r e s s e s t h e murine c l a s s II M H C l - E m o l e c u l e ( G e r m a i n a n d Quill, 1986; Ruberti et al., 1992) a n d w a s a g e n e r o u s gift d  from Dr. W . Jefferies (Biotechnology laboratory, University of British C o l u m b i a ) . It w a s a l s o maintained D M E M + 1 0 % F C S . All antibodies w e r e u s e d a s purified Ig. T h e antibodies w h i c h r e c o g n i z e the murine cell s u r f a c e I C A M - 1 ( l g G ) , I C A M - 2 ( l g G ) , C D 1 1 a ( l g G ) a n d C D 4 9 d ( l g G ) 2a  2a  2b  2b  h a v e b e e n d e s c r i b e d in chapter 2 . T h e rat anti-mouse V C A M - 1 ( A 4 2 9 , l g G ) w a s 2 a  p u r c h a s e d from P h a r m i n g e n ( S a n Diego, C A ) . T h e biotinylated m A b s to l - A  d  (AMS-  3 2 . 1 , l g G ) , l - E ( A M S - 1 6 , l g G ) , B7-1 ( 1 G 1 0 , l g G ) , a n d B 7 - 2 ( G L 1 , l g G ) w e r e d  2 a  2 a  2 a  2 a  a l s o p u r c h a s e d from P h a r m i n g e n . A l l c o m m e r c i a l antibodies w e r e d i a l y z e d against 1 X P B S to r e m o v e s o d i u m a z i d e . T h e hybridoma cell lines that p r o d u c e rat a n t i - m o u s e C D 2 4 ( M 1 / 6 9 . 1 6 . 1 1 , A T C C TIB 125, l g G ) (Springer et al., 1978) a n d murine anti-rat 2 b  109  IgK ( R G 7 / 9 . 1 , A T C C T I B 1 6 9 , l g G ) w e r e obtained from A m e r i c a n T y p e 2 b  Culture  C o l l e c t i o n (Rockville, M D ) .  3:2.4 Transfection of l-E* L cells with murine ICAM-1 and ICAM-2 cDNAs T h e murine I C A M - 1 (Horley et al., 1989) and I C A M - 2 c D N A s in the e x p r e s s i o n vector p B C M G S N e o ( K a r a s u y a m a et al., 1990) w e r e transfected into R T 1 0 . 3 cells by the poly-L-ornithine method (Dong et al., 1993). Transfectants w e r e s e l e c t e d in D M E M containing  1 0 % F C S a n d G 4 1 8 (500 |ag/ml, C a n a d i a n Life T e c h n o l o g i e s ) .  transfectants  Bulk  that e x p r e s s e d high levels of I C A M - 1 a n d I C A M - 2 w e r e isolated by  panning directly with purified anti-ICAM-1 or a n t i - I C A M - 2 m A b immobilized o n petri d i s h e s ( F a l c o n 1 0 0 1 , B e c t o n Dickinson).  T h e isolated cells w e r e e x p a n d e d a n d  e x p r e s s i o n levels of various cell surface m o l e c u l e s w e r e tested b y flow cytometry a s d e s c r i b e d in t h e previous chapter.  C e l l s (3 X 10 ) w e r e stained with I C A M - 1 , I C A M - 2 , 5  V C A M - 1 , or C D 2 4 antibodies (4 (ag/ml).  T h e s e c o n d a r y stain w a s T I B 1 6 9 - F I T C  (described in 3:2.3) a n d d e a d cells w e r e stained with propidium iodide (2 u.g/ml). T h e cells w e r e a l s o stained with biotinylated  l - A , l - E , B 7 - 1 , a n d B 7 - 2 antibodies a n d d  d  counter-stained with streptavidin-FITC. T h e stained cells w e r e a n a l y z e d o n a F A C S t a r ( B e c t o n Dickinson).  3:2.5 Cell adhesion assay Adhesion  of P M A - a c t i v a t e d  splenic T cells to t h e I C A M - 1  and ICAM-2  transfected R T 1 0 . 3 cells w a s a s s e s s e d a s d e s c r i b e d in the previous chapter.  110  3:2.6 Proliferation assay of primary allogeneic response S p l e n i c T cells w e r e isolated from C 3 H / H e (H-2 ) mice (2-10 months old) using k  nylon w o o l a s d e s c r i b e d in the previous chapter (Julius et al., 1973). L cells (H-2 ) a n d k  RT10.3 (H-2  k  e x p r e s s i n g l - E ) cells w e r e harvested with P B S + 2.5 m M E D T A a n d d  irradiated with 12,000 rads using a Philips R T 2 5 0 X - r a y m a c h i n e . S p l e n i c T cells (2.5 X 1 0 ) w e r e c o m b i n e d with varying n u m b e r s of irradiated stimulator cells in 2 0 0 uJ of 5  R P M I 1 6 4 0 + 5 % F C S + 5 X 10~ M p - m e r c a p t o e t h a n o l ( P - M E ) in 96-well flat bottom 5  plates ( F a l c o n 3 0 7 2 , B e c t o n Dickinson). A n t i b o d i e s for inhibition s t u d i e s w e r e u s e d at 4 u.g/ml. T h e plates w e r e cultured for 4 , 5, or 6 d a y s at 37°C in a 5 % C 0 atmosphere.  2  humidified  T h e cultures w e r e then pulsed with 1 u.Ci/well of [methyl- H]-thymidine 3  ( H - T d R ) ( D u P o n t , B o s t o n , M A ) for a further 8 hrs a s a m e a s u r e of newly s y n t h e s i z e d 3  DNA.  T h e radiolabeled cells w e r e then harvested onto filters a n d the radioactivity o n  the filters w a s m e a s u r e d in a p-plate liquid scintillation counter ( L K B W a l l a c 1 2 0 5 , T u r k u , Finland).  T h e results a r e e x p r e s s e d a s m e a n of triplicate  experiments  ±  s t a n d a r d error m e a n (cpm + S E M ) . In a s e p a r a t e s e t of e x p e r i m e n t s , 2.5 X 1 0 s p l e n i c 5  T cells w e r e c o m b i n e d with 1.25 X 1 0 irradiated untransfected 4  10  3  R T 1 0 . 3 cells a n d 5 X  L cells e x p r e s s i n g either I C A M - 1 ( L : I C A M - 1 ) or I C A M - 2 ( L : I C A M - 2 ) from the  previous  chapter.  T h e proliferative  r e s p o n s e to the mixed  measured a s described above.  111  cell stimulation w a s  3:2.7 Secondary stimulation assay S p l e n i c T cells (1.0 X 1 0 ) from C 3 H / H e mice w e r e cultured with irradiated 7  R T 1 0 . 3 transfectant cells (8 X 1 0 ) in 5 ml of R P M 1 1 6 4 0 + 5 % F C S + 5 X 10" M p - M E 5  5  in 6-well flat bottom plates ( F a l c o n 3 0 4 6 , B e c t o n Dickinson) for 6 d a y s .  T h e T cells  w e r e then h a r v e s t e d a n d w a s h e d three times with 10 ml R P M 1 1 6 4 0 + 5 % F C S . T h e T cells w e r e incubated without a n y stimulus for overnight in R P M 1 1 6 4 0 + 5 % F C S + 5 X 10"  5  M p-ME.  2 X 1 0 of t h e s e T cells from e a c h primary stimulation w e r e then 5  c o m b i n e d with 3 X 1 0 irradiated (7500 rads) B A L B / c (H-2 ), C 3 H / H e (H-2 ), or 5  d  k  C 5 7 B L / 6 (H-2 ) s p l e e n cells in 9 6 well round bottom plates ( F a l c o n 3 0 7 7 , B e c t o n b  Dickinson) in 2 0 0 ul of R P M 1 1 6 4 0 + 5 % F C S + 5 X 10" M p - M E a n d incubated for 4 5  d a y s . T cell proliferation to the s e c o n d a r y stimulus w a s m e a s u r e d a s d e s c r i b e d for the primary a l l o g e n e i c r e s p o n s e .  Inhibition studies u s e d purified C D 1 1 a antibody at a  concentration of 4 jag/ml.  3:3 R e s u l t s 3:3.1 Expression of ICAM-1 and ICAM-2 on RT10.3 cells R T 1 0 . 3 cells (l-E -transfected L cells) e x p r e s s i n g murine I C A M - 1 or I C A M - 2 d  w e r e g e n e r a t e d by the transfection of the appropriate c D N A s , followed by p a n n i n g with o n e of the a n t i - I C A M m A b s . F l o w cytometric a n a l y s i s of the transfected cells s h o w e d that t h e e x p r e s s i o n levels of I C A M - 1 a n d I C A M - 2 w e r e equivalent o n t h e respective cells (Figure 13a). T h e r e w a s a low level of e n d o g e n o u s I C A M - 2 e x p r e s s i o n o n t h e untransfected a n d the I C A M - 1 - t r a n s f e c t e d cells.  112  F o u r other potential  costimulatory  A) no 1 ° A b  ICAM-1 A b  ICAM-2 A b  VCAM-1 Ab  HSA Ab  CO  F i g u r e 1 3 Flow cytometric analysis of RT10.3 cells. R T 1 0 . 3 cells transfected with t h e murine I C A M - 1 a n d I C A M - 2 c D N A w e r e a n a l y z e d for various cell s u r f a c e m o l e c u l e s . A ) T h e top p a n e l of histograms represents I C A M - 1 , I C A M - 2 , V C A M - 1 , a n d M 1 / 6 9 e x p r e s s i o n o n t h e transfected R T 1 0 . 3 cells a s determined by indirect i m m u n o f l u o r e s c e n c e . B) T h e bottom p a n e l of h i s t o g r a m s (next page) represents e x p r e s s i o n of M H C c l a s s II l-A a n d l-E m o l e c u l e s , a s well a s the cotimulatory m o l e c u l e s B7-1 a n d B 7 - 2 .  (figure l e g e n d o n p r e v i o u s p a g e )  m o l e c u l e s , V C A M - 1 , B 7 - 1 , B 7 - 2 , a n d heat stable antigen ( H S A , C D 2 4 ) , w e r e not detected o n t h e s e cells (Figure 13a and 13b).  T h e cells a l s o e x p r e s s e d equivalent  levels of l - E (Figure 13b). I-A w a s not detected on the surface of the R T 1 0 . 3 cells. d  d  3:3.2 Adhesion of PMA-activated splenic T cells to RT10.3 cells In order to determine the relative binding capacities of the I C A M s o n the transfected R T 1 0 . 3 cells, the a d h e s i o n of P M A - a c t i v a t e d s p l e n i c T cells to the R T 1 0 . 3 cells w a s determined.  Approximately 1 8 % of T cells a d h e r e d to untransfected cells  w h e r e a s e x p r e s s i o n of I C A M - 1 a n d I C A M - 2 o n R T 1 0 . 3 cells i n c r e a s e d a d h e s i o n to 8 4 % a n d 6 7 % respectively (Figure 14). T h e I C A M - 1 - and I C A M - 2 - m e d i a t e d a d h e s i o n w a s inhibited by the anti-LFA-1 m A b w h e r e a s the isotype control antibody ( a n t i - C D 4 9 d m A b ) had no effect on a d h e s i o n . T h e anti-ICAM-1 m A b inhibited T cell a d h e s i o n to the I C A M - 1 - t r a n s f e c t e d R T 1 0 . 3 cells and the a n t i - I C A M - 2 m A b inhibited t h e a d h e s i o n to I C A M - 2 - t r a n s f e c t e d R T 1 0 . 3 cells. T h e s e results indicate that I C A M - 1 a n d I C A M - 2 o n the transfected R T 1 0 . 3 cells are functional a n d readily mediated T cell a d h e s i o n . T h e low level of e n d o g e n o u s I C A M - 2 a p p e a r s to be too low to contribute significantly to the overall a d h e s i o n .  3:3.3 Primary allogeneic response of splenic T cells against RT10.3 cells T h e role of I C A M - 2 in a T cell r e s p o n s e against a l l o g e n e i c c l a s s II M H C w a s examined  (Figure  15).  Murine  splenic T cells from C 3 H / H e m i c e  (H-2 ) k  were  stimulated with I C A M - 1 - a n d ICAM-2-transfected R T 1 0 . 3 cells ( H - 2 e x p r e s s i n g l - E ) . k  115  d  0  25  50 75 cells bound (%)  100  F i g u r e 1 4 Adhesion of splenic T cells to RT10.3 cells expressing ICAM-1 and ICAM-2. R T 1 0 . 3 cells w e r e grown in 96-well flat bottom wells. T o e a c h well, 1 0 P M A - a c t i v a t e d s p l e n i c T cells labeled with c a l c e i n A M w e r e a d d e d with the appropriate blocking antibody (4 |ig/ml). T h e binding a s s a y a n d w a s h e s w e r e performed a s d e s c r i b e d previously. R e s u l t s a r e e x p r e s s e d a s a m e a n of triplicate wells ± S E M . 5  116  Irradiated RT10.3 cells H-2k:l-Ed  F i g u r e 15 Schematic representation of T cell stimulation by allogeneic MHC class II and ICAM-2. Murine s p l e n i c T cells from C 3 H / H e m i c e (H-2 ) w e r e purified by a nylon wool c o l u m n a n d c o m b i n e d with irradiated stimulator cells (RT10.3) e x p r e s s i n g I C A M - 1 o r I C A M - 2 . T h e R T 1 0 . 3 cells (H-2 ) e x p r e s s the allogeneic M H C c l a s s II m o l e c u l e l - E . After 5 d a y s , the cells w e r e p u l s e d with [ H]-TdR and the proliferative r e s p o n s e s w e r e e x a m i n e d a s d e s c r i b e d in Materials a n d M e t h o d s . k  k  3  d  S i n c e the R T 1 0 . 3 cells are of C 3 H / H e origin, the C 3 H / H e T cells will r e s p o n d to the allogeneic l - E allogeneic  d  M H C e x p r e s s e d on the surface of the R T 1 0 . 3 cells.  response,  determined  by  [ H]-TdR  incorporation,  3  was  The  optimal  achieved  by  incubating 2.5 X 1 0 s p l e n i c T cells with 1.5 X 1 0 R T 1 0 . 3 cells (Figure 16) for 5 d a y s 5  4  (Figure 17). T h e a l l o g e n e i c r e s p o n s e pattern to the R T 1 0 . 3 cells is similar r e g a r d l e s s of w h e t h e r the cells are I C A M - t r a n s f e c t e d or untransfected.  U n d e r t h e s e conditions,  the T cell r e s p o n s e to untransfected R T 1 0 . 3 cells (nearly 2 0 0 0 0 cpm) w a s substantially higher than the control (less than 2 0 0 cpm) in w h i c h no R T 1 0 . 3 cells w e r e a d d e d (Figure 18).  T h e e x p r e s s i o n of I C A M - 1 and I C A M - 2 on R T 1 0 . 3 cells  significantly  e n h a n c e d the T cell stimulation by 2.5-3 fold. A n t i - L F A - 1 m A b inhibited the stimulation of T cells by I C A M - 1 - and ICAM-2-transfected R T 1 0 . 3 cells, w h e r e a s it had little effect on the stimulation with the untransfected R T 1 0 . 3 cells.  T h e T cell stimulation with  untransfected  A s well, the  R T 1 0 . 3 cells w a s  LFA-1-independent.  