MECHANISM OF DELAYED HYPERSENSITIVITY REACTIONS In V i t r o and In Vivo Studies of the Possible Role of Certain Lymphokines i n the Development of Delayed Hype r s e n s i t i v i t y Reactions by FOOK CHUEN WONG L B. Sc., National Taiwan University, 1961 M. Sc., University of B r i t i s h Columbia, 1970 A THESIS SUBMITTED IN THE REQUIREMENTS DOCTOR OF PARTIAL FULFILLMENT OF FOR THE DEGREE OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES The Department of Pathology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1977 © Fook Chuen Wong, 1977 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis . for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pathology The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date Feb. 7, 1978 ABSTRACT The aim of the present study was to determine how lympho-kines could exert t h e i r b i o l o g i c a l action on the skin during the course of the delayed h y p e r s e n s i t i v i t y reactions. Four groups of experiments were conducted to inves t i g a t e : ( 1 ) The production and the separation of lymphokines, ( 2 ) the protease a c t i v i t y of lympho-kines and the e f f e c t of lymphokines on the kinin-forming system, ( 3 ) the e f f e c t of lymphokines on mast c e l l s and p l a t e l e t s , and ( 4 ) the e f f e c t of enzyme-treated lymphokines on the skin inflammatory reaction. Guinea pig lymph node lymphocytes, stimulated by either the s p e c i f i c antigen DNP-BGG or by concanavalin A, were used to generate lymphokines. Parameters for te s t i n g lymphokine a c t i v i t i e s were those of migration i n h i b i t o r y factor (MIF) and skin reactive factor (SRF). Separation of MIF and SRF from the lymphokine preparation by gel f i l t r a t i o n , electrophoresis and f r a c t i o n a l p r e c i p i t a t i o n with ammonium sulphate was unsuccessful, which indicated that the physi-cal properties of MIF and SRF were s i m i l a r . Lymphokine preparations contained l i t t l e or no neutral and a c i d i c protease a c t i v i t i e s . K i n i n was not released from fresh plasma when incubated with lympho-kines and lymphokines did not activate Hageman factor and p r e k a l l i -k r e i n d i r e c t l y , i n d i c a t i n g that lymphokines did not contain any detectable activator of the kinin-forming system. Lymphokine did not cause the release of histamine from the mast c e l l i n v i t r o , and did not induce p l a t e l e t aggregation. Insoluble gel-enzymes were chosen for the treatment of lymphokines because they could be completely removed by c e n t r i f u -gation and f i l t r a t i o n . MIF and SRF a c t i v i t i e s were completely destroyed by the incubation with chymotrypsin-agarose and thus confirmed that MIF and SRF are proteins. Neuraminidase-agarose abolished the MIF a c t i v i t y , i n d i c a t i n g that the terminal s i a l i c acid residues were necessary for MIF a c t i v i t y and that MIF i s a glycoprotein. However, neuraminidase-agarose treated lymphokine retained most of i t s skin r e a c t i v i t y , suggesting that the skin inflammatory reaction induced by lymphokines i s not mainly due to MIF. No experiment provided conclusive evidence as to whether MIF and SRF a c t i v i t i e s were due to d i f f e r e n t substances, or were due to one substance with d i f f e r e n t a c t i v i t i e s . - i v -TABLE OF CONTENTS Page TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES ix LIST OF APPENDICES x LIST OF FIGURES x i ABBREVIATIONS xiv ACKNOWLEDGEMENTS xv INTRODUCTION 1 LITERATURE REVIEW 6 I CHARACTERISTICS OF DELAYED-TYPE HYPERSENSITIVITY 6 II LYMPHOKINES - THE MEDIATORS OF DELAYED-TYPE HYPERSENSITIVITY 9 1. Lymphokine Production _n V i t r o 9 ( i ) C e l l types for the production of lymphokine... 