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Divergent mechanisms utilized by SOCS3 to mediate IL-10 inhibition of TNF-a and nitric oxide production.. Qasimi, Pooran 2005-12-31

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DIVERGENT M E C H A N I S M S UTILIZED B Y S0CS3 TO M E D I A T E IL-10 INHIBITION OF TNF-a A N D NITRIC OXIDE PRODUCTION B Y MACROPHAGES by  P O O R A N QASIMI B . S c . and B . A S c , S i m o n Fraser U n i v e r s i t y , 2 0 0 0  A THESIS SUBMITTED IN PARTIAL F U L L F I L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in  THE F A C U L T Y OF G R A D U A T E STUDIES E X P E R I M E N T A L MEDICINE  THE UNIVERSITY OF BRITISH C O L U M B I A April 2005  © P o o r a n Q a s i m i , 2005  Abstract T h e c y t o k i n e , i n t e r l e u k i n - 1 0 ( I L - 1 0 ) , i n h i b i t s a c t i v a t i o n o f macrophages b y activators s u c h as l i p o p o l y s a c c h r i d e ( L P S ) . H o w e v e r the m e c h a n i s m b y w h i c h I L - 1 0 interferes w i t h L P S s i g n a l l i n g is s t i l l unclear. O n e w e l l - c h a r a c t e r i z e d s i g n a l l i n g p a t h w a y activated b y I L - 1 0 is that o f Stat3 ( S i g n a l transducer and activator o f transcription), w h i c h is essential for the antip r o l i f e r a t i v e and a n t i - i n f l a m m a t o r y actions o f I L - 1 0 o n macrophages. In our search for I L - 1 0 i n d u c e d , Stat3-regulated genes, w e f o u n d a candidate b e l o n g i n g to S O C S (suppressor o f c y t o k i n e s i g n a l l i n g ) f a m i l y o f negative regulators o f c y t o k i n e s i g n a l l i n g . U s i n g mutant I L - 1 0 receptor and d o m i n a n t negative Stat3, w e s h o w that I L - 1 0 induces S O C S 3 message and p r o t e i n i n a Stat3-dependent manner.  H o w e v e r m e r e e x p r e s s i o n o f S O C S 3 p r o t e i n i n macrophages was  not sufficient to i n h i b i t T N F - a p r o t e i n p r o d u c t i o n i n response to L P S , suggesting that a d d i t i o n a l I L - 1 0 - i n d u c e d signals are required. A n d i n d e e d w e f i n d I L - 1 0 s t i m u l a t i o n induces p h o s p h o r y l a t i o n o f tyrosine 204 o f S O C S 3 protein. In order to determine the r o l e o f S O C S 3 i n I L - 1 0 i n h i b i t i o n o f m a c r o p h a g e a c t i v a t i o n , w e d e r i v e d c e l l lines f r o m S O C S 3 " and S O C S 3 " 7  + /  m i c e . S O C S 3 " " macrophages respond to L P S i n a m a n n e r s i m i l a r to w i l d - t y p e c e l l s , but I L - 1 0 is 7  less effective i n i n h i b i t i n g L P S - i n d u c e d T N F - a and N O p r o d u c t i o n i n the S O C S 3 " " c e l l s as 7  c o m p a r e d to S O C S 3 " macrophages. + /  c D N A restored I L - 1 0 responsiveness.  Reconstitution o f S O C S 3 " cells w i t h a wild-type S O C S 3 7  In order to determine w h i c h S O C S 3 d o m a i n is important  i n I L - 1 0 s i g n a l l i n g , S O C S 3 " macrophages w e r e reconstituted w i t h v a r i o u s S O C S 3 d o m a i n 7  mutants. I L - 1 0 r e q u i r e d a l l d o m a i n s o f S O C S 3 p r o t e i n for the i n h i b i t i o n o f T N F - a p r o t e i n expression. H o w e v e r , for i n h i b i t i o n o f T N F - a m R N A e x p r e s s i o n , I L - 1 0 does not seem to require the K I R d o m a i n o f S O C S 3 protein. In contrast, o n l y the t w o tyrosine residues, 2 0 4 a n d 2 2 1 , located i n the S O C S - b o x d o m a i n are r e q u i r e d for I L - 1 0 i n h i b i t i o n o f L P S - i n d u c e d i N O S  ii  p r o t e i n e x p r e s s i o n and subsequent N O p r o d u c t i o n . T h e s e studies demonstrate the i m p o r t a n c e o f S O C S 3 p r o t e i n i n the a n t i - i n f l a m m a t o r y a c t i o n o f I L - 1 0 and that i n h i b i t i o n o f N O and T N F - a b y I L - 1 0 depends o n different d o m a i n s o f S O C S 3 . C h a r a c t e r i z a t i o n o f S O C S 3 s i g n a l l i n g d o m a i n s and its i m m e d i a t e d o w n s t r e a m targets w i l l a l l o w d e v e l o p m e n t o f therapeutic strategies, w h i c h replicate the b e n e f i c i a l anti- i n f l a m m a t o r y a c t i o n o f I L - 1 0 .  in  T A B L E OF CONTENTS , Abstract  1 1  T a b l e o f Contents  i  List o f Tables  v  List o f Figures  v  v  l  List o f Abbreviations  V 1 1  Acknowledgements  x  C H A P T E R 1: I n t r o d u c t i o n  1  1.1 M a c r o p h a g e s  1  1.2 L P S s i g n a l l i n g  9  1.3 I L - 1 0 s i g n a l l i n g  16  1.4 S O C S 3 p r o t e i n  22  C H A P T E R 2: M a t e r i a l s and M e t h o d s  32  C H A P T E R 3: R e s u l t s  39  C H A P T E R 4: D i s c u s s i o n  60  C H A P T E R 5: C o n c l u s i o n and Future D i r e c t i o n s  73  Bibliography  76  iv  LIST OF TABLES T a b l e 1: R e q u i r e m e n t for different S O C S 3 d o m a i n s i n m e d i a t i n g I L - 1 0 i n h i b i t i o n o f v a r i o u s m a c r o p h a g e responses. (Page 69)  v  LIST OF FIGURES  F i g u r e 1: S c h e m a t i c representation o f r e g u l a t i o n o f i N O S p r o t e i n b y L P S a n d I L - 1 0 i n macrophages. (Page 7) F i g u r e 2: S c h e m a t i c representation o f L P S s i g n a l l i n g . (Page 12) F i g u r e 3: S c h e m a t i c representation o f I L - 1 0 s i g n a l l i n g . (Page 19) F i g u r e 4: T h e alternative names and d o m a i n structures o f v a r i o u s m e m b e r s o f the S O C S f a m i l y . (Page 23) F i g u r e 5: Structure and m o d e l o f kinase i n h i b i t i o n b y J A B / S O C S 1 and C I S 3 / S O C S 3 . (Page 25) F i g u r e 6: T h e S O C S - b o x targets proteins for p r o t e a s o m a l degradation b y several m e c h a n i s m s . (Page 27) F i g u r e 7: I n d u c t i o n o f S O C S 3 message b y I L - 1 0 i n a Stat3-dependent manner. (Page 40) F i g u r e 8: I n d u c t i o n o f S O C S 3 p r o t e i n b y I L - 1 0 . (Page 4 1 ) F i g u r e 9: E c t o p i c e x p r e s s i o n o f S O C S 3 p r o t e i n is not sufficient i n i n h i b i t i o n to c o m p l e t e l y i n h i b i t T N F - a p r o t e i n p r o d u c t i o n i n response to L P S . (Page 43) F i g u r e 10: I L - 1 0 induces p h o s p h o r y l a t i o n o f S O C S 3 p r o t e i n at tyrosine 2 0 4 i n the S O C S - b o x d o m a i n . (Page 45) F i g u r e 11: I L - 1 0 i n h i b i t i o n o f T N F - a p r o t e i n e x p r e s s i o n requires S O C S 3 d u r i n g the early phase o f s i g n a l l i n g . (Page 47) F i g u r e 12: I L - 1 0 i n h i b i t s e x p r e s s i o n o f T N F - a m R N A i n a S O C S 3 - d e p e n d e n t manner. (Page 48) F i g u r e 13 A :  S c h e m a t i c representation o f v a r i o u s d o m a i n mutants. (Page 50)  F i g u r e 1 3 B : R e c o n s t i t u t i o n o f S O C S 3 " cells w i t h W T and mutant S O C S 3 . (Page 51) 7  F i g u r e 14: I L - 1 0 requires a l l d o m a i n s o f S O C S 3 p r o t e i n for i n h i b i t i o n o f T N F - a p r o t e i n p r o d u c t i o n . (Page 53) F i g u r e 15: E x c l u d i n g K I R d o m a i n , I L - 1 0 requires a l l d o m a i n s o f S O C S 3 protein for i n h i b i t i o n o f T N F - a m R N A e x p r e s s i o n . (Page 55) F i g u r e 16: Y 2 0 4 and 221 o f S O C S 3 protein is important for i n h i b i t i o n o f N O p r o d u c t i o n b y I L - 1 0 . (Page 57) F i g u r e 17: I L - 1 0 requires Y 2 0 4 / 2 2 1 o f S O C S 3 for i n h i b i t i o n o f i N O S p r o t e i n e x p r e s s i o n . (Page 59) F i g u r e 18: S c h e m a t i c representation o f the m e c h a n i s m b y w h i c h I L - 1 0 m a y be i n h i b i t i n g L P S s i g n a l l i n g p a t h w a y . (Page 70)  vi  LIST OF ABBREVIATIONS  AAMO  A l t e r n a t i v e l y activated m a c r o p h a g e  Akt  V - A k t m u r i n e v i r a l oncogene h o m o l o g y 1  AP-1  A c t i v a t o r Protein-1  APC  A n t i g e n presenting c e l l  ATP  A d e n o s i n e triphosphate  Ca  Calcium  CD14  M o n o c y t e differentiation antigen C D 14  CD80/86  Co-stimulatory molecule C D 8 0 / 8 6  c-Jun  c - J u n m i t o g e n activated k i n a s e  CAMO  C l a s s i c a l l y activated m a c r o p h a g e  CREB  c y c l i c A M P r e s p o n s i v e element b i n d i n g p r o t e i n  DNA  Deoxyribonucleic acid  ECL  Enhanced Chemiluminescence  EDTA  Ethylene-diamine-tetraacetic acid  ELISA  E n z y m e - l i n k e d i m m u n o s o r b e n t assay  ERK  E x t r a c e l l u l a r - s i g n a l regulated k i n a s e  FACS  Fluorescence-activated cell sorting/cell-scanning  GFP  G r e e n fluorescence p r o t e i n  GM-CSF  G r a n u l o c y t e m a c r o p h a g e c o l o n y s t i m u l a t i n g factor  GP130  G l y c o p r o t e i n 130 ( I L - 6 receptor)  HO-1  Heme-oxygenase-1  ICAM  Intercellular a d h e s i o n m o l e c u l e  IFN-A,  Interferon-A,  IKB  IKB p r o t e i n  IKK  IKB p r o t e i n k i n a s e vii  IL-1  Interleukin-1  IL-6  Interleukin-6  IL-8  Interleukin-8  IL-10  Interleukin-10  IL-12  Interleukin-12  ILK  Integrin-linked kinase  iNOS  inducible nitric oxide  JAK  Janus kinase  JNK  c-Jun-N-terminal kinase  KIR  Kinase inhibitory region  KO  Knock-out  LPS  Lipopolysaccharide  LJJF  L e u k e m i a i n h i b i t o r y factor  MO  Macrophage  MHC  Major histocompatibality complex  MAPK  M i t o g e n activated p r o t e i n k i n a s e  MD-2  M D - 2 protein  MTP  Macrophage inflammatory protein  MKK  M A P K kinase  MyD88  M y e l o i d differentiation p r i m a r y response gene 88  NF-KB  N u c l e a r factor-KB  NMDA  N-mefhyl-D-aspartate  NO  Nitric oxide  p38 M A P K  p38 m i t o g e n activated p r o t e i n k i n a s e  PBS  Phosphate buffered saline  PGE2  Prostaglandin E 2  PI3-K  P h o s p h o t i d y l i n o s i t o l 3-kinase  viii  PKA  P r o t e i n kinase A  PKC  P r o t e i n kinase C  PMSF  Phenylmethanosulfonly fluoride  RNA  Ribonucleic acid  ROO-  R e a c t i v e o x y g e n intermediates  SAPK  Stress activated p r o t e i n k i n a s e  SDS  S o d i u m d o d e c y l sulphate  SH2  Src h o m o l o g y 2  SOCS  Suppressor o f c y t o k i n e s i g n a l l i n g  SR  Scavenger receptor  STAT  S i g n a l transducer and activator o f t r a n s c r i p t i o n  TCF/LEF-1  T - c e l l / l y m p h o i d enhancer factor-1  Thr  Threonine  TIR  T o l l - i n t e r l e u k i n - 1 receptor  TIRAP  T I R d o m a i n - c o n t a i n i n g adaptor p r o t e i n  TLR4  T o l l - l i k e receptor-4  TNF-a  T u m o r necrosis f a c t o r - a  TNFR  T N F receptor  TRAF6  T N F - r e c e p t o r associated factor-6  Tyr  Tyrosine  ng/mL  N a n a g r a m s per m i l l i l i t e r  Hg/mL  M i c r o g r a m s per m i l l i l i t r e  U/mL  E n z y m a t i c unit per m i l l i l i t r e  WT  Wild-type  Y204  T y r o s i n e 2 0 4 residue  Y221  T y r o s i n e 221 residue  Acknowledgements I w o u l d l i k e to thank m y supervisor, D r . A l i c e M u i , for g i v i n g m e the o p p o r t u n i t y to be part o f her l a b o r a t o r y and g a i n this i n v a l u a b l e experience. I w o u l d l i k e to also thank m y s u p e r v i s o r y c o m m i t t e e , D r . V i n c e D u r o n i o , and D r . M i c h a e l C o x , for a l l their scientific advise, t i m e and c o n s i d e r a t i o n w h i c h a l l o w e d m e to beat deadlines. I w o u l d l i k e to thank D r . B i l l S a h l a n d D r . C h r i s O n g for their support. L a s t but not least, I w o u l d l i k e to extend m y gratitude to m y colleagues i n D r . M u i ' s lab : A l i G h a n i p o u r , A n d r e w M i n g L u m , R u p i n d e r D h e s i and Irfan M o l e d i n a . M y s p e c i a l thanks and gratitude goes to A n d r e w M i n g L u m , w h o h e l p e d m e t r e m e n d o u l s y w i t h a l l the northern data. A l s o , thanks to D r . A k i h i k o Y o s h i m u r a for p r o v i d i n g us w i t h the S O C S 3 d o m a i n mutants, and D r . N i c h o l a s C a c a l a n o for p r o v i d i n g us w i t h the p h o s p h o - s p e c i f i c S O C S 3 antibodies. A s w e l l , thanks to the T r a n s p l a n t T r a i n e e p r o g r a m for p r o v i d i n g f u n d i n g for m y project. T h i s experience w o u l d not have b e e n the same w i t h o u t the support o f m y f a m i l y and friends, so a s p e c i a l thanks goes to t h e m for their constant encouragment and faith i n m e .  x  \  \  CHAPTER 1: Introduction 1.1  Macrophages: Functions T h e i m m u n e system is c o m p o s e d o f m a n y interdependent c e l l types that h a v e  s p e c i a l i z e d functions a n d c o l l e c t i v e l y protect the b o d y f r o m bacterial, parasitic, v i r a l infections and g r o w t h o f t u m o u r c e l l s . A m o n g these, the m a c r o p h a g e i s o n e o f the m o s t p l e i o t r o p i c , e x h i b i t i n g a b r o a d range o f b i o l o g i c a l functions, i n c l u d i n g p r o - and/or a n t i - i n f l a m m a t o r y activities a n d p h a g o c y t o s i s [4]. T h e y also e n g u l f and ingest f o r e i g n particles (innate i m m u n e response) a n d present these antigens to T - c e l l s (acquired i m m u n e response) a n d thus are often referred to as antigen-presenting c e l l s ( A P C ) [5]. T h i s i s an important first step i n the i n i t i a t i o n o f an a c q u i r e d i m m u n e response, w h i c h they regulate t h r o u g h a v a r i e t y o f c y t o k i n e s a n d c o s t i m u l a t o r y m o l e c u l e s [6]. M a c r o p h a g e differentiation a n d a c t i v a t i o n states are greatly i n f l u e n c e d b y e n v i r o n m e n t a l signals i n c l u d i n g , m i c r o b i a l antigens a n d c y t o k i n e s [5, 7, 8 ] . T h u s the nature a n d consequence o f m a c r o p h a g e a c t i v a t i o n i s different a m o n g different m a c r o p h a g e p o p u l a t i o n s (e.g. bone m a r r o w - d e r i v e d , h u m a n b l o o d m o n o c y t e s , tissue macrophages f r o m different sites, a n d macrophages isolated f r o m i n f l a m m a t o r y lesions o r w o u n d s ) a n d depends o n w h i c h c y t o k i n e s are present [4].  1.1.1  Macrophages: Types O v e r the past years distinct m a c r o p h a g e subsets h a v e b e e n characterized. T h e s e subsets  i n c l u d e c l a s s i c a l l y activated macrophages ( C A M $ ) , alternatively activated macrophages ( A A M $ ) , a n d T y p e - 2 activated macrophages [9-11]. T h e best-studied are the C A M $ , w h i c h are i n d u c e d b y p r o - i n f l a m m a t o r y m i c r o b i a l m o l e c u l e s s u c h as l i p o p o l y s a c c h a r i d e ( L P S ) i n a T h i T c e l l ( c e l l - m e d i a t e d i m m u n e response) c y t o k i n e e n v i r o n m e n t (e.g. Interferon-y ( f F N - y ) , t u m o u r  1  necrosis f a c t o r - a ( T N F - a ) ) and release i n f l a m m a t o r y and/or m i c r o b i c i d a l products [9, 12]. T h e s e c e l l s are d i s t i n g u i s h e d b y their a b i l i t y to p r o d u c e n i t r i c o x i d e ( N O ) i n a d d i t i o n to their increased e x p r e s s i o n o f major h i s t o c o m p a t i b i l i t y c o m p l e x ( M H C ) class II a n d C D 8 6 , and their enhanced antigen-presenting c a p a c i t y [13, 14]. B y i n c r e a s i n g o x i d a t i v e burst and N O release, C A M $ p l a y an important r o l e i n p r o t e c t i o n against i n t r a c e l l u l a r pathogens [12]. T h e y also exert anti-proliferative and c y t o t o x i c activities, p a r t l y due to their a b i l i t y to secrete N O and p r o i n f l a m m a t o r y c y t o k i n e s ( T N F - a , interleukin-1 ( I L - 1 ) , I L - 6 ) [9, 12, 15].  T h e o b s e r v a t i o n that the d e v e l o p m e n t o f C A M $ is i n h i b i t e d b y T h 2 T - c e l l c y t o k i n e s , lead to c h a r a c t e r i z a t i o n o f alternatively activated m a c r o p h a g e s ( A A M $ ) , w h i c h have a different p h y s i o l o g i c a l f u n c t i o n [16]. T h e y do not p r o d u c e I L - 1 2 , but secrete I L - 1 0 a n d d r i v e preferentially a T h 2 T - c e l l - l i k e (antibody-mediated) i m m u n e response [10, 11, 17]. T h e y also express arginase a c t i v i t y , an e n z y m e that competes w i t h i n d u c i b l e n i t r i c o x i d e synthase ( i N O S ) for L - a r g i n i n e [10]. A r g i n a s e converts L - a r g i n i n e to urea and L - o r n i t h i n e thus s h u n t i n g the substrate a w a y f r o m the N O p r o d u c t i o n p a t h w a y ( F i g . 1).  has been s h o w n to i n f l u e n c e  the differentiation o f T - c e l l s , and determine whether i m m u n o - s t i m u l a t i o n versus i m m u n o suppression or protective i m m u n e responses versus i m m u n o - p a t h o l o g y responses o c c u r [12, 15, 18, 19].  C o - s t i m u l a t i o n o f L P S activated macrophages t h r o u g h the F e y receptor results i n i n h i b i t i o n o f I L - 1 2 synthesis and increased amounts o f I L - 1 0 as c o m p a r e d to L P S s t i m u l a t i o n alone [10, 2 0 ] . F u r t h e r m o r e , these c e l l s s w i t c h f r o m t r i g g e r i n g a T h l T - c e l l response d o m i n a t e d b y I F N - y secretion, to the i n d u c t i o n o f a T h 2 T - c e l l response t y p i f i e d b y I L - 4 secretion and increased a n t i b o d y responses, m a i n l y o f the I g G l isotype [10, 2 1 ] . T h e s e c e l l s are c a l l e d T y p e  2  2-activated m a c r o p h a g e s .  T h e y e x h i b i t f u n c t i o n a l s i m i l a r i t i e s to C A M $ and A A M ^ .  they p r o d u c e T N F - a , I L - 1 and I L - 6 , but u n l i k e  Like  T y p e 2-activated m a c r o p h a g e s  do not p r o d u c e I L - 1 2 , but secrete I L - 1 0 and d r i v e p r e f e r e n t i a l l y a T h 2 T - c e l l - l i k e i m m u n e response [10, 11, 17]. T h i s phenotype is s i m i l a r to  w i t h the o n l y difference b e i n g that  T y p e 2-activated c e l l s do not express arginase a c t i v i t y [10]; suggesting that m a c r o p h a g e p o p u l a t i o n s w i t h o v e r l a p p i n g phenotypes and/or functions exist. T h u s the c l a s s i f i c a t i o n o f the distinct m a c r o p h a g e p o p u l a t i o n s is s t i l l operational due to absence o f d e f i n i t i v e m o l e c u l a r markers.  