low  level  of  e n d o g e n o u s I C A M - 2 e x p r e s s i o n did not a p p e a r to h a v e a n y stimulatory effect o n the T cells s i n c e the a n t i - C D 1 1 a m A b did not h a v e any affect on the b a c k g r o u n d r e s p o n s e . A n t i - I C A M - 1 a n d a n t i - I C A M - 2 m A b s a l s o specifically inhibited T cell stimulation with the transfected  R T 1 0 . 3 cells e x p r e s s i n g the  relevant  ICAMs,  although  inhibition by t h e s e m A b s w e r e lower than that of the anti-LFA-1 m A b . (anti-CD49d) did not h a v e a n effect on the stimulation. possibility proliferative  that the  R T 1 0 . 3 cells w e r e  behaving  the  levels  Control m A b  In order to eliminate  as feeder  of  cells a n d  the  supplying  cytokines to the T cells, 4 day supernatants from irradiated a n d n o n -  118  60  100  1000 10000 Number of stimulator cells  100000  F i g u r e 16 Dose response of splenic T cells to RT10.3 expressing ICAM-1 or ICAM2. S p l e n i c T cells (2.5 x 10 ) were cultured with increasing n u m b e r s of e a c h irradiated R T 1 0 . 3 transfectant. Proliferative r e s p o n s e s were m e a s u r e d o n d a y 5 a n d e x p r e s s e d a s a m e a n of triplicate wells ± S E M . 5  119  60  o..-. RT10.3 RT10.3:ICAM-1 RT10.3-.ICAM-2  - 40E Q.  c o  a •  mmm >  CO  o  Q.  o o  £ 20 OC "O H  I  CO  4  5 6 Days in culture  7  8  F i g u r e 17 Kinetics of the allogeneic T cell response to ICAM-1- or ICAM-2transfected RT10.3 cells. S p l e n i c T cells (2.5 x 10 ) w e r e cultured with e a c h type of irradiated R T 1 0 . 3 transfectant (1.5 x 10") for the indicated periods. T h e proliferative T cell r e s p o n s e s w e r e determined a s d e s c r i b e d previously. 5  120  80  • RT10.3  responses  + CD11a Ab  + ICAM-1 Ab  + ICAM-2 Ab  + CD49d Ab  F i g u r e 18 Effects of antibodies on allogeneic T cell response to RT10.3 cells. S p l e n i c T cells (2.5 x 10 ) w e r e cultured with irradiated I C A M - 1 - o r I C A M - 2 - t r a n s f e c t e d R T 1 0 . 3 cells (1.5 x 10 ) in the p r e s e n c e of the indicated antibodies (4 |ig/ml). Proliferative r e s p o n s e s w e r e m e a s u r e d o n day 5 a s d e s c r i b e d previously a n d e x p r e s s e d a s a m e a n of triplicate cultures + S E M . s  4  irradiated R T 1 0 . 3 cells (transfected and untransfected) w e r e incubated with the T cells. T h e T cells did not proliferate to any notable level (all w e r e under 1000 cpm).  3:3.4 Allogeneic response to mixed stimulator cells T h e r e are two p o s s i b l e explanations for the i n c r e a s e d T cell r e s p o n s e to I C A M 1 a n d I C A M - 2 e x p r e s s i o n o n R T 1 0 . 3 cells.  O n e explanation is that e x p r e s s i o n of  I C A M - 1 a n d I C A M - 2 i n c r e a s e s the L F A - 1 - d e p e n d e n t physical interaction of T cells to the R T 1 0 . 3 cells a n d therefore results in e n h a n c e d T c R recognition of p e p t i d e : M H C complex.  T h e other p o s s i b l e explanation is that  cells provide  costimulatory  through the T c R .  the I C A M s present o n t h e R T 1 0 . 3  signals which augment  the primary  signal  transmitted  In order to distinguish between t h e s e two p o s s i b l e m e c h a n i s m s , T  cells w e r e c o m b i n e d with untransfected I C A M - 1 o r I C A M - 2 (Figure 19).  R T 1 0 . 3 cells a n d L cells transfected  with  If the I C A M - 1 a n d I C A M - 2 m o l e c u l e s w e r e actually  transmitting costimulatory signals, then the two signals m a y be spatially s e p a r a t e d a n d T cell proliferation s h o u l d still b e o b s e r v e d . E  d  Untransfected R T 1 0 . 3 cells e x p r e s s i n g I-  w e r e c o m b i n e d with L cells e x p r e s s i n g I C A M - 1 ( L : I C A M - 1 ) or I C A M - 2 ( L : I C A M - 2 )  but not l - E , a n d u s e d to stimulate splenic T cells from C 3 H / H e m i c e (H-2 ). T h e d  k  mixture of t h e s e cells w e r e able to stimulate T cells a b o v e the level of control cultures in w h i c h untransfected L cells plus R T 1 0 . 3 cells w e r e u s e d to stimulate T cells (Figure 20).  T h e p r e s e n c e of untransfected L cells did not i n c r e a s e the T cell r e s p o n s e to  untransfected R T 1 0 . 3 cells.  H o w e v e r , the p r e s e n c e of L : I C A M - 1 a n d L : I C A M - 2 with  untransfected R T 1 0 . 3 cells greatly i n c r e a s e d the T cell r e s p o n s e a b o v e the control  122  F i g u r e 19 Schematic representation of T cell response to mixed stimulators. T h e stimulatory role of I C A M - 2 in a l l o g e n e i c T cell r e s p o n s e w a s e x a m i n e d u s i n g a mixture of two populations of stimulator cells s e p a r a t e l y e x p r e s s i n g I C A M - 2 a n d l - E . Untransfected R T 1 0 . 3 ( H - 2 : l - E ) cells which d o not e x p r e s s ICAM-1 o r I C A M - 2 are c o m b i n e d with L cells (H-2 ) transfected with I C A M - 2 a n d u s e d to stimulate T cells (H-2 ). d  k  k  d  k  media only! UCAM-2 UCAM-1 L cells RT10.3 + L:ICAM-2 RT10.3 + L:ICAM-1 RT10.3 + L cells RT10.3:ICAM-2 RT10.3:ICAM-1 RT10.3  6 Figure 20  Stimulation  20 40 60 80 [3H]-TdR incorporation (cpm/1000)  of T cells with RT10.3  and ICAM-2-transfected  L cells.  Irradiated untransfected R T 1 0 . 3 cells (1.25 x 10") w e r e c o m b i n e d with irradiated L c e l l s (5 x 10 ) transfected with ICAM-1 ( L I C A M - 1 ) or I C A M - 2 ( L I C A M - 2 ) a n d i n c u b a t e d with 2.5 x 1 0 s p l e n i c T cells. A s negative controls, T cells w e r e incubated without stimulator cells (media alone), or with L cells, L I C A M - 1 , or L I C A M - 2 without R T 1 0 . 3 cells. I C A M - 1 - t r a n s f e c t e d R T 1 0 . 3 ( R T 1 0 . 3 : I C A M - 1 ) a n d I C A M - 2 - t r a n s f e c t e d R T 1 0 . 3 ( R T 1 0 . 3 : I C A M - 2 ) w e r e a l s o u s e d a s stimulators in positive controls. T h e proliferative r e s p o n s e s of T cells w e r e m e a s u r e d on d a y 5 a n d e x p r e s s e d a s a m e a n of triplicate cultures + S E M . 3  5  124  culture. A l t h o u g h the T cell r e s p o n s e to L I C A M - 1 a n d L I C A M - 2 w a s slightly greater than that a g a i n s t untransfected L cells, the r e s p o n s e s to R T 1 0 . 3 + L : I C A M - 1 a n d R T 1 0 . 3 + L I C A M - 2 w e r e still greater than the additive effect of r e s p o n s e s to R T 1 0 . 3 cells a n d to L I C A M - 1 or L I C A M - 2 . T h e s e results indicate that I C A M - 1 a n d I C A M - 2 on R T 1 0 . 3 cells not only c a n e n h a n c e the physical interaction b e t w e e n T cells a n d R T 1 0 . 3 cells, but that they c a n a l s o deliver an e s s e n t i a l costimulatory m o l e c u l e through L F A - 1 on T cells.  3:3.5 Secondary  stimulation  A b s e n c e of a costimulatory signal able to a u g m e n t the primary s i g n a l from the T c R l e a d s to a n a n e r g i c state in which the T cells are functionally p a r a l y z e d upon s u b s e q u e n t restimulation with the appropriate antigen.  In order to e x a m i n e w h e t h e r  I C A M - 1 a n d I C A M - 2 mediated s i g n a l s are sufficient to avert a n a n e r g i c state, T cells stimulated with R T 1 0 . 3 transfected with I C A M - 1 or I C A M - 2 w e r e later c o m b i n e d with a l l o g e n e i c A P C s a n d the T cell r e s p o n s e w a s m e a s u r e d (Figure 21).  S p l e n i c T cells  from C 3 H / H e m i c e (H-2 ) stimulated with R T 1 0 . 3 cells ( H - 2 e x p r e s s i n g l - E ) for 6 d a y s k  k  d  w e r e h a r v e s t e d a n d s u b s e q u e n t l y rechallenged with irradiated s p l e e n c e l l s from B A L B / c ( H - 2 ) , C 5 7 B L / 6 (H-2 ), or C 3 H / H 3 (H-2 ) mice. T cells stimulated with I C A M d  b  k  transfected R T 1 0 . 3 cells in primary culture d i s p l a y e d a vigorous r e s p o n s e to B A L B / c s p l e e n cells in the s e c o n d a r y stimulation (Figure 22).  In contrast, T c e l l s stimulated  with untransfected R T 1 0 . 3 cells in primary culture did not r e s p o n d significantly to B A L B / c s p l e e n cells.  T cells r e c o v e r e d from primary cultures, r e g a r d l e s s of I C A M  125  primary allogeneic response  •  6 days  RT10.3 (H-2 :I-E ) k  d  C3H/He (H-2 ) k  overnight culture in media only  irradiated spleen cells  4 days  l H]-TdR uptake 3  C3H/He (H-2 ) BALB/c (H-2 ) C57BL/6 (H-2 ) K  d  b  Schematic representation of secondary stimulation of T cells. S p l e n i c T c e l l s  Figure 21  (H-2 ) are cultured with irradiated R T 1 0 . 3 transfectants in a primary a l l o g e n e i c r e s p o n s e for 6 d a y s . T h e T cells are then transfered into fresh m e d i a without stimulators for overnight. T h e T c e l l s are then c o m b i n e d with irradiated s p l e e n cells from either a C 3 H / H e m o u s e (H-2 ), a B A L B / c m o u s e (H-2 ), o r a C 5 7 B L / 6 (H-2 ) a n d cultured for 4 d a y s before examining t h e proliferative response. k  d  b  k  Secondary stimulus:  media BALB/c B A L B / c (+Ab) C57BL/6 C 5 7 B L / 6 (+Ab) C3H/He  medial BALB/c B A L B / c (+Ab) 1  C57BL/6 C 5 7 B L / 6 (+Ab) C3H/He  Primary stimulus- RT10.3:ICAM-2  m e d i a ZZZ3I BALB/c B A L B / c (+Ab)  77? \  T  f  C 5 7 B L / 6 y///////y///77777. C 5 7 B L / 6 (+Ab) C3H/He 50  100  150  [ H ] - T d R i n c o r p o r a t i o n (cpm/1000) 3  F i g u r e 22 Secondary responses of T cells. S p l e n i c T cells were stimulated with R T 1 0 . 3 , R T 1 0 . 3 : I C A M - 1 , or R T 1 0 . 3 : I C A M - 2 cells for 6 d a y s . T h e T cells from e a c h r e s p o n s e w e r e then harvested a n d cultured ovdernight in m e d i a before being c o m b i n e d with irradiated s p l e e n cells from the indicated strains of m i c e in roundbottom wells (2 x 1 0 T cells + 3 x 10 s p l e e n cells). A n t i - C D 1 1 a antibody w a s a d d e d w h e r e indicated by (+Ab) at a concentration of 4 u.g/ml. Proliferation w a s e x a m i n e d o n d a y 4 a n d e x p r e s s e d a s a m e a n of triplicate cultures + S E M . 5  5  127  e x p r e s s i o n o n t h e stimulator  cells, s h o w e d e q u a l a n d substantial  r e s p o n s e s to  C 5 7 B L / 6 s p l e e n cells but not to s y n g e n e i c C 3 H / H e s p l e e n cells. T h i s indicated that the T cells w e r e functional a n d had remained viable in culture.  T h e r e s p o n s e s in the  s e c o n d a r y stimulation w e r e inhibited by the anti-LFA-1 m A b .  T h e s e results indicate  that T cells stimulated b y untransfected R T 1 0 . 3 cells in primary cultures b e c a m e specifically n o n - r e s p o n s i v e to H - 2  d  cells.  T h e s e a n e r g i c cells w e r e still viable s i n c e  they w e r e a b l e to r e s p o n d to a n t i - C D 3 cross-linking (3 u.g/ml) with P M A (5 ng/ml) [ R T 1 0 . 3 : (3.52 ± .23) x 1 0 ICAM-2:  5  c p m ; R T 1 0 . 3 I C A M - 1 : (3.15 ± .37) x 1 0  (2.95 + .14) x 1 0 cpm]. 5  In contrast,  5  cpm; RT10.3  primary stimulation with  ICAM-  transfected R T 1 0 . 3 cells greatly e n h a n c e d the T cell r e s p o n s e s to a l l o g e n e i c B A L B / c s p l e e n cells in s e c o n d a r y stimulation.  3:4 Discussion T h e functional role of cell surface I C A M - 2 in t h e stimulation of T cells with a l l o g e n e i c c l a s s II M H C w a s e x a m i n e d in this chapter.  In contrast to I C A M - 1 , w h o s e  role in T cell activation h a s b e e n d e m o n s t r a t e d in various s y s t e m s (van S e v e n t e r et al., 1990; v a n S e v e n t e r etal., 1 9 9 1 a ; D a m l e etal., 1992b; D a m l e etal., 1992c), the role of I C A M - 2 h a s not b e e n thoroughly investigated. T h e functional role of I C A M - 2 in T cell activation h a s b e e n previously investigated using a n t i - I C A M - 2 m A b to inhibit the T cell r e s p o n s e or testing the effects of recombinant I C A M - 2 / F  c  fusion proteins o n T cells  stimulated by a n t i - T c R cross-linking (Damle et al., 1 9 9 2 a ; D a m l e et al., 1992b). In this study w e h a v e u s e d murine fibroblast L cells transfected with l - E  128  d  and ICAM-1 or  I C A M - 2 . T h e s e cells are able to act a s A P C s and c a n stimulate a l l o g e n e i c T cells from C 3 H / H e (H-2 ) mice. T h i s s y s t e m has a n u m b e r of a d v a n t a g e s . k  First, the function of  cell s u r f a c e I C A M - 2 , not purified recombinant proteins, c a n be d e t e r m i n e d .  