10 ( i i ) In v i t r o lymphocyte ac t i v a t o r for release of lymphokines 13 2. Nature of Lymphokines 14 3. Ch a r a c t e r i s t i c s of the Lymphokines 16 ( i ) Migration Inhibitory Factor (MIF) 16 ( i i ) Chemotactic Factor (CF) 20 ( i i i ) Lymphotoxin (LT) 21 (i v ) Macrophage a c t i v a t i n g factor (MAF) 23 (v) Mitogenic factor (MF) 23 (vi) Skin reactive factor (SRF) 24 4. Lymphokine Production In Vivo 26 III PROBLEMS IN CORRELATION OF IN VITRO LYMPHOKINE ACTIVITY WITH DELAYED-TYPE HYPERSENSITIVITY IN VIVO 27 1. MIF and SRF 27 2. SRF and Plasma Mediator-producing Systems 28 - v -Page MATERIALS AND METHODS 30 I PREPARATION OF LYMPHOKINES 30 1. Animals 30 2. Preparation of Antigen 31 3. Mitogen 31 4. Preparation of Animals 31 a. S e n s i t i z a t i o n with DNP-BGG 31 b. S e n s i t i z a t i o n with Freund's Adjuvant .31 5. Culture Medium 32 6. Lymphocyte Preparation 32 7. P u r i f i c a t i o n of Lymphocytes from LN C e l l s 32 II LYMPHOKINE PRODUCTION IN VITRO 34 1. S p e c i f i c Antigen A c t i v a t i o n 34 2. Non-specific A c t i v a t i o n of Lymphocytes 34 III SEPARATION OF LYMPHOKINES 36 1. Fractionation of Sephadex G-100 36 2. Polyacrylamide Gel Electrophoresis 37 3. Salt F ractionation of Lymphokines 39 IV ASSAYS FOR LYMPHOKINES.. 40 1. Skin Inflammatory Test for SRF A c t i v i t y 40 a. By Measuring Erythema 40 b. By Measuring the Increase i n Vascular Permeability 41 2. I n h i b i t i o n of Macrophage Migration Test for MIF A c t i v i t y 41 3. Histology 44 V PROTEOLYTIC ACTIVITY IN LYMPHOKINE SUPERNATANTS 45 1. Acid Protease A c t i v i t y 45 2. Neutral Protease A c t i v i t y 45 VI LYMPHOKINES EXPOSED TO HAGEMAN FACTOR, PREKALLIKREIN OR FRESH PLASMA 46 1. Separation of Hageman Factor and P r e k a l l i k r e i n 46 a. Globulin F r a c t i o n 47 b. Anionic exchange chromatography 47 - v i -Page 2. P r e k a l l i k r e i n Assay 48 3. Assay for Hageman Factor 50 4. Further P u r i f i c a t i o n of P r e k a l l i k r e i n 51 a. Rechromatography on DEAE-Sephadex (DEAE-II) 51 b. CM-Sephadex C-50 cation exchange chromatography.. 51 5. Further P u r i f i c a t i o n of Hageman Factor 52 6. Lymphokine Exposed to HF and P r e k a l l i k r e i n 52 7. Bioassay for Ki n i n Release from Fresh Plasma Exposed to the Lymphokines 52 VII EFFECT OF LYMPHOKINES ON MAST CELLS 54 1. E f f e c t of Lymphokine on Mesenteric Mast C e l l s of Guinea Pigs 54 2. E f f e c t of Lymphokine on Isolated Mast C e l l s of the Rat 54 VIII EFFECT OF LYMPHOKINES ON PLATELETS 56 IX ENZYME TREATMENT OF LYMPHOKINE PREPARATIONS 57 1. Preparation of Immobilized Enzymes 57 a. Preparation of Chymotrypsin-agarose 57 b. Preparation of Clostridium Perfringens Neuraminidase-agarose 58 c. Preparation of Control Neuraminidase-agarose 59 d. Determination of enzymatic a c t i v i t i e s 59 ( i ) Chymotrypsin-agarose 59 ( i i ) Neuraminidase-agarose 60 2. The Treatment of Lymphokines with Enzymes 60 a. Lymphokine Treated with Soluble Neuraminidase.... 60 b. Lymphokine Treated with Chymotrypsin-agarose 62 c. Lymphokine Treated with Neuraminidase-agarose.... 62 RESULTS 64 I PRODUCTION OF LYMPHOKINES 64 II SEPARATION OF LYMPHOKINES 64 1. Gel F i l t r a t i o n 64 2. Polyacrylamide Gel Electrophoresis 65 3. F r a c t i o n a l P r e c i p i t a t i o n with Ammonium Sulphate 65 - v i i -Page III PROTEASE ACTIVITY OF LYMPHOKINE 66 IV LYMPHOKINES EXPOSED TO HAGEMAN FACTOR, PREKALLIKREIN AND FRESH PLASMA 67 1. Separation of P r e k a l l i k r e i n and Hageman Factor 68 2. Lymphokines exposed to Hageman Factor and P r e k a l l i k r e i n 68 3. Biossay for Kinin Release from Fresh Plasma Exposed to Lymphokines 69 V EFFECT OF LYMPHOKINES ON MAST CELLS 69 VI EFFECT OF LYMPHOKINES ON PLATELETS 70 VII ENZYME TREATMENT OF LYMPHOKINES 70 1. Ef f e c t of Soluble Neuraminidase on Lymphokine A c t i v i t y 71 2. E f f e c t of Insoluble Enzyme-Agarose on Lymphokine A c t i v i t y 71 DISCUSSION 74 I PRODUCTION OF LYMPHOKINES 74 II SEPARATION OF LYMPHOKINES 76 1. Gel F i l t r a t i o n 76 2. Polyacrylamide Gel Electrophoresis 77 III EFFECTS OF BIOLOGICAL PRODUCTS ON LYMPHOKINE ASSAYS 78 IV PROTEASE ACTIVITY OF LYMPHOKINES 80 V LYMPHOKINES EXPOSED TO FRESH PLASMA, HAGEMAN FACTOR AND PREKALLIKREIN 83 VI EFFECTS OF LYMPHOKINES ON MAST CELLS 85 VII EFFECTS OF LYMPHOKINES ON PLATELETS 86 VIII ENZYME TREATMENT OF LYMPHOKINES 87 1. Chymotrypsin-agarose 87 2. Neuraminidase-agarose 87 - v i i i -Page IX PROBLEMS ON THE STUDIES OF THE MECHANISM OF LYMPHOKINE EFFECTS ON MACROPHAGE 94 X POSSIBLE MECHANISM OF DELAYED HYPERSENSITIVITY REACTIONS - A HYPOTHESIS 96 SUMMARY AND CONCLUSION 101 BIBLIOGRAPHY 105 - ix -LIST OF TABLES TABLE Page 1 Comparison of MIF a c t i v i t y of lymphokine produced by unpurified and p u r i f i e d LN lymphocytes stimu-lated with Con A and DNP-BGG antigen 64a 2 Gel electrophoresis of the Sephadex G-100 fractions from supernatant of LN lymphocytes cultured with Con A 65a 3 Protease a c t i v i t y of lymphokine and lysosomal prepar-ations 66a 4 E f f e c t of b i o l o g i c a l products on lymphokine assays.. 