1.1.2  Effectors and regulatory products of macrophages A c t i v a t i o n o f m a c r o p h a g e s b y rFN-y or bacterial c e l l products s u c h as L P S i n d u c e s a  n u m b e r o f i m m u n o l o g i c responses s u c h as p r o d u c t i o n o f p r o - i n f l a m m a t o r y mediators (e.g. I L - 1 , I L - 6 , T N F - a , c h e m o k i n e s , m y e l o p e r o x i d a s e etc). A c t i v a t e d m a c r o p h a g e s also i n d u c e m i c r o b i o c i d a l a c t i v i t y (e.g. release o f reactive o x y g e n and n i t r o g e n intermediates), l y m p h o c y t e a c t i v a t i o n (antigen p r o c e s s i n g and presentation) and tissue r e m o d e l l i n g (e.g. p r o d u c t i o n o f elastase/collegenase/hyalruronidase e n z y m e s ) [11]. T h e s e responses are important for macrophages to exert their functions i n k i l l i n g bacteria, parasites, v i r a l - i n f e c t e d c e l l s and t u m o u r c e l l s and p r o v i d i n g T - c e l l help.  (i)  Interleukin-1 (IL-1) I L - 1 is p r o d u c e d p r i m a r i l y b y activated macrophages, but c a n also be m a d e b y other  cells. I L - 1 has a n u m b e r o f p h y s i o l o g i c a l actions i n c l u d i n g i n d u c i n g p r o d u c t i o n o f tissue factor thus t r i g g e r i n g the b l o o d - c l o t t i n g cascade, d e c r e a s i n g b l o o d pressure and i n d u c i n g fever. B u t the p r e d o m i n a n t i m m u n e f u n c t i o n o f I L - 1 is to enhance the a c t i v a t i o n o f T - c e l l s i n response to antigens and thus initiate an adaptive i m m u n e response [22]. T h e r e is an increase i n p r o d u c t i o n 3  o f I L - 2 and its receptor b y T - c e l l s i n response to I L - 1 , w h i c h i n turn augments the a c t i v a t i o n o f the T - c e l l s i n an autocrine m a n n e r [22]. I L - 1 also induces e x p r e s s i o n o f I F N - y b y T - c e l l s . T h i s effect o n T - c e l l a c t i v a t i o n b y I L - 1 is m i m i c k e d b y T N F - a w h i c h is another c y t o k i n e secreted b y activated macrophages,  (ii)  Interleukin-6 (IL-6) I L - 6 is a c y t o k i n e i n the h e m a t o p o i e t i n f a m i l y [23] p r o d u c e d b y activated m a c r o p h a g e s ,  but also b y fibroblasts, e n d o t h e l i a l c e l l s and activated T - h e l p e r c e l l s [24]. Its functions range f r o m k e y roles i n acute-phase p r o t e i n i n d u c t i o n to B and T - c e l l g r o w t h and differentiation [25, 2 6 ] . U n l i k e I L - 1 , I L - 2 a n d T N F - a , I L - 6 does not i n d u c e c y t o k i n e e x p r e s s i o n , but rather augments the responses o f i m m u n e c e l l s to other c y t o k i n e s [27]. I L - 6 acts i n s y n e r g y w i t h I L - 1 and T N F - a i n m a n y i m m u n e responses, i n c l u d i n g T - c e l l a c t i v a t i o n [28]. I L - 6 induces t r a n s c r i p t i o n o f v a r i o u s proteins through the three major s i g n a l t r a n s d u c t i o n p a t h w a y s ; p r o t e i n k i n a s e C , c A M P / p r o t e i n kinase A , and the c a l c i u m release p a t h w a y [29]. I L - 6 stimulates the acute-phase reaction, w h i c h alerts the innate i m m u n e system and protects against tissue damage [30]. T h i s results i n the increases synthesis o f the t w o major acute-phase proteins, C - r e a c t i v e p r o t e i n ( C R P ) , w h i c h increases the rate o f p h a g o c y t o s i s o f bacteria, and s e r u m a m y l o i d A ( S A A ) . S i m i l a r l y , it also increases the synthesis o f f i b r i n o g e n , an important c l o t t i n g agent. T h e l o c a l acute phase r e a c t i o n leads to a systemic reaction, w h i c h i n c l u d e s : fever, i n c r e a s e d erythrocyte sedimentation rate, increased secretion o f g l u c o c o r t i c o i d s , and the a c t i v a t i o n o f the c o m p l e m e n t a n d c l o t t i n g cascades. [31] D y s r e g u l a t i o n i n I L - 6 p r o d u c t i o n has b e e n s h o w n to lead to septic s h o c k and pathogenesis o f m a n y diseases and a u t o i m m u n e disorders, s u c h as l i v e r a u t o i m m u n e disease  [32-34].  4  (iii)  Chemokines C h e m o k i n e s are a m o n g the m o s t abundant proteins p r o d u c e d b y an activated  m a c r o p h a g e . Interleukin-8 ( I L - 8 ) , m o n o c y t e c h e m o t a c t i c protein-1 ( M C P - 1 ) and m a c r o p h a g e i n f l a m m a t o r y p r o t e i n - l a ( M l P - l a ) are the major l e u k o c y t e chemoattractants p r o d u c e d d u r i n g a bacterial i n f e c t i o n b y r e c r u i t i n g neutrophils, T - c e l l s and m o r e m a c r o p h a g e s to the site [35]. I L 8 also stimulates neutrophils to degranulate. (iv)  T u m o r Necrosis Factor-a (TNF-a)  T N F - a is a p l e i o t r o p i c i n f l a m m a t o r y c y t o k i n e p r o d u c e d b y several types o f cells, but p r i m a r i l y b y activated m o n o c y t e s / m a c r o p h a g e s [36]. It is an acute phase p r o t e i n , w h i c h initiates a cascade o f c y t o k i n e s and increases v a s c u l a r p e r m e a b i l i t y , thereby r e c r u i t i n g m a c r o p h a g e and neutrophils to a site o f i n f e c t i o n [37]. B i o l o g i c a l effects o f this m o l e c u l e i n c l u d e i n d u c t i o n o f apoptosis, c y t o l y s i s o f t u m o r c e l l s , a c t i v a t i o n o f p o l y m o r p h o n u c l e a r ( P M N ) l e u k o c y t e s , a n t i v i r a l a c t i v i t y and i n d u c t i o n o f I L - 1 [38, 3 9 ] . It possesses b o t h g r o w t h s t i m u l a t i n g and g r o w t h i n h i b i t o r y properties. F o r instance, d u r i n g i n f l a m m a t i o n , T N F - a i n d u c e s n e u t r o p h i l p r o l i f e r a t i o n , but u p o n b i n d i n g to the T N F R - 5 5 receptor it causes n e u t r o p h i l apoptosis [40]. S t i m u l a t e d macrophages p r o d u c e m e m b r a n e - b o u n d 27 k d T N F - a , w h i c h c a n either b i n d d i r e c t l y to T N F R - 5 5 and T N F R - 7 5 receptors t h r o u g h c e l l - t o - c e l l contact, but the m a j o r i t y o f T N F - a p r o t e i n undergoes cleavage and b i n d s i n its s o l u b l e f o r m [41]. T N F - a secreted b y the m a c r o p h a g e causes p r o d u c t i o n o f tissue factor r e s u l t i n g i n b l o o d c l o t t i n g w h i c h serves to c o n t a i n the i n f e c t i o n [42, 4 3 ] . I n the absence o f T N F - a , m i c e infected w i t h gram-negative bacteria are susceptible to sepsis [44]. B u t h i g h levels o f T N F - a correlate w i t h increased r i s k o f m o r t a l i t y [37] and T N F - a also seems to be a central m e d i a t o r i n v a r i o u s pathologies [43]. A few s u c h e x a m p l e s i n c l u d e : septic shock, cancer, A I D S , transplantation rejection, m u l t i p l e 5  sclerosis, diabetes, r h e u m a t o i d arthritis, C r o h n ' s D i s e a s e , trauma, m a l a r i a , m e n i n g i t i s , i s c h e m i a reperfusion injury, and adult respiratory distress s y n d r o m e .  (v)  Nitric Oxide (NO) M a c r o p h a g e s use the c y t o t o x i c properties o f N O to e l i m i n a t e parasites, bacteria and  other p o t e n t i a l l y infectious particles [45]. In the vasculature, N O reacts w i t h i r o n i n the active site o f the e n z y m e g u a n y l y l c y c l a s e ( G C ) , s t i m u l a t i n g it to p r o d u c e the i n t r a c e l l u l a r m e d i a t o r c y c l i c G M P ( c G M P ) that i n turn enhances the release o f neurotransmitters r e s u l t i n g i n s m o o t h m u s c l e r e l a x a t i o n and v a s o d i l a t i o n [45]. N O t o x i c i t y is l i n k e d to its a b i l i t y to c o m b i n e w i t h s u p e r o x i d e anions (0 ~) 2  to f o r m p e r o x y n i t r i t e ( O N O O ) , an o x i d i z i n g free r a d i c a l that c a n cause  D N A fragmentation and l i p i d o x i d a t i o n [45]. N O is also a m e d i a t o r i n i n f l a m m a t o r y diseases s u c h as r h e u m a t i s m and arthritis. T h e e n z y m e , i n d u c i b l e n i t r i c o x i d e synthase ( i N O S ) , catalyzes the p r o d u c t i o n o f N O f r o m L - a r g i n i n e ( F i g . 1). W i t h i n macrophages, L - a r g i n i n e c a n be m e t a b o l i z e d b y t w o different p a t h w a y s that result i n the p r o d u c t i o n of: (i) L - c i t r u l l i n e and N O b y i N O S ; and ( i i ) u r e u m and L-ornithine b y arginase. T h e r e g u l a t i o n o f i N O S - a r g i n a s e b a l a n c e b y T h l T - c e l l i m m u n e mediators ( I F N - 7 or L P S ) and T h 2 T - c e l l c y t o k i n e s ( I L - 4 and I L - 1 0 ) i n distinct m a c r o p h a g e p o p u l a t i o n s reflects their p o l a r i z a t i o n to either c l a s s i c a l l y or alternatively activated macrophages.  Studies have s h o w n that l o w b l o o d pressure i n d u c e d b y  septic shock, as w e l l as i n f l a m m a t i o n associated w i t h the d e v e l o p m e n t o f arthritis and k i d n e y disease is reversed w i t h L - N M M A (an i N O S i n h i b i t o r ) treatment [46]. T r a n s g e n i c m i c e unable to generate N O d u r i n g the i m m u n e responses d i s p l a y a r e d u c e d i n f l a m m a t o r y response [47]. I L - 1 0 i n h i b i t s N O p r o d u c t i o n b y either i n d u c i n g u p r e g u l a t i o n o f arginase e x p r e s s i o n , thus shunting a w a y the L - a r g i n i n e substrate f r o m the i N O S p a t h w a y , or b y i n h i b i t i n g e x p r e s s i o n o f i N O S p r o t e i n itself.  6  Figure 1. Schematic representation of regulation of iNOS protein by LPS and IL-10 in macrophages. L P S activates N F K B p a t h w a y w h i c h leads to i n d u c t i o n o f i N O S protein. i N O S p r o t e i n uses L - a r g i n i n e as a substrate to synthesize n i t r i c o x i d e and c a r l i n e , o n the other h a n d the e n z y m e arginase, i n d u c e d b y I L - 1 0 , shunts the L - a r g i n i n e a w a y f r o m the i N O S p a t h w a y and degrades it into products s u c h as L - o r n i t h i n e and urea. I L - 1 0 c a n also i n h i b i t i N O S p r o t e i n d i r e c t l y , either b y i n h i b i t i n g its message or p r o t e i n expression.  vi)  Myeoldperoxidase (MPO) M P O , an i r o n - c o n t a i n i n g protein, is f o u n d i n the a z u r o p h i l i c granules o f n e u t r o p h i l i c  p o l y m o r p h o n u c l e a r l e u k o c y t e s ( P M N s ) and i n the l y s o s o m e s o f m o n o c y t e s i n h u m a n s [11]. Its major role is to a i d i n m i c r o b i a l k i l l i n g . M o n o c y t e s lose their M P O a c t i v i t y d u r i n g c o n v e r s i o n to tissue macrophages, therefore m i c r o b i c i d a l and c y t o t o x i c a c t i v i t y o f m a c r o p h a g e s is dependent m a i n l y o n reactive o x y g e n intermediates ( R O I ) , N O and other substances w h i c h are s i m i l a r to those i n neutrophils [48-51]. H o w e v e r , macrophages m a y acquire M P O f r o m their e n v i r o n m e n t  7  (e.g. f r o m ingested n e u t r o p h i l s ) to participate i n their c y t o t o x i c m e c h a n i s m s . M P O m a y have a r o l e i n atherosclerosis, carcinogenesis, and degenerative n e u r o l o g i c a l diseases.  (vii)  Prostanoids  U p o n p h a g o c y t o s i s , macrophages release a r a c h i d o n i c a c i d f r o m esterified g l y c e r o l p h o s p h o l i p i d s o f the c e l l m e m b r a n e s [52, 5 3 ] . It is i m m e d i a t e l y m e t a b o l i z e d into p r o i n f l a m m a t o r y agents c a l l e d prostanoids [ e s p e c i a l l y p r o s t a g l a n d i n E 2 ( P G E ) and p r o s t a c y c l i n ( P G I ) ] . These factors i n d u c e v a s o d i l a t a t i o n , act s y n e r g i s t i c a l l y w i t h c o m p l e m e n t c o m p o n e n t C 5 a and L T B , mediate fever and m y a l g i a i n response to I L - 1 , i n the c o m b i n a t i o n w i t h b r a d y k i n i n a n d h i s t a m i n e they contribute to erythema, edema, and p a i n i n d u c t i o n .  (viii)  Extracellular Proteases  M a c r o p h a g e s not o n l y secrete c y t o t o x i c and i n f l a m m a t o r y mediators, but they also release substances p a r t i c i p a t i n g i n tissue r e o r g a n i z a t i o n , w h i c h i n c l u d e e n z y m e s , s u c h as h y a l u r o n i d a s e , elastase, and collagenase. H y a l u r o n i d a s e reduces tissue v i s c o s i t y and a l l o w s for greater spreading o f m a t e r i a l i n tissue spaces b y d e s t r o y i n g h y a l u r o n i c a c i d , an important c o m p o n e n t o f c o n n e c t i v e tissue. S i m i l a r l y , elastase and collagenase e n z y m e s are i n v o l v e d i n degradation o f c o l l a g e n and elastin, the b a s i c c o m p o n e n t s o f c o n n e c t i v e tissue, r e s u l t i n g i n d i s o r g a n i z a t i o n o f extracellular m a t r i x , w h i c h is important for the integrity o f the c e l l s .  8  1.2  Macrophage Activation through LPS Signalling Pathway L i p o p o l y s a c c h a r i d e ( L P S ) is an integral c o m p o n e n t o f gram-negative b a c t e r i a l c e l l w a l l ,  w h i c h elicits a b r o a d s p e c t r u m o f b i o l o g i c a l activities [4]. L P S causes a c t i v a t i o n o f m o n o c y t e s / m a c r o p h a g e s , w h i c h are then able to r e c o g n i z e and k i l l f o r e i g n bacterial m i c r o o r g a n i s m s [54]. L P S also induces p r o d u c t i o n o f endogenous m e d i a t o r s s u c h as T N F - a , I L - 1 , I L - 6 , I L - 8 , N O , s u p e r o x i d e anions, and l i p i d m e d i a t o r s [55, 5 6 ] . H o w e v e r , e x c e s s i v e amounts o f p r o - i n f l a m m a t o r y c y t o k i n e s m a y result i n fatal s e p t i c / e n d o t o x i c shock. W h e n c y t o k i n e p r o d u c t i o n increases to s u c h an extent that it escapes the l o c a l i n f e c t i o n , or w h e n i n f e c t i o n enters the b l o o d s t r e a m , sepsis d e v e l o p s [56, 5 7 ] . S y s t e m a t i c e d e m a results i n l o w b l o o d v o l u m e , h y p o - p r o t e i n a n e m i a , neutropenia and then n e u t r o p h i l i a [44]. V i c t i m s o f septic s h o c k experience fever, f a l l i n g b l o o d pressure, m y o c a r d i a l suppression, d e h y d r a t i o n , acute r e n a l failure, tissue damage a n d then respiratory arrest [42]. T i s s u e damage is brought o n b y the loss o f b l o o d f l o w , w h i c h i n turn increases the p r o d u c t i o n o f N O and l e a d i n g to a further f a l l i n b l o o d pressure. F a t a l i t y due to o r g a n failure m a y o c c u r . Therefore, i n order to understand h o w to m a i n t a i n regulated m a c r o p h a g e f u n c t i o n d u r i n g i n f l a m m a t o r y responses, it is important to study the m o l e c u l a r m e c h a n i s m s that b o t h u n d e r l i e and l i m i t L P S s i g n a l l i n g .  1.2.1  LPS Signalling L P S consists o f four regions that are structurally a n d f u n c t i o n a l l y distinct: the O -  specific c h a i n , the outer core, the inner core and the l i p i d A m o i e t y [4]. T h e c o n s e r v e d l i p i d A r e g i o n is r e s p o n s i b l e for the m a j o r i t y o f b i o l o g i c a l activities o f L P S [58]. T h e three m o l e c u l e s expressed o n the surface o f macrophages that are k n o w n to b i n d to l i p i d A m o i e t y o f L P S i n c l u d e : C D 1 4 , the m a c r o p h a g e scavenger receptor ( S R ) , and the [32 ( C D 1 1 / C D 1 8 ) l e u k o c y t e  9  integrins [59]. H o w e v e r , the s i g n a l l i n g receptor for L P S consists o f t o l l - l i k e receptor-4 ( T L R 4 ) and t w o other e x t r a c e l l u l a r subunits, the g l y c o s y l p h o s p h a t i d y l i n o s i t o l - l i n k e d C D 14 and s o l u b l e M D 2 p r o t e i n , w h i c h increases the affinity o f L P S b i n d i n g [60-63] ( F i g . 2). T h e serum protein, L P S b i n d i n g p r o t e i n ( L B P ) , accelerates the h i g h affinity b i n d i n g o f L P S to C D 14 because it c a t a l y t i c a l l y transfers L P S m o n o m e r s f r o m aggregates ( i n c l u d i n g b a c t e r i a l m e m b r a n e s ) to C D 14 [64-66]. T h e i m p o r t a n c e o f C D 14 w a s s h o w n b y experiments u s i n g b l o c k i n g m o n o c l o n a l antibodies to C D 1 4 r e s u l t i n g i n i n h i b i t i o n i n the a b i l i t y o f L P S to stimulate phagocytes [59]. A s o l u b l e fragment o f C D 14 ( s C D 1 4 ) facilitates the a c t i v a t i o n o f c e l l s that do not express m e m b r a n e C D 14, s u c h as endothelial c e l l s [67-69]. M i c e deficient i n C D 14 are resistant to e n d o t o x i n s h o c k after L P S challenge [70, 71]. It is g e n e r a l l y accepted that the interaction b e t w e e n l i p i d A and C D 14 is significant i n c e l l u l a r a c t i v a t i o n o f L P S , but since C D 14 l a c k s a transmembrane and c y t o p l a s m i c d o m a i n , it must associate w i t h another transmembrane m o l e c u l e to transduce signals into the c e l l [57]. T h e transmembrane s i g n a l l i n g subunit for the L P S receptor c o m p l e x is T L R 4 and its d i s c o v e r y has greatly contributed to the understanding o f the m o l e c u l a r basis o f L P S r e c o g n i t i o n and L P S - i n d u c e d s i g n a l l i n g events [72]. T h e i n v o l v e m e n t o f T L R 4 i n L P S - i n d u c e d s i g n a l l i n g events w a s demonstrated u s i n g L P S - h y p o r e s p o n s i v e m i c e , w h i c h have either a p o i n t m u t a t i o n i n T L R 4 [73, 74] or are T L R 4 - d e f i c i e n t [62]. A t h i r d extracellular c o m p o n e n t o f the L P S receptor is M D - 2 . E x p r e s s i o n o f M D - 2 , as w e l l as T L R 4 , is r e q u i r e d for reconstituting L P S responsiveness i n the n o n - m a c r o p h a g e , B a / F 3 , c e l l [75], a n d also h u m a n e m b r y o n i c k i d n e y ( H E K ) 293 c e l l s [76]. D o w n - r e g u l a t i o n o f c e l l surface e x p r e s s i o n o f T L R 4 / M D 2 c o m p l e x i n m o u s e peritoneal m a c r o p h a g e s correlates w i t h e n d o t o x i n tolerance, a state o f L P S unresponsiveness  [77].  