Second,  the stimulation of primary T cells and not c l o n e d T cell lines c a n be e x a m i n e d , s i n c e the T cell r e s p o n s e to a l l o g e n e i c c l a s s II M H C in primary stimulation is strong e n o u g h for detection.  Third, stimulation of T cells with A P C s , not by a n t i - T c R / C D 3 c r o s s -  linking, is being studied w h i c h is more physiologically relevant. T c R / C D 3 is estimated to be 1 0 - 1 0 3  et al., 1 9 9 1 ; W e b e r et al., 1992).  4  T h e affinity of m A b for  fold higher than that of T c R for A g / M H C (Matsui  T h e difference in affinity m a y be important w h e n T  c e l h A P C interactions are e x a m i n e d .  Finally, l-E -transfected L cells ( R T 1 0 . 3 ) do not d  e x p r e s s B 7 or H S A , two cell surface m o l e c u l e s demonstrated to deliver costimulatory s i g n a l s to T cells (Linsley et al., 1 9 9 1 ; G i m m i et al., 1 9 9 1 ; Liu et al., 1992).  Therefore,  the functional role of murine I C A M - 2 on surrogate A P C s in T cell stimulation c a n be e x a m i n e d in the a b s e n c e of t h e s e important costimulatory m o l e c u l e s . T h i s study h a s demonstrated that I C A M - 2 e x p r e s s e d on R T 1 0 . 3 cells is not only able to mediate L F A - 1 - d e p e n d e n t T cell a d h e s i o n but a l s o plays a significant role in the activation  of resting cells.  S p l e n i c T cells (H-2 ) k  mount  a relatively  low  but  significant proliferative r e s p o n s e to untransfected R T 1 0 . 3 cells (H-2 ) e x p r e s s i n g l - E . k  d  H o w e v e r , t h o s e stimulated T cells s e e m to b e c o m e u n r e s p o n s i v e w h e n c h a l l e n g e d with H - 2  d  s p l e e n cells from B A L B / c mice in a s e c o n d a r y stimulation.  T h i s d o e s not  s e e m to be d u e to cell death in the primary culture b e c a u s e they are able to r e s p o n d to a n t i - C D 3 / P M A stimulation and to third party allogeneic s p l e e n cells from C 5 7 B L / 6 mice  129  (H-2 ). b  In fact, the a n t i - H - 2  s e c o n d a r y r e s p o n s e to H - 2  r e s p o n s e of t h e s e cells w a s m u c h higher than  s p l e e n cells.  d  mount a s e c o n d a r y a n t i - H - 2  b  d  the  Therefore, the failure of t h e s e T cells to  r e s p o n s e cannot be e x p l a i n e d by the lack of clonal  e x p a n s i o n of the a n t i - H - 2 reactive T cells in the primary culture a n d implies a n active d  m e c h a n i s m to induce a n anergic state. In contrast, the e x p r e s s i o n of I C A M - 1 or I C A M 2 o n R T 1 0 . 3 cells greatly e n h a n c e s the stimulation of T cells in a n L F A - 1 - d e p e n d e n t m a n n e r in primary cultures. T h i s stimulation a l s o prevents T cell u n r e s p o n s i v e n e s s to s u b s e q u e n t a l l o g e n e i c stimulation.  T h u s , T cells stimulated with I C A M - t r a n s f e c t e d  R T 1 0 . 3 cells in primary cultures display a vigorous r e s p o n s e to H - 2  d  s p l e e n cells in  s e c o n d a r y stimulation. T h e T cell r e s p o n s e to R T 1 0 . 3 cells is reflected in the a d h e s i o n profile of P M A activated s p l e n i c T cells to R T 1 0 . 3 cells. Therefore, the e n h a n c e d r e s p o n s e of T cells to I C A M - t r a n s f e c t e d R T 1 0 . 3 cells m a y be a result of improved T cell a d h e s i o n to A P C s w h i c h w o u l d facilitate more efficient T c R recognition of A g / M H C .  H o w e v e r , this study  h a s d e m o n s t r a t e d that I C A M - 1 or I C A M - 2 and l - E e x p r e s s e d on s e p a r a t e L cells are d  still able to stimulate T cells. T h i s is further indicative of the costimulatory potential of I C A M - 2 a s well a s I C A M - 1 on A P C s . Similar experiments h a v e a l s o s h o w n that I C A M 1 c a n provide a costimulatory signal (van S e v e n t e r et al., 1991b).  T cells are able to  proliferate w h e n the primary antigen-specific signal and the costimulatory signal are spatially s e p a r a t e (Dubey et al., 1995). T h e s e findings are consistent with the results from this study indicating that cell surface I C A M - 1 a n d I C A M - 2 provide a n e c e s s a r y costimulatory signal w h i c h a u g m e n t s the signal provided by e n g a g e m e n t of the T c R  130  with A g b o u n d to M H C . Other groups h a v e s h o w n that purified I C A M - 1 a n d I C A M - 2 c a n provide potent costimulatory signals to anti-CD3-activated T cells. H o w e v e r , this study d e m o n s t r a t e s that I C A M - 2 on A P C s c a n a l s o act a s a potential molecule.  costimulatory  T h e s e results demonstrate that I C A M - 2 a n d a l l o g e n e i c c l a s s II M H C  e x p r e s s i o n is sufficient for T cell activation in addition to rescuing the T cell from a state of a l l o - u n r e s p o n s i v e n e s s . T h e question a s to whether T c R - d e r i v e d a n d costimulatory s i g n a l s m a y be present on s e p a r a t e cells has b e e n a d d r e s s e d by s e v e r a l g r o u p s . A l t h o u g h the results from this study a n d by others (van S e v e n t e r et al., 1990; v a n S e v e n t e r et al.,  1991)  indicate that the two signals may be present on s e p a r a t e cells, other g r o u p s h a v e a r g u e d a n d d e m o n s t r a t e d the opposite (Galvin et al., 1992; Liu a n d J a n e w a y , 1992; J a n e w a y a n d Bottomly, 1994). T h e s e investigators argue that the two s i g n a l s must be on the s a m e cell for an optimal T cell r e s p o n s e .  If the s i g n a l s could be present on  s e p a r a t e cells, then autoreactive T cells could b e c o m e activated on a regular b a s i s . T h i s is not o b s e r v e d a n d the question a s to whether the two s i g n a l s c a n be delivered by two cells requires further investigation to determine if the two cell stimulation is a reflection of the tissue culture conditions. T h e costimulatory signal generated by the interaction b e t w e e n B 7 on A P C s a n d C D 2 8 on T cells h a s b e e n extensively studied (Linsley a n d Ledbetter, 1 9 9 3 ; B o u s s i o t i s et al., 1996).  T h i s study s u g g e s t s that I C A M - 2 a s well a s I C A M - 1 m a y provide a n  alternative costimulatory pathway for T cells w h e n A P C s d o not e x p r e s s B 7 . H o w e v e r , the e x a c t nature of the I C A M - m e d i a t e d signal a p p e a r s to be unclear.  131  B o u s s i o t i s et al.  (1993) h a v e previously demonstrated that N I H 3 T 3 cells transfected with h u m a n I C A M 1 a n d D R 7 w e r e able to induce T cell proliferation in primary cultures.  H o w e v e r , the  stimulated T cells w e r e not able to respond to N I H 3 T 3 cells transfected with D R 7 a n d I C A M - 1 or B 7 in s e c o n d a r y stimulations.  This apparently contradicts the results from  our study w h i c h indicates that e x p r e s s i o n of I C A M - 1 or I C A M - 2 a n d a l l o g e n e i c c l a s s II M H C provides a d e q u a t e stimulation for T cells in the a v e r s i o n of a n a n e r g i c state. H o w e v e r , the fact that w e u s e d s p l e e n cells, instead of transfected N I H 3 T 3 cells, a s stimulators in the s e c o n d a r y culture may help to explain the a n o m a l y .  Professional  A P C s present in a s p l e e n cell population e x p r e s s i n g B 7 , I C A M s , a s well a s c l a s s II M H C m a y be r e s p o n s i b l e for the s e c o n d a r y stimulation in our s y s t e m . T h i s s e c o n d a r y stimulation is L F A - 1 - d e p e n d e n t a s demonstrated by the inhibition using a n t i - C D 1 1 a mAb.  W h e t h e r the b l o c k a d e is occurring at the level of a d h e s i o n or costimulation is  uncertain.  W h a t is c l e a r is that p r e s e n c e of I C A M - 1 or I C A M - 2 in primary culture is  a b l e to r e s c u e T cells from a n state of u n r e s p o n s i v e n e s s to a l l o g e n e i c s p l e e n cells in a s e c o n d a r y stimulation. costimulation  It h a s b e e n previously s h o w n by o n e group that I C A M - 1  in a primary  culture  e n h a n c e s B 7 r e s p o n s i v e n e s s in a  secondary  r e s p o n s e (Damle etal., 1992b). Although  the  profile  of cytokines  r e l e a s e d in t h e s e e x p e r i m e n t s  was  not  e x a m i n e d , it h a s b e e n investigated by others (Boussiotis et al., 1993; B o u s s i o t i s et al., 1994; S e m n a n i et al., 1994). W h e n T cells receive a costimulatory signal v i a I C A M - 1 , IL-2 is r e l e a s e d . signal.  T h i s h a s a l s o b e e n o b s e r v e d w h e n B7-1 provides the s e c o n d a r y  If I C A M - 1 and B7-1 are together providing the costimulation, the level of IL-2  132  r e l e a s e d i n c r e a s e s drastically if either are providing the signal a l o n e ( D u b e y et 1995).  H o w e v e r , the cytokine profile for I C A M - 2 has not b e e n e x a m i n e d .  al.,  If IL-2 is  detected with I C A M - 2 stimulation, it will provide further support that I C A M - 2 c a n in fact act a s a c l a s s i c costimulatory molecule. It m a y a l s o display other cytokines reflecting the  unique  immune  response  initiated  by  the  distribution  pattern  of  ICAM-2.  C o s t i m u l a t i o n by L F A - 1 :ICAM-1 interaction h a s s h o w n that G M - C S F is a l s o r e l e a s e d ( S e m n a n i et al., 1994). W h e r e a s , costimulation by L F A - 3 : C D 2 results in IL-5 r e l e a s e in addition to IL-2. The  finding that murine I C A M - 2 on A P C s m a y function a s a costimulatory  m o l e c u l e for T cells in the a b s e n c e of B 7 or H S A has important implications to antigen presentation  by  cell types  not  normally  c o n s i d e r e d to  be  'professional' A P C s .  Inflammatory cytokines s u c h a s IFN-y are known to induce c l a s s II M H C e x p r e s s i o n on s o m e non-lymphoid cells that do not e x p r e s s B 7 or H S A ( O ' C o n n e l l a n d E d i d i n , 1990; Farrar a n d S c h r e i b e r , 1993).  S o m e of t h e s e cells, including endothelial cells,  constitutively e x p r e s s I C A M - 2 .  It is p o s s i b l e that those cells m a y function in antigen  presentation under certain conditions. function  T h e capacity for a n endothelial cell line to  a s a n A P C h a s b e e n reported  (St. Louis et  al.,  1993).  In  addition,  demonstration that the two required signals for T cell activation m a y be delivered by two s e p a r a t e cells a l s o raises the possibility that C A M s m a y not be a n a b s o l u t e requirement o n a n A P C . A cell with antigenic peptides a n d M H C on the s u r f a c e but no costimulatory a d h e s i o n proteins might still be able to elicit a T cell r e s p o n s e if a n adjacent cell e x p r e s s e s the appropriate costimulatory m o l e c u l e s .  133  Furthermore, our  results r a i s e s the possibility that non-professional A P C s e x p r e s s i n g c l a s s II M H C a n d I C A M s but not B 7 or H S A m a y play a role in graft rejection a n d a u t o i m m u n e d i s e a s e s . M i c e treated with anti-LFA-1 a n d anti-ICAM-1  m A b s while  undergoing  a cardiac  allograft w e r e found to be tolerant to s u b s e q u e n t skin allograft (Isobe et al., Isobe a n d lhara, 1993).  1992;  Further studies are required to a s s e s s the functional role of  I C A M - 2 in vivo a s to whether a n t i - I C A M - 2 m A b m a y be able to block a host v e r s u s graft r e s p o n s e to t i s s u e s s u c h a s the endothelium.  134  3:5  References  B e r g N N a n d O s t e r g a a r d H L (1995) Characterization of intercellular a d h e s i o n molecule-1 (ICAM-1 )-augmented degranulation by cytotoxic T cells. I C A M - 1 a n d antiC D 3 must b e c o - l o c a l i z e d for optimal a d h e s i o n a n d stimulation. J. Immunol. 1 5 5 : 1 6 9 4 . 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Cell 42:81. 1  139  Chapter 4 R o l e of I C A M - 2 in l e u k o c y t e t r a n s e n d o t h e l i a l m i g r a t i o n  4:1 Introduction T h e i m m u n e s y s t e m is c o m p r i s e d of a n integrated distinct cells a n d o r g a n s .  c o m p l e x of functionally  L e u k o c y t e s m o v e continuously throughout the b o d y using  the b l o o d s t r e a m a n d lymphatic v e s s e l s a s pathways.  