70a 5 E f f e c t of enzyme on lymphokine a c t i v i t y 71a - x -LIST OF APPENDICES APPENDIX Page 1 Analysis of variance of MIF a c t i v i t y to the lymphokine production from unpurified and p u r i f i e d lymphocytes 124 2 Analysis of variance of MIF a c t i v i t y of lympho-kine treated with neuraminidase-agarose 125 0 - x i -LIST OF FIGURES FIGURE Page 1 Instruments for lymphokine studies 32a 2 E l u t i o n pattern of lymphokine supernatant on c a l i -brated Sephadex G-100 column. 64b 3 I n h i b i t i o n of Macrophage migration by each f r a c -t i o n from Sephadex G-100 64c 4 I n h i b i t i o n of macrophage migration by each e l e c t r o -phoretic f r a c t i o n 65b 5 Skin inflammatory reaction at 4 hours after the intradermal i n j e c t i o n of 0.1 ml of macrophage ly s o -somal preparation and lymphokine i n the sk i n of guinea pig 66b 6 Skin inflammatory reaction at 4 hours after the intradermal i n j e c t i o n of human PMN lysosomal preparation i n the sk i n of guinea p i g 67a 7 Chromatographic separation of rabbit plasma globu-l i n s on DEAE-Sephadex A-50 68a > 8 Chromatographic separation on DEAE-sephadex A-50 of fractions containing Hageman factor (pre-PKA) 68b 9 Chromatography on DEAE-Sephadex A-50 of p r e k a l l i k r e i n pooled from the f i r s t DEAE-Sephadex column as shown i n Figure 7 68c 10 Chromatography on CM-Sephadex C-50 of p r e k a l l i k r e i n pooled from the second DEAE-Sephadex column as shown in Figure 9 68d - x i i -FIGURES Page 11 A c t i v a t i o n of Hageman factor by k a o l i n and lympho-kine 68e 12 The release of k i n i n from fresh plasma exposed to lymphokine and k a o l i n 69a 13 The release of histamine from suspension of i s o -l a t e d mast c e l l s of the rats 69b 14 E f f e c t of adding lymphokine and ADP on the o p t i c a l density of p l a t e l e t - r i c h plasma 69c 15 E f f e c t of neuraminidase on the c a p i l l a r y migration of peritoneal macrophages from normal guinea pig . . . 70b 16 Hemorrhagic skin inflammatory reaction at 4 hours a f t e r the intradermal i n j e c t i o n of Cl_. perfringens neuraminidase 70 c 17 Heat s t a b i l i t y of soluble and immobilized neuramini-dase of CI. perfringens 70d 18 Standard curve for the hydrolysis of alpha-casein by chymotrypsin 71a * 19 Standard curve for the hydrolysis of NAN-lactose by the neuraminidase of CI. perfringens 71b 20 MIF and SRF a c t i v i t i e s a f t e r incubation with immo-b i l i z e d agarose-bound enzymes 72a - x i i i -FIGURES Page 21 Skin inflammatory reactions at 4 hours af t e r the intradermal i n j e c t i o n of 0.1 ml of lymphokine and enzyme-treated lymphokines i n the dorsal skin of guinea pig 72b 22 E f f e c t of immobilized enzyme-agarose on lymphokine for macrophage migration i n h i b i t i o n 72c 23 Deep dermis of reaction s i t e at 4 hours af t e r the intradermal i n j e c t i o n of 0.1 ml of the test solutions i n the skin of guinea pig 73a 24 Skin inflammatory reactions at 4 hours after the intradermal i n j e c t i o n of 0.1 ml Con A solution at d i f f e r e n t concentrations i n the skin of guinea pig. 79a 25 . Possible Mechanism of Delayed Hype r s e n s i t i v i t y Reaction 96a - x i v -ABBREVIATIONS BAEe Benzoyl Arginine E t h y l Ester CF Chemotactic Factor CFA Complete Freund's Adjuvant Con A Concanavalin A DFP Diisopropyl Fluorophosphate DH Delayed H y p e r s e n s i t i v i t y DNP-BGG Dinitrophenylated Bovine Gamma-globulin FCS F e t a l Calf Serum HF Hageman Factor LAF Lymphocyte A c t i v a t i