10  A f t e r L P S b i n d s its receptor c o m p l e x , T L R 4 associates w i t h the i n t r a c e l l u l a r adaptor proteins, M y d 8 8 and T I R A P proteins [22]. M y d 8 8 recruits the I R A K k i n a s e w h i c h associates w i t h the adaptor p r o t e i n T R A F 6 , l e a d i n g to a c t i v a t i o n o f the M A P 3 - k i n a s e s T a k l and M E K K 1 w h i c h phosphorylates and activates the I K B k i n a s e ( I K K ) c o m p l e x [78, 79]. I K K p h o s p h o r y l a t i o n o f IKB o n s e r i n e u b i q u i t i n ligase c o m p l e x , E 3 R S  23  I k B  targets I K B for r e c o g n i t i o n and u b i q u i t i n a t i o n b y the E 3 [80] and degradation b y 2 6 S proteasome. I K B regulates the  a c t i v a t i o n o f the N u c l e a r factor K B ( N F K B ) , w h i c h is a transcription factor sequestered i n the c y t o p l a s m b y I K B p r o t e i n [79]. I K B p h o s p h o r y l a t i o n b y I K K results i n release o f N F K B f r o m the N F K B / I K B c o m p l e x . T h i s p h o s p h o r y l a t i o n leads to the exposure o f the nuclear l o c a l i z a t i o n signals ( N L S ) o n the N F K B subunits and the subsequent t r a n s l o c a t i o n o f the m o l e c u l e to the nucleus [81].  11  Figure 2.  Schematic representation of LPS signalling.  A f t e r L P S b i n d s its receptor c o m p l e x , T L R 4 associates w i t h the intracellular adaptor proteins, M y d 8 8 and T I R A P proteins. M y d 8 8 recruits the I R A K kinase w h i c h associates w i t h the adaptor protein T R A F 6 , l e a d i n g to activation o f the M A P 3 - k i n a s e s T a k l and M E K K 1 / 4 , P K R , c r a f and P I 3 - k i n a s e . IKB p h o s p h o r y l a t i o n b y I K K results i n release o f N F K B from the N F K B / I K B c o m p l e x . T h i s p h o s p h o r y l a t i o n leads to the exposure o f the nuclear l o c a l i z a t i o n signals ( N L S ) o n the N F K B subunits and the subsequent translocation o f the m o l e c u l e to the nucleus, w h e r e transcription o f regulated genes is i n d u c e d .  12  1.2.2  Targets of L P S Signalling  a)  N F K B activation There are five N F K B subunit f a m i l y m e m b e r s that f o r m d i m e r s : R e l A (p65), p 5 0 , R e l B ,  c - R e l , and p 5 2 ; and they d i m e r i z e i n v a r i o u s c o m b i n a t i o n s w h i c h h a v e d i f f e r i n g D N A b i n d i n g affinity and transactivation potential [82]. T h e m o s t c o m m o n and best-characterized f o r m o f N F K B is the p 6 5 / p 5 0 heterodimer, w h i c h b i n d s to a consensus sequence ( 5 ' G G G A C T T T C C - 3 ' ) i n the p r o m o t e r o f v a r i o u s genes and activates their transcription [82]. F o r instance, N F K B induces the t r a n s c r i p t i o n o f v a r i o u s interleukins (e.g. I L - 1 ) , c y t o k i n e s (e.g. T N F - a ) and i n d u c i b l e n i t r i c o x i d e synthase ( i N O S ) [83]. N F K B p l a y s an important r o l e i n the r e g u l a t i o n o f i m m u n e responses, i n f l a m m a t i o n , c e l l - c y c l e p r o g r e s s i o n , c e l l apoptosis, oncogenesis and v a r i o u s a u t o i m m u n e diseases [84]. T h e a c t i v a t i o n o f N F K B is thought to be part o f a stress response as it is activated b y a v a r i e t y o f s t i m u l i that i n c l u d e T N F - a , I L - 1 , l y m p h o k i n e s , and U V [85]. P y r o l l i d i n e dithiocarbamate (an i n h i b i t o r o f N F K B D N A b i n d i n g a c t i v i t y ) c o m p l e t e l y i n h i b i t s c y t o k i n e p r o d u c t i o n , s h o w i n g that a c t i v a t i o n o f N F K B is essential for p r o i n f l a m m a t o r y m e d i a t o r p r o d u c t i o n [39]. b)  P K B activation L P S s t i m u l a t i o n o f p r o t e i n k i n a s e B ( P K B ) c a n also activate I K K c o m p l e x b y  p h o s p h o r y l a t i o n o f I K K at threonine 23 [86, 87]. A d d i t i o n a l l y , it has been s h o w n that P K B stimulates the transactivation a c t i v i t y o f the R e l A / p 6 5 t h r o u g h i n d u c t i o n o f I K K and p38 M A P K [14]. S u p p r e s s i o n o f I K K a c t i v i t y b y I L - 1 0 has b e e n s h o w n to lead to i n a c t i v a t i o n o f L P S and T N F - a i n d u c e d N F K B e x p r e s s i o n [88].  13  c)  ILK activation L P S also activates i n t e g r i n - l i n k e d kinase ( I L K ) , an ankyrin-repeat c o n t a i n i n g  serine/threonine p r o t e i n kinase, w h o s e a c t i v i t y is regulated b y c e l l u l a r levels o f phosphatidylinositol-3,4,5-trisphosphate ( P I P 3 ) i n a p h o s p h a t i d y l i n o s i t o l - 3 ' - k i n a s e ( P I 3 - K ) dependent m a n n e r [89]. I L K induces p h o s p h o r y l a t i o n and a c t i v a t i o n o f p r o t e i n kinase B ( P K B ) and I K B , l e a d i n g to a c t i v a t i o n o f N F K B , w h i c h then activates i N O S [90]. B e s i d e s r e g u l a t i o n o f N F K B i n response to L P S , I L K also regulates the a c t i v i t y o f transcription factors s u c h as Pc a t e n i n e - T C F / L E F - 1 ( T - c e l l / l y m p h o i d enhancer factor), A P - 1 (adaptor protein-1) [91] and C R E B ( c A M P responsive element b i n d i n g protein) [92].  1.2.3  Activation of mitogen-activated kinases (MAPKs) O t h e r important events i n L P S s i g n a l l i n g i n c l u d e a c t i v a t i o n o f the M A P kinases: J N K  [85], E R K 1 / 2 [93] and p38 M A P K [94]. T h e c o n t r i b u t i o n o f these pathways to e x p r e s s i o n o f the p r o t o t y p i c p r o i n f l a m m a t o r y c y t o k i n e T N F - a has been w e l l studied. (i)  JNK  T N F - a t r a n s c r i p t i o n is regulated b y the c - J u n - N - t e r m i n a l k i n a s e ( J N K ) . A c t i v a t i o n o f J N K is necessary for p h o s p h o r y l a t i o n o f activating transcription factor 2 ( A T F - 2 ) and c - J u n (c-Jun m i t o g e n activated kinase) w h i c h c o m p l e x e s w i t h the transcription factor C R E B to support transcription o f T N F - a [85]. F u n c t i o n a l analysis o f T N F - a promoter, s h o w e d enhancer elements i n the r e g i o n c o n t a i n i n g C R E B and N F - K B sites to be required for o p t i m a l transcription o f the T N F - a gene i n response to L P S [85]. B u t concerted p a r t i c i p a t i o n o f c - J u n c o m p l e x e s and p50/p65 are required for the m a x i m a l L P S i n d u c t i o n o f the T N F - a p r o m o t e r [85].  14  )  ERK1/2 L P S activates E R K t h r o u g h the serine/threonine k i n a s e , T p l 2 , and the E R K p a t h w a y is r e q u i r e d for c y t o p l a s m i c transport o f T N F - a m R N A [93]. T h e 3'-untranslated r e g i o n ( U T R ) o f the T N F - a transcript contains a w e l l - c h a r a c t e r i z e d type II A U - r i c h element ( A R E ) i n v o l v e d i n b o t h translational c o n t r o l and m R N A s t a b i l i t y [94, 9 5 ] . L P S - i n d u c e d T p l 2 / E R K - t r a n s d u c e d signals target the A U - r i c h element i n the 3 ' U T R o f the T N F - a R N A and controls its c y t o p l a s m i c m R N A transport [93]. T p ^ ' m i c e also demonstrate resistance to L P S / D - G a l a c t o s a m i n e - i n d u c e d s h o c k due to post-transcriptional defect i n the i n d u c t i o n o f T N F - a b y L P S [93]. (iii)  p38 M A P K  p 3 8 M A P K regulates translation o f T N F - a p r o t e i n [94]. L P S a c t i v a t i o n o f p 3 8 M A P K is essential for r e m o v i n g the T N F - a m R N A translational b l o c k and a l l o w i n g association o f T N F a m R N A w i t h p o l y r i b o s o m e s , the site o f active p r o t e i n translation [94]. T h i s r e g u l a t i o n is dependent o n the presence o f the A R E i n the 3 ' U T R .  15  1.3  Biological Activities of I L - 1 0 A s d i s c u s s e d p r e v i o u s l y , the persistence o f i n f l a m m a t o r y processes often results i n tissue  damage, or e v e n fatality; r e s u l t i n g i n disorders s u c h as: r h e u m a t o i d arthritis, C r o h n ' s disease, and septic s h o c k [96]. T h u s the i m m u n e system is faced w i t h a permanent c h a l l e n g e : h o w to c o n t r o l i n f e c t i o n w h i l e l i m i t i n g tissue damage. H e n c e , a n t i - i n f l a m m a t o r y m e c h a n i s m s are m a n d a t o r y for host s u r v i v a l . A m o n g the factors that l i m i t i n f l a m m a t o r y responses, the antii n f l a m m a t o r y c y t o k i n e , I L - 1 0 , is one o f the m o s t important. I L - 1 0 is a k e y regulator o f b o t h innate and a c q u i r e d i m m u n i t y . P r o d u c e d b y activated B - c e l l s , keratinocytes, m o n o c y t e s and macrophages, I L - 1 0 w a s i n i t i a l l y detected as a T h 2 T - c e l l product that i n h i b i t e d the p r o l i f e r a t i o n , d e v e l o p m e n t and f u n c t i o n o f T h l T - c e l l s . T h e m o l e c u l a r c l o n i n g o f I L - 1 0 and subsequent studies u t i l i z i n g r e c o m b i n a n t c y t o k i n e revealed that a l t h o u g h I L - 1 0 exerted direct effects o n T - c e l l s [97, 9 8 ] , its major site o f action w a s the activated m a c r o p h a g e [99]. A c t i v a t i o n o f macrophages b y I F N - y or L P S induces a n u m b e r o f i m m u n e responses, m a n y o f w h i c h are i n h i b i t e d b y I L - 1 0 [100]. I L - 1 0 suppresses p r o d u c t i o n o f p r o i n f l a m m a t o r y mediators: c y t o k i n e s , i n c l u d i n g T N F - a [101], I L - 1 [102] I L - 6 ; and c h e m o k i n e s s u c h as M I P l a [103], M C P - l a n d I L - 8 [104]. It also i n h i b i t s p h a g o c y t o s i s [3] b y i n h i b i t i n g reactive o x y g e n and n i t r o g e n intermediate p r o d u c t i o n i n m o n o c y t e s / m a c r o p h a g e s and neutrophils [101]. I n a d d i t i o n to l i m i t i n g and t e r m i n a t i n g i n f l a m m a t o r y responses, I L - 1 0 also regulates g r o w t h and/or differentiation o f B c e l l s , N K c e l l s , c y t o t o x i c and helper T - c e l l s , mast c e l l s , granulocytes, d e n d r i t i c c e l l s , keratinocytes, and e n d o t h e l i a l c e l l s [100]. B y i n h i b i t i n g e x p r e s s i o n o f c o s t i m u l a t o r y m o l e c u l e s s u c h as C D 8 0 / 8 6 , M H C II and I C A M - 1 , I L - 1 0 m a k e s macrophages a p o o r A P C [105-108]. I L - 1 0 also i n h i b i t s the p r o d u c t i o n o f I L - 1 2 , w h i c h together w i t h the i n h i b i t i o n o f other c o - s t i m u l a t o r y m o l e c u l e s suppresses p r i m a r y a l l o a n t i g e n - s p e c i f i c T - c e l l  16  responses [20, 109]. I L - 1 0 induces a l o n g l a s t i n g state o f non-responsiveness (anergy) i n T c e l l s , w h i c h cannot be reversed b y I L - 2 or b y s t i m u l a t i o n w i t h a n t i - C D 3 and a n t i - C D 2 8 [110]. T h e s e i n h i b i t o r y effects o f I L - 1 0 are not due to n o n - s p e c i f i c , g l o b a l s u p p r e s s i o n o f c e l l u l a r m e t a b o l i s m , since p r o d u c t i o n o f the a n t i - i n f l a m m a t o r y m e d i a t o r s s u c h as I L - 1 R antagonist and s o l u b l e T N F - a receptor are enhanced b y I L - 1 0 . V a r i o u s a n i m a l m o d e l studies have substantiated the  in vivo i m p o r t a n c e o f an anti-  i n f l a m m a t o r y r o l e for I L - 1 0 . Targeted d i s r u p t i o n o f the I L - 1 0 gene results i n m i c e c h a r a c t e r i z e d b y i n f l a m m a t o r y b o w e l disease [111] and exaggerated i m m u n e reactions w h e n c h a l l e n g e d w i t h antigen or L P S [112]. A d m i n i s t r a t i o n o f I L - 1 0 , o n the other h a n d , ameliorates disease i n s u c h m o d e l s o f e n d o t o x e m i a [113], transplantation [114] and a u t o i m m u n i t y [115]. I n h u m a n s , the presence o f elevated endogenous I L - 1 0 is a p o s i t i v e p r o g n o s t i c v a r i a b l e i n a u t o i m m u n e disease [116] and a l l o g e n e i c transplant patients [117]. I L - 1 0 has r e c e n t l y entered c l i n i c a l trials for the treatment o f h u m a n i n f l a m m a t o r y b o w e l disease [118].  1.3.1  IL-10 Signalling Pathways in Macrophages A s s h o w n i n F i g u r e 3, the I L - 1 OR consists o f at least t w o subunits [119-122]. B o t h  b e l o n g to the type II c y t o k i n e receptor s u p e r - f a m i l y , w h i c h i n c l u d e a l l the I F N receptor proteins [123] and b o t h are necessary for I L - 1 0 s i g n a l transduction. T h e p r i m a r y l i g a n d - b i n d i n g c o m p o n e n t designated I L - l O R a , b i n d s I L - 1 0 w i t h h i g h affinity and i n the presence o f I L - 1 0 associates w i t h an a c c e s s o r y subunit c a l l e d I L - l O R p ( C R F 2 - 4 ) . L i k e other m e m b e r s o f the class II c y t o k i n e receptor f a m i l y , b o t h I L - l O R a and  IL-10RP possess  a juxtamembrane box 1 motif  important for i n t e r a c t i o n w i t h m e m b e r s o f the Janus f a m i l y kinases ( J a k l / T y k 2 ) [123]. L i g a n d i n d u c e d h e t e r o - d i m e r i z a t i o n o f the receptor chains results i n a c t i v a t i o n o f I L - l O R a - b o u n d J a k l  17  and I L - l O R p - b o u n d T y k 2 . A c t i v a t e d Jak kinases p h o s p h o r y l a t e I L - 1 0 R a o n t w o c y t o p l a s m i c t y r o s i n e residues ( Y 4 2 7 / 4 7 7 o f m I L - l O R l ; Y 4 4 6 / 4 9 6 o f h I L - l O R l ) [123] and create d o c k i n g sites for latent c y t o p l a s m i c transcription factors o f the Stat f a m i l y [124].  I n the case o f the I L -  1 0 R , Stat3 is recruited to these p h o s p h o t y r o s y l residues [125] and b e c o m e s p h o s p h o r y l a t e d b y receptor-bound Jak kinases. S t a t l also b e c o m e s tyrosine p h o s p h o r y l a t e d , but not to the same extent as Stat3 and the m e c h a n i s m o f receptor recruitment is not clear. U p o n p h o s p h o r y l a t i o n , Stat3 and S t a t l hetero- and/or h o m o d i m e r i z e , translocate into the n u c l e u s , b i n d specific sequences i n the promoters o f target genes and stimulate t r a n s c r i p t i o n [125, 126]. Studies u s i n g mutant I L - 1 0 R 1 l a c k i n g the t y r o s i n e necessary for Stat3 a c t i v a t i o n , d o m i n a n t - i n t e r f e r i n g Stat3 and a c o u m e r m y c i n d i m e r i z a b l e Stat3, h a v e s h o w n that the Stat3 p a t h w a y is essential for the anti-proliferative a c t i o n o f I L - 1 0 o n m a c r o p h a g e c e l l g r o w t h [127]. T h e absolute requirement o f Stat3 i n m e d i a t i n g the a n t i - i n f l a m m a t o r y effects o f I L - 1 0 has b e e n demonstrated [128]. Stat3 p a t h w a y is also essential for the a b i l i t y o f I L - 1 0 to i n h i b i t L P S i n d u c e d p r o d u c t i o n o f the p r o i n f l a m m a t o r y c y t o k i n e T N F - a [129].  1.3.2  Genes regulated by I L - 1 0 t h r o u g h Stat3 p a t h w a y I L - 1 0 has been s h o w n to i n d u c e e x p r e s s i o n o f certain genes i n a Stat3-dependent  manner.  T h e s e genes i n c l u d e the c e l l c y c l e i n h i b i t o r s , p l 9  I N 1 C 4 d  [130], heme-oxygenase-1 ( H O -  1) [131], nuclear p r o t e i n B c l 3 [ 1 3 2 ] , w h i c h is a m e m b e r o f IKB p r o t e i n f a m i l y h a r b o r i n g a n k y r i n repeat d o m a i n s and suppressor o f c y t o k i n e s i g n a l l i n g 3 ( S O C S 3 ) , w h i c h w i l l be d i s c u s s e d i n m o r e detail o n page 3 1 .  18  IL-10  Figure 3. Schematic representation of IL-10 signaling. I L - 1 0 receptor contains t w o subunits, a andp\ w h i c h interact w i t h m e m b e r s o f the Janus f a m i l y kinases ( J a k l / T y k 2 ) . L i g a n d - i n d u c e d h e t e r o d i m e r i z a t i o n o f the receptor chains results i n a c t i v a t i o n o f J a k l and T y k 2 , w h i c h then p h o s p h o r y l a t e I L - l O R a o n t w o c y t o p l a s m i c tyrosine residues ( Y 4 2 7 / 4 7 7 o f m I L - l O R a ; Y 4 4 6 / 4 9 6 o f h I L - l O R a ) and create d o c k i n g sites for Stat3 t r a n s c r i p t i o n factor, w h i c h is recruited to these p h o s p h o t y r o s y l residues a n d b e c o m e s p h o s p h o r y l a t e d b y receptor-bound J a k kinases. U p o n p h o s p h o r y l a t i o n , Stat3 h o m o d i m e r i z e s , translocate into the n u c l e u s , b i n d specific sequences i n the p r o m o t e r s o f target genes and stimulate t r a n s c r i p t i o n  19  1.3.3  Targets of IL-10 in Inhibition of LPS Signalling  a)  Inhibition of N F K B activation A l t h o u g h several studies have reported i n h i b i t o r y a c t i o n o f I L - 1 0 o n either the N F K B  [22, 7 8 - 8 0 , 107, 133-135] or M A P kinase pathways [39], the m e c h a n i s m s g o v e r n i n g this process is s t i l l unclear. I L - 1 0 pretreatment i n h i b i t s L P S and T N F - a i n d u c e d N F K B D N A - b i n d i n g b y s p e c i f i c a l l y d i s r u p t i n g the p 6 5 / p 5 0 heterodimer c o m p l e x i n h u m a n P B M C [133] and m u r i n e m a c r o p h a g e s [135]. T N F - a gene t r a n s c r i p t i o n is suppressed b y I L - 1 0 [136, 137], p o s s i b l y b y interfering w i t h a c t i v a t i o n o f N F K B [88, 133, 138]. I n a l v e o l a r macrophages, I L - 1 0 stabilizes IKB p r o t e i n b y d e l a y i n g its L P S - m e d i a t e d degradation and r e s u l t i n g i n d e l a y e d n u c l e a r translocation o f the p65 subunit [139]. T N F - a i n d u c e d I L - 8 p r o d u c t i o n is regulated t h r o u g h an N F i c B - d e p e n d e n t m e c h a n i s m [140], w h i c h is also i n h i b i t e d b y I L - 1 0 t h r o u g h i n h i b i t i o n o f N F K B t r a n s c r i p t i o n a l a c t i v i t y [104]. Therefore, N F i c B - d e p e n d e n t t r a n s c r i p t i o n a l r e g u l a t i o n is a target for the a n t i - i n f l a m m a t o r y actions o f I L - 1 0 [135]. H o w e v e r , i n contrast, other studies have demonstrated that I L - 1 0 is unable to suppress L P S - i n d u c e d a c t i v a t i o n o f N F K B D N A b i n d i n g i n h u m a n m o n o c y t e s [141]. These c o n f l i c t i n g results m a y be due to different c e l l types used i n the studies ( h u m a n P B M C a n d m u r i n e macrophages versus h u m a n m o n o c y t e s ) .  