T h e y circulate throughout the  b l o o d s t r e a m a n d c r o s s capillary networks into various lymphoid a n d non-lymphoid t i s s u e s ( P i c k e r a n d Butcher, 1 9 9 2 ; B e v i l a c q u a , 1 9 9 3 ; C a r l o s a n d H a r l a n ,  1994).  L y m p h o c y t e s re-enter the vasculature v i a the efferent lymphatic c h a n n e l s s u c h a s the t h o r a c i c a n d m e s e n t e r i c ducts. This e n s u r e s that the entire functional repertoire of the i m m u n e s y s t e m c a n effectively survey the host a n d m a x i m i z e the i m m u n e r e s p o n s e to the p a t h o g e n .  V e r y few, if any, i m m u n e r e s p o n s e s are initiated in t h e b l o o d s t r e a m .  T h e r e f o r e , the entry of leukocytes into lymph n o d e s is a k e y regulatory step in the normal function of the i m m u n e s y s t e m . T h e m o l e c u l a r characterization of leukocyte a d h e s i o n to the e n d o t h e l i u m h a s m a d e it c l e a r that the v a s c u l a r lining plays a n active role in p r o c e s s e s s u c h a s inflammation a n d recirculation in the i m m u n e r e s p o n s e . L e u k o c y t e s c a n b e recruited to sites of inflammation by chemoattractant gradients (Furie et al., 1 9 9 1 ; K a v a n a u g h et al., 1 9 9 1 ; O p p e n h e i m e r - M a r k s et al., 1991). A n upregulation of a d h e s i o n m o l e c u l e s at the site of inflammation  c o i n c i d e s with the e n h a n c e d transendothelial migration of  l e u k o c y t e s (Kishimoto et al., 1 9 9 0 ; L u s c i n k a s et al., 1 9 9 1 ; D o b r i n a et al., 1991).  140  Circulating lymphocytes are imported into s e c o n d a r y lymphoid o r g a n s s u c h a s lymph n o d e s a n d P e y e r ' s p a t c h e s through  physiologically distinct post-capillary v e n u l e s ,  t e r m e d high endothelial v e n u l e s ( H E V ) . T h e s e v e n u l e s are called H E V b e c a u s e they are lined with tall, c u b o i d a l , a n d metabolically active endothelial cells.  T h i s is in  contrast to normal endothelial cells which line the rest of the v a s c u l a t u r e . T h e H E V in the  secondary  lymphoid  tissues  provide  a  port  of  exit  for  normal  circulating  l y m p h o c y t e s to gain entry into the lymphatic s y s t e m . A s e q u e n c e of a d h e s i v e events which allows leukocytes to migrate out of the b l o o d s t r e a m into the extravascular s p a c e s h a s b e e n identified (Harlan et al.,  1992).  V e n o u s flow transports the leukocytes throughout the v a s c u l a t u r e resulting in r a n d o m contact with the v e s s e l wall. After the initial contact, s o m e of the l e u k o c y t e s a p p e a r to roll along the endothelial surface ( Z i m m e r m a n et al., 1992). T h i s s l o w s the p a s s a g e of the leukocyte allowing for s u b s e q u e n t a d h e s i v e interactions.  Following the rolling, the  l e u k o c y t e s a d h e r e more efficiently to the endothelium taking on a flattened a n d s p r e a d out m o r p h o l o g y (Smyth et al., 1993). After a firm attachment, the l e u k o c y t e s c a n then crawl o v e r the endothelial surface s e a r c h i n g for an o p e n i n g a n d s q u e e z e b e t w e e n endothelial cells (diapedesis) (Smith, 1992; S t o s s e l , 1993). O n c e in the subendothelial tissue, the leukocyte c a n migrate to a n a r e a of inflammation or to s e c o n d a r y or tertiary lymphoid t i s s u e s . T h r e e families  of  adhesion  receptors  mediate  the  various  extravasation p r o c e s s ( B e v i l a c q u a , 1993; C a r l o s a n d H a r l a n , 1994).  s t a g e s of  T h e selectin  family of proteins are involved in the initial a d h e s i o n w h i c h results in rolling  141  the  under  conditions of v e n o u s flow (von A n d r i a n et al., 1 9 9 1 ; L a w r e n c e a n d Springer, 1 9 9 1 ; A b a s s i et  1 9 9 3 ; L e y et al.,  al.,  1993; L a w r e n c e a n d Springer, 1993).  Adhesion  b e t w e e n the (3 integrins a n d Ig-like counter-receptors m e d i a t e s the firm a d h e s i o n (von 2  A n d r i a n et al., 1 9 9 1 ; Muller a n d W e i g l , 1992; Butcher, 1 9 9 1 ; Springer, 1994).  The  s a m e pair of a d h e s i o n m o l e c u l e s a n d P E C A M - 1 a l s o mediate the transendothelial migration (Muller et al., 1993; Bird et al., 1993; V a p o r c i y a n et al., 1993). through the s u b e n d o t h e l i a l tissue is mediated by the extracellular matrix c o m p o n e n t s (Hakkert et al.,  Migration  integrins a d h e s i o n with  1 9 9 1 ; Kuijpers et al.,  1993).  The  s p e c i f i c m o l e c u l e s involved in e a c h step d e p e n d s on m a n y factors including activation state of both the leukocyte a n d the endothelium, leukocyte type, a n d p r e s e n c e of activators (Butcher, 1 9 9 1 ; Springer, 1994). T h e role of various C A M s in transendothelial migration h a s b e e n e x a m i n e d both with  antibody  blocking studies a s well  a s knockout  mice.  The  LFA-1 :ICAM-1  interaction h a s b e e n e x a m i n e d a n d found to play a critical role in migration (Furie et al., 1 9 9 1 ; K a v a n a u g h et al., 1 9 9 1 ; O p p e n h e i m e r - M a r k s et al., 1991).  Not only d o e s it  m e d i a t e neutrophil migration a c r o s s a n endothelial barrier, it a l s o a s s i s t s l y m p h o c y t e s in the p r o c e s s . M i c e deficient for I C A M - 1 (Xu et al., 1994) or L F A - 1 (Schmits et al., 1996) a l s o display altered migratory patterns.  L y m p h o c y t e s a n d neutrophils play  important roles in normal recirculation a s well a s inflammation.  Both of t h e s e  p r o c e s s e s utilize c o m m o n m o l e c u l e s . H o w e v e r , the antibody against L F A - 1 is a b l e to block transendothelial migration more effectively than the antibody against I C A M - 1 , s u g g e s t i n g that I C A M - 1 is not the only L F A - 1 ligand utilized in the transendothelial  142  migration p r o c e s s (Furie et al., 1 9 9 1 ; K a v a n a u g h et al., 1 9 9 1 ; O p p e n h e i m e r - M a r k s et al., 1991). A l t h o u g h antibodies to ICAM-1 block lymphocyte binding to a n d migration across  non-lymphoid  endothelial  cells, they  interactions ( M a y a n d A g e r , 1992; A g e r , 1994).  h a v e no effect  on l y m p h o c y t e : H E V  B a s e d on the constitutive endothelial  e x p r e s s i o n of I C A M - 2 (de F o u g e r o l l e s et al., 1991), it is a p o s s i b l e c a n d i d a t e a s a n alternate L F A - 1 ligand in leukocyte migration a c r o s s endothelial barriers.  However,  the role of I C A M - 2 in transendothelial migration a c r o s s lymphoid a n d non-lymphoid endothelial cells h a s not b e e n e x a m i n e d . T h e objective of the work presented in this chapter w a s to e x a m i n e the potential role of I C A M - 2 in transendothelial migration of l y m p h o c y t e s a n d neutrophils,  two  leukocytic cell types that play key roles in recirculation a n d inflammation. T h e first step in e x a m i n i n g the role w a s to set up a a s s a y s y s t e m that quantitates the migrated l e u k o c y t e s . A n endothelial cell line w a s grown on a porous m e m b r a n e w h i c h provided a n effective barrier s u c h that the leukocytes a b o v e the endothelial cells could not p a s s i v e l y c r o s s the endothelial cells. I C A M - 1 - a n d I C A M - 2 - t r a n s f e c t e d cells w e r e a l s o e x a m i n e d for their ability to mediate migration in this s y s t e m .  4:2 M a t e r i a l s a n d m e t h o d s  4:2.1  Animals C 3 H / H e m i c e u s e d in this study w e r e bred at the Joint A n i m a l Facility of the  B . C . C a n c e r R e s e a r c h C e n t r e from the founders p u r c h a s e d from J a c k s o n L a b o r a t o r i e s (Bar Harbor, M E ) .  143  4:2.2 Cell lines and antibodies T h e murine endothelial cell line S V E C 4 . 1 0 , derived from S V 4 0 infection of peripheral lymph n o d e s t r o m a , w a s maintained in D M E M containing 5 % F C S a n d h a s b e e n d e s c r i b e d previously ( O ' C o n n e l l a n d E d i d i n , 1990). T h e S V E C 4 . 1 0 line h a s b e e n s h o w n to retain the morphology a n d functional characteristics of normal endothelial cells.  T h e murine l y m p h o m a cell line TIL1 w a s derived from a tumor initiated in  C 3 H / H e m i c e by inoculation with IL-7-producing F s a - R f i b r o s a r c o m a cells ( M c B r i d e et al., 1992).  T h e non-adherent tumor infiltrating l y m p h o c y t e s w e r e e x p a n d e d a n d  s e p a r a t e d from the adherent fibroblastoid cells.  TIL1 a n d S V E C 4 . 1 0 cells w e r e a  g e n e r o u s gift from Dr. G Dougherty (Terry F o x Laboratory, B . C . C a n c e r R e s e a r c h C e n t e r ) . TIL1 cells w e r e maintained in D M E M containing 1 0 % F C S . All antibodies w e r e u s e d a s purified Ig. T h e antibodies w h i c h r e c o g n i z e the murine cell s u r f a c e antigens I C A M - 1 ( l g G ) , I C A M - 2 ( l g G ) , 2a  (lgG ), 2b  2a  LFA-1 (lgG ), 2b  VLA-4  a n d V C A M - 1 ( l g G ) have b e e n d e s c r i b e d in the previous two c h a p t e r s . T h e 2a  rat antibody R B 6 - 8 C 5 ( l g G ) which r e c o g n i z e s the murine granulocyte antigen Gr-1 2b  h a s b e e n d e s c r i b e d previously (Hestdal et al., 1991) a n d w a s p u r c h a s e d from P h a r m i n g e n . T h e rat antibodies which r e c o g n i z e m o u s e P E C A M - 1 ( C D 3 1 ) ( M E C 1 3 . 3 , l g G ) a n d m o u s e C D 2 5 ( 3 C 7 , l g G ) w e r e a l s o p u r c h a s e d from P h a r m i n g e n . T h e 2 a  2 b  h y b r i d o m a cell lines that p r o d u c e anti-mouse C D 4 4 ( K M 8 1 , A T C C T I B 2 4 1 , l g G ) 2 a  ( M i y a k e et al., 1990) a n d anti-mouse M a c - 1 ( M 1 / 7 0 . 1 5 . 1 1 . 5 , A T C C T I B 128, l g G ) 2 b  144  (Springer et al., 1979) were obtained from American Type Culture Collection (Rockville, MD).  4:2.3 Expression of ICAM-1 and ICAM-2 on SVEC4.10 cells  The murine ICAM-1 (Horley et al., 1989) and ICAM-2 cDNAs in the expression vector pBCMGSNeo (Karasuyama et al., 1990) were transfected into SVEC4.10 cells by the poly-L-ornithine method (Dong et al., 1993). Transfectants were selected in DMEM containing 5% FCS and G418 (500 ug/ml). Bulk transfectants that expressed high levels of ICAM-1 and ICAM-2 were isolated by panning directly with purified antiICAM-1 or anti-ICAM-2 mAb immobilized on petri dishes (Falcon 1001) as described in Chapter 3. The isolated cells were expanded and expression levels of various surface molecules were tested by flow cytometry as described in previous chapters. The secondary stain was GaRlgG-FITC (Cooper Biomedical) and dead cells were stained with 2 u,g/ml propidium iodide.  4:2.4  Immunoprecipitation  The SVEC4.10 cells and their transfectants were grown for 2 days and the cells were harvested as described for flow cytometric analysis. The cells were then washed and labeled with biotin as described previously (von Boxberg et al., 1990). The plasma membranes were solubilized and incubated with either anti-ICAM-1 mAb or anti-ICAM2 mAb coupled to Affi-Gel 10 beads (from chapter 2) for 4 hours at 4°C. The beads were then washed and the bound fraction washed eluted as described. The fraction  145  w a s then subjected to 1 0 % S D S - P A G E a n d transferred to a n Immobilon-P m e m b r a n e (Millipore) w h i c h w a s probed with streptavidin-horseradish p e r o x i d a s e . T h e filter w a s then i n c u b a t e d in a n e n h a n c e d c h e m i l u m i n e s c e n c e solution ( A m e r s h a m ) a n d e x p o s e d to X - r a y film for 2-8 minutes.  4:2.5 Isolation of murine bone marrow neutrophils B o n e marrow cells w e r e obtained by flushing out m i c e tibia a n d f e m u r s with HBSS (Ca /Mg + +  free) containing 0 . 1 % B S A (Hart et al., 1986).  + +  T h e cells w e r e  p a s s e d through a 2 7 g a u g e needle to obtain a single cell s u s p e n s i o n before being c o m b i n e d with a hypotonic T r i s - a m m o n i u m chloride solution to lyse erythrocytes. T h e pelleted cells w e r e w a s h e d with C a  + +  /Mg  + +  free  HBSS  + 0 . 