n g Factor LBTI Lima Bean Trypsin I n h i b i t o r LN Lymph Node LNPF Lymph Node Permeability Factor LT Lymphotoxin MAF Macrophage A c t i v a t i n g Factor MF Mitogenic Factor MIF Migration I n h i b i t o r y Factor NAN-Lactose N-acetyl-neuramin-lactose PAF P l a t e l e t Aggregation Factor PBS Phosphate Buffer Saline PEC Peritoneal Exudate C e l l s PMN Polymorphonuclear leukocytes PPD P u r i f i e d Protein Derivative of Tuberculin PPP P l a t e l e t Poor Plasma PRP P l a t e l e t Rich Plasma SAS Saturated Ammonium Sulfate SRF Skin Reactive Factor TBS Tris- B u f f e r Saline TCA T r i c h l o r o a c e t i c Acid TEMED Tetramethylethylene Diamine - xv -ACKNOWLEDGEMENTS The author would l i k e to express his sincere gratitude to Dr. W. H. Chase, Professor of Pathology, University of B r i t i s h Columbia, for his continuous guidance throughout the planning and execution of the experiments as well as during the preparation of thi s manuscript. I also wish to thank Drs. R. H. Pearce, J. Levy, and J. P. W. Thomas, the members of the Ph.D. Committee for th e i r encourage-ment and advice throughout the programme. It i s my pleasure to express thanks to Dr. D. F. Hardwick, Professor and Head of the Department of Pathology, Un i v e r s i t y of B r i t i s h Columbia, for providing the f a c i l i t i e s used i n this study. In addition, the members of the fa c u l t y and s t a f f of the Department of Pathology are thanked for providing constant support over the duration of this study. The author i s indebted to the Medical Research Council for f i n a n c i a l support i n the form of a four year studentship. 1 INTRODUCTION The immune response induced by the introduction of foreign substances (antigens) into man and animal takes two basic forms: (1) Humoral immunity, mediated by antibodies which s p e c i f i c a l l y bind with the antigen; and (2) cell-mediated immunity, mediated by " s e n s i t i z e d " lymphocytes. The l a t t e r i s characterized by the delayed h y p e r s e n s i t i v i t y reaction (DH). As an example, when tuberculin i s injected intradermally into a tub e r c u l i n - p o s i t i v e host, a slow inflammatory reaction w i l l develop within 24-28 hours at the s i t e of i n j e c t i o n . M i c r o s c o p i c a l l y , i t i s characterized by a mononuclear c e l l i n f i l t r a t i o n predominantly composed of lymphocytes and c e l l s derived from blood monocytes (Cohen and McCluskey, 1973). Lymphocytes appear to i n i t i a t e the DH reaction and are responsible for i t s immunological s p e c i f i c i t y (Dvorak, 1974). Correlates of the delayed h y p e r s e n s i t i v i t y state are now assayed i _ v i t r o . It can be shown that the i n t e r a c t i o n of " s e n s i t i z e d " lymphocytes with s p e c i f i c antigen y i e l d s soluble mediators (lymphokines) which have several b i o l o g i c a l a c t i v i t i e s . The observation by Dumonde et a l . , (1969) that the i n j e c t i o n of f l u i d s containing these mediators into normal guinea pig skin would e l i c i t a reaction s i m i l a r to the delayed h y p e r s e n s i t i v i t y skin reaction suggested that these substances may pa r t i c i p a t e i n the DH response. 2 Lymphokines may mediate a number of reactions by t h e i r e f f e c t s on other c e l l s or i n t e r a c t i o n with other proteins to form b i o l o g i c a l l y active substances. They include at least the following f a c t o r s : Migration Inhibitory Factor (MIF), Skin Reactive Factor (SRF), Macrophage A c t i v a t i n g Factor (MAF), Chemotactic Factor (CF), Lymphotoxin (LT) and Mitogenic Factor (MF) (David and David, 1972). It i s apparent that many of these substances, by the nature of t h e i r b i o l o g i c a l a c t i v i t i e s , could be involved i n e l i c i t i n g an inflam-matory reaction. However, up t i l l now, no soluble lymphokine product i s a v a i l a b l e i n a highly p u r i f i e d form. Therefore attempts to explain the mechanism of delayed h y p e r s e n s i t i v i t y are only con-j e c t u r a l . It i s not clear to what extent the various