b)  Inhibiting production of other pro-inflammatory mediators I L - 1 0 interferes w i t h L P S - i n d u c e d p r o d u c t i o n o f p r o - i n f l a m m a t o r y mediators at the  l e v e l o f t r a n s c r i p t i o n or m R N A stability, p r o t e i n translation or p r o t e i n s t a b i l i t y [137-139, 142, 143].  Incubation o f a l v e o l a r macrophages w i t h I L - 1 0 results i n the steady state m R N A l e v e l s o f  T N F - a , I L - 1 a and I L - 1 [3 and i n h i b i t i o n o f their gene e x p r e s s i o n [139]. In m u r i n e macrophages, I L - 1 0 acts p r i m a r i l y t h r o u g h post-transcriptional m e c h a n i s m s b y d e s t a b i l i z i n g T N F - a m R N A  20  [142, 144] or b y i n h i b i t i n g gene translation v i a suppressing the a c t i v a t i o n o f p 3 8 M A P K [94]. O f p a r t i c u l a r note, I L - 1 0 w a s recently s h o w n to suppress T N F - a translation b y i n h i b i t i n g the a s s o c i a t i o n o f T N F - a m R N A w i t h p o l y r i b o s o m e s i n a m e c h a n i s m also dependent o n the presence o f an intact A R E i n the 3 ' - U T R [94].  Studies h a v e also s h o w n that some o f the i n t r a c e l l u l a r m e c h a n i s m s b y w h i c h I L - 1 0 i n h i b i t s p r o i n f l a m m a t o r y c y t o k i n e p r o d u c t i o n b y L P S - a c t i v a t e d macrophages are dependent o n de n o v o p r o t e i n synthesis [3, 104, 142]. F o r instance, treatment w i t h c y c l o h e x i m i d e , w h i c h b l o c k s p r o t e i n synthesis, a n t a g o n i z e d IL-10-dependent i n h i b i t i o n o f T N F - a , I L - l p and I L - 6 m R N A e x p r e s s i o n and c y t o k i n e release, suggesting an i n v o l v e m e n t o f n e w l y s y n t h e s i z e d proteins i n I L 10 a c t i o n [88, 133, 142].  c)  Inhibition of receptor expression  S i g n a l l i n g p a t h w a y s are also i n h i b i t e d b y suppression o f receptor e x p r e s s i o n , thus the p o s s i b i l i t y that I L - 1 0 i n h i b i t s L P S s i g n a l l i n g b y i n h i b i t i n g e x p r e s s i o n o f T L R 4 has been e x a m i n e d i n m o n o c y t e s and p o l y m o r p h o n u c l e a r l e u k o c y t e s , w h e r e I L - 1 0 has been s h o w n to i n h i b i t u p r e g u l a t i o n o f T L R 4 m R N A i n response to L P S [145].  21  1.4  Suppressor of Cytokine Signalling 3 ( S O C S 3 ) In our search for I L - 1 0 i n d u c e d , Stat3-regulated genes, w e f o u n d a candidate b e l o n g i n g  to the S O C S (Suppressors o f C y t o k i n e S i g n a l l i n g ) f a m i l y o f negative regulators o f c y t o k i n e s i g n a l i n g ( F i g . 4). T h e S O C S proteins are a f a m i l y o f S t a t - i n d u c i b l e genes v a r i o u s l y referred to as C I S ( c y t o k i n e - i n d u c i b l e S H 2 protein) [146-148], S O C S [149] or S S I (Stat-induced Stati n h i b i t o r ) [150, 151]. T h e first m e m b e r o f S O C S , C I S 1 , w a s c l o n e d as a n early gene i n d u c e d b y v a r i o u s c y t o k i n e s , a c t i n g v i a the c o n s e r v e d i n t r a c e l l u l a r r e g i o n o f their respective receptors. T h e e n c o d e d p r o t e i n h a d a d o m a i n that differed i n a m i n o a c i d sequence f r o m a l l the k n o w n S H 2 d o m a i n s . C I S p l a y s an important r o l e i n i n h i b i t i o n o f e r y t h r o p o i e t i n a n d I L - 3 receptor s i g n a l l i n g [152]. T h e s e c o n d m e m b e r o f the f a m i l y w a s c l o n e d b y three independent research groups and the p r o t e i n w a s g i v e n three different names: 1) E n d o et a l , i s o l a t e d a single p o s i t i v e c l o n e f r o m a c D N A l i b r a r y , e x p r e s s i n g a J a k - b i n d i n g protein, J A B , capable o f interacting w i t h the Jak k i n a s e d o m a i n [147]; 2) N a k a et a l , d i s c o v e r e d Stat3-induced Stat3 i n h i b i t o r ( S S I ) b y u s i n g m o n o c l o n a l antibodies against a sequence m o t i f f o u n d i n the S H 2 d o m a i n o f Stat3 [151]; and 3) Starr et a l , d i s c o v e r e d a p r o t e i n capable o f suppressing c y t o k i n e s i g n a l transduction, S O C S 1 [40]. S O C S 1 and S O C S 3 have b e e n s h o w n to be essential for p r o p e r r e g u l a t i o n o f interferon-y and I L - 6 responses, r e s p e c t i v e l y [152 1999].  22  SOCS member; KIR SH2.  SOCS BOX  CIS/CIS 1  c c c  SOCS1/IAB/SSI1 SOCS2/CIS2/SSI2  S0GS3/CIS3 /S $13 SOCS4  SOCS5/CIS6 2Z:  SOCS6/CIS4 SOC37/CIS7  Figure 4. The alternate names and domain structures of various members of the SOCS family.  The kinase inhibitory region (KIR) of SOCS 1 and SOCS3 is dashed. This figure is adaptedfromLarsen, L. et al, 2002 [2].  23  1.4.1  Conserved Domains of S O C S Proteins  a)  K I R and S H 2 domains S O C S proteins are a f a m i l y o f seven proteins that possess a C - t e r m i n a l , 70 a m i n o a c i d  r e g i o n o f h o m o l o g y referred to as the S O C S b o x as w e l l as an S H 2 d o m a i n [153] as s h o w n i n F i g u r e 4. H o w e v e r , S O C S 1 and S O C S 3 are different f r o m the rest o f their f a m i l y m e m b e r s since they not o n l y h a v e no introns, but also possess a 12 a m i n o a c i d extended S H 2 d o m a i n that constitute a k i n a s e i n h i b i t o r y r e g i o n ( K I R ) [153]. S O C S proteins have also b e e n f o u n d to i n h i b i t the catalytic a c t i v i t y o f Jak and n o n - J a k f a m i l y kinases [154] and m a y act o n other types o f s i g n a l l i n g m o l e c u l e s [155] through their K I R d o m a i n s . S O C S 1 and S O C S 3 co-precipitate w i t h Jaks u p o n c y t o k i n e s t i m u l a t i o n , and are able to i n h i b i t the k i n a s e a c t i v i t y o f these, a l t h o u g h w i t h d i f f e r i n g affinity and k i n e t i c s [147, 152]. F i g u r e 5 s h o w s a schematic representation o f the structure and m o d e l o f k i n a s e i n h i b i t i o n b y S O C S 1 and S O C S 3 . S O C S 3 has w e a k e r affinity for b i n d i n g to the Y 1 0 0 7 o f J a k 2 , r e s i d i n g inside an a c t i v a t i o n l o o p o f the k i n a s e d o m a i n , J H 1 [152]. It is suggested that the p r i m a r y b i n d i n g for S O C S 3 m a y not be Jak, but other m o l e c u l e s w i t h i n the s i g n a l l i n g cascade, s u c h as the p h o s p h o r y l a t e d c y t o k i n e receptor or Stat proteins [148]. S O C S 3 has b e e n s h o w n to interact t h r o u g h its S H 2 d o m a i n w i t h p h o s p h o - t y r o s i n e sites o n the receptors for the c y t o k i n e that i n d u c e d it. These i n c l u d e the receptors for I L - 6 ( Y 7 5 7 / Y 7 5 9 ) [156], i n s u l i n [157], erythropoietin ( Y 4 0 1 ) [158] and l e p t i n ( Y 9 8 5 ) [159].  24  A kinase inhibitory region (KIR)  extended SH2 •ubdomain (ESS)  CH domain (SOCS box)  JAB/SOCS-1  F i g u r e 5. S t r u c t u r e a n d m o d e l o f k i n a s e i n h i b i t i o n b y J A B / S O C S 1 a n d C I S 3 / S O C S 3 . ( A ) S c h e m a t i c m o d e l o f the functions o f J A B a n d C I S 3 d o m a i n s . T h e b o l d h i g h l i g h t e d circles represent the essential a m i n o acids i n the kinase i n h i b i t o r y r e g i o n ( K I R ) and the extended S H 2 s u b d o m a i n . ( B ) T h e m o d e l o f J H 1 a c t i v a t i o n and i n h i b i t i o n b y J A B / S O C S 1. B i n d i n g o f J A B to the a c t i v a t i o n l o o p prevents the access o f substrates and/or A T P to the catalytic p o c k e t T h i s figure is adapted f r o m Y a s u k a w a , H . et a l , 2 0 0 0 [1].  25  b)  SOCS-box domain T h e c o n s e r v a t i o n o f the S O C S - b o x d o m a i n i n a l l S O C S proteins indicates that it p l a y s  a n important role i n the p h y s i o l o g i c a l a c t i o n o f these proteins. A l l S O C S proteins have been f o u n d to associate t h r o u g h their S O C S - b o x w i t h E l o n g i n s B and C [153]. T h e E l o n g i n B C c o m p l e x w a s i n i t i a l l y i d e n t i f i e d as components o f the m a m m a l i a n t r a n s c r i p t i o n factor SIII, w h i c h w h e n interacting w i t h a t h i r d p r o t e i n ( E l o n g i n A ) , c a n increase the o v e r a l l e l o n g a t i o n rate o f R N A p o l y m e r a s e II i n v i t r o [160]. E l o n g i n B is u b i q u i t i n - l i k e p r o t e i n and E l o n g i n C is s i m i l a r i n sequence to S k p l protein, w h i c h t h r o u g h p r o t e i n - p r o t e i n i n t e r a c t i o n regulates c e l l u l a r processes, s u c h as c e l l c y c l e , transcription and d e v e l o p m e n t [161]. E l o n g i n A stabilizes the E l o n g i n B C c o m p l e x and the m o t i f b y w h i c h E l o n g i n A r e c o g n i z e s E l o n g i n C has a m i n o a c i d s i m i l a r i t y to the S O C S - b o x . T h i s E l o n g i n s B C c o m p l e x recruits C u l l i n - 2 ( C u l - 2 ) , R b x l , and the E 2 u b i q u i t i n - c o n j u g a t i n g e n z y m e and this forms the E 3 u b i q u i t i n - l i g a s e [162]. T h e p r o x i m i t y o f this u b i q u i t i n ligase to S O C S - r e c r u i t e d s i g n a l l i n g m o l e c u l e s c a n result i n the latter's u b i q u i t i n a t i o n and degradation [163, 164]. F i g u r e 6 s h o w s a schematic representation o f several m e c h a n i s m s used b y S O C S - b o x to target proteins for degradation. T h e role o f E l o n g i n s B C i n S O C S f u n c t i o n h o w e v e r is not e n t i r e l y clear. V a r i o u s reports suggest that a s s o c i a t i o n o f S O C S p r o t e i n w i t h E l o n g i n s B C serves to protect [162, 165] rather than accelerate proteasome-mediated degradation [160]. Interaction o f S O C S - b o x w i t h E l o n g i n B C c o m p l e x has been s h o w n to m a r k e d l y increase the s t a b i l i t y o f the S O C S 1 p r o t e i n and protect it f r o m p r o t e a s o m a l degradation [162, 166]. A study b y H a n a d a et a l , has s h o w n that relative to S O C S 3 w i l d type controls, T - c e l l s e x p r e s s i n g S O C S 3 p r o t e i n w i t h a mutant S O C S b o x d o m a i n , express s i g n i f i c a n t l y l o w e r levels o f S O C S 3 p r o t e i n due to d e s t a b i l i z a t i o n o f the  26  protein, w h i c h is reversed w i t h o v e r - e x p r e s s i o n o f E l o n g i n B C [165]. T h i s d i c h o t o m y reflects v a r i a b l e abilities o f different S O C S / E l o n g i n s c o m p l e x e s to recruit an u b i q u i t i n - l i g a s e a c t i v i t y .  Figure 6. The SOCS-box targets proteins for proteasomal degradation by several mechanisms. ( A ) A f t e r S O C S b i n d i n g to J A K , e l o n g i n B and C are recruited to the S O C S - b o x , and the u b i q u i t i n - h o m o l o g y d o m a i n o n S O C S - b o x is r e c o g n i n z e d b y the proteasome. ( B ) E l o n g i n s B a n d C b i n d to the S O C S - b o x and recruit an E 3 ligase, transferring u b i q u i t i n to the S O C S p r o t e i n a n d targeting the c o m p l e x for proteasomal degradation. U B L , u b u q u i t i n l i k e protein. T h i s figure is adapted f r o m L a r s e n , L . et a l , 2 0 0 2 [2].  27  1.4.2  Tyrosine Phosphorylation of SOCS3 Protein Jaks and receptor tyrosine kinases c a n p h o s p h o r y l a t e S O C S 3 at t w o t y r o s i n e residues,  2 0 4 and 2 2 1 , w i t h i n the c o n s e r v e d S O C S - b o x d o m a i n s w h i c h then a l l o w s it to b i n d to the S H 2 d o m a i n o f the R a s i n h i b i t o r p i 20 R a s G A P l e a d i n g to i n h i b i t i o n o f S T A T 5 a c t i v a t i o n but m a i n t a i n i n g E R K a c t i v a t i o n [167, 168]. S i n c e S O C S proteins i n h i b i t g r o w t h factor responses; tyrosine p h o s p h o r y l a t i o n o f S O C S 3 c a n ensure c e l l s u r v i v a l and c e l l c y c l e p r o g r e s s i o n (proliferation) t h r o u g h R a s pathway. R e c e n t l y , the same group have s h o w n that tyrosine p h o s p h o r y l a t i o n o f S O C S 3 at Y 2 0 4 / Y 2 2 1 a l l o w s it to interact w i t h adaptor proteins N c k - 1 and C r k - L , and thus m o d u l a t e their d o w n s t r e a m s i g n a l i n g [174]. A n o t h e r study has s h o w n that S O C S 3 tyrosine p h o s p h o r y l a t i o n regulates p r o t e i n s t a b i l i t y and E l o n g i n B C interaction [169]. T y r o s i n e p h o s p h o r y l a t i o n decreased S O C S 3 p r o t e i n h a l f - l i f e b y d i s r u p t i n g the interaction b e t w e e n S O C S 3 and E l o n g i n B C , w h i l e S O C S 3 / S O C S 1 c h i m e r a (3/1/3), w h i c h b o u n d m u c h m o r e s t r o n g l y to E l o n g i n B C , w a s s i g n i f i c a n t l y m o r e stable than w i l d - t y p e S O C S 3 [169]. T h u s a p h o s p h o - S O C S 3 p r o t e i n m a y serve different functions d e p e n d i n g o n the s t i m u l i , t i m e and l o c a t i o n i n the c e l l .  1.4.3  Role of SOCS3 in Inflammatory Diseases In case o f i n f l a m m a t o r y diseases s u c h as r h e u m a t o i d arthritis, ulcerative c o l i t i s and  C r o h n ' s disease, S O C S 3 is h i g h l y expressed i n affected tissues [165]. In patients w i t h C r o h n ' s disease, there is constitutive a c t i v a t i o n o f Stat3 i n T - c e l l s f r o m c o l o n m u c o s a due to i n a b i l i t y o f S O C S 3 to i n h i b i t I L - 6 i n d u c e d p h o s p h o r y l a t i o n o f Stat3 [170]. T h e m e c h a n i s m u n d e r l y i n g the i n a b i l i t y o f S O C S 3 to i n h i b i t I L - 6 - i n d u c e d Stat3 a c t i v a t i o n i n these patients is not k n o w n . T r a n s g e n i c m i c e e x p r e s s i n g a d o m i n a n t interfering S O C S 3 p r o t e i n e x h i b i t a m o r e potent Stat3  28  a c t i v a t i o n a n d a m o r e severe c o l i t i s than c o m p a r e d to the w i l d - t y p e littermates [165]. T h e s e results suggest that S O C S 3 acts as a negative feedback regulator i n i n f l a m m a t o r y diseases, a n d therefore S O C S 3 m a y be u t i l i z e d b y I L - 1 0 to exert its a n t i - i n f l a m m a t o r y actions.  29  The focus of this thesis Hypothesis:  S O C S 3 mediates I L - 1 0 d e a c t i v a t i o n o f macrophages b y targeting  s i g n a l l i n g proteins activated b y L P S .  T h e o v e r a l l objective o f this p r o p o s a l is to define at a m o l e c u l a r l e v e l the m e c h a n i s m b y w h i c h I L - 1 0 - i n d u c e d s i g n a l l i n g events interfere w i t h those o f m a c r o p h a g e activators a n d characterize the r o l e they p l a y i n m e d i a t i n g I L - 1 0 ' s a n t i - i n f l a m m a t o r y action. T h e i m p o r t a n c e o f the Stat3 p a t h w a y i n the a n t i - i n f l a m m a t o r y a c t i o n o f I L - 1 0 has b e e n established a n d some o f the Stat3-regulated gene i n d u c t i o n events i n v o l v e d i n m a c r o p h a g e d e a c t i v a t i o n b y I L - 1 0 has b e e n i d e n t i f i e d [132]. P r e l i m i n a r y experiments e x a m i n i n g the I L - 1 0 responsiveness o f v a r i o u s candidate genes s h o w e d that S O C S 3 , a m e m b e r o f a f a m i l y o f negative regulators o f c y t o k i n e s i g n a l l i n g , i s regulated i n a Stat3-dependent m a n n e r a n d m a y b e important i n m e d i a t i n g I L - 1 0 ' s a n t i - i n f l a m m a t o r y action.  In m o s t other c y t o k i n e receptor systems, the Stat p a t h w a y interacts w i t h a n d i s m o d u l a t e d b y the a c t i o n o f other pathways. P r e l i m i n a r y observations suggest that S O C S 3 m a y be i n v o l v e d i n the m e c h a n i s m b y w h i c h I L - 1 0 antagonizes L P S a c t i o n [3]. I n this study, w e set out to e x a m i n e the m o d u l a t i o n o f L P S - i n d u c e d c y t o k i n e p r o d u c t i o n b y d e t e r m i n i n g the stage at w h i c h c y t o k i n e p r o d u c t i o n (i.e. transcription, translation) i s affected b y S O C S 3 expression. M i c e disrupted for S O C S 3 gene are e m b r y o n i c a l l y lethal due to i m p a i r e d p l a c e n t a l d e v e l o p m e n t [22]. A n a l y s i s o f these m i c e s h o w e d that d u r i n g trophoblast giant c e l l differentiation, S O C S 3 n o r m a l l y functions to m o d u l a t e l e u k e m i a i n h i b i t o r y factor ( L I P ) s i g n a l l i n g . S i m i l a r l y , tetraploid rescued S O C S 3 ~ m i c e d i e w i t h i n 3 w e e k s o f b i r t h because o f cardiac m o n o c y t e h y p e r t r o p h y _ /  caused b y a loss o f S O C S 3 - m e d i a t e d i n h i b i t i o n o f L I F a n d C T - 1 s i g n a l l i n g [169]. T h u s , i n  30  order to study the effects o f loss o f S O C S 3 p r o t e i n o n I L - 1 0 s i g n a l l i n g , w e d e r i v e d m a c r o p h a g e c e l l lines f r o m S O C S 3 " fetal l i v e r c e l l s [152]. W e have also investigated w h i c h d o m a i n o f _ /  S O C S 3 p r o t e i n is important for i n h i b i t i o n o f L P S s i g n a l l i n g p a t h w a y i n response to I L - 1 0 b y reconstituting S O C S 3 " m a c r o p h a g e s w i t h W T and mutant S O C S 3 . Therefore, d e t e r m i n i n g the 7  p o i n t at w h i c h S O C S 3 interferes w i t h L P S s i g n a l l i n g p a t h w a y s and i d e n t i f y i n g the s p e c i f i c S O C S 3 r e g i o n responsible for this action w i l l g i v e insights into the m e c h a n i s m b y w h i c h S O C S 3 mediates i n h i b i t i o n o f m a c r o p h a g e a c t i v a t i o n and define n e w targets for S O C S 3 action.  31  CHAPTER 2: Material/Methods 2.1  Reagents  A l l reagents w e r e obtained f r o m S i g m a ( O a k v i l l e , C a n a d a ) unless o t h e r w i s e i n d i c a t e d . T N F - a E L I S A k i t s w e r e obtained from B D P h a r m i n o g e n ( M i s s i s s a u g a , C a n a d a ) . A n t i b o d i e s to S O C S 3 p r o t e i n a n d p h o s p h o - S O C S 3 p r o t e i n (phospho T y r 2 0 4 / 2 2 1 ) w e r e k i n d l y s u p p l i e d b y D r . N i c h o l a s C a c a l a n o ( U C L A ) . E r k a n t i b o d y w a s from C e l l S i g n a l l i n g ( M i s s a s s a u g a , C a n a d a ) a n d i N O S a n t i b o d y w a s from Santa C r u z (Santa C r u z , U S A ) . I L - 1 0 w a s k i n d l y s u p p l i e d b y D r . K e v i n M o o r e ( D N A X R e s e a r c h Institute).  