1 % B S A a n d then  s u s p e n d e d in 3 ml of 4 5 % P e r c o l l (density = 1.0575 g/ml; P h a r m a c i a ) in C a  + +  /Mg  + +  free  H B S S . T h i s w a s l o a d e d onto a P e r c o l l density gradient in a 15 ml p o l y c a r b o n a t e tube. T h e gradient c o n s i s t e d of 2 ml of 6 2 % (density = 1.0776 g/ml), 5 5 % (density = 1.0693 g/ml), a n d 5 0 % (density = 1.0634 g/ml) P e r c o l l in C a  + +  /Mg  + +  free H B S S  layered  s u c c e s s i v e l y onto 3 ml of a n 8 1 % P e r c o l l c u s h i o n (density = 1.1002 g/ml). gradient w a s then centrifuged at 1600 g for 3 0 min at 10°C.  The  T h e cells b a n d i n g  b e t w e e n t h e 6 2 % a n d 8 1 % layers w e r e harvested with a P a s t e u r pipette a n d w a s h e d twice with C a  + +  /Mg  + +  free H B S S . C e l l purity w a s a s s e s s e d by flow cytometry using the  R B 6 - 8 C 5 antibody.  146  4:2.6 Adhesion and transmigration assays A d h e s i o n of leukocytes to S V E C 4 . 1 0 m o n o l a y e r s w a s quantitated a s d e s c r i b e d before in t h e previous two chapters. Calcein  A M a n d tested  for their  TIL1 cells a n d neutrophils w e r e l a b e l e d with ability  m o n o l a y e r s e x p r e s s i n g I C A M - 1 or I C A M - 2 .  to a d h e r e  to S V E C 4 . 1 0  subconfluent  Blocking antibodies w e r e a d d e d 15 min  prior to the addition of the labeled leukocytes to the monolayer-containing wells. Transmigration  studies w e r e  performed  using  DMEM  ( D M E M p ) w h i c h c a n interfere with C a l c e i n A M f l u o r e s c e n c e . performed in 2 4 well plates (Falcon 3 0 4 7 , B e c t o n Dickinson).  without  p h e n o l red  These assays were E a c h well c o n t a i n e d a  transmigration insert ( F a l c o n 3 0 9 7 , B e c t o n Dickinson) with D M E M p " + 5 % F C S in both the u p p e r a n d lower c h a m b e r s . T h e transmigration insert c o n s i s t s of a plastic circular shell with a n inert porous polycarbonate filter (8 u.m pores) at t h e bottom.  SVEC4.10  a n d their transfectants w e r e trypsinized a n d 1 0 cells w e r e a d d e d inside the insert. 5  T h e filter m e m b r a n e s w e r e precoated with fibronectin (100 u.l of 2 0 ucj/ml in P B S ; S i g m a C h e m i c a l C o . ) for 1 hr before the endothelial cells w e r e a d d e d . T h e S V E C 4 . 1 0 cells w e r e a b l e to grow a s a m o n o l a y e r o n the porous m e m b r a n e without falling into the lower c h a m b e r . After a 36-40 hr incubation at 37°C, fresh D M E M p " + 5 % F C S w a s a d d e d to both the top a n d lower c h a m b e r s a n d C a l c e i n A M labeled l e u k o c y t e s (2 x 10 ) w e r e gently a d d e d to the top c h a m b e r . 5  T h e plates w e r e then p l a c e in a n  incubator for v a r i o u s times. T h e inserts w e r e then r e m o v e d a n d the bottom s i d e of the m e m b r a n e w a s gently w a s h e d with D M E M p " + 5 % F C S . T h e extent of transmigration w a s d e t e r m i n e d by the level of yellow f l u o r e s c e n c e in the bottom of the 24-well plate.  147  4:2.7  Examination of endothelial monolayer  permeability  T h e endothelial m o n o l a y e r grown on the transmigration inserts w a s e x a m i n e d for permeability. T h e S V E C 4 . 1 0 m o n o l a y e r w a s grown on the inserts a s d e s c r i b e d for transmigration a s s a y s .  T h e m e d i a w a s a l s o replaced before the a s s a y .  c h a m b e r , yellow fluorescently  labeled b e a d s (0.98  um  In the top  Fluorospheres, Molecular  P r o b e s , E u g e n e , O R ) w e r e a d d e d in a v o l u m e of 300 uJ ( 0 . 1 % solids by volume). a s s a y s w e r e a l l o w e d to  p r o c e e d like transmigration  assays  a n d the  The  degree  of  permeability w a s determined by the yellow f l u o r e s c e n c e at the bottom of the well.  4:2.8  Simultaneous quantitation of yellow and red  fluorescence  In order to determine whether varying a m o u n t s of red a n d y e l l o w fluorescent d y e c o u l d be quantitated independently, different combination of filters w e r e e m p l o y e d . In a 96-well plate, C a l c e i n A M - l a b e l e d b o n e marrow neutrophils (yellow) w e r e a d d e d at varying concentrations (2.5 x 1 0 , 5 x 1 0 , 7.5 x 1 0 , 1 x 10 ) in 100 ul of D M E M p " + 4  4  4  5  5 % F C S . F o r e a c h cell concentration, varying a m o u n t s of red-labeled b e a d s (1 u.m Fluoresbrite P C R e d m i c r o s p h e r e s ; P o l y s c i e n c e s Inc., Warrington, P A ) w e r e a l s o p r e s e n t ( 0 . 0 2 5 % , 0 . 0 5 % , 0 . 0 7 5 % , 0 . 1 0 % solids/volume). T h e yellow f l u o r e s c e n c e in the wells w a s determined using the B filter for both excitation (k= 4 8 5 ± 1 0 nm) a n d e m i s s i o n (X= 5 3 0 ± 12.5 nm) readings. T h e red f l u o r e s c e n c e w a s e x a m i n e d using the D filter for excitation (k= 590 ± 10 nm) a n d the E filter for e m i s s i o n (k= 6 4 5 ± 2 0 nm). All fluorescent  readings w e r e taken with a C y t o F l u o r 2 3 0 0  148  F l u o r e s c e n t reader  (Millipore)  a n d the software  program  CytoCalc  (version  01.00.04).  Neutrophil  transmigration w a s a s s e s s e d in the p r e s e n c e of red-labeled b e a d s . Inserts containing the S V E C 4 . 1 0 m o n o l a y e r s w a s e s t a b l i s h e d a s before.  However, 2 x 1 0 Calcein A M 5  labeled neutrophils w e r e then c o m b i n e d with 0 . 1 0 % red-labeled b e a d s a n d a d d e d to the migration inserts.  A t various time points, the inserts w e r e r e m o v e d a n d yellow  f l u o r e s c e n c e (transmigration) a n d red f l u o r e s c e n c e (permeability) w e r e a s s e s s e d for e a c h well.  4:3 R e s u l t s 4:3.1 Expression of ICAM-1 and ICAM-2 on SVEC4.10  cells  T h e S V E C 4 . 1 0 endothelial cell line d o e s not e x p r e s s e n d o g e n o u s I C A M - 1 or I C A M - 2 . T h i s murine cell line w a s transfected with the I C A M - 1 or I C A M - 2 c D N A s a n d p a n n e d with t h e appropriate antibody.  F l o w cytometric a n a l y s i s r e v e a l e d that the  levels of e x p r e s s i o n of the two murine I C A M s w e r e equivalent (Figure 2 3 ) . T h e a d h e s i o n m o l e c u l e V C A M - 1 w a s a l s o not detected o n t h e s u r f a c e of S V E C 4 . 1 0 cells. In F i g u r e 2 4 , t h e immunoprecipitation reveals that the I C A M - 1 a n d I C A M - 2 m o l e c u l e s are of t h e appropriate s i z e , 9 0 - 9 5 k D a n d 5 0 - 5 5 k D respectively, indicating that there w a s n o a b n o r m a l glycosylation patterns in the S V E C 4 . 1 0 cell line.  4:3.2 Time course of diffusion T h e endothelial cells w e r e grown to confluency o n the m e m b r a n e inserts. A l t h o u g h the p r e s e n c e of m e d i u m ( D M E M p " + 5 % F C S ) in the insert m a d e it difficult to  149  nol°Ab  ICAM-1 A b  ICAM-2 A b  VCAM-1 A b  PECAM-1 A b  CD44Ab  SVEC4.10  IW  UB  iW  TO  IW  IW  lino ""W  SVEC4.10: ICAM-1 *T  SVEC4.10: ICAM-2 Iffi W "to ' i n  Figure 23  Itt'  F / c w cytometric analysis of SVEC4.10 cells. T h e endothelial cell line S V E C 4 . 1 0 w a s transfected with the I C A M - 1 ( S V E C : I C A M - 1 ) or I C A M - 2 ( S V E C : I C A M - 2 ) a n d subjected to flow cytometry u s i n g the indicated antibodies a n d F(ab') goat anti-rat I g G - F I T C a s the s e c o n d a r y stain. I C A M - 1 , I C A M - 2 , V C A M - 1 , P E C A M - 1 , a n d C D 4 4 e x p r e s s i o n a r e shown. 2  ICAM-2 Ab 0 1 2  ICAM-1 Ab 0 1 2  -214  -111  74  -46  •30  Figure 24  Immunoprecipitation of ICAM-1 and ICAM-2 from transfected SVEC4.10 cells. Untransfected a n d ICAM-transfected S V E C 4 . 1 0 cells were s u r f a c e biotinylated a n d I C A M - 1 a n d ICAM-2 were immunoprecipitated using the appropriate antibody c o n j u g a t e d to b e a d s . T h e s a m p l e s were subjected to S D S - 1 0 % P A G E ( S V E C 4 . 1 0 in lane 0, S V E C 4 . 1 0 : I C A M - 1 in lane 1, S V E C 4 . 1 0 : I C A M - 2 in lane 2) a n d detected by W e s t e r n blotting using peroxidase-conjugated streptavidin.  151  v i s u a l i z e t h e c o n f l u e n c y of the monolayer, the permeability could still b e e x a m i n e d using fluorescently-labeled b e a d s . T h e S V E C 4 . 1 0 m o n o l a y e r s a p p e a r to b e intact a s d e m o n s t r a t e d by the time c o u r s e in Figure 2 5 . T h e b e a d s diffuse efficiently a c r o s s the m e m b r a n e in the insert w h e n S V E C 4 . 1 0 cells a r e not present. endothelial cells are present, the b e a d s d o not effectively  H o w e v e r , w h e n the  c r o s s the m o n o l a y e r . T h e  S V E C 4 . 1 0 cells form tight junctions with adjacent cells that d o not allow t h e p a s s i v e diffusion of 0.98 u,m b e a d s .  4:3.3 Transmigration of TIL1 cells across an endothelial monolayer A preliminary test to e x a m i n e whether the s e t up would actually support the transmigration of TIL1 cells w a s carried out. C a l c e i n A M labeled TIL1 cells w e r e a d d e d into the inserts containing S V E C 4 . 1 0 m o n o l a y e r s . After v a r i o u s time intervals, the inserts w e r e r e m o v e d from the wells a n d the migrated cells w e r e quantitated by the m e a s u r e m e n t of yellow f l u o r e s c e n c e . readily (Figure 26).  TIL1 cells diffuse a c r o s s the m e m b r a n e filter  H o w e v e r , untransfected S V E C 4 . 1 0 cells ( I C A M - 1 " , ICAM-2") w e r e  a b l e to form a n i m p e r m e a b l e barrier a c r o s s the porous m e m b r a n e in w h i c h f e w TIL1 cells ( 1 1 % of total cells) w e r e detected in the lower c h a m b e r after transmigration.  ICAM-1-  and ICAM-2-transfected S V E C 4 . 1 0  cells w e r e  promote migration of TIL1 cells a c r o s s the endothelial monolayer.  migration.  difference  in the kinetics  T h e migration  of I C A M - 1 - m e d i a t e d  T h e r e is a n  and ICAM-2-mediated  of TIL1 cells a p p e a r s faster through  152  a b l e to  In both c a s e s ,  approximately 3 8 % of total TIL1 cells h a d migrated after 16 hours. apparent  16 hours of  SVEC4.10  cells  Time (hr)  Figure 25  Kinetics of bead diffusion across SVEC monolayer.  Fluorescently  l a b e l e d b e a d s (0.98 urn diameter) were a d d e d in the top c h a m b e r of migration inserts with a n d without the various S V E C 4 . 1 0 transfectant m o n o l a y e r s grown on the p o r o u s m e m b r a n e . T h e inserts w e r e r e m o v e d at various times to a s s e s s the diffusion of b e a d s in the bottom c h a m b e r . T h e results from e a c h time point are e x p r e s s e d a s a m e a n of duplicate wells + S E M .  153  0$  0  ,  5  ,  10 Time (hr)  ,  15  20  F i g u r e 26 Ability of TIL 1 cells to utilize ICAM-1 and ICAM-2 for migration across the SVEC4.10 monolayer. TIL1 cells labeled with c a l c e i n A M w e r e a d d e d to the top c h a m b e r of the migration insert. T h e migration insert w a s in p l a c e until the indicated t i m e s afterwhich the insert w a s r e m o v e d a n d migration w a s a s s e s s e d by the amount of f l u o r e s e c e n c e in the bottom c h a m b e r . All points are e x p r e s s e d a s a m e a n of duplicate wells ± S E M .  154  e x p r e s s i n g I C A M - 1 than I C A M - 2 in the initial 8 hours. H o w e v e r , over longer periods of time (16 hours), the total n u m b e r of migrated TIL1 cells is e q u a l . T h i s s y s t e m not only d e m o n s t r a t e s that it is a d e q u a t e in quantitating transendothelial migration, it a l s o d e m o n s t r a t e s that I C A M - 1 a n d I C A M - 2 are able to mediate the migration p r o c e s s in a n L F A - 1 - d e p e n d e n t m a n n e r (Figure 27).  4:3.4 Isolation of neutrophils B o n e marrow neutrophils isolated by density centrifugation w e r e a s s e s s e d for purity using the R B 6 - 8 C 5  antibody.  F l o w cytometry  population w a s ~ 9 0 % pure (Figure 28).  T h e population a l s o d i s p l a y e d t h e identical  positive staining pattern for I C A M - 2 , L F A - 1 , a n d M a c - 1 . (CD31)and l g G  2 b  revealed that the isolated  Isotype controls for l g G  2 a  ( C D 2 5 ) w e r e negative.  4:3.5 Neutrophil binding to SVEC4.10 cells Isolated neutrophils w e r e labeled with C a l c e i n A M a n d tested for their ability to a d h e r e to t h e S V E C 4 . 1 0 cells a n d their transfectants.  