lymphokine a c t i v i t i e s are manifestations of the same or r e l a t e d substances. It i s also uncertain whether the lymphokines which are active i n v i t r o have the same a c t i v i t i e s i n the i n t a c t animal since the substances or t h e i r actions may be modified by i n h i b i t o r s and/or "cofactors". Among the lymphokines, MIF i s one of the most thoroughly investigated. It was f i r s t described by Rich and Lewis (1932). This r e l a t i v e l y simple assay has been shown to correlate with DH i n vi v o . However, the precise r o l e of MIF i n DH i s a question of great i n t e r e s t . Bennet and Bloom (1968) showed that i n j e c t i o n of MIF-rich fract i o n s into the skin of normal guinea pigs was followed by the development of an erythematous and indurated reaction which appeared 3 within 3-4 hours. Other workers have obtained si m i l a r results and further, have shown that this skin reactive factor has a molecular weight of 39,000 and could be destroyed by pepsin (Pick _ t a l . , 1969; M a i l l a r d e_ a l . , 1972). MIF i s a glycoprotein with a mole-cular weight of 35,000-50,000 and i s stable after heating to 56°C for 30 minutes (David and David, 1972). Neuraminidase and chymo-tr y p s i n could completely abolish MIF a c t i v i t y (Remold and David, 1971). Nevertheless, i t remains to be shown unequivocally that MIF and not another material i n the supernatant proteins causes the skin reactions. Experiments were undertaken to elucidate the involvement of SRF i n the DH reaction. More s p e c i f i c a l l y , these were designed to determine whether or not SRF can be i s o l a t e d as an separate e n t i t y from the MIF a c t i v i t y . In addition studies were done to determine whether manifestations of the DH reaction are due to the d i r e c t action of lymphokines or rather act through secondary ef f e c t o r s such as p l a t e l e t s or mast c e l l s . F i r s t l y , the SRF and MIF a c t i v i t i e s of the d i f f e r e n t f r a c t i o n s obtained from the Sephadex column and gel electrophoresis were compared. In addition, immobilized neuraminidase and chymo-trypsin-agarose which were prepared by coupling the soluble enzymes to CNBr-activated Sepharose were chosen for enzymatic studies on SRF and MIF a c t i v i t i e s . There were two reasons for using these i n s o l -uble enzymes: 4 (1) S t a b i l i t y of SRF a c t i v i t y to neuraminidase and chymotrypsin has not yet been reported; and (2) they can be completely removed by centrifugation and f i l t r a t i o n after the incubation of lymphokines with the insoluble enzymes. It has been reported that s e n s i t i z e d lymphocytes could release p r o t e o l y t i c enzymes. Havemann et a l . , (1972) demonstrated that human MIF-rich supernatant contained esterase a c t i v i t y and that the MIF a c t i v i t y could be blocked by di i s o p r o p y l fluoro-phosphate (DFP), a serine esterase i n h i b i t o r . This suggested that MIF acts as a protease. Houck et a l . , (1973) determined that lymphokine from mouse lymphocytes as well as lymph node permeability factor (LNPF) possessed cathepsin D-like protease and suggested that both lympho-kine and LNPF had skin reactive a c t i v i t i e s which were due to proteo-l y t i c enzymes. With the appropriate substrates among the normal plasma proteins, the p r o t e o l y t i c enzymes yielded cleavage products with mediator a c t i v i t i e s . David and Becker (1974) found that DFP or other known organophosphorus esterase i n h i b i t o r s did not a f f e c t guinea p i g MIF a c t i v i t y . Thus, experiments which determined whether or not lymphokines produced from guinea p i g lymphocytes have neutral or acid protease a c t i v i t y , and whether the SRF a c t i v i t y could be due to protease were also undertaken i n t h i s study. 