2.2  Cell Culture  J774.1 m u r i n e m a c r o p h a g e c e l l l i n e ( A m e r i c a n T y p e T i s s u e C u l t u r e C o l l e c t i o n ) w e r e c u l t u r e d i n D u b e l c c o ' s M o d i f i e d E a g l e ' s M e d i u m ( D M E M ) s u p p l e m e n t e d w i t h 9 % ( v / v ) fetal c a l f s e r u m ( F C S ) o n tissue culture grade dishes. M a c r o p h a g e s w e r e c e l l lines also d e r i v e d from e m b r y o n i c fetal l i v e r hematopoetic p r o g e n i t o r c e l l s to generate S O C S 3 " a n d S O C S 3 " i m m o r t a l i z e d + /  7  m a c r o p h a g e c e l l s ( k i n d l y p r o v i d e d b y D r . James Ihle, St. Jude C h i l d r e n ' s h o s p i t a l , T N ) . T h e s e c e l l s w e r e g r o w n i n Iscoves M o d i f i e d D u b e l c c o ' s M e d i u m ( E V I D M ) s u p p l e m e n t e d w i t h 9 % ( v / v ) F C S a n d 5 % C 1 2 7 c o n d i t i o n e d m e d i a (contains C o l o n y - S t i m u l a t i n g F a c t o r - 1 ) . A l l c e l l s were g r o w n at 3 7 ° C a n d 5 % CO2 i n a standard tissue culture incubator.  2.3  CaCh Competent cells  E . c o l i c e l l s , D H 5 a , w e r e g r o w n i n 2 m l l i q u i d b r o t h ( L B ) m e d i u m o v e r n i g h t i n a 3 7 ° C shaker. T h e next day, i n o c u l a t e d 100 m l L B - m e d i u m w i t h 1 m l o f o v e r n i g h t culture a n d g r e w u n t i l O D 6 0 0 = 0 . 4 5 or a little less ( u s u a l l y takes about a n hour, i f O D d i d not r e a c h then c h e c k e d e v e r y 10 m i n u t e s ) . T h e culture w a s c h i l l e d i n a flask o n i c e for about 15-30 minutes. T h e centrifuge tubes were p r e c h i l l e d o n i c e . T h e culture w a s spun d o w n c o l d at 4 0 0 0 r p m (2000g) for 10 32  m i n u t e s and the pellet w a s resuspended i n 1/3 o f o r i g i n a l v o l u m e w i t h 100 m M i c e c o l d CaCi2 and incubated o n i c e for 30 minutes. T h e tubes w e r e spun c o l d at 3 0 0 0 r p m (1500 g) for 10 minutes. T h e pellet w a s then resuspended i n 1/25 o f o r i g i n a l v o l u m e w i t h i c e c o l d 100 m M C a C l 2 / 1 5 % g l y c e r o l . T h e bacterial culture w a s then incubated at 4 ° C for a few hours. B a c t e r i a l c e l l s w e r e a l i q u o t e d into p r e c h i l l e d tubes (200 ul/tube) and stored at - 8 0 ° C . ( M o l e c u l a r C l o n i n g M a n u a l , S h a m b r o o k et al) 2.4  E . C o l i Transformation  T h e CaCi2 competent E . c o l i c e l l s (DH5o;) were t h a w e d o n i c e and used at 5 0 u l per transformation. 3-5 u l o f p l a s m i d was added to the c e l l s and they w e r e kept o n i c e for 1 hour. C e l l s w e r e put i n heat bath set at 4 2 ° C for 2 minutes and then put o n i c e for 2 minutes, p r i o r to the a d d i t i o n o f 2 m l o f p r e - w a r m e d L B ( 3 7 ° C ) and i n c u b a t i o n at 3 7 ° C i n c u b a t o r for 1 h o u r to a l l o w the bacteria to recover. T u b e s w e r e then spun d o w n at 12000 r p m and the supernatant w a s aspirated off. T h e bacterial pellet w a s resuspended i n 50 u l o f L B . T h e n L B + A m p i c i l l i n (100 u g / m L ) agar plates w e r e streaked w i t h 50 u l o f b a c t e r i a l culture. C o l o n i e s w e r e p i c k e d w i t h a u t o c l a v e d t o o t h p i c k s to inoculate 4 m l o f L B + A m p i c i l l i n (100 u g / m L ) s o l u t i o n . T h e cultures w e r e g r o w n i n a 3 7 ° C shaker overnight for a p p r o x i m a t e l y 16 hours. ( M o l e c u l a r C l o n i n g M a n u a l , S h a m b r o o k et al) 2.5  Plasmid D N A prep (miniprep)  D N A w a s isolated u s i n g M i n i p r e p k i t f r o m Q i a g e n ( M a r y l a n d , U S A ) . T h e bacterial s o l u t i o n w a s centrifuged i n e p p e n d o r f tubes at 14000 r p m for a few seconds. L B w a s aspirated o f f and b a c t e r i a l pellet w a s re-suspended i n 2 0 0 u l P I buffer (25 m M T r i s p H 8.0, 10 m M E D T A p H 8.0, R N a s e also added) b y m i x i n g up and d o w n . T h e n 2 0 0 u l o f P 2 buffer (0.2 N N a O H , 1% S D S ) w a s added to l y s e the bacteria. T h e tubes w e r e m i x e d t h o r o u g h l y a n d 3 5 0 u l o f N 3 buffer  33  (3 M K O A C , 3 M H O A C p H 4.8-5.5, ethanol w a s also added) w a s added. E a c h tube w a s i m m e d i a t e l y m i x e d gently to a l l o w K  +  i o n precipitate S D S w i t h g e n o m i c D N A . T h e tubes w e r e  s p u n at 12000 r p m for 10 m i n and the supernatants ( - 7 5 0 u l ) were transferred onto c o l u m n s w i t h c o l l e c t i o n tubes. T h e c o l u m n s were spun at 12000 r p m for 1 m i n and 7 5 0 u l o f 7 0 % ethanol w a s added to the c o l u m n . T h e c o l u m n s w e r e s p u n at 12000 r p m for 1 m i n and the f l u i d i n the c o l l e c t i o n tubes w e r e p o u r e d out so that c o l u m n s c o u l d be s p u n for another 1 m i n to ensure a l l the r e s i d u a l ethanol has been spun out o f the c o l u m n s . T h e c o l u m n s w e r e transferred into e p p e n d o r f tubes and D N A w a s eluted from c o l u m n s b y a d d i t i o n o f 50 u l o f water. A f t e r 1 m i n , the c o l u m n s w e r e spun at 12000 r p m for 1 m i n . T h e D N A c o n c e n t r a t i o n w a s based o n spectrophotometer measurements at 2 6 0 n m .  2.6  Retrovirus Infection  M y c - t a g g e d S O C S 3 constructs i n p M X - I R E S - E G F P ( p r o v i d e d b y D r . A k i h i k o Y o s h i m u r a ) w e r e transfected into P l a t - E v i r a l p a c k a g i n g c e l l l i n e , u s i n g l i p o s o m a l - m e d i a t e d transfection. P l a t - E c e l l s w e r e plated at 3 X 1 0 c e l l s per plate. T h e next day, 0 . 9 u l o f 4 0 m M l i p o s o m e were 6  added to 2 0 0 p i o f a u t o c l a v e d water i n one tube a n d 3 p g o f D N A to 2 0 0 p i o f water i n another tube. T h e t w o solutions were m i x e d and i n c u b a t e d @ R T for 25 m i n u t e s a n d 2 m l o f D M E M w a s added to the m i x t u r e and added to the P l a t - E c e l l s for 6 hours. T h e n 2 m l o f 9 % D M E M w a s added to rescue the c e l l s from starvation. T h e next day, m e d i a w a s aspirated o f f the P l a t - E c e l l s and 2 m l o f 9 % I M D M supplemented w i t h 5 % C 1 2 7 c o n d i t i o n e d m e d i a w a s added to the cultures. T h e next day, v i r u s - c o n t a i n i n g supernatant w a s c o l l e c t e d , centrifuged t w i c e for 10 m i n at 15000 r p m and added to the S O C S 3 " " c e l l s ( 2 X 1 0 S O C S 3 " " c e l l s i n 6 c m plates). Infected 7  6  7  c e l l s e x p r e s s i n g G F P w e r e detected w i t h a fluorescence m i c r o s c o p e . G F P e x p r e s s i o n w a s observable 1 d a y after i n f e c t i o n , but was m o r e evident o n the s e c o n d d a y after i n f e c t i o n . T h e s e  34  c e l l s w e r e sorted based o n G F P e x p r e s s i o n u s i n g a F l u o r e s c e n c e A c t i v a t e d C e l l Sorter. A f t e r sorting, these c e l l s w e r e g r o w n i n 9 % I M D M s u p p l e m e n t e d w i t h 5 % C 1 2 7 c o n d i t i o n e d m e d i a .  2.7  Determination of protein expression by Western Blot Analysis  Parental J 7 7 4 . 1 , S O C S 3 " , and S O C S 3 " macrophages ( 5 X 1 0 ) w e r e treated w i t h m I L - 1 0 (100 + /  7  6  n g / m L ) , L P S (10 n g / m L ) , or I L - 1 0 + L P S at the concentrations i n d i c a t e d , w a s h e d w i t h c o l d phosphate-buffered saline ( P B S ) , and l y s e d i n 2 0 0 u l o f buffer (150 n M N a C l , 5 0 m M T r i s - H C l , 2 m M EDTA, ImMNaV0  4  , I m M N a F and 1 % N P 4 0 ) s u p p l e m e n t e d w i t h C o m p l e t e Protease  I n h i b i t o r C o c k t a i l ( R o c h e , M o n t r e a l , C a n a d a ) . C e l l lysates w e r e c o l l e c t e d f o l l o w i n g centrifugation at 15000 r p m for 10 minutes to r e m o v e the n u c l e i a n d the p r o t e i n c o n c e n t r a t i o n w a s m e a s u r e d u s i n g B C A m e t h o d . A l i q u o t s o f c e l l lysate c o n t a i n i n g 100 u g o f p r o t e i n w e r e b o i l e d i n I X s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e g e l electrophoresis ( S D S - P A G E ) s a m p l e buffer and then subjected to S D S - P A G E . Proteins w e r e subsequently electro-transferred onto a P V D F m e m b r a n e ( B i o R a d , C a n a d a ) . M e m b r a n e s w e r e b l o c k e d w i t h T r i s - b u f f e r e d saline ( T B S ) 3 % B S A at r o o m temperature for 1 hr, then i n c u b a t e d w i t h the appropriate p r i m a r y antibodies i n T B S - 3 % B S A o v e r n i g h t o n a shaker. T h e m e m b r a n e s w e r e then w a s h e d several times w i t h T B S - 0 . 0 5 % T w e e n buffer and incubated w i t h f l u o r e s c e n t l y - l a b e l e d ( A l e x o r F l o u r 680) secondary antibodies ( M o l e c u l a r P r o b e s , O r e g a n , U S A ) for p r o t e i n detection u s i n g the L i c o r O d y s s e y system. T h e f o l l o w i n g antibodies w e r e u s e d at 1:1000 d i l u t i o n : a n t i - S O C S 3 [168], p h o s p h o - s p e c i f i c antibodies to Y 2 0 4 / Y 2 2 1 o n S O C S 3 [168], a n t i - i N O S and a n t i - E R K .  2.8  Determination of TNF-a protein levels by Enzyme-Linked Immuno-sorbent Assay (ELISA)  S O C S 3 " and S O C S 3 " macrophages w e r e plated at 2 X 1 0 c e l l / w e l l i n I M D M c o n t a i n i n g 9 % + /  7  5  s e r u m s u p p l e m e n t e d w i t h 5 % C 1 2 7 c o n d i t i o n e d m e d i a i n 24 w e l l tissue culture plates and  35  g r o w n overnight. T h e next day, the m e d i a was c h a n g e d to D M E M c o n t a i n i n g 9 % s e r u m and cells w e r e stimulated w i t h L P S (100 n g / m L ) or L P S + m I L - 1 0 (10 n g / m L ) for 2 hours. T h e supernatants w e r e c o l l e c t e d and a n a l y z e d for the presence o f T N F - a ! p r o t e i n u s i n g m o u s e - T N F a E L I S A k i t . T h e 96 w e l l E L I S A plates w e r e coated o v e r n i g h t w i t h 50 p i o f T N F - a capture a n t i b o d y d i l u t e d 1:250 i n c o a t i n g buffer ( I m M NaHCC>3, 3 m M NaCC»3). T h e w e l l s w e r e w a s h e d 5 times w i t h P B S / t w e e n - 2 0 (0.05%) and incubated w i t h 100 p i o f assay diluent ( 1 0 % F C S i n 1 X P B S ) for 1 h o u r to b l o c k n o n - s p e c i f i c b i n d i n g . 100 o f samples w e r e added to each w e l l and incubated for 2 hours at R T . T h e w e l l s w e r e w a s h e d 5 times and i n c u b a t e d w i t h 50 p i o f biotin-conjugated detection a n t i b o d y (1:250) for 1 hour. T h e w e l l s w e r e w a s h e d 5 times and incubated w i t h 50 p i o f avidin-conjugated H R P e n z y m e (1:250) for 30 minutes. T h e n w e l l s are w a s h e d 7 times (30 sec each) and f i n a l l y incubated w i t h 100 p i o f substrate. T h e substrate used w a s O - P h e n y l e n e d i a m i n e tablet ( S i g m a , St. L o u i s , U S A ) d i s s o l v e d i n phosphate-citrate buffer at final concentration o f 0.4 m g m l , and activated b y a d d i t i o n o f 0 . 0 3 % h y d r o g e n p e r o x i d e . T h e E L I S A plates w e r e d e v e l o p e d i n the dark for 30 minutes. T h e r e a c t i o n was stopped w i t h 100 p i o f 3 M S u l f u r i c a c i d . U s i n g a n E L I S A plate reader, the w e l l s w e r e read at 4 9 2 n m . T h e standard c u r v e w a s obtained b y u s i n g T N F - a (100 u g / m L ) w i t h the top concentration o f 2 0 0 0 p g / m L . T r i p l i c a t e samples w e r e assayed so that statistics c o u l d be a p p l i e d to calculate p-values (p-value < 0.01 indicates s i g n i f i c a n c e ) .  2.9  Detection of Nitrite production  N i t r i t e p r o d u c t i o n w a s measured b y the G r e i s s m e t h o d [127]. S O C S 3 " and S O C S 3 " + /  / _  macrophages w e r e plated at 2 X 1 0 c e l l / m L i n I M D M c o n t a i n i n g 9 % s e r u m supplemented w i t h 5  5 % C I 2 7 c o n d i t i o n e d m e d i a i n 24 w e l l tissue culture plates and g r o w n overnight. T h e next day, the m e d i a w a s c h a n g e d to D M E M c o n t a i n i n g 9 % serum and c e l l s w e r e stimulated w i t h either  36  L P S ( l O O n g m l ) or L P S + I L - 1 0 (10 n g / m L ) for 24 hours. T h e supernatants w e r e c o l l e c t e d a n d a n a l y z e d for the presence o f nitrite u s i n g the G r e i s s assay. S e v e n t y - f i v e p i o f the culture supernatant w a s c o m b i n e d w i t h 75 u l o f greiss reagent: 1% ( v / v ) s u l f a n i l a m i d e , 0 . 1 % ( v / v ) n a p h t h y l e t h y l e n e d i a m i n e h y d r o c h l o r i d e , 2 . 5 % ( v / v ) p h o s p h o r i c a c i d , a n d water, a n d i n c u b a t e d at r o o m temperature for 5 m i n . A b s o r b a n c e at 550 n m w a s m e a s u r e d b y an E L I S A plate reader. S o d i u m N i t r i t e (1 m M ) w a s u s e d to generate a standard c u r v e w i t h top c o n c e n t r a t i o n o f 2 5 0 n M . T r i p l i c a t e samples w e r e assayed so that statistics c o u l d be a p p l i e d to c a l c u l a t e p - v a l u e s (p-value < 0.01 indicates s i g n i f i c a n c e ) .  2.10  Determination of TNF-a RNA expression by Northern Blot Analysis  S O C S 3 " a n d S O C S 3 " c e l l s w e r e treated for 1-2 hours w i t h L P S (100 n g / m L ) or L P S + I L - 1 0 + /  7  (10 n g / m L ) . C e l l s w e r e harvested i n 1 m l o f T r i Z o l . C h l o r o f o r m (400 u l ) w a s added, a n d then samples w e r e s h a k e n v i g o r o u s l y for 30 seconds a n d incubated for 15 m i n u t e s at r o o m temperature ( R T ) . T h e samples w e r e then centrifuged ( 1 4 0 0 0 r p m ) for 10 m i n u t e s (4 ° C ) a n d the top aqueous phase w a s transferred into R N a s e free 1.5 m l e p p e n d o r f f tubes. T h e samples w e r e v o r t e x e d after a d d i t i o n o f 5 0 0 u l o f c o l d i s o p r o p a n o l a n d i n c u b a t e d for 15 m i n ( R T ) a n d centrifuged ( 1 4 0 0 0 r p m ) to precipitate the R N A .  T h e supernatant w a s aspirated o f f a n d 1 m l o f  c o l d 7 5 % ethanol w a s added to w a s h the pellet. S a m p l e s w e r e centrifuged at 11500 r p m for 10 m i n . T h e supernatant w a s aspirated o f f a n d the pellet w a s left at R T for 10 m i n to air dry. O n c e the pellet w a s d r y , it w a s resuspended i n 10 u l o f R N a s e - f r e e water. F o r m a l d e h y d e - g e l l o a d i n g buffer ( f o r m a l d e h y d e , f o r m a m i d e , M O P S , 5 0 % g l y c e r o l , I m M E D T A , 0 . 2 5 % b r o m o p h e n o l b l u e , . 2 5 % x y l e n e c y a n o l F F ) w a s added a n d R N A denatured b y h e a t i n g for 10 m i n u t e s at 5 5 ° C . E q u i v a l e n t amounts o f R N A w e r e r e s o l v e d b y electrophoresis o n 1% agarose g e l c o n t a i n i n g 3 7 % f o r m a l d e h y d e , b l o t t e d onto a n y l o n m e m b r a n e , a n d c r o s s - l i n k e d b y exposure to U V light.  37  The membranes were then prehybridized, hybridized, and washed according to standard procedures (Molecular Cloning Manual, Shambrook et al). The T N F - a and G A P D H probes were radiolabeled with [a- P] dCTP by the random priming method.  2.11  Statistical Analysis  Data are expressed as the mean + SE. J M P I N 4 software program was used to perform One-way A N O V A analysis and p-values < 0.01 were considered significant.  38  C H A P T E R 3: Results 3.1  Induction of SOCS3 message by IL-10 in a Stat3-dependent manner. In a p r e l i m i n a r y s u r v e y o f S O C S f a m i l y m e m b e r s , w e f o u n d that I L - 1 0 treatment o f  J774.1 c e l l s induces S O C S 3 m R N A expression. S O C S 3 m R N A is i n d u c e d b y I L - 1 0 as e a r l y as 30 m i n and this e x p r e s s i o n is sustained for as l o n g as 10 hours post s t i m u l a t i o n ( F i g . 7a). I n order to investigate the r o l e o f the Stat p a t h w a y i n I L - 1 0 r e g u l a t i o n o f S O C S 3 , a d o m i n a n t i n h i b i t o r y f o r m ( l a c k i n g the C - t e r m i n a l transactivation d o m a i n , AStat) w a s constructed and r e t r o v i r a l l y transduced into J774.1 cells. E x p r e s s i o n o f A S t a t 3 i n h i b i t e d the a b i l i t y o f I L - 1 0 to i n d u c e S O C S 3 m R N A ( F i g . 7b). I n contrast, A S t a t l d i d not i n h i b i t I L - 1 0 i n d u c t i o n o f S O C S 3 i n these c e l l s ( F i g . 7e) i n d i c a t i n g that S O C S 3 m R N A e x p r e s s i o n is Stat3-dependent. N e x t w e w a n t e d to determine whether the t w o tyrosines ( Y 4 2 7 / 4 7 7 for m I L - l O R ; Y 4 4 6 / 4 9 6 for M L - 1 0 R ) i n the c y t o p l a s m i c d o m a i n o f the I L - 1 0 receptor ( I L - 1 OR) w e r e r e q u i r e d for S O C S 3 i n d u c t i o n . W e expressed either the w i l d - t y p e ( W T ) or h I L - l O R : T y r  F F  mutant ( l a c k i n g b o t h Y 4 4 6 and  Y 4 9 6 ) i n J 7 7 4 cells. C e l l s were treated w i t h h u m a n I L - 1 0 i n the presence o f a b l o c k i n g antim l L - l O R a n t i b o d y to prevent h I L - 1 0 s t i m u l a t i o n o f the endogenous m o u s e I L - 1 OR. A s s h o w n i n F i g . 7 c , a l t h o u g h the W T I L - 1 OR s i g n a l i n g w a s able to i n d u c e S O C S 3 e x p r e s s i o n , the tyrosine mutant w a s not ( F i g . 7d).  39  SOCS3  GAPDH I  c  p  0.5  m  i  2  5  1  0  0.5  1  2  5  10  * mm m m  •  m  c. 1 ^  w  d.  s^te  •  ^^fe  tfft- S B mm  e.  ^tfM^  €P I P  Parental  ASTAT3  -mm wm. mm wm  b.  hours  m  ML-10R: WT  MM  ML-10R: Tyr  Wt  ASTAT1  Figure 7. Induction of S O C S 3 message by IL-10 in a Stat3-dependent manner. Parental J774.1 cells or cells expressing a dominant Statl (AStatl), dominant negative Stat3 (AStat3), wild-type hIL-10 (hIL10R:WT) or tyr-null (hIL10R:TyrFF) were treated for the indicated times with LPS or LPS + IL-10. (Cell lines described in O'Farrell et al E M B O J 17:1006, 1998 [3]). The R N A blot was analyzed by Northern analysis for SOCS3 mRNA expression and G A P D H m R N A to confirm equal R N A loading in each sample.  