A s s e e n in F i g u r e 2 9 , the  neutrophils a d h e r e d to the untransfected S V E C 4 . 1 0 cells ( 4 7 % of total input cells). However,  I C A M - 1 a n d I C A M - 2 e x p r e s s i o n o n the endothelial cell line i n c r e a s e d  binding to 8 4 % a n d 7 6 % respectively. This i n c r e a s e d a d h e s i o n w a s L F A - 1 - s p e c i f i c a s d e m o n s t r a t e d by the a n t i - C D 1 1 a antibody which inhibited  binding to b a c k g r o u n d  levels. T h e control antibody against C D 4 9 d has no effect on a d h e s i o n .  155  40 •  migration  SVEC4.10  + CD11 a Ab  + CD49d Ab  SVEC4.10 cells expressing ICAM-1 or ICAM-2 are able to media transendothelial migration in an LFA-1-dependent manner. TIL1 cells l a b  Figure 27  c a l c e i n A M w e r e e x a m i n e d for their ability to migrate a c r o s s I C A M - 1 - a n d I C A M - transfected S V E C 4 . 1 0 m o n o l a y e r s . Antibodies were a d d e d to TIL1 cells 2 0 min prior to addition of cells to the monolayer. T h e migration w a s allowed to p r o c e e d for 6 hrs. T h e results are e x p r e s s e d a s a m e a n of duplicate wells ± S E M .  156  LOG FLUORESCENCE INTENSITY  F i g u r e 28 Expression of adhesion molecules on mouse bone marrow neutrophils. M o u s e neutrophils isolated by differential centrifugation from b o n e marrow w e r e a n a l y z e d by flow cytometry. Neutrophils (2.5 x 10 ) w e r e stained with t h e indicated primary antibody (750 ng in 100 (il final volume). T h e s a m p l e s w e r e then counters t a i n e d with F ( a b ' ) goat anti-rat I g G - F I T C . T h e ordinate represents the relative cell n u m b e r a n d the a b s c i s s a represents the log f l u o r e s c e n c e intensity. 5  2  157  100  binding  •  SVEC4.10  •  SVEC4.10:ICAM-1  +CD11aAb  + CD49d Ab  Figure 29 Neutrophil binding to SVEC4.10 monolayers. C a l c e i n A M labeled neutrophils (1 x 10 ) w e r e a d d e d to wells with untransfected S V E C 4 . 1 0 m o n o l a y e r s a n d I C A M - 1 - a n d I C A M - 2 - t r a n s f e c t e d m o n o l a y e r s . T h e neutrophils w e r e a l l o w e d to bind for 8 min a n d w a s h e d a s d e s c r i b e d previously. B l o c k i n g anitbodies w e r e a d d e d (4 |Lig/ml) 15 min prior to addition of the neutrophils to the wells. R e s u l t s are p r e s e n t e d a s a m e a n of triplicate wells + S E M . 5  158  4:3.6 Neutrophil transendothelial migration across SVEC4.10  monolayers  T h e e x p r e s s i o n of I C A M - 1 a n d I C A M - 2 greatly a s s i s t e d the transendothelial migration of neutrophils a c r o s s S V E C 4 . 1 0 cells (Figure 30). E x p r e s s i o n of I C A M - 1 a n d I C A M - 2 allows a n i n c r e a s e in neutrophil migration (both 4 6 % of total input cells after 8 hours) c o m p a r e d with untransfected S V E C 4 . 1 0 cells (15%).  Transmigration occurs  through untransfected S V E C 4 . 1 0 cells at a slower rate than the transfected cells. T h e migration profile of I C A M - 1 - m e d i a t e d migration is very similar to that of I C A M - 2 in both rate a n d a b s o l u t e migration at e a c h time point. p r o c e s s is s h o w n to b e partially  In Figure 3 1 , t h e migration  L F A - 1 - d e p e n d e n t with the a n t i - C D 1 1 a antibody  inhibition bringing the level of migration to almost b a c k g r o u n d levels (18%).  The  control antibody against C D 4 9 d had no effect.  4:3.7 Simultaneous quantitation of migration and permeability It is p o s s i b l e that s o m e of the migrated neutrophils a r e leaking through holes o p e n e d up in t h e endothelial m o n o l a y e r by previously migrated neutrophils.  To  e x a m i n e this possibility, C a l c e i n A M - l a b e l e d neutrophils c o m b i n e d with red fluorescent b e a d s w e r e a d d e d onto the endothelial m o n o l a y e r bearing m e m b r a n e .  T h e first step  in s u c h a n e x a m i n a t i o n is to e n s u r e that the filter setup will not allow t h e yellow f l u o r e s c e n c e of the neutrophils to interfere with the red f l u o r e s c e n c e of the b e a d s . U s i n g filter D for excitation (X= 590 ± 10 nm) a n d filter E for e m i s s i o n (X= 6 4 5 ± 2 0 nm), it w a s p o s s i b l e to m e a s u r e red f l u o r e s c e n c e without a n y interference from the yellow cells ( T A B L E 2).  U s i n g filter B for both excitation (X= 4 8 5 ± 10 nm) a n d  159  100  no monolayer SVEC4.10 SVEC4.10:ICAM-1  ^ 75-  SVEC4.10.-ICAM-2  X  co E 50  /  25  T "  4  Time (hr)  8  10  12  F i g u r e 3 0 Neutrophil migration across SVEC4.10 monolayers. Migration of c a l c e i n A M l a b e l e d neutrophils a c r o s s S V E C 4 . 1 0 a n d I C A M - t r a n s f e c t e d m o n o l a y e r s w a s a s s e s s e d a s d e s c r i b e d for TIL1 cells. T h e migration at e a c h time point is e x p r e s s e d a s a m e a n of duplicate wells + S E M .  160  c o  +-*  • MB  CO  rj) E  3  Cl)  c  migration  + CD11 a Ab  + CD49d Ab  F i g u r e 31 Antibody blocking of neutrophil migration across SVEC4.10 monolayers. C a l c e i n A M l a b e l e d neutrophils were incubated with antibodies (4 ug/ml) in H B S S + 5 % F C S for 2 0 min prior to addition to the migration inserts. T h e a s s a y w a s a l l o w e d to p r o c e e d for 6 h r s a n d the results a r e e x p r e s s e d a s a m e a n of duplicate wells + SEM.  161  TABLE 2 Red bead fluorescence does not interfere with yellow cell fluorescence.  C a l c e i n A M l a b e l e d neutrophils w e r e c o m b i n e d with varying c o n c e n t r a t i o n s of red fluorescently labeled b e a d s (0.98 pirn diameter) in a v o l u m e of 1 0 0 pJ of H B S S + 5 % F C S . T h e s e c o m b i n a t i o n s w e r e p l a c e d in 9 6 well plates a n d the f l u o r e s c e n c e readings a r e e x p r e s s e d a s a m e a n of triplicate wells ± S E M . F o r e a c h combination, the yellow f l u o r e s c e n c e reading (determined with Filter B for both excitation a n d e m i s s i o n ) is the top n u m b e r in the table below a n d the red f l u o r e s c e n c e reading (determined with Filter D for excitation a n d Filter E for e m i s s i o n ) is the bottom italicized n u m b e r in p a r e n t h e s e s .  0.025% beads  0.05% beads  0.075% beads  0.10% beads  30 + 2  38 + 2  41 + 1  52 + 4  60 + 2  (45 ± 1)  (109 ± 1)  (185 ±2)  854 + 63  876 + 50  (45 ± 1)  (109 + 2)  (181 + 4)  (248 ± 8)  (345 + 8)  1552 + 72  1523 + 58  1509 + 27  1478 + 65  1509 + 11  no beads  no cells  2.5 x 10 cells 4  5.0 x 10 cells 4  7.5 x 10 cells 4  1.0 x 10 cells 5  893 + 42  (249 ±4) 887 + 84  (332 + 14) 881 + 15  (45 ± 1)  (109 ±2)  (176 ±5)  (262 ±3)  (328 + 5)  2409 + 39  2367 + 34  2287 + 93  2374 + 41  2307 + 85  (45 ± 1)  (108 ±3)  (182 + 3)  (264 ±5)  (339 ±8)  2911+16  2816 + 59  2743 + 62  2792 + 113  2744 + 96  (45 ± 1)  (107 ±2)  (179 + 3)  (258 ±6)  (336 ± 4)  e m i s s i o n (A= 530 + 12.5 nm), the yellow f l u o r e s c e n c e could be m e a s u r e d without interference from the red b e a d s .  U s i n g this combination of filters, it is p o s s i b l e to  e x a m i n e migration a n d permeability simultaneously.  T h e time c o u r s e of neutrophil  migration in the p r e s e n c e of red b e a d s (Figure 32a) is identical to that without b e a d s (Figure 30).  T h e b e a d s , in the p r e s e n c e of migrating neutrophils, d o not a p p e a r to  m o v e through the m e m b r a n e (Figure 32b).  This s u g g e s t s that previously migrated  neutrophils d o not leave gaping holes in the endothelial m o n o l a y e r a n d s u b s e q u e n t migration is not d u e to p a s s i v e diffusion through the e x p o s e d m e m b r a n e p o r e s .  4:4  Discussion  T h e importance of the L F A - 1 glycoprotein in the a d h e s i o n a n d extravasation of leukocytes  across  endothelial  cells  has  been  documented  in  many  systems  ( B e v i l a c q u a , 1993; C a r l o s a n d H a r l a n , 1994). V a r i o u s g r o u p s h a v e d e m o n s t r a t e d that L F A - 1 on l e u k o c y t e s interacts with I C A M - 1 on endothelial cells in the p r o c e s s of transendothelial migration (Furie etal., 1 9 9 1 ; K a v a n a u g h et al., 1 9 9 1 ; O p p e n h e i m e r M a r k s et al., 1991).  In certain studies, the role of the L F A - 1 : I C A M - 1 interaction h a s  b e e n attributed to the actual migration a c r o s s the endothelial barrier.  H o w e v e r , the  I C A M - 1 c o m p o n e n t , a s determined by antibody inhibition, c a n be s m a l l e r than the LFA-1 component.  T h i s has b e e n attributed to the utilization of a n alternate L F A - 1  counter-receptor present on endothelial cells.  I C A M - 2 , present on endothelial cells,  m a y m e d i a t e the L F A - 1 a d h e s i o n . H o w e v e r , the p r e c i s e role of I C A M - 2 in leukocyte  163  B)  OA  1  1  1  1  1  2  3  4  5  6  7  80-T  1  8  9 .  Time (hr) F i g u r e 3 2 Simultaneous assessment of neutrophil migration and SVEC4.10 monolayer permeability. C a l c e i n A M labeled neutrophils w e r e c o m b i n e d with red fluorescently labeled b e a d s (0.98 |im) a n d a d d e d to S V E C 4 . 1 0 m o n o l a y e r s . Both migration of neutrophils a n d diffusion of b e a d s a c r o s s the m o n o l a y e r s w a s a s s e s s e d a s d e s c r i b e d in t h e Materials a n d M e t h o d s . A ) T h e time c o u r s e of neutrophil migration a c r o s s t h e m o n o l a y e r is s h o w n . B) T h e time c o u r s e of b e a d diffusion a c r o s s the m o n o l a y e r is s h o w n .  164  transmigration is still unclear. T h e p u r p o s e of this work w a s to e x a m i n e the possibility of w h e t h e r I C A M - 2 on endothelial cells could assist leukocyte transmigration. T h e work d o n e in this chapter revolved around establishing a n in vitro s y s t e m to determine  if  transmigration.  ICAM-2  expression  on  endothelial  cells  would  allow  leukocyte  Initial c o n c e r n s about the permeability of the endothelial m o n o l a y e r  w e r e d i s m i s s e d w h e n it w a s s h o w n that the fluorescently-labeled b e a d s c o u l d not to diffuse a c r o s s the monolayer.  Endothelial cells transfected with the I C A M - 2 c D N A  w e r e a b l e to permit migration of both a T cell line (TIL1) a n d b o n e marrow neutrophils. The  i n c r e a s e d migration  in the  I C A M - t r a n s f e c t e d endothelial  d e p e n d e n t a s d e m o n s t r a t e d by the a n t i - C D 1 1 a antibody.  cells w a s  LFA-1-  T h e s e experiments were  preliminary a n d the c o m p l e t e specificity w a s not d e m o n s t r a t e d with the anti-ICAM-1 a n d a n t i - I C A M - 2 antibody inhibition. A s well, the inhibition by the a n t i - C D 1 1 b a n d antiC D 1 8 antibodies w a s not e x a m i n e d . T h e migration of leukocytes a c r o s s the endothelial barrier is a multistep p r o c e s s w h i c h p r o c e e d s by the s u c c e s s i v e formation of a d h e s i v e b o n d s b e t w e e n receptors on l e u k o c y t e s a n d counter-receptors on endothelial cells.  Interference with a n y of the  a d h e s i v e interactions w o u l d inhibit all s u b s e q u e n t interactions. T h e migration a s s a y in this c h a p t e r is a static m o d e l s y s t e m in w h i c h no flow w a s applied to mimic v e n e o u s flow.  T h e r e f o r e , rolling of leukocytes, mediated by selectins, is unlikely to play a role.  It is difficult to determine from t h e s e results whether I C A M - 2 on endothelial cells allows more efficient  leukocyte a d h e s i o n or is directly  involved  in their  transmigration.  H o w e v e r , previous studies h a v e s h o w n that L F A - 1 : I C A M - 1 interaction play a critical  165  role in the actual migration p r o c e s s ( K a v a n a u g h et al., 1 9 9 1 ; O p p e n h e i m e r - M a r k s et al., 1991). T cells are a b l e to bind endothelial cells in a n I C A M - 1 - i n d e p e n d e n t manner. T h e s e cells remain bound with the addition of blocking antibodies but are not a b l e to migrate a s they would if the antibodies w e r e not a d d e d .  