5 Pharmacological studies on SRF by M a i l l a r d et a l . , (1972) indicated that SRF a c t i v i t y was i n h i b i t e d by polybrene and protamine s u l f a t e which counteract the a c t i v a t i o n of Hageman factor i n the kinin-forming system. On the other hand, t h i s a c t i v i t y was enhanced by sodium diethyl-dithiocarbamate which i s an i n h i b i t o r of k i n i n -ase. This suggested that SRF may be related to the a c t i v a t i o n of Hageman factor i n the kinin-forming system. The kinin-forming system consists of Hageman factor, p r e k a l l i k r e i n and kininogen (Cochrane and Wuepper, 1971). Activated Hageman factor activates p r e k a l l i k r e i n to form k a l l i k r e i n which, i n turn, cleaves kininogen to produce k i n i n . K i n i n i s a potent mediator of increase i n vas-cular permeability and causes the contraction of guinea pig ileum (Rocha e S i l v a , 1970). In order to determine whether or not guinea pig lymphokine can d i r e c t l y activate the kinin-forming system, p a r t i a l l y p u r i f i e d Hageman factor and p r e k a l l i k r e i n which were i s o l a t e d from rabbit plasma were interacted with the lymphokine. It i s well known that lymphokines can a f f e c t the a c t i v i t y of macrophages, lymphocytes, neutrophils, eosinophils and f i b r o b l a s t s (Cohen, 1976). Since both p l a t e l e t and mast c e l l s are involved i n the inflammatory response (Bloom, 1974; Zucker, 1974), the f i n a l series of experiments i n this study was undertaken to determine whether or not lymphokines can a f f e c t the a c t i v i t i e s of p l a t e l e t s and mast c e l l s . 6 LITERATURE REVIEW I CHARACTERISTICS OF DELAYED-TYPE HYPERSENSITIVITY Immunity to foreign antigens i n man and animals results from at least two types of immune response: (1) Humoral immunity which i s mediated by antibodies, and (2) cell-mediated immunity which i s mediated by the sensi-t i s e d lymphoid c e l l s . The c l a s s i c model for the study of cell-mediated immunity i s the delayed hypersensitivy reaction (DH). The skin tuberculin reaction i s a t y p i c a l example for DH. The c h a r a c t e r i s t i c s of DH have been reviewed previously by many investigators (Turk, 1967; Bloom and Chase, 1967; Cohen and McCluskey, 1973; Cohen, 1977). B r i e f l y , DH can be characterized by the following: (1) The development of delayed skin inflammatory reaction at the s i t e of the i n j e c t i o n of a s p e c i f i c antigen into a previously s e n s i t i z e d i n d i v i d u a l . The maximal response i s reached i n 24-28 hours. 7 (2) The i n f i l t r a t i o n of mononuclear c e l l s which is com-posed predominantly of lymphocytes and macrophages at the test s i t e . (3) The adoptive transfer of the reaction with s e n s i t i z e d lymphoid c e l l s to a normal r e c i p i e n t . (4) The production of several mediator materials (lympho-kines) upon exposure of s e n s i t i z e d lymphoid c e l l s to the s p e c i f i c antigen (Salvin and Neta, 1975). The l a s t two phenomena have been a c t i v e l y investigated i n attempts to elucidate the mechanism of the DH reaction. Chase (1945) f i r s t demonstrated the c e l l u l a r transfer of DH i n the guinea pi g . He found that normal guinea pigs would react to tuberculin when vi a b l e lymphoid c e l l s from s e n s i t i z e d animals were trans-ferred. Heated c e l l s , frozen and thawed c e l l s or blood serum were unable to e f f e c t the transfer. Many laboratories confirm his r e s u l t s (Cummings et. a l . , 1947; Kitcheimer and Weiser, 1947; Stavitsky, 1948; Metaxas and Metaxas-Buehler, 1955). The bulk of the experimental evidence indicates that when l a b e l l e d donor se n s i -t i z e d lymphoid c e l l s are transferred to a normal r e c i p i e n t , only a small number (2-8%) of these l a b e l l e d c e l l s are found i n the spe c i -8 f i c skin reaction s i t e s (Turk, 1962; McClusky et a l . , 1963; Turk and Oort, 1963) while the majority of the c e l l s are of r e c i p i e n t o r i g i n (Cohen et a l . , 1967). It has been proposed that DH reactions are triggered by the l o c a l i n t e r a c t i o n of antigen with an antibody-like receptor on the surface of the s e n s i t i z e d lymphocytes, presumably T c e l l s (Cohen, 1976). These previously s e n s i t i z e d lymphoid c e l l s