40  FF  3.2  Induction of SOCS3 protein by IL-10. We then determined whether IL-10 induces SOCS3 protein expression, since mRNA  expression does not always correlate with protein levels. SOCS3 protein is known to be very unstable, so to prevent SOCS3 protein degradation via the 26S proteasome, J774.1 cells were pretreated with 3 nM of MG132 (a proteasome inhibitor) for 30 minutes. These cells then were stimulated with LPS+IL-10 or IL-10 at various times (Fig. 8). SOCS3 protein expression was observed in response to IL-10 by 1 hour (Fig. 8b). Interestingly, LPS+IL-10 treatment induces SOCS3 protein even more rapidly (Fig. 8c) compared to IL-10 alone. LPS alone was not able to induce SOCS3 protein at any time point (Fig. 8a).  " Non-specific band  a. LPS  SOCS-3 (33 kDa)  Ctl  15  30  60  120  240 ( Non-specific band  ""*ftlj|*miii..i. :  b.  IL-10  •  Ctl  15  30  60  i  120  mm  SOCS-3 (33 kDa)  240 (• • Non-specific band  C.  LPS+IL-10 SOCS-3 (33 kDa)  Ctl  15  30  60  120  240 (min)  Figure 8. Induction of SOCS3 protein by IL-10. J774.1 cells were treated with control buffer or (a) 10 ng/mL LPS, (b) 100 ng/mL IL-10 or (c) 10 ng/mL LPS with 100 ng/mL IL-10 for the indicated length of time. Lysates were made IX in SDS-PAGE sample buffer, resolved by SDS-PAGE and subjected to immunoblot analysis with antibody to SOCS3 protein. The top bands are non-specific showing equivalent protein levels in each sample. These are results from 3 independent experiments. 41  3.3  Ectopic expression of SOCS3 protein is not sufficient to completely inhibit TNF-a production in response to LPS. O n c e it w a s d e t e r m i n e d that S O C S 3 m R N A w a s i n d u c e d b y I L - 1 0 i n a Stat3-dependent  manner, w e e c t o p i c a l l y expressed S O C S 3 p r o t e i n i n J774.1 m u r i n e c e l l s . B o t h c o n s t i t u t i v e l y e x p r e s s i n g S O C S 3 and parental J 7 7 4 c e l l s w e r e s t i m u l a t e d w i t h L P S + I L - 1 0 for 10 hours and their supernatants w e r e c o l l e c t e d for T N F - a d e t e r m i n a t i o n b y E L I S A . F i g u r e 9 s h o w s that J774.1 c e l l s c o n s t i t u t i v e l y e x p r e s s i n g S O C S 3 p r o t e i n m a k e less T N F - a p r o t e i n i n response to L P S as c o m p a r e d to the c o n t r o l group. N o t a b l y h o w e v e r , c o m p l e t e i n h i b i t i o n o f T N F - a production required addition o f IL-10.  42  20000 f  g> 15000  + Parental  LPS + IL10  SOCS 3  F i g u r e 9. E c t o p i c e x p r e s s i o n o f SOCS3 p r o t e i n is n o t s u f f i c i e n t to c o m p l e t e l y i n h i b i t T N F - a p r o d u c t i o n i n r e s p o n s e to L P S . W e e c t o p i c a l l y expressed S O C S 3 p r o t e i n i n J774.1 m u r i n e c e l l s . These c e l l s were stimulated w i t h L P S + I L - 1 0 and their supernatant w a s tested for T N F - a l e v e l s after 10 hrs v i a an E L I S A . ( T h i s E L I S A assay w a s carried out b y D r . A l i c e M u i )  43  3.4  IL-10 induces phosphorylation of SOCS3 protein at tyrosine 204 in the SOCS-box domain. S i n c e m e r e e x p r e s s i o n o f S O C S 3 protein i n macrophages w a s not sufficient to i n h i b i t  T N F - a : p r o t e i n p r o d u c t i o n i n response to L P S , this suggests that a d d i t i o n a l I L - 1 0 signals are required. It has been s h o w n that S O C S 3 p r o t e i n is p h o s p h o r y l a t e d i n response to c y t o k i n e s , g r o w t h factors and b y several f a m i l i e s o f kinases, i n c l u d i n g Jaks and receptor tyrosine kinases [168]. J774.1 c e l l s w e r e pretreated w i t h 10 u M N a V 0  3  and 3 u M M G 1 3 2 (a proteasome  i n h i b i t o r ) for 30 minutes p r i o r to s t i m u l a t i o n w i t h 100 n g / m L o f L P S + I L - 1 0 (100 n g / m L ) or I L 10 alone. I L - 1 0 w a s able to i n d u c e p h o s p h o r y l a t i o n o f S O C S 3 p r o t e i n at Y 2 0 4 ( F i g . 10a) at 2 hours post s t i m u l a t i o n . T h e b l o t w a s re-probed for p r o t e i n S O C S 3 a n t i b o d y ( F i g . 10c), and later for E r k 1/2 p r o t e i n ( F i g . l O d ) to c o n f i r m equal p r o t e i n l o a d i n g . P h o s p h o - s p e c i f i c a n t i b o d y raised against Y 2 0 4 w a s tested for its s p e c i f i c i t y . T h e p h o s p h o - s p e c i f i c Y 2 0 4 a n t i b o d y detected S O C S 3 p r o t e i n i n c e l l s e x p r e s s i n g W T or S O C S 3  Y 2 2 1 F  p r o t e i n , but not i n S O C S 3 " or _ /  Y204F  SOCS3  , Z U H r  m a c r o p h a g e s ( F i g . l O e ) . T h e same b l o t w a s reprobed w i t h a n t i - S O C S 3 a n t i b o d y to  c o n f i r m the presence o f S O C S 3 p r o t e i n i n these samples ( F i g . l O g ) . A n o t h e r b l o t w a s p r o b e d for p h o s p h o - Y 2 2 1 , and there were bands i n I L - 1 0 treated samples ( F i g . 10b). H o w e v e r , the p h o s p h o - s p e c i f i c Y 2 2 1 a n t i b o d y d i d not s h o w s p e c i f i c i t y ( F i g . l O f ) .  44  Q_  a.  o + WT  Anti-PY204  KO Y204F Y221F Anti-PY 204  b.  Anti-PY221  C.  Anti-SOCS3  d.  Anti-Erkl/2  Anti-PY 221  Anti-SOCS3  Figure 10. IL-10 induces phosphorylation of SOCS3 protein at tyrosine 204 in the SOCS-box domain. J774.1 cells were pretreated with 10 u M NaV03 and 5 u M MG132 proteosome inhibitor for 1 hour at 37°C. Then the samples were treated with control buffer or 100 ng/mL LPS + 100 ng/ml IL-10 or 100 ng/mL IL-10 alone for 2 hrs before preparation of cell lysates. Lysates were made I X in SDS-PAGE sample buffer, resolved by SDS-PAGE and subjected to immunoblot analysis with antibodies to phospho-tyr 204 SOCS3 (PY204), phospho-tyr 221 SOCS3 (PY221) or SOCS3 protein. The same blot was reprobed for Erk protein to confirm equal protein loading in each sample. SOCS " cells and SOCS"" reconstituted with cDNA for SOCS3 (WT), Y204F or Y221F mutants of SOCS3 were treated LPS+IL-10 for 2 hrs and samples were treated as above. The samples were subjected to immunoblot analysis with antibodies to PY204 (lOe) and PY221 (101). The blots were then reprobed with anti-SOCS3 antibody to confirm presence of SOCS3 protein (lOg). This experiment was carried out 3 independent times. 7  45  3.5  IL-10  inhibition of T N F - a protein expression requires  S O C S 3  during the early  phase of signaling. T N F - a is a p r i m a r y m e d i a t o r o f n u m e r o u s i m m u n o l o g i c functions, i n c l u d i n g i n f l a m m a t i o n and r e g u l a t i o n o f i m m u n e proliferative and a c t i v a t i o n responses. W e chose to study T N F - a as one o f the readouts for an activated m a c r o p h a g e due to f o l l o w i n g reasons: 1) T N F - a p r o d u c t i o n is a h a l l m a r k characteristic o f m a c r o p h a g e a c t i v a t i o n , 2) T N F - a e x p r e s s i o n i s regulated at m u l t i p l e l e v e l s and 3) S i g n a l transduction m e c h a n i s m s i n v o l v e d i n this r e g u l a t i o n are w e l l - c h a r a c t e r i z e d . I n order to determine the potential role o f S O C S 3 p r o t e i n i n I L - 1 0 i n h i b i t i o n o f T N F - a p r o t e i n expression, w e e x a m i n e d p r o d u c t i o n o f T N F - a i n response to L P S + I L - 1 0 after 2 hours, i n the S O C S 3 " vs S O C S " " macrophages. I L - 1 0 w a s able to i n h i b i t + /  7  p r o d u c t i o n o f T N F - a i n S O C S " c e l l s and not i n S O C S 3 " " macrophages ( F i g . 11, p a n e l A ) . T o + /  7  c o n f i r m that the i n h i b i t i o n i n these c e l l s are t r u l y due to the absence o f S O C S 3 p r o t e i n , w e e x a m i n e d the p r o d u c t i o n o f T N F - a i n S O C S3" " macrophages reconstituted w i t h S O C S 3 c D N A 7  v i a r e t r o v i r a l transduction. S O C S 3 " " c e l l s reconstituted w i t h S O C S 3 c D N A regained their 7  responsiveness to I L - 1 0 and w e r e able to i n h i b i t T N F - a p r o d u c t i o n s i m i l a r to S O C S " + /  macrophages ( F i g . 11, p a n e l A ) . B u t w h e n S O C S 3 " and S O C S 3 " " c e l l s w e r e treated w i t h + /  7  L P S + I L - 1 0 for 4 hours, I L - 1 0 w a s able to i n h i b i t T N F - a p r o d u c t i o n b o t h i n S O C S 3 " and + /  S O C S 3 " c e l l s ( F i g . 11, p a n e l B ) . T h e s e results suggest that the dependence o f I L - 1 0 o n S O C S 3 7  for i n h i b i t i o n o f T N F - a p r o d u c t i o n occurs o n l y d u r i n g the e a r l y phase o f I L - 1 0 s i g n a l l i n g .  46  A. unstimulated LPS(100ng/mL) LPS+IL-10 (lOng/mL)  LPS SOCS3  362+4.9  147+1.4  SOCS3  350+6.0  282+1.4  372+4.2  128+2.3  SOCS3 -'- : W T  S0CS3  SOCS3 " 7  L+10  SOCSS-^WT  B. unstimulated L P S (100 ng/mL)  o  LPS+IL-10 (lOng/mL)  y 3 C  P.  LPS S0CS3 "  1847+1.3  249±1.4  S 0 C S 3 -'-  1073±2.5  152+1,2  1306±1.9  57+1.0  +/  S0CS3 - :WT ;  SOCS3  S0CS3-'-  L+10  SOCS3- :WT A  Figure 11. IL-10 inhibition of TNF-a protein expression requires SOCS3 during the early phase of signaling. M a c r o p h a g e c e l l lines w e r e d e r i v e d from w i l d - t y p e o r S O C S 3 " b o n e m a r r o w d e r i v e d / _  m a c r o p h a g e s . T h e reconstituted c e l l s are the S O C S 3 " c l o n e w h i c h has b e e n transduced _ /  w i t h the c D N A for S O C S 3 . C e l l s w e r e s t i m u l a t e d w i t h 100 n g / m L L P S ± 10 n g / m L I L - 1 0 for ( A ) 2 hrs a n d ( B ) 4 hrs. T N F - a l e v e l s i n the supernatants w e r e measured b y E L I S A . T h e tables above c o n t a i n the absolute l e v e l s o f T N F - a p r o t e i n ( p g / m L ) for each set o f data. T h i s data represents 1 o f 4 experiments.  47  3.6  IL-10 inhibits expression of TNF-a mRNA in a SOCS3-dependent manner. Since we observed inhibition o f T N F - a protein production b y I L - 1 0 i n a S 0 C S 3 -  dependent m a n n e r ( F i g . 11), w e then e x a m i n e d the e x p r e s s i o n o f T N F - a m R N A i n response to L P S + I L - 1 0 at 2 hours. I L - 1 0 w a s able to i n h i b i t T N F - a m R N A e x p r e s s i o n i n the S O C S " + /  macrophages ( F i g . 12a, p a n e l A ) , but not i n S O C S 3 " macrophages ( F i g . 12b, panel A ) . T h e 7  same blot w a s reprobed for G A P D H to c o n f i r m equal l o a d i n g ( F i g . 12, panel B ) . O n c e again, I L - 1 0 d i d not require S O C S 3 p r o t e i n to i n h i b i t T N F - a m R N A e x p r e s s i o n at 4 hours (data not shown).  B  A  GAPDH  TNF-a A  r LPS a.  b-  SOCS3 + /  0  1 2  \  r LPS  LPS+IL-10 1 2  A  ^ LPS+IL-10  1 2 mm .tarn 1 2 mm hours • H0| mm* VP  VP  S O C S 3 -'-  Figure 12. IL-10 inhibits expression of TNF-a mRNA in a SOCS3-dependent manner. S O C S 3 " and SOCS3~'~ macrophages w e r e treated w i t h 100 n g / m L o f L P S + 100 n g / m L + /  I L - 1 0 for 1 and 2 hrs. S a m p l e s were c o l l e c t e d i n T r i Z o l and R N A was extracted and r e s o l v e d b y 1% agarose gel and then subjected to R N A blot analysis w i t h probes for T N F - a . P a n e l B s h o w s the same blot w a s reprobed for G A P D H to c o n f i r m equal R N A i n each sample. T h i s experiment w a s p e r f o r m e d 5 times.  48  3.7  Reconstitution of S O C S 3  cells with W T and mutant S O C S 3 .  SOCS proteins have three conserved regions: SOCS-box, SH2 and KIR domains. We retfovirally transduced SOCS3 " macrophage cell clones with wild type or mutant SOCS3 7  cDNAs (as described by Yoshimura, A et al in JBC 2000) using infection methods devised for high efficiency gene transfer into macrophage cells [127, 130]. Figure 13A shows a schematic representation of these various SOCS3 domain mutants. The cells were sorted based on GFP expression. Lysates prepared from these cells were examined via western analysis to confirm expression of SOCS3 protein (Fig. 13B, lanes 3-9). Samples then were also prepared from the parental SOCS3 " and SOCS3" " cells (Fig. 13B, lanes 1-2) treated with 100 ng/mL of IL-10 for +/  7  1 hour to induce expression of endogenous SOCS3 protein. The expression of each SOCS3 protein was confirmed to be similar to each other and similar to levels of endogenous SOCS3 protein induced by IL-10.  49  a. S0CS3 . WT  I  KIR  KIR  b. S0CS3 . SH2  c. SOCS3 ": K I R 7  d. S0CS3 . SOCS-box  e. SOCS3 ": Y204F 7  f. SOCS3 ": Y221F 7  Y204  Y221  Y204  Y221  R71E  SH2 d o m a i n  KIR  SH2 d o m a i n  KIR  Y221  SH2 domain  L22D  KIR  Y204  Y204F  Y221  Y204  Y221F  SH2 d o m a i n  SH2 d o m a i n  F i g u r e 13 A . S c h e m a t i c r e p r e s e n t a t i o n o f v a r i o u s S O C S 3 p r o t e i n m u t a n t s .  The SOCS3 ": SH2 and SOCS3 ": KIR have a pointtation in the domains, while the SOCS3 ": SOCS-box has a completely deleted domain. The SOCS3" ": Y204F and SOCS3 ": Y221F have a point mutation in one of the tyrosine residues in the SOCS-box domain. 7  7  _/  7  7  50  1  2  3  4  5  6  7  8  9  33 kDa (SOCS-3)  00  CO  &o  GO  Figure 13B. Reconstitution of SOCS3 " cells with WT and mutant SOCS3. S 0 C S 3 " c e l l s (lane 1) a n d S O C S 3 " c e l l s (lane 2) w e r e treated w i t h 100 n g / m L L P S + I L - 1 0 + /  7  for 1 h o u r to i n d u c e e x p r e s s i o n o f endogenous S O C S 3 p r o t e i n . T h e reconstituted cells (lane 3-9) are the S O C S 3 ~~ c e l l s w h i c h have b e e n transduced w i t h an e m p t y vector, c D N A for S O C S 3 , S H 2 d o m a i n mutant ( R 7 1 E ) , K I R d o m a i n mutant ( L 2 2 D ) , S O C S - b o x d e l e t i o n ( D C 4 0 ) , Y 2 0 4 F o r Y 2 2 1 F , w h i c h c o n s t i t u t i v e l y express the m y c - t a g g e d S O C S 3 p r o t e i n as described i n the M a t e r i a l and M e t h o d s . L y s a t e s w e r e m a d e I X i n S D S - P A G E sample buffer, r e s o l v e d b y S D S - P A G E a n d subjected to i m m u n o b l o t analysis w i t h a n t i b o d y to S O C S 3 protein. T h e same b l o t w a s reprobed w i t h anti-erk a n t i b o d y to s h o w equal p r o t e i n present i n each sample. T h e s e results are representative o f 3 independent experiments.  51  3.8  IL-10 requires all domains of SOCS3 protein for inhibition of T N F - a protein production. O n c e it w a s c o n f i r m e d that a l l the S O C S 3 " m a c r o p h a g e s reconstituted w i t h v a r i o u s 7  S O C S 3 d o m a i n mutants express c o m p a r a b l e l e v e l s o f S O C S 3 p r o t e i n , experiments w e r e c o n d u c t e d to determine w h i c h one o f these d o m a i n s are important i n the i n h i b i t o r y effects o f I L 10 o n T N F - a p r o t e i n p r o d u c t i o n . C e l l s w e r e s t i m u l a t e d w i t h 100 n g / m L o f L P S + I L - 1 0 (10 n g / m L ) for 2 hours. T h e l e v e l s o f T N F - a p r o t e i n i n culture supernatant w e r e d e t e r m i n e d v i a an E L I S A . F i g u r e 14 s h o w s that I L - 1 0 r e q u i r e d a l l d o m a i n s o f S O C S 3 p r o t e i n for i n h i b i t i o n o f T N F - a protein production.  52  100 LPS+IL-10  (3 O  O  60  io.  a  ti Z fx  40 20 0  CN  o o 00  U O  >  00  o  O' oo  oo  u  O  ai  X oo  5  ^ '  m oo <_> O oo  00  O  00  00  O O  00  00  oo U O oo  IX,  o r*1  oo U O oo  OO  U O  00  S0CS3 -  SOCS.V  n  S0CS3"'  lxlO"  6  lxlO"  7  Vector  9xl0"  7  0.80  WT  0.80  SH2  9xl0-  5  0.06  KIR  4xl0"  6  0.08  SOCSbox  6xl0'  5  Y204F  3xl0"  5  0.18  7  Y221F  lxlO"  6  0.07  7  +/  lxlO"  6  7 6  0.06  7 7 7 7  CN  X  o  Cells  00  O O oo  Figure 14. IL-10 requires all domains of SOCS3 protein for inhibition of TNF-a protein production. S O C S 3 ~ , S O C S 3 " and the S O C S 3 " " reconstituted cells (as d e s c r i b e d i n F i g . 13) w e r e +  7  7  treated w i t h 100 n g / m L L P S and 10 n g / m L I L - 1 0 for 2 hours. T h e supernatants w e r e c o l l e c t e d to be a n a l y z e d for T N F - a secretion b y E L I S A . T h i s e x p e r i m e n t w a s c a r r i e d out 7 independent times and p - v a l u e s w e r e c a l c u l a t e d u s i n g o n e - w a y A N O V A a n a l y s i s to determine the s i g n i f i c a n c e o f the difference b e t w e e n each S O C S 3 " " reconstituted cells 7  relative to S O C S 3 " or S O C S 3 " cells. These p - v a l u e s are listed i n the table, a p - v a l u e < + /  7  0.01 w a s c o n s i d e r e d significant.  53  3.9  Excluding the KIR domain, IL-10 requires all domains of  SOCS3 protein for  inhibition of TNF-a mRNA expression. In order to determine the d o m a i n o f S O C S 3 i n v o l v e d i n I L - 1 0 i n h i b i t i o n o f T N F - a m R N A , S O C S 3 " a n d S O C S " macrophages, as w e l l as a l l the S O C S 3 " m a c r o p h a g e s + /  7  7  reconstituted w i t h v a r i o u s S O C S 3 d o m a i n mutants w e r e treated w i t h 100 n g / m L o f L P S + I L 1 0 (10 n g / m L ) for 2 hours and total R N A w a s prepared for a N o r t h e r n analysis. S O C S 3 " : W T 7  c e l l s r e g a i n e d their responsiveness to I L - 1 0 and w e r e able to i n h i b i t T N F - a m R N A e x p r e s s i o n , w h i l e the S O C S 3 " : V e c t o r b e h a v e d s i m i l a r to parental S O C S 3 " c e l l s ( F i g . 15c, d, r e s p e c t i v e l y ) . 7  7  F i g u r e 15 also s h o w s that T N F - a m R N A e x p r e s s i o n is i n h i b i t e d i n S O C S 3 " : K I R (15g), but not 7  i n S O C S 3 " : S O C S - b o x ( F i g . 15e), S O C S 3 " : S H 2 ( F i g . 15f), S O C S 3 " : Y 2 0 4 F ( F i g . 15h), and 7  7  7  S O C S 3 " : Y 2 2 1 F ( F i g . 15i) macrophages. R e p r o b i n g the northern b l o t for G A P D H m R N A 7  c o n f i r m e d equal R N A l o a d i n g .  54  F i g u r e 1 5 . E x c l u d i n g K I R d o m a i n , I L - 1 0 r e q u i r e s a l l d o m a i n s o f SOCS3 p r o t e i n for i n h i b i t i o n of T N F - a m R N A expression. SOCS3 \ SOCS3 + /  / _  a n d the S O C S 3 - / - reconstituted c e l l s (as d e s c r i b e d i n F i g . 13) w e r e  treated w i t h 100 n g / m L L P S and 10 n g / m L I L - 1 0 for 2 hours. T h e R N A w a s c o l l e c t e d i n T r i Z o l a n d s a m p l e s w e r e r e s o l v e d b y 1% agarose g e l a n d subjected to R N A b l o t analysis w i t h probe for T N F - a . T h e same b l o t w a s re-probed for G A P D H m R N A to c o n f i r m equal R N A i n each sample. T h e bands w e r e quantitated u s i n g a p h o s p h o i m a g e r , a n d the ratios o f T N F - a m R N A / G A P D H m R N A w e r e p l o t t e d as histograms (right-hand panel). T h i s e x p e r i m e n t w a s c a r r i e d out 4 t i m e s .  