T h e e x p e r i m e n t s in this  project d o not e x a m i n e whether the i n c r e a s e d transmigration by I C A M - 2 e x p r e s s i o n is a direct result of the migration step or simply d u e to i n c r e a s e d a d h e s i o n . m o l e c u l e s h a v e a l s o b e e n e x a m i n e d for the exact role they play in the process.  Other  migration  P E C A M - 1 ( C D 3 1 ) h a s b e e n s h o w n to be involved in m o n o c y t e migration  (Muller et al., 1993). Addition of anti-CD31 antibody d o e s not alter m o n o c y t e binding to the endothelial cells but d o e s arrest the m o n o c y t e motility at the junction b e t w e e n adjacent endothelial cells. It is very likely that I C A M - 2 e x p r e s s i o n on the endothelial cells i n c r e a s e s the binding capacity.  If I C A M - 2 ,  like I C A M - 1 ,  c a n mediate  the  migration p r o c e s s , addition of the a n t i - I C A M - 2 antibody after the initial binding h a s o c c u r r e d s h o u l d inhibit further migration. L e u k o c y t e s adherent on endothelial cells a p p e a r to crawl o v e r the  luminal  s u r f a c e s e a r c h i n g for intercellular junctions to s q u e e z e b e t w e e n ( B e v i l a c q u a , 1 9 9 3 ; C a r l o s a n d H a r l a n , 1994).  T h e leukocytes m o v e a c r o s s the endothelial cells to the  a b l u m i n a l s u r f a c e a n d on to the extravascular tissue.  T h e d i a p e d e s i s involves  intercellular junction d i s a s s e m b l y a n d m a y leave g a p s in the e n d o t h e l i u m . T h e s e g a p s m a y facilitate s u b s e q u e n t d i a p e d e s i s u n l e s s quickly repaired. A s d e m o n s t r a t e d by the preliminary e x p e r i m e n t s in the chapter, neutrophil migration a c r o s s the m o n o l a y e r is not a c c o m p a n i e d by p a s s i v e b e a d (red fluorescent) diffusion.  166  SVEC4.10 Although  this d o e s not eliminate the possibility that holes are still present in the endothelial cell m o n o l a y e r , they are n o n e t h e l e s s not big e n o u g h to allow 1 u.m b e a d s to p a s s through. S i n c e neutrophils are 10-20 u.m in diameter, they are m u c h bigger than a n y g a p p o s s i b l y left in the m o n o l a y e r a n d their migration must be a n active p r o c e s s involving neutrophil s h a p e distortion. T h e L F A - 1 : I C A M interactions are not the only m o l e c u l a r pairings involved in leukocyte migration.  In e x a m i n i n g the leukocyte-endothelial cell a d h e s i o n s , it h a s  b e c o m e a p p a r e n t that other a d h e s i o n p a t h w a y s play prominent roles in leukocyte migration.  In fact, t h e s e pathways are all s u p e r i m p o s e d upon e a c h other a n d m a n y  factors dictate w h i c h m a y be more significant than the others in v a r i o u s situations. T h e activation state of the endothelium is o n e s u c h factor.  U n d e r inflammatory conditions,  I C A M - 1 is upregulated on endothelial cells a n d leukocytes h a v e a stronger preference of I C A M - 1 o v e r I C A M - 2 under s u c h conditions (Dustin et al., 1986; Dustin et al., 1988; Dustin a n d Springer, 1991). Other m o l e c u l e s s u c h a s V C A M - 1 are a l s o upregulated in inflammation  a n d m a y alter the a d h e s i v e capacity of the endothelium ( R i c e a n d  B e v i l a c q u a , 1989; R i c e et al., 1990; C a r l o s et al., 1990). T h e combination of different molecular  pairings b e t w e e n  leukocytes inflammatory  migrate  out  conditions,  leukocytes a n d endothelium  of the  bloodstream  neutrophils  are  the  in  one  first  c a n determine  tissue  or  leukocytes  another. present,  whether Under whereas  l y m p h o c y t e s a n d m o n o c y t e s m a k e up the majority of migrating l e u k o c y t e s at later time points (Munro et al., 1989; L e u n g et al., 1 9 9 1 ; Norris et al., 1 9 9 1 ; B e v i l a c q u a , 1 9 9 3 ; A g e r , 1994). M o l e c u l e s involved in this p r o c e s s , a s determined by antibody inhibition,  167  include L F A - 1 , M a c - 1 , a n d I C A M - 1 .  U n d e r normal recirculation, neutrophils d o not  migrate out of the bloodstream, but lymphocytes a n d m o n o c y t e s exhibit s p o n t a n e o u s migration a c r o s s endothelium.  M o l e c u l e s involved in this a s determined by genetically  deficient m i c e include the selectin family m e m b e r s ( M a y a d a s et al., 1 9 9 3 ; L a b o w et al., 1994; T e d d e r et al., 1995).  I C A M - 2 h a s not b e e n e x a m i n e d in normal recirculation.  H o w e v e r , this study indicates that p r e s e n c e of I C A M - 2 on endothelial m o n o l a y e r s d o e s promote lymphocyte (TIL1) migration in an L F A - 1 - d e p e n d e n t m a n n e r .  A l t h o u g h the  a n t i - I C A M - 2 antibody inhibition h a s not yet b e e n d o n e , it d o e s a p p e a r that I C A M - 2 m a y play a role in normal recirculation. T h e filter setup u s e d to distinguish b e t w e e n red a n d yellow f l u o r e s c e n c e c a n a l s o be u s e d to determine w h i c h cell type m a y preferentially utilize w h i c h m o l e c u l e for migration.  In a h e t e r o g e n e o u s cell population, similar to the  b l o o d s t r e a m , different cells may be labeled with red a n d yellow fluorescent d y e s a n d tested for s i m u l t a n e o u s migration. T h e ability of antibodies against a d h e s i o n m o l e c u l e s on both leukocytes a n d endothelial cells to block migration provides an attractive opportunity to treat various d i s e a s e states. inhibit a  A n t i b o d i e s against L F A - 1 , M a c - 1 , a n d I C A M - 1 h a v e b e e n s h o w n to  s e r i e s of  inflammatory  r e s p o n s e s (Carlos a n d  Harlan,  1994).  When  neutrophils are the major c a u s e of tissue d a m a g e , t h e s e antibodies in c o m b i n a t i o n or sometimes  alone  can  decrease  the  extent  of  damage.  In  the  case  of  i s c h e m i a / r e p e r f u s i o n , t h e s e antibodies have b e e n s h o w n to at least partially prevent the d a m a g e after a c a r d i a c or renal transplant (Cosimi et al., 1990; H a r l a n et al., 1992; Isobe et al., 1992). V a s c u l a r o c c l u s i o n initiates the d a m a g e to the e n d o t h e l i u m w h i c h  168  is further magnified after reperfusion by activation of the inflammatory s y s t e m a n d a d h e s i o n of neutrophils. T h e antibodies are able to block the neutrophil a d h e s i o n a n d thus inhibit the s u b s e q u e n t d a m a g e . T h e antibodies are a l s o a b l e to block the i m m u n e r e s p o n s e g e n e r a t e d by a n organ allograft. the e n d o t h e l i u m in the allograft.  O n e site of attack by the i m m u n e s y s t e m is  S i n c e endothelial cells e x p r e s s I C A M - 2 , they m a y be  a target for neutrophil attachment.  A s well, endothelial cells h a v e b e e n s h o w n to  g e n e r a t e a T cell r e s p o n s e (St. Louis et al., 1993) which m a y be I C A M - 2 - d e p e n d e n t . T h i s p r o v i d e s a g o a l for immune s y s t e m modulation in w h i c h the a n t i - I C A M - 2 antibody m a y inhibit both neutrophil a d h e s i o n a n d the T cell r e s p o n s e to e n d o t h e l i u m .  169  4:5 References A b a s s i O , Kishimoto T K , Mclntire L V , A n d e r s o n D C , a n d Smith C W (1993) E-selectin s u p p o r t s neutrophil rolling under conditions of flow. J. Clin. Invest. 9 2 : 2 7 1 9 . A g e r A (1994) L y m p h o c y t e recirculation and homing: roles of a d h e s i o n m o l e c u l e s a n d chemoattractants. Trends Cell Biol. 4 : 3 2 6 . B e v i l a c q u a M P (1993) Immunol. 11:767.  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Med. 1 8 1 : 2 2 5 9 .  have  V a p o r c i y a n A A , D e L i s s e r H M , Y a n H C , M e n d i g u r e n II, Thorn S R , J o n e s M L , W a r d P A , a n d A l b e l d a S M (1993) Involvement of platelet-endothelial cell a d h e s i o n molecule-1 in neutrophil recruitment in vivo. Science 2 6 2 : 1 5 8 0 . v o n A n d r i a n U H , C h a m b e r s J D , M c E v o y L M , B a r g a t z e R F , Arfors K E , a n d B u t c h e r E C (1991) T w o step m o d e l of leukocyte-endothelial interaction in inflammation: distinct roles for L E C A M - 1 a n d the leukocyte (3 integrins in vivo. Proc. Natl. Acad. Sci. USA 88:7538. 2  v o n B o x b e r g Y , W u t z R, a n d S c h w a r z U (1990) U s e of the biotin-avidin s y s t e m for labelling, isolation a n d characterization of neural cell-surface proteins. Eur. J. Biochem. 1 9 0 : 2 4 9 .  174  X u H , G o n z a l o J A , St. Pierre Y , Williams IR, K u p p e r T S , C o t r a n R S , S p r i n g e r T A , a n d G u t i e r r e z - R a m o s J C (1994) L e u k o c y t o s i s a n d resistance to septic s h o c k in intercellular a d h e s i o n m o l e c u l e 1-deficient mice. J. Exp. Med. 180:95. Z i m m e r m a n G A , Prescott S M , and Mclntyre T M (1992) Endothelial cell interaction with g r a n u l o c y t e s : Tethering and signalling m o l e c u l e s . Immunol. Today 13:93.  175  Chapter 5  5:1  Discussion  T h e physiological function of I C A M - 2 is the least understood of the three L F A - 1 counter-receptors.  T h e role of the L F A - 1 :ICAM-1 interaction  has been examined  extensively a n d found to participate in virtually every a d h e s i o n - d e p e n d e n t  function  a s s a y e d (Martz, 1987; Springer, 1990; Dustin a n d Springer, 1991). T h e L F A - 1 : I C A M - 3 interaction h a s a l s o b e e n studied a n d found to play a role in T cell activation F o u g e r o l l e s et al., 1994).  (de  However, the role of I C A M - 2 in the i m m u n e r e s p o n s e h a s  not b e e n clarified. T h e g o a l of this thesis w a s to characterize further the functional role of the L F A - 1 : I C A M - 2 interaction.  T h e first objective w a s to isolate the murine I C A M - 2  c D N A a n d u s e it a s a tool to e x a m i n e e x p r e s s i o n , tissue distribution, a n d binding capacity.  T h e next objective w a s to investigate p o s s i b l e functional roles of I C A M - 2 .  T h e c D N A w a s u s e d to test the p o s s i b l e role of I C A M - 2 in T cell activation  and  leukocyte transendothelial migration. T h e cloning of the murine I C A M - 2 c D N A involved a unique P C R - b a s e d strategy c o m b i n i n g s e q u e n c e information of similar m o l e c u l e s .  T h e unifying feature in the  clustering of s e q u e n c e data w a s the Ig d o m a i n structure (Williams, 1987; W i l l i a m s a n d B a r c l a y , 1988).  T h i s structural feature is present in m o l e c u l e s w h i c h function within  the i m m u n e s y s t e m . A n t i g e n recognition is the fundamental f o c u s of most m e m b e r s in the immunoglobulin superfamily.  However, Ig-like m o l e c u l e s h a v e b e e n identified in  certain invertebrate s p e c i e s indicating that early a n c e s t o r s of t h e s e d o m a i n s w e r e  176  present prior to the specialization of the i m m u n e s y s t e m . Ig superfamily m e m b e r s a r e a l s o present in insects; t h e s e m o l e c u l e s a r e involved in the n e r v o u s s y s t e m a n d function in a x o n g u i d a n c e a n d fasciculation (Harrelson a n d G o o d m a n , 1988).  T h e Ig  d o m a i n m a y h a v e b r a n c h e d out a n d w a s widely adopted in evolution b e c a u s e of its stability conferred by the d i s u l p h i d e - b o n d e d p-strand structure (Williams a n d B a r c l a y , 1 9 8 8 ; Hunkapiller a n d H o o d , 1989).  It is c o m m o n l y a c c e p t e d that Ig-like s e q u e n c e s  w e r e derived by g e n e duplication a n d d i v e r g e n c e from a primordial d o m a i n .  The  d i v e r g e n c e o c c u r r e d to a level s u c h that only the b a s i c structure r e m a i n e d . In terms of function, t h e s e Ig-related interaction.  structures  probably played a primordial  role in cell:cell  F r o m a n early role in cell a d h e s i o n , the cell surface m o l e c u l e s b e c a m e  likely c a n d i d a t e s to control the behaviour of cells in the d e v e l o p m e n t of a n i m m u n e system.  In t h e invertebrate Caenorhabditis elegans, certain d e v e l o p i n g neural cells  u n d e r g o a p r o g r a m m e d cell death followed by p h a g o c y t o s i s (Horvitz et al., 1 9 8 2 ; H e d g e c o c k et al., 1983). In s o m e c a s e s , the neural cell is d e s t r o y e d by recognition of a neighboring cell.  