are postulated to reach the reaction s i t e by a random process (Najarian and Feldman, 1963). The actual mechanism of i n t e r a c t i o n of antigen with se n s i -t i z e d c e l l s and the generation of the DH reaction remain obscure. In the i n v i t r o tissue culture system, the i n t e r a c t i o n of antigen with s e n s i t i z e d lymphocytes re s u l t s i n the release of pharmacological mediators, lymphokines, into the nutrient medium. These mediators may be the factors which are released by s e n s i t i z e d lymphocytes i n the l o c a l skin test s i t e . These factors could be responsible for the l o c a l a t t r a c t i o n of a non-specific population of monocuclear c e l l s as well as for the vascular changes observed (Lawrence and Landy, 1969). 9 II LYMPHOKINES - THE MEDIATORS OF DELAYED-TYPE HYPERSENSITIVITY 1. Lymphokine Production In V i t r o Over the past ten years, studies of DH have s h i f t e d from _n vivo to in v i t r o systems i n an attempt to eliminate the complexities of the i n vivo m i l i e u . The production of lymphokines by lymphocytes as an i n v i t r o correlate of the DH reaction has been reviewed by many investigators (Bloom and Jimenez, 1970; David and David, 1972; Pick and Turk, 1972; Granger, 1972; David, 1975; Cohen, 1976). It has been unequivocably demonstrated in v i t r o that the i n t e r a c t i o n of se n s i t i z e d lymphocytes with antigen produces a va r i e t y of b i o l o g i c a l mediators such as migration i n h i b i t o r y f a c t o r , chemotactic factor, mitogenic factor and skin reactive f a c t o r . Generally, these lymphokine a c t i v i t i e s have been evaluated i n i n v i t r o system. The release of lymphokines from the activatedlymphocytes i s dependent upon the following two conditions: ( i ) ( i i ) types of c e l l s present, and the method of a c t i v a t i o n of the c e l l s . 10 ( i ) C e l l types for the production of lymphokine DH reaction has been generally assumed to be mediated by T lymphocytes (T c e l l s ) and lymphokines are assumed to be T - c e l l products. This assumption comes from the production of MIF by lymphocytes from patients with agammaglobulinemia (Rocklin fit a l . . 1970) and agammaglobulinemic chickens completely devoid of B c e l l s were able to produce chemotactic factor as well as normal chickens i n response to Con A (Altman and Kirchner, 1972).It i s now clear that with the recent development of techniques to i s o l a t e pure populations of T and B c e l l s , both B and T c e l l s can produce various lymphokines including MIF (Yoshida et a l . , 1973; Rocklin et a l . , 1974; Bloom et a l . , 1975), chemotactic factor (Wahl et a l . , 1974; Wahl et a l . , 1975), and mitogenic factor (Mackler et. a l . , 1974; Wahl et a l . , 1975). Rocklin et a l . , (1974, 1975) found that T c e l l s produced MIF and MF, but B c e l l s produce MIF only. Further, Wahl et a l . , (1974) observed that a c t i v a t i o n of B c e l l s to produce chemotactic lymphokine occurred subsequent to i n t e r a c t i o n of B c e l l surface receptors for Ig, Fc and C^ with an t i - I g , antigen-antibody complex and antigen-antibody-C complex, a l l of which f a i l e d to ac t i v a t e T c e l l s . However, Bloom and Shevack (1975) indicated that although MIF could be produced by B and T c e l l s , B c e l l s were incapable of being stimulated by antigen i n the absence of T c e l l s . 11 Recent studies have shown that macrophages as accessory c e l l s may f a c i l i t a t e the development of cell-mediated immunity. There i s evidence that macrophages can enhance the production of MIF (Nelson and Leu, 1975; Ohishi and Onoue, 1975), lymphotoxin (Folch et a l . , 1973), monocyte chemotactic factor (Wahl et a l . , 1974) and macrophage a c t i v a t i o n (Wahl et. a l . , 1975). P r o l i f e r a t i o n of lymphocytes i n response to s p e c i f i c antigens and mitogens has also been shown to be dependent upon the presence of macrophages (Blaese et. a l . , 1972; Waldon e t _ l . , 1973; Nelson and Leu, 1975; Yoshinaga et a l . , 1975; Nelson and Leu, 1975; Yoshinaga e t a l . , 1975; Rosenstreich et al., 1976). Wahl e t a l . , (1975) found that T c e l l s were macrophage dependent i n t h e i r response to thymus-dependent antigens and to T - c e l l mitogen such as Con A for production of chemotactic factor, whereas B - c e l l mitogen and thymic independent antigens such as polymerized f l a g e l l i n are macrophage independent. Osteoclast a c t i v a t i o n factor, produced by PHA.