55  3.10  Y204/Y221 of SOCS3 protein is important for inhibition of NO production by IL-10. W e then e x a m i n e d whether i n h i b i t i o n o f N O p r o d u c t i o n b y I L - 1 0 is also S O C S 3 -  dependent.  Parental S O C S 3 " and S O C S 3 + /  / _  macrophages w e r e treated w i t h 100 n g / m L o f  L P S + I L - 1 0 (10 n g / m L ) for 2 4 hours, and the l e v e l s o f N O i n the supernatants were d e t e r m i n e d u s i n g the G r e i s s assay. I L - 1 0 w a s able to i n h i b i t N O p r o d u c t i o n i n S O C S 3 " c e l l s , but not + /  S O C S 3 " c e l l s . T o ensure that this effect is due s o l e l y to the absence o f S O C S 3 p r o t e i n , S O C S 3 7  /_  c e l l s reconstituted w i t h S O C S 3 c D N A w e r e tested as w e l l and F i g u r e 16 s h o w s that  r e c o n s t i t u t i o n w i t h w i l d - t y p e S O C S 3 restored responsiveness to I L - 1 0 , w h i l e the S O C S 3 " c e l l s 7  reconstituted w i t h an e m p t y v e c t o r behave l i k e parental S O C S 3 " c e l l s . N e x t , S O C S 3 " " c e l l s 7  7  reconstituted w i t h v a r i o u s S O C S 3 d o m a i n mutants w e r e e x a m i n e d i n order to determine w h i c h o f these regions are important i n I L - 1 0 ' s a b i l i t y to i n h i b i t N O p r o d u c t i o n . O n l y m u t a t i o n o f Y 2 0 4 and Y 2 2 1 i n the S O C S - b o x d o m a i n abrogated the a b i l i t y o f I L - 1 0 to i n h i b i t N O p r o d u c t i o n ( F i g . 16).  56  r 1 LPs+iL-10 cells  S'OCS3 -  B O  SOCS3'  2xl0-  3 -O O  Vector  6xl0  WT  0.70  SH2  a,  O x  40-  a* co  o CO  >  y> o «3  00 o o co  co co o co  CO  co O O co  CO  U O  O O 00  CO  (1*  o  +/  SOCS3--  6  2xl0"  6  0.70  6  7 7  2xl0"  5  7  0.60  5xl0"  3  7  KIR  0.20  9xl0"  5  SOCSbox  0.20  3xl0"  4  Y204F  4x10°  0 90  Y221F  lxlO"  0.90  7 7 7  4  /  x o co U O  a o co  CO  CO  U O co  Figure 16. Y204 and Y221 of SOCS3 protein is important for inhibition of NO production by IL-10. S O C S 3 " , S O C S 3 " a n d the S O C S 3 " reconstituted c e l l s (as d e s c r i b e d i n F i g . 13) w e r e + /  7  7  treated w i t h 100 n g / m L L P S and 10 n g / m L I L - 1 0 for 24 hrs. T h e supernatants w e r e c o l l e c t e d to be a n a l y z e d for N O p r o d u c t i o n v i a nitrite assay. T h i s e x p e r i m e n t w a s c a r r i e d out 7 independent times a n d p - v a l u e s w e r e c a l c u l a t e d u s i n g O n e - w a y A N O V A to determine the s i g n i f i c a n c e o f the difference b e t w e e n S O C S 3 ' " reconstituted c e l l s relative to S O C S 3 " o r 7  + /  S O C S 3 " " c e l l s . T h e s e p - v a l u e s are listed i n the table, a p - v a l u e o f < 0.01 w a s c o n s i d e r e d 7  significant.  57  3.11  IL-10 requires Y204 and Y221 of SOCS3 for inhibition of iNOs protein expression. S i n c e w e o b s e r v e d N O i n h i b i t i o n b y I L - 1 0 i n a S O C S 3 - d e p e n d e n t m a n n e r ( F i g . 16), w e  investigated whether the i n h i b i t i o n o f N O b y I L - 1 0 is due to d o w n r e g u l a t i o n o f i n d u c i b l e n i t r i c o x i d e synthase ( i N O S ) , w h i c h catalyzes the p r o d u c t i o n o f N O f r o m L - a r g i n i n e . Parental S O C S 3 " a n d S O C S 3 " c e l l s , as w e l l as the S O C S 3 " reconstituted w i t h v a r i o u s S O C S 3 + /  /  7  constructs (as m e n t i o n e d above) w e r e treated w i t h 100 n g / m L o f L P S + L + 1 0 (10 n g / m L ) for 24 hours and c e l l lysates  Were prepared for d e t e r m i n a t i o n o f i N O S p r o t e i n b y i m m u n o b l o t analysis.  F i g u r e 17 s h o w s that I L - 1 0 w a s able to i n h i b i t e x p r e s s i o n o f i N O S p r o t e i n i n S O C S 3 " b u t not + /  i n S O C S 3 " c e l l s and this i n h i b i t i o n r e q u i r e d b o t h Y 2 0 4 and Y 2 2 1 i n the S O C S - b o x d o m a i n . 7  T h e blots w e r e re-probed w i t h a n t i b o d y against E R K 1 / 2 to c o n f i r m e q u a l amount o f p r o t e i n w a s l o a d e d for each sample.  58  SOCS+/r  Vector  socs-/r  i  r  Reconstituted WT i  Quantitation of iNOS protein expression  r  100  0 LPS L+10 0 LPS L+10 0 LPS L+10 0 LPS L+10 iNOS  SOCS-box I  SH2 I I  KIR I  I  Y204F I  Y221F 1 I  I  HHHHHH 1 i I | •i :  I  0 LPS L+10 0 LPS L+10 0 LPS L+10 0 LPS L+10 0 LPS L+10 iNOS  .„_  ^  *.  r  erk  F i g u r e 17. IL-10 r e q u i r e s Y 2 0 4 a n d Y 2 2 1 o f S O C S 3 f o r i n h i b i t i o n o f i N O s p r o t e i n expression. S O C S 3 ~ , S O C S 3 " a n d the S O C S 3 " " reconstituted cells (as d e s c r i b e d i n F i g . 13) w e r e treated + /  7  7  w i t h 100 n g / m L L P S and 10 n g / m L I L - 1 0 for 24 hrs. L y s a t e s w e r e prepared i n I X i n S D S - P A G E sample buffer, r e s o l v e d b y S D S - P A G E a n d subjected to i m m u n o b l o t analysis w i t h a n t i b o d y to i N O S p r o t e i n . T h e same b l o t w a s reprobed w i t h a n t i b o d y to erk p r o t e i n to c o n f i r m equal p r o t e i n l o a d i n g i n each sample. T h e bands w e r e quantitated b y densitometry and the ratios for i N O S protein/erk p r o t e i n w e r e plotted as histograms (right-hand panel). T h e s e are results f r o m 3 independent experiments.  59  CHAPTER 4: Discussion T h e innate i m m u n e s y s t e m initiates l o c a l and sometimes s y s t e m i c i n f l a m m a t o r y responses that alert the b o d y to the presence o f potential threats and guides the d e v e l o p m e n t o f subsequent adaptive i m m u n e responses. H e n c e there are t w o faces to the i n f l a m m a t o r y process; w h i l e o n one h a n d i n f l a m m a t i o n is u s u a l l y h e l p f u l and protective to the host, i f left u n c h e c k e d , the e x c e s s i v e amount o f i n f l a m m a t o r y c y t o k i n e s c o u l d cause tissue destruction, p h y s i o l o g i c a l changes and c o m p l i c a t i o n s s u c h as septic shock. It is s t i l l p o o r l y u n d e r s t o o d h o w an i n i t i a l , b e n e f i c i a l host response to i n f e c t i o n m e d i a t e d b y i n f l a m m a t o r y mediators, c a n sometimes progress to a t o x i c s y s t e m i c reaction. L P S is a potent activator o f m a c r o p h a g e s and this causes release o f v a r i o u s p r o - i n f l a m m a t o r y mediators s u c h as T N F - a , N O , I L - 1 , I L - 6 and I L - 8 , a l l o f w h i c h are suppressed b y I L - 1 0 [100]. H o w e v e r , the m e c h a n i s m b y w h i c h I L - 1 0 i n h i b i t s L P S s i g n a l l i n g p a t h w a y is not defined as o f yet. I L - 1 0 induces e x p r e s s i o n o f m a n y proteins i n a Stat3-dependent manner.  O n e candidate p r o t e i n w h o s e r e g u l a t i o n is b y I L - 1 0 i n a Stat3-  dependent m a n n e r is S O C S 3 . S O C S f a m i l y o f proteins are t r a n s c r i p t i o n a l l y activated b y a b r o a d range o f extracellular l i g a n d s and functions i n a c l a s s i c a l feedback l o o p to regulate s i g n a l transduction t h r o u g h m u l t i p l e c y t o k i n e and g r o w t h factor receptors [157, 158, 171]. These proteins w e r e i n i t i a l l y c l o n e d as c y t o k i n e - i n d u c i b l e i m m e d i a t e - e a r l y genes that c o u l d i n h i b i t a c t i v a t i o n o f Stat proteins and b i o l o g i c a l responses to several c y t o k i n e s [40, 147, 151].  W e and others have f o u n d that I L - 1 0 r a p i d l y induces S O C S 3 m R N A i n macrophages ( F i g . 7a) [105] and i n neutrophils [172]. T h i s i n d u c t i o n occurs i n a Stat3 ( F i g . 6 7 b , 7c) and Y 4 4 6 / Y 4 9 6 ( h u m a n - I L - l O R a ) dependent m a n n e r ( F i g . 7 d , 7e). S O C S 3 p r o t e i n is i n d u c e d b y I L - 1 0 alone as e a r l y as 1 h o u r ( F i g . 8b). H o w e v e r , stronger S O C S 3 p r o t e i n e x p r e s s i o n is o b s e r v e d i n macrophages treated w i t h L P S + I L - 1 0 ( F i g . 8c) and the same is true for S O C S 3  60  m R N A (data not s h o w n ) . T h i s enhancement m a y be due to p o s t - t r a n s c r i p t i o n a l s t a b i l i z a t i o n o f the m R N A or post-trahslational r e g u l a t i o n o f S O C S 3 p r o t e i n [173]. O t h e r investigators h a v e reported L P S i n d u c t i o n o f S O C S 3 p r o t e i n , h o w e v e r this i n d u c t i o n w a s o b s e r v e d o n l y after 3 hours post s t i m u l a t i o n [173], at w h i c h t i m e autocrine c y t o k i n e s l i k e I L - 1 0 are b e i n g p r o d u c e d . In o u r hands h o w e v e r , L P S treatment d i d not result i n i n d u c t i o n o f S O C S 3 p r o t e i n at a n y o f the t i m e points e x a m i n e d . S i n c e mere e x p r e s s i o n o f S O C S 3 p r o t e i n i n macrophages w a s not sufficient to i n h i b i t T N F - a p r o t e i n p r o d u c t i o n i n response to L P S , this suggests that a d d i t i o n a l I L - 1 0 signals s u c h as tyrosine p h o s p h o r y l a t i o n , p r o t e i n m o d i f i c a t i o n or a c t i v a t i o n o f other proteins m a y be r e q u i r e d . S O C S 3 has been reported to be p h o s p h o r y l a t e d b y activated J A K s , S r c f a m i l y kinases, and receptor tyrosine kinases (eg. E P O R , P D G F R ) at Y 2 0 4 and Y 2 2 1 i n the c o n s e r v e d S O C S - b o x m o t i f . S O C S 3 c a n b i n d R a s G A P , an i n h i b i t o r o f R a s , w h e n p h o s p h o r y l a t e d b y P D G F , r e s u l t i n g i n E R K a c t i v a t i o n important for c e l l s u r v i v a l despite its i n h i b i t i o n o f Stat5 p h o s p h o r y l a t i o n due to i n h i b i t i o n o f I L - 2 s i g n a l l i n g p a t h w a y [168]. T y r o s i n e p h o s p h o r y l a t i o n o f S O C S 3 c a n ensure c e l l s u r v i v a l and c e l l c y c l e p r o g r e s s i o n (proliferation) t h r o u g h the R a s p a t h w a y i n this c e l l system. I L - 1 0 i n d u c e d p h o s p h o r y l a t i o n o f S O C S 3 p r o t e i n at Y 2 0 4 w i t h i n 2 hours post s t i m u l a t i o n o f J774.1 macrophages ( F i g . 10a and 10b, r e s p e c t i v e l y ) . T h e s p e c i f i c i t y o f the p h o s p h o - s p e c i f i c antibodies r a i s e d against Y 2 0 4 / Y 2 2 1 residues w e r e tested o n S O C S 3 " : Y 2 0 4 F 7  and S O C S 3 : Y 2 2 1 F macrophages. A  U n f o r t u n a t e l y , w e w e r e o n l y able to c o n f i r m the s p e c i f i c i t y  o f the p h o s p h o - s p e c i f i c 2 0 4 a n t i b o d y ( F i g . l O e ) . T h e p h o s p h o - s p e c i f i c 221 a n t i b o d y also reacted w i t h S O C S 3 " : Y 2 2 1 F c e l l s ( l O f ) , so it either cross-reacts w i t h the S O C S 3 p r o t e i n or 7  SOCS3  P Y 2 0 4  . B y reconstituting S O C S 3 " macrophages w i t h the S O C S 3 7  61  Y 2 0 4 F / Y 2 2 1 F  d o u b l e mutant,  w e w i l l be able to e x c l u d e the latter p o s s i b i l i t y . Therefore, b y i m m u n o b l o t analysis w e cannot c o n c l u d e whether I L - 1 0 induces p h o s p h o r y l a t i o n at tyrosine 221 o f S O C S 3 protein. H o w e v e r , our analysis w i t h the S O C S 3 " : Y 2 2 1 F macrophages suggest that this residue is i n d e e d - /  important for I L - 1 0 i n h i b i t i o n o f T N F - a and N O p r o d u c t i o n . R e c e n t l y it has b e e n s h o w n that S O C S 3 p h o s p h o r y l a t i o n at Y 2 2 1 , a l l o w s S O C S 3 to b i n d N e k and C r k - L , adaptor proteins, and recruit N e k to activated receptor tyrosine kinases and modulates N e k tyrosine p h o s p h o r y l a t i o n i n fibroblasts, thus r e g u l a t i n g adaptor p r o t e i n s i g n a l transduction [174]. N e k S H 3 d o m a i n s are i n v o l v e d i n a c t i v a t i n g the I N K and p 3 8 M A P k i n a s e cascades [175], and C r k - L S H 3 d o m a i n s are i n v o l v e d i n a c t i v a t i n g E R K and I N K p a t h w a y [176]. Therefore, p h o s p h o r y l a t i o n o f S O C S 3 causes m o d u l a t i o n i n the r e g u l a t i o n o f proteins that interact w i t h S O C S 3 protein, and thus affecting the d o w n s t r e a m events. H e n c e it is p o s s i b l e that I L - 1 0 i n d u c e s p h o s p h o r y l a t i o n o f S O C S 3 protein, w h i c h a l l o w s S O C S 3 p r o t e i n to interact and i n h i b i t proteins that effect transcription and translation o f i n f l a m m a t o r y mediators, s u c h as T N F - a and N O . I n h i b i t i o n o f T N F - a p r o d u c t i o n b y I L - 1 0 has been attributed to effects o n N F K B a c t i v a t i o n [88], M A P K s i g n a l l i n g p a t h w a y [94], rate o f t r a n s c r i p t i o n [177], m R N A s t a b i l i t y [142], and translational e f f i c i e n c y [94]. W e n o w s h o w that T N F - O ! p r o t e i n p r o d u c t i o n and m R N A e x p r e s s i o n w a s i n h i b i t e d b y I L - 1 0 i n a S O C S 3 - d e p e n d e n t manner, but o n l y up to 2 hours post s t i m u l a t i o n ( F i g . 11, p a n e l A and F i g . 12, r e s p e c t i v e l y ) . W e d i d not see a n y requirement for S O C S 3 p r o t e i n at 4 hours post s t i m u l a t i o n ( F i g . 11, panel B ) , so w e c o n c l u d e that p r i o r to 2 hours, I L - 1 0 i n h i b i t s L P S s i g n a l l i n g i n S O C S 3 - d e p e n d e n t manner, but after 2 hours there m a y be other Stat3-dependent or independent p a t h w a y s that c o m e into p l a y .  62  4.1.  SH2 domain of SOCS3 protein is important in inhibition of TNF-a protein production and mRNA expression by IL-10. I L - 1 0 requires the S H 2 d o m a i n o f S O C S 3 p r o t e i n for i n h i b i t i o n o f L P S - i n d u c e d T N F - a  p r o t e i n p r o d u c t i o n ( F i g . 14) and m R N A e x p r e s s i o n ( F i g . 15f). P o s s i b l e m e c h a n i s m s b y w h i c h this m a y happen is d i s c u s s e d b e l o w . A l t h o u g h S O C S 1 b i n d s d i r e c t l y to the a c t i v a t i o n l o o p o f Jaks through its S H 2 d o m a i n , the S H 2 d o m a i n o f S O C S 3 b i n d s to c y t o k i n e receptor. S H 2 d o m a i n o f C I S stably associates w i t h the t y r o s i n e - p h o s p h o r y l a t e d c y t o p l a s m i c part o f E P O and I L - 3 receptors [178]. Y 4 0 1 o f E P O receptor is an essential t y r o s i n e site for b o t h S O C S 3 - S H 2 d o m a i n b i n d i n g and Stat5 a c t i v a t i o n , suggesting that S O C S 3 i n h i b i t s a c t i v i t y o f Stat5 t h r o u g h its S H 2 d o m a i n [146]. M u t a t i o n i n the S H 2 d o m a i n enhances the L I F - d e p e n d e n t Stat3 and E P O dependent Stat5 transcriptional a c t i v i t y [165]. T h e S H 2 d o m a i n o f S O C S 3 has b e e n s h o w n to b i n d to Y 7 5 7 / 7 5 9 o f g p l 3 0 , Y 9 8 5 o f the l e p t i n receptor, and Y 4 0 1 o f the E P O receptor, s o m e o f w h i c h are the same b i n d i n g sites for S H 2 - c o n t a i n i n g tyrosine phosphatase 2 ( S H P 2 ) [83, 158, 159, 179]. S i n c e S O C S 3 i n h i b i t s g p l 3 0 s i g n a l l i n g and S H P 2 p r o m o t e s g p l 3 0 s i g n a l l i n g t h r o u g h a c t i v a t i o n o f m i t o g e n - a c t i v a t e d p r o t e i n kinases, thus it is p o s s i b l e that S O C S 3 suppresses aspects o f g p l 3 0 s i g n a l l i n g b y c o m p e t i n g w i t h S H P 2 for receptor b i n d i n g . H e n c e , i n S O C S 3 " 7  m a c r o p h a g e s reconstituted w i t h S H 2 d o m a i n mutant, it is l i k e l y that I L - 1 0 is unable to i n h i b i t T N F - a p r o t e i n ( F i g . 14) and m R N A e x p r e s s i o n ( F i g . 15f), because loss o f S H 2 d o m a i n o f S O C S 3 m a y a l l o w for S H 2 d o m a i n o f S H P 2 or s o m e other S H 2 - c o n t a i n i n g p r o t e i n to b i n d L P S s i g n a l l i n g targets s u c h as J N K , and p r o m o t e transcription o f T N F - a and subsequent T N F - a p r o t e i n e x p r e s s i o n b y targeting other M A P K s .  63  4.2.  KIR domain of S O C S 3 protein is important in inhibition of T N F - a protein production by IL-10. T h e K I R d o m a i n o f S O C S 3 p r o t e i n is important for i n h i b i t i o n o f L P S - i n d u c e d T N F - a  p r o t e i n p r o d u c t i o n b y I L - 1 0 ( F i g . 14). In other systems, S O C S 3 has been s h o w n to b i n d to the t y r o s i n e - p h o s p h o r y l a t e d peptide Y 7 5 9 o f I L - 6 receptor ( g p l 3 0 ) , t h r o u g h its S H 2 d o m a i n , w h i c h brings its K I R d o m a i n i n p r o x i m i t y to J a k 2 and causes its i n h i b i t i o n . S O C S 3 has been s h o w n to s p e c i f i c a l l y b i n d to tyrosine 1007 i n the a c t i v a t i o n l o o p o f J a k 2 , the p h o s p h o r y l a t i o n o f w h i c h is r e q u i r e d for its activation. T h u s , S O C S 3 m i g h t i n h i b i t Jak k i n a s e a c t i v i t y t h r o u g h the pseudosubstrate, K I R , b y r e c r u i t i n g and b i n d i n g to a c r i t i c a l p h o s p h o - t y r o s i n e at the intracellular part o f a c y t o k i n e receptor. M u t a t i o n i n the K I R d o m a i n enhances the L I F dependent Stat3 and E P O - d e p e n d e n t Stat5 transcriptional a c t i v i t y [165]. A p o i n t m u t a t i o n i n the K I R d o m a i n o v e r c o m e s the i n h i b i t o r y effect o f b o t h S O C S 3 and J A B p r o t e i n and transgenic m i c e expressing S O C S 3 mutated i n K I R d o m a i n s h o w m o r e potent Stat3 a c t i v a t i o n and a m o r e severe c o l i t i s i n d u c e d w i t h dextran sulphate s o d i u m ( D S S ) [180]. It is p o s s i b l e that i n S O C S 3 " : 7  W T macrophages, K I R d o m a i n o f S O C S 3 is able to i n d u c e a translational suppression o f T N F a p r o t e i n b y i n h i b i t i n g an upstream kinase a c t i v i t y , but m a y be K I R d o m a i n ' s r o l e is not extended to transcriptional regulation, w h i c h w o u l d e x p l a i n w h y i n S O C S " : K I R , i n h i b i t i o n o f 7  T N F - a p r o t e i n p r o d u c t i o n ( F i g . 14), but and not T N F - a m R N A e x p r e s s i o n ( F i g . 15g) b y I L - 1 0 is d i s a b l e d . T h u s one potential target is the p38 M A P K p a t h w a y w h i c h has been s h o w n to be responsible for translational c o n t r o l o f T N F - a p r o t e i n [93].  