If this primitive natural killer cell specificity could b e modified to  include a requirement of a pathogen infection of cells, then a n i m m u n e s y s t e m m a y have evolved. T h e structural conservation of Ig-like d o m a i n s through evolution s u g g e s t s that they a r e stable configurations which c a n assist in m a n y cellular functions.  The  m a i n t e n a n c e of I C A M m o l e c u l e s a c r o s s s p e c i e s indicates that they play critical roles in the i m m u n e s y s t e m . A m i n o acid s e q u e n c e a n a l y s i s h a s revealed that t h e a m i n o acid identity b e t w e e n h u m a n a n d murine I C A M - 2 (60%) is higher than that for I C A M - 1  177  (53%).  In e x a m i n i n g c r o s s - s p e c i e s binding, it w a s found that h u m a n L F A - 1 is a b l e to  a l s o bind murine I C A M - 1 a n d I C A M - 2 in addition to the h u m a n counter-parts ( J o h n s t o n et al., 1990; X u et al., 1992). H u m a n : m u r i n e hybrid cells h a v e s h o w n that a  L  a n d p of 2  h u m a n a n d m o u s e are a b l e to p r o m i s c u o u s l y c o - a s s o c i a t e a n d form i n t e r s p e c i e s a p c o m p l e x e s detected at the cell s u r f a c e (Marlin et al.,  1986; L a r s o n a n d Springer,  1990). T h e s e hybrid c o m p l e x e s w e r e u s e d in c r o s s - s p e c i e s binding a s s a y s a n d found that the h u m a n a : m u r i n e p c o m p l e x is c a p a b l e of binding h u m a n I C A M - 1 . 2  L  Various  r e s i d u e s in both L F A - 1 a n d the I C A M counter-receptors h a v e b e e n c o n s e r v e d in both the  mouse and human genome.  Alteration  of glutamic  acid at r e s i d u e 34  and  glutamine at residue 73 in h u m a n I C A M - 1 disrupts the ability to bind L F A - 1 (Staunton et al., 1990).  Interestingly, both of t h e s e residues are a l s o present in the other four  c h a r a c t e r i z e d h u m a n a n d murine I C A M s .  In addition, the crystal structure of h u m a n  I C A M - 2 h a s r e v e a l e d that the glutamic acid residue lies in a p-strand w h i c h interact with the  l-domain  of L F A - 1 ( C a s a s n o v a s et  al.,  1997).  may  The degree  of  s e q u e n c e c o n s e r v a t i o n is therefore high e n o u g h to u s e it a s a b a s i s for c r o s s - s p e c i e s cloning of I C A M - l i k e m o l e c u l e s .  In addition to the cloning of murine I C A M - 2 of this  thesis, c a n i n e I C A M - 1 h a s a l s o b e e n P C R amplified b a s e d on h u m a n a n d I C A M - 1 s e q u e n c e s (Smith et  al.,  1991).  murine  T h i s m a y provide a s i m p l e method  for  identifying yet u n c h a r a c t e r i z e d I C A M - l i k e m o l e c u l e s . T h e g e n o m i c organization of the murine I C A M - 2 g e n e confirms its a s s i g n m e n t a s a m e m b e r of the Ig superfamily.  A s with other m e m b e r s of this family, there is  g o o d correlation b e t w e e n the intron/exon organization a n d the structural d o m a i n s of  178  the protein (Williams a n d B a r c l a y , 1988; Hunkapiller a n d H o o d , 1989).  T h e leader  peptide, the two Ig-like d o m a i n s , a n d the t r a n s m e m b r a n e / c y t o p l a s m i c d o m a i n s are e n c o d e d by four e x o n s s e p a r a t e d by three type I introns.  T h e s e type I introns are  c o m m o n l y found b e t w e e n Ig-like e x o n s of other m e m b e r s of the Ig superfamily.  A  uniform intron p h a s e is important for the evolution of the Ig superfamily b e c a u s e it e n s u r e s that m o l e c u l e s with multiple Ig-like d o m a i n s c a n b e restructured by e x o n duplication a n d shuffling without altering the reading frame.  G e n e duplication a n d  d i v e r g e n c e from a primordial d o m a i n are the likely origin of Ig-related m o l e c u l e s . T h e c o r r e s p o n d e n c e b e t w e e n protein d o m a i n s a n d e x o n structure, a s well a s the uniform p h a s e of the introns supports this theory of evolution. ray crystallography studies.  Further support c o m e s from x-  E v i d e n c e h a s s h o w n that s p l i c e sites in g e n e s usually  m a p to a m i n o a c i d r e s i d u e s located at the protein s u r f a c e (Craik et al., 1982). has  been  confirmed  with  the  crystallographic  structure  of  the  Ig  This  domains  of  i m m u n o g l o b u l i n s a n d H L A m o l e c u l e s (Bjorkman et al., 1987; A l z a r i et al., 1988). T h e g e n o m i c I C A M - 2 c l o n e isolated from this project did not contain e n o u g h sequence  u p s t r e a m of transcription  to a s s e s s the  regulatory  region  adequately.  H o w e v e r , another group h a s a n a l y z e d this region more thoroughly (Xu et al., 1992). T h e y h a v e found that this region contains a T A T A - l i k e s e q u e n c e , a n inverted C A A T box, a n d a c o n s e n s u s transcription initiation s e q u e n c e . H o w e v e r , no binding sites for the transcription factors NF-KB a n d A P - 1 w e r e identified a s they w e r e for the I C A M - 1 5' u p s t r e a m region ( V o r a b e r g e r et  al.,  1991).  T h i s is not surprising s i n c e I C A M - 2  e x p r e s s i o n is not inducible a n d the transcription factors NF-KB a n d A P - 1 are involved  179  in g e n e induction (Angel et al., 1987; E d b r o o k e et al., 1989). T h e only c a s e s in w h i c h I C A M - 2 e x p r e s s i o n is elevated is on neoplastic cells ( R o o s , 1 9 9 1 ; Ellis et al., R e n k o n e n et al., 1992). islands have  1992;  I C A M - 2 a p p e a r s to be a constitutively e x p r e s s e d g e n e . C p G  b e e n s h o w n to be a s s o c i a t e d with the  5' region  of  constitutively  e x p r e s s e d g e n e s a n d methylation c a n play a n important role in g e n e e x p r e s s i o n (Bird, 1986; G a r d i n e r - G a r d e n a n d Frommer, 1987; C e d a r , 1988).  Constitutively e x p r e s s e d  g e n e s contain C p G islands which are unmethylated through all s t a g e s of the cell c y c l e . W h e t h e r methylation plays a role in I C A M - 2 e x p r e s s i o n remains to be s e e n . specific factors regulating the s o m e w h a t limited distribution remain to be identified.  Tissue-  pattern of I C A M - 2 a l s o  O n e group has s h o w n that the h u m a n I C A M - 2 promoter is  able to function in the murine s y s t e m in a similar tissue-specific m a n n e r o b s e r v e d in the h u m a n s y s t e m ( C o w a n etal., 1996). T h e e x i s t e n c e of a subfamily of Ig-related m o l e c u l e s ( I C A M - 1 , - 2 , a n d -3) with the ability to bind L F A - 1 attests to the importance of t h e s e a d h e s i o n p a t h w a y s .  An  inability to m a k e t h e s e interactions in vivo results in the clinical s y m p t o m s a s s o c i a t e d with L A D ( A n d e r s o n a n d Springer, 1987). T h e functional significance of e a c h of t h e s e interactions  h a s b e e n e x a m i n e d to varying d e g r e e s .  contact is the least understood.  H o w e v e r , the L F A - 1 : I C A M - 2  B a s e d on the structural a n d functional h o m o l o g y with  I C A M - 1 a n d I C A M - 3 , I C A M - 2 w a s tested for its ability to a s s i s t in T cell activation. T cell activation using purified I C A M - 2 protein h a s s h o w n that it c a n stimulate proliferation  ( D a m l e et al.,  1 9 9 2 a ; D a m l e et al.,  1992b).  Every LFA-1-dependent  r e s p o n s e a p p e a r s to be greater for I C A M - 1 than for I C A M - 2 .  180  It w a s initially thought  that the p r e f e r e n c e of I C A M - 1 over I C A M - 2 by L F A - 1 w a s d u e to the s m a l l e r s i z e of the I C A M - 2 (only two d o m a i n s ) protein not being able to extend its binding site a s c l o s e to L F A - 1 .  H o w e v e r , the difference m a y actually be qualitative.  A  modified  v e r s i o n of I C A M - 2 with a n extra five d o m a i n s from C D 3 1 h a s not s h o w n any difference in proliferation from the native I C A M - 2 molecule.  It m a y be that L F A - 1 c a n exist in  multiple activated conformational states (Binnerts et al., 1994).  E v i d e n c e for this h a s  b e e n p r e s e n t e d in w h i c h L F A - 1 c a n r e c o g n i z e I C A M - 1 but cannot r e c o g n i z e I C A M - 3 . H o w e v e r , L F A - 1 recognizing I C A M - 3 is able to still bind to I C A M - 1 . T h e role of L F A - 1 : I C A M - 2 interaction in T cell proliferation to alloantigen h a s b e e n e x a m i n e d . T h e r e s p o n s e c a n be inhibited by antibodies against either m o l e c u l e a n d thus providing a potential target for immune s y s t e m modulation acceptance. survival.  A n t i b o d i e s h a v e b e e n administered The  question  of  whether  the  previously for  antibodies  are  t o l e r a n c e state d e p e n d s on e a c h specific situation.  allograft  i n c r e a s e d graft  merely  i m m u n o s u p p r e s s i v e a g e n t s or whether they are assisting in the  in  non-specific  induction  of  a  T h e u s e of L F A - 1 a n d I C A M - 1  m A b s h a s b e e n d o c u m e n t e d to i n c r e a s e the survival of v a r i o u s allografts. In o n e c a s e involving c a r d i a c allograft in mice, t h e s e two antibodies together w e r e a b l e to prolong the survival of the graft (Isobe et al., 1992).  In addition, t h e s e m i c e w e r e a b l e to  a c c e p t a n allo-skin graft indicating that the i m m u n e s y s t e m had b e c o m e tolerant to the allotype of the graft. T h e next step would be to e x a m i n e if the I C A M - 2 antibody could a l s o i n c r e a s e the survival of an allograft. the  engraftment  is  immunosuppressive  181  If this is p o s s i b l e , then w h e t h e r the nature of or  immunotolerant  could  be  examined.  E n d o t h e l i u m in solid o r g a n s e x p r e s s I C A M - 2 a n d c a n be a site of a n  immune  response.  prolong  A n t i b o d i e s m a y be able to block the i m m u n e r e s p o n s e a n d  engraftment. T h e role of I C A M - 2 in transendothelial migration w a s e x a m i n e d .  M y work  s u g g e s t s that I C A M - 2 present on endothelial cells a s s i s t s neutrophil migration in a n L F A - 1 - d e p e n d e n t manner.  S i n c e neutrophils are the first leukocytic cell type  migrate the endothelium at a r e a s of inflammation  to  (Bevilacqua, 1993; Carlos and  H a r l a n , 1994; A g e r , 1994), I C A M - 2 m a y a l s o play a role in inflammation a s well a s recirculation.  A n t i - I C A M - 2 treatment  may  inhibit  the  migration  and  thus  the  inflammatory r e s p o n s e . T h e e x p r e s s i o n of I C A M - 2 on platelets ( D i a c o v o et al., 1994) m a y a l s o be tied in with migration. A l t h o u g h post-neutrophil migration did not indicate that there w e r e any large holes in the endothelial monolayer, it did not e x c l u d e the possibility that the m o n o l a y e r had n o n e t h e l e s s b e e n d a m a g e d . T h e migration p r o c e s s involves junction d i s a s s e m b l y a n d it would be difficult to imagine that the junction w o u l d immediately r e a s s e m b l e .  T h e holes would h a v e to be repaired in order to  prevent v a s c u l a r l e a k a g e . O n e possibility is that the I C A M - 2 on platelets m a y a d h e r e to the trailing e n d of a migrating leukocyte a n d stop at the m o n o l a y e r initiating the c a s c a d e of e v e n t s required to mediate w o u n d repair a n d acting like a " v a s c u l a r b a n d aid". I C A M - 2 deficient mice m a y provide the a n s w e r to this p o s s i b l e role of I C A M - 2 on platelets.  182  The  functional  role  of  ICAM-2  has  remained  elusive.  The  experiments  d e s c r i b e d in this t h e s i s h a v e attempted to s h e d s o m e light on this issue. I h a v e s h o w n that ICAM-2 c a n a s s i s t in T cell activation by providing a costimulatory s i g n a l .  It c a n  a l s o m e d i a t e neutrophil migration a c r o s s a n endothelial monolayer. Therefore, I C A M 2 m a y play a significant role in i m m u n e and inflammatory r e s p o n s e s . 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