64  4.3.  SOCS-box domain is important in inhibition of TNF-a protein production and mRNA expression by IL-10. I L - 1 0 requires S O C S - b o x d o m a i n o f S O C S 3 p r o t e i n for i n h i b i t i o n o f L P S - i n d u c e d T N F -  a p r o t e i n p r o d u c t i o n ( F i g . 14) and m R N A e x p r e s s i o n ( F i g . 15e). I n a d d i t i o n to interference w i t h kinase a c t i v i t y and b i n d i n g to other s i g n a l l i n g m o l e c u l e s , S O C S c a n also target associated proteins t h r o u g h i n t e r a c t i o n w i t h the S O C S - b o x for proteasome-mediated degradation. T h e S O C S - b o x d o m a i n has no catalytic a c t i v i t y and mediates p h y s i o l o g i c a l effects o f S O C S 3 t h r o u g h p r o t e i n - p r o t e i n interactions [181]. A m o d e l is p r o p o s e d w h e r e the target m o l e c u l e s c o n t a i n i n g p h o s p h o - t y r o s i n e m i g h t b e c o m e a substrate o f the p r o t e o l y t i c m a c h i n e r y b y b i n d i n g to S O C S : after b i n d i n g o f S O C S , the S O C S - b o x acts as a n adaptor m o l e c u l e , b r i n g i n g into its c o m p l e x E l o n g i n B C [160, 163]. T h e target p r o t e i n is then u b i q u i t i n a t e d through recruitment o f the E 3 ligase. D u r i n g the subsequent p r o t e o l y t i c association, the substrate and the associated S O C S proteins m a y be destroyed, and the c e l l is r e a d y to r e s p o n d o n c e a g a i n i f the s t i m u l i are s t i l l present [163]. E v i d e n c e exists that Jaks c a n be i m m u n o p r e c i p i t a t e d i n a c o m p l e x c o n t a i n i n g S O C S 1 , E l o n g i n B and E l o n g i n C [162]. It has been s h o w n that I L - 6 - i n d u c e d e x p r e s s i o n o f S O C S 3 is sustained i n the presence o f the proteasome i n h i b i t o r , L L n L , i m p l y i n g that S O C S 3 p r o t e i n m a y be r a p i d l y targeted for p r o t e a s o m a l degradation s o o n after its i n d u c t i o n [163]. In support o f these results, u s i n g a n t i - u b i q u i t i n antibodies, it has been s h o w n that a posttranslational m o d i f i e d f o r m o f C I S p r o t e i n ( - 3 7 k D a ) exists i n a d d i t i o n to the p r e d i c t e d i n v i t r o translated p r o t e i n size o f C I S ( - 3 3 k D a ) . T h i s 37 k D a f o r m accumulates i n the presence o f the proteasome i n h i b i t o r s , L L n L and lactacystin, but r a p i d l y degrades w h e n the p r o t e i n synthesis is b l o c k e d b y c y c l o h e x a m i d e [162, 178, 182]. S O C S p r o t e i n e x p r e s s i o n c a n also i n h i b i t E P O  65  receptor and Stat5 p h o s p h o r y l a t i o n , w h i c h is not seen i n the presence o f the proteasome i n h i b i t o r s i n d i c a t i n g proteasome i n v o l v e m e n t i n i n a c t i v a t i o n o f b o t h E P O - r e c e p t o r and Stat5 [182]. S i n c e S O C S - b o x has been s h o w n to be i n v o l v e d i n b o t h s t a b i l i z i n g and d e g r a d i n g its interacting m o l e c u l e s , it is p o s s i b l e that it m a y serve to s t a b i l i z e the IKB protein, thus i n h i b i t i n g N F K B p a t h w a y . I n a l v e o l a r macrophages, I L - 1 0 stabilizes IKB p r o t e i n b y d e l a y i n g its L P S m e d i a t e d degradation a n d r e s u l t i n g i n d e l a y e d n u c l e a r t r a n s l o c a t i o n o f the p 6 5 subunit [139]. It is also p o s s i b l e that S O C S - b o x m a y interact w i t h other L P S s i g n a l l i n g proteins s u c h as p 3 8 M A P K , I N K or I K K and cause their degradation i n response to I L - 1 0 , w h i c h w o u l d also result i n i n h i b i t i o n o f N F K B p a t h w a y ( i n v o l v e d i n t r a n s c r i p t i o n o f T N F - a and i N O S ) . T h i s m a y e x p l a i n the loss o f i n h i b i t i o n at T N F - a : p r o t e i n p r o d u c t i o n ( F i g . 14) and T N F - a m R N A e x p r e s s i o n ( F i g . 15e) i n macrophages expressing S O C S - b o x mutant. B u t w e do not observe i n h i b i t i o n o f i N O S p r o t e i n e x p r e s s i o n b y I L - 1 0 i n macrophages e x p r e s s i n g S O C S - b o x mutant, p o s s i b l y due to different m e c h a n i s m s c o m i n g into p l a y at different times. M a c r o p h a g e s w e r e s t i m u l a t e d w i t h L P S + I L - 1 0 for 2 hours for T N F - a experiments, w h i l e c e l l s w e r e stimulated for 2 4 hours for the i N O S p r o t e i n i m m u n o b l o t . F o r i n h i b i t i o n o f T N F - a p r o t e i n i n response to L P S , the role o f S O C S - b o x m a y be c r i t i c a l for suppression o f the targets o f I L - 1 0 , but for i n h i b i t i o n o f i N O S p r o t e i n e x p r e s s i o n , S O C S - b o x d o m a i n m a y o n l y p l a y a secondary role, w h i c h c a n be compensated for b y the other d o m a i n s w h e n S O C S - b o x d o m a i n is i n a c c e s s i b l e to I L - 1 0 .  4.4.  Y204 and Y221 of SOCS3 are important in inhibition of NO production and iNOS protein expression by IL-10. N O mediates the a b i l i t y o f macrophages to k i l l or i n h i b i t the g r o w t h o f t u m o u r c e l l s ,  bacteria, fungi and parasites [45]. B u t this r e g u l a t i o n must be t i g h t l y c o n t r o l l e d since unregulated N O p r o d u c t i o n causes f a l l i n b l o o d pressure ( v a s o d i l a t i o n ) , w h i c h p o t e n t i a l l y  66  results i n i n j u r y to host tissue. H e r e w e s h o w that I L - 1 0 i n h i b i t s N O p r o d u c t i o n i n S O C S 3 dependent m a n n e r and o n l y requires Y 2 0 4 a n d Y 2 2 1 o f S O C S 3 p r o t e i n to be intact ( F i g . 16). W e further e x a m i n e d the effect o f S O C S 3 i n response to I L - 1 0 o n i N O S , the e n z y m e that is r e s p o n s i b l e for p r o d u c t i o n o f N O . I L - 1 0 i n h i b i t i o n o f i N O S p r o t e i n e x p r e s s i o n reflected the results obtained for N O p r o d u c t i o n . i N O S p r o t e i n e x p r e s s i o n w a s i n h i b i t e d b y I L - 1 0 i n S O C S 3 dependent m a n n e r and o n l y r e q u i r i n g the Y 2 0 4 a n d Y 2 2 1 o f S O C S 3 p r o t e i n ( F i g . 17). T h i s o b s e r v a t i o n is supported b y another study c a r r i e d out b y O ' F a r r e l l et a l , w h e r e they have demonstrated that I L - 1 0 i n h i b i t s N O p r o d u c t i o n and i N O S p r o t e i n e x p r e s s i o n i n a Stat3dependent m a n n e r [127].  Interestingly I L - 1 0 is able to i n h i b i t N O p r o d u c t i o n and i N O S p r o t e i n e x p r e s s i o n w h e n the entire S O C S - b o x d o m a i n is deleted ( S O C S 3 " : S O C S - b o x ) , but not w h e n there is a 7  p o i n t m u t a t i o n i n either o f the t w o tyrosine residues, 2 0 4 ( S O C S 3 " : Y 2 0 4 F ) and 221 ( S O C S 3 " " : 7  7  Y 2 2 1 F ) , l o c a t e d i n the S O C S - b o x d o m a i n ( F i g . 16 and F i g . 17). T h i s interesting o b s e r v a t i o n c o u l d be e x p l a i n e d b y the f o l l o w i n g m o d e l : w h e n the S O C S - b o x d o m a i n is deleted, S O C S 3 is not able to interact w i t h E l o n g i n B / C c o m p l e x and target proteins for u b i q u i t i n a t i o n , but the other d o m a i n s o f S O C S 3 ( S H 2 / K I R d o m a i n s ) are s t i l l capable o f c a r r y i n g out i n h i b i t o r y effects o n p r o t e i n X ( i n the L P S s i g n a l i n g p a t h w a y ) . F o r instance, S O C S 3 p r o t e i n c a n i n h i b i t the a s s o c i a t i o n o f p r o t e i n X w i t h other m o l e c u l e s b y interacting w i t h the p h o s p h o - t y r o s i n e s o f p r o t e i n X t h r o u g h its S H 2 d o m a i n , or i f p r o t e i n X is a k i n a s e , then S O C S 3 c a n suppress its k i n a s e a c t i v i t y v i a the K I R d o m a i n . H o w e v e r , i n S O C S 3 " : Y 2 0 4 F and S O C S 3 " : Y 2 2 1 F /  7  m a c r o p h a g e s , S O C S - b o x d o m a i n is intact and c a n s t i l l associate w i t h E l o n g i n B / C c o m p l e x , but S O C S 3 cannot be p h o s p h o r y l a t e d b y I L - 1 0 since there is p o i n t m u t a t i o n i n either Y 2 0 4 or Y221.  T h u s w h e n S O C S 3 / E l o n g i n B C c o m p l e x interacts w i t h a target p r o t e i n X, it m a y  67  enhance the stability of protein X rather than degrading it becauseY204/Y221 cannot be phosphorylated by IL-10 and thus cannot be targeted for ubiquitination. Thus, when SOCS3 protein interacts with Elongin B/C and there is no "degradation signal" (ie. phosphorylation by IL-10), then this interaction serves to stabilize the SOCS3 protein and its associated protein. As well, this makes the other domains of SOCS3 protein unavailable and inaccessible for other inhibitory mechanisms, since they are in complex with Elongin B/C. Therefore we observe complete loss of inhibitory effects of IL-10 on N O production and iNOS protein expression in SOCS3" ": Y204F and SOCS3" : Y221F SOCS3, but not when SOCS-box domain is deleted. 7  A  68  4.5. Summary In this study we have successfully demonstrated the importance of SOCS3 protein in mediating the anti-inflammatory action of IL-10, which is also supported by the SOCS3 overexpression study [3]. Our results also show that inhibition of TNF-a and NO production by IL10 is mediated by different domains of SOCS3 protein, which suggests that there are different mechanisms that come into play in response to IL-10 (Fig. 18). Table 1 summarized the requirement for different SOCS3 domains in mediating IL-10 inhibition of TNF-a protein production and mRNA expression, as well as NO production and iNOS protein expression.  Table 1. Summary of the requirement for different SOCS3 domains in mediating IL-10 inhibition of various macrophage responses. SOCS3 domain  TNF-a protein production  TNF-a mRNA  NO production  iNOS protein expression  KIR  Yes  No  No  No  SH2  Yes  Yes  No  No  SOCS-box  Yes  Yes  No  No  Y204  Yes  Yes  Yes  Yes  Y221  Yes  Yes  Yes  Yes  69  Figure 18. Schematic representation of the mechanism by which IL-10 may be inhibiting L P S signaling pathway. I L - 1 0 induces S O C S 3 protein expression, w h i c h then inhibits L P S signaling at multiple levels b y different domains. I L - 1 0 requires S H 2 domain, S O C S - b o x domain, Y 2 0 4 and Y 2 2 1 o f S O C S 3 protein to inhibit T N F - a m R N A expression possibly b y interfering w i t h N F K B protein activation. S O C S 3 requires a l l its domains for inhibition o f T N F - a protein release b y I L - 1 0 , possibly b y interfering either w i t h the M A P K s , or IKB protein stability or receptor tyrosine activity. I L - 1 0 requires Y 2 0 4 and Y 2 2 1 o f S O C S 3 protein inhibition o f i N O S protein expression b y I L - 1 0 , w h i c h is possibly either through inhibition o f I L K pathway or N F K B pathway, and ultimately inhibition o f i N O S protein expression leads to inhibition o f N O production. f o r  70  4.6  O t h e r Studies  O u r hypothesis that S O C S 3 is i n v o l v e d i n the a n t i - i n f l a m m a t o r y a c t i o n o f I L - 1 0 disagrees w i t h c o n c l u s i o n s reached b y L e e and C h a u [131]. T h e y o b s e r v e d n o effect o f S O C S 3 d e l e t i o n (through antisense o l i g o n u c l e o t i d e s ) o n the a b i l i t y o f I L - 1 0 to i n h i b i t T N F - a p r o d u c t i o n . H o w e v e r , their e x p e r i m e n t a l system differs f r o m ours i n that they a d d I L - 1 0 to c e l l s 4 hours p r i o r to L P S c h a l l e n g e . W e s h o w that I L - 1 0 requires S O C S 3 o n l y i n the e a r l y phase (< 2 hours) o f i n h i b i t i o n o f T N F - a p r o t e i n p r o d u c t i o n . In their s y s t e m another I L - 1 0 - i n d u c e d gene c a l l e d h e m e oxygenase-1 ( H O - 1 ) appears to be central to m e d i a t i n g m a c r o p h a g e d e a c t i v a t i o n . H O - 1 is not i n d u c e d u n t i l 3 hours after I L - 1 0 s t i m u l a t i o n and m a x i m a l l e v e l s are not a c h i e v e d u n t i l 2 4 hours; w h i c h is w h y they chose to add I L - 1 0 four hours p r i o r to L P S s t i m u l a t i o n . W e and others [3, 94] have c h o s e n a s y s t e m w h e r e I L - 1 0 and L P S are added s i m u l t a n e o u s l y since the p h y s i o l o g i c a l target o f I L - 1 0 is the activated m a c r o p h a g e . T h e r e s t i n g m a c r o p h a g e is different f r o m the L P S activated m a c r o p h a g e , and it m i g h t be reasonable to expect different m e c h a n i s m s to c o m e into p l a y under different circumstances. I n contrast, studies carried out b y J u n g et a l s h o w e d that a l t h o u g h H O - 1 gene is upregulated b y I L - 1 0 ( i n D N A array), a s p e c i f i c H O - 1 i n h i b i t o r z i n c - p r o t o p o r p h y r i n ( Z n P P ) w a s not able to abrogate I L - 1 0 i n h i b i t i o n o f L P S i n d u c e d T N F - a p r o t e i n p r o d u c t i o n [183]. T h i s suggests that other I L - 1 0 regulated genes are m e d i a t i n g the a n t i - i n f l a m m a t o r y effects o f I L - 1 0 . O u r d e m o n s t r a t i o n o f a r o l e o f S O C S 3 i n I L - 1 0 i n h i b i t i o n o f m a c r o p h a g e f u n c t i o n is also i n apparent contrast to the results obtained b y H . Y a s u k a w a et a l , w h e r e they have s h o w n that w h e n S O C S 3 gene is disrupted i n m a c r o p h a g e s , there is n o affect o n I L - 1 0 ' s a b i l i t y to i n h i b i t T N F - a as c o m p a r e d to S O C S 3 w i l d - t y p e macrophages [173]. O n c e again, their s y s t e m  71  is v e r y different, as they treated their cells w i t h L P S + I L - 1 0 for d u r a t i o n o f 5 to 25 hours and then e x a m i n e d for affect o n T N F - a p r o d u c t i o n . A s w e have s h o w n , I L - 1 0 does not require S O C S 3 to i n h i b i t T N F - a p r o d u c t i o n d u r i n g the late phase o f s i g n a l i n g (> 2 hours) ( F i g . 11, p a n e l B ) and m R N A e x p r e s s i o n (data not s h o w n ) . T h u s S O C S 3 p r o t e i n is important d u r i n g the e a r l y i n h i b i t o r y a c t i o n o f I L - 1 0 o n T N F - a e x p r e s s i o n b y a m a c r o p h a g e , w h i l e other r e g u l a t o r y m e c h a n i s m s c o m e into p l a y at later times. H o w e v e r , i n support o f a role for S O C S 3 i n I L - 1 0 i n h i b i t i o n o f i n f l a m m a t i o n S u z u k i et al have demonstrated that interference w i t h S O C S 3 function i n a transgenic m o u s e enhanced the m o u s e ' s s u s c e p t i b i l i t y to intestinal i n f l a m m a t i o n suggesting that the a b l a t i o n o f S O C S 3 does i n d e e d interfere w i t h the n o r m a l negative r e g u l a t o r y a c t i o n o f I L - 1 0 i n c o n t r o l o f c o l o n i n f l a m m a t i o n [180].  72  CHAPTER 5 : Future Directions  O n e o f the p r i o r i t i e s i n further understanding the m e c h a n i s m b y w h i c h S O C S 3 mediates the a n t i - i n f l a m m a t o r y a c t i o n o f I L - 1 0 is to i d e n t i f y the proteins w h i c h interact w i t h the v a r i o u s d o m a i n s o f S O C S 3 . T h e effect o f S O C S 3 c o u l d be e x a m i n e d i n response to I L - 1 0 o n a c t i v i t y o f L P S s i g n a l l i n g m o l e c u l e s s u c h as I L K , I K K , I K B , and P K B . w i l l m a k e use o f glutathione-S-transferase  Studies w i l l be d e s i g n e d w h i c h  (gst) f u s i o n proteins c o n t a i n i n g the different regions  o f S O C S 3 (for e x a m p l e , S O C S - b o x d o m a i n , S H 2 d o m a i n and the N - t e r m i n a l d o m a i n w h i c h i n c l u d e s the 22 a m i n o a c i d K I R d o m a i n . I n case o f the K I R d o m a i n , the candidate p r o t e i n c o u l d be a k i n a s e , s u c h as the src tyrosine kinase f a m i l y m e m b e r L y n , w h i c h is activated b y L P S and i n h i b i t e d b y I L - 1 0 i n m o n o c y t e s [184]. A s w e l l , the tyrosine p h o s p h o r y l a t i o n sites 204 a n d 221 located i n the S O C S - b o x d o m a i n are f o u n d to be important i n supporting I L - 1 0 i n h i b i t i o n o f T N F - a p r o d u c t i o n and m R N A e x p r e s s i o n , as w e l l as N O p r o d u c t i o n and i N O S p r o t e i n expression. E x p e r i m e n t s w i l l be d e s i g n e d to i d e n t i f y the proteins that associate w i t h these p h o s p h o t y r o s y l sites u s i n g b i o t i n y l a t e d p h o s p h o p e p t i d e c o r r e s p o n d i n g to the relevant tyrosine p h o s p h o r y l a t i o n site as the affinity reagent. T h e b i o t i n y l a t e d phosphopeptide w i l l be p u l l e d d o w n u s i n g streptavidin agarose and the associated proteins w i l l be v i s u a l i z e d b y s i l v e r staining. I f the bands cannot be i d e n t i f i e d t h r o u g h a k n o w n antibody, then proteins w i l l be p u r i f i e d for m i c r o s e q u e n c e analysis. O n c e i d e n t i f i e d , the interaction o f these k n o w n or n o v e l proteins w i t h intact S O C S 3 p r o t e i n c a n be c o n f i r m e d b y c o - i m m u n o p r e c i p i t a t i o n studies and their role i n I L - 1 0 s i g n a l l i n g tested b y R N A i - m e d i a t e d k n o c k - d o w n or o v e r - e x p r e s s i o n studies. F i n a l l y u s i n g m i c r o - a r r a y analysis, w e  73  c o u l d identify other L P S - i n d u c e d genes that are i n h i b i t e d b y I L - 1 0 i n a S O C S 3 - d e p e n d e n t manner.  74  Concluding remarks W e have d e f i n i t i v e l y s h o w n the importance o f S O C S 3 p r o t e i n i n the a n t i - i n f l a m m a t o r y a c t i o n o f I L - 1 0 and also have s h o w n that i n h i b i t i o n o f N O and T N F - a b y I L - 1 0 is m e d i a t e d b y different d o m a i n s o f S O C S 3 protein, w h i c h suggests that there are different m e c h a n i s m s that c o m e into p l a y i n response to I L - 1 0 . T h i s is the first d e m o n s t r a t i o n o f alternate m e c h a n i s m s o f a c t i o n o f S O C S 3 p r o t e i n o n divergent p a t h w a y s activated b y the same s t i m u l i . 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