-activated lymphocytes has been found to be macrophage-dependent (Horton et a l . , 1974). Oppenheim and Rosenstreich (1976) described i n t h e i r review that the early a c t i v a t i o n of T c e l l s by both antigens and mitogens required macrophages and very few are required to demonstrate t h i s a c t i v a t i o n . The mechanism by which macrophages exert lymphocyte-activating functions remains unclear. Several d i f f e r e n t mechanisms have been proposed. 1 2 (1) Macrophages may enhance lymphocyte activity by promoting lymphocyte v i a b i l i t y (Chen and Hirsch, 1972; Mosier and Pierce, 1972). (2) Lymphocyte activating factor (IAF) released from macrophages has been found to promote lymphocyte proliferation (Gery and Waksman, 1972; Yoshinaga et a l . , 1975; Diamantstein and Ulmer, 1976; Calderon et_ a l . , 1975). Yoshinaga et a l . , (1975) demonstrated that purified macrophages or culture supernatants following three hours of incubation of macrophages caused a similar enhancement of DNA synthesis by thymocytes. As a result, they suggested that LAF might produce some changes on the thymocyte surface resulting in unmasking of hidden receptor sites or in rearrangement of the ce l l surface to form clusters. Calderon et a l . , (1975) reported similar results and further indicated that LAF had a molecular weight between 15,000 to 21,000 and also increased the plaque-forming c e l l response to both IgG and IgM classes. (3) Macrophages can take up the antigen, retain i t in an immunogenic form and present i t to to the lymphocyte resulting in lymphocyte activation (Unanue, 1972). 13 (4) Macrophages also regulate immune responses by presenting surface-bound antigen i n a more immunogenic fashion to T c e l l s . This function is dependent on d i r e c t macrophage-lymphocyte contact (Oppenheim and Rosenstreich, 1976). Even T - c e l l mitogens, which presumably in t e r a c t d i r e c t l y with T c e l l s , are much more stimulatory when on the surface of a macrophage (Rosenstreich et a l . , 1976). ( i i ) In v i t r o lymphocyte activator for release of lymphokines Agents that activate lymphocytes to release lymphokines can be c l a s s i f i e d into the following two major types: (a) S p e c i f i c antigens: antigens activated the lymphocytes only from previously s e n s i t i z e d donors. (b) Non-specific mitogens: mitogens such as Con A and PHA can activate n o n - s p e c i f i c a l l y the lymphocytes from non-sensitized donors. A v a r i e t y of antigens and mitogens capable of inducing lymphokine production have been reviewed by Bloom (1971) and Granger (1972). PPD, bovine gamma-globulin, hapten-protein conjugates, streptokinase-streptodornase, Con A and PHA have been generally used. It i s widely agreed by most investigators that nonspecific 14 mitogens induce higher le v e l s of lymphokines than s p e c i f i c antigens due to t h e i r capacity to activate more of the lymphoid c e l l s than s p e c i f i c antigens. In a rough temporal sense, lymphokine can be released into the medium after a c t i v a t i o n between 2-92 hours following the a c t i v a t i o n of lymphocytes. Williams and Granger (1969) found that lymphotoxin was released as early as 2 hours after human lymphocytes were stimulated with PHA. Bennett and Bloom (1967) demonstrated that the release of MIF by PPD-stimulated guinea pig c e l l s began within 6 hours of culture. Further, the production of MIF might continue for 4 days with d a i l y changes of medium (Bloom and Bennet, 1968). Usually, MIF, as well as SRF was produced by mitogen or antigen-stimulated lymphocytes within 24 hours of incubation (Pick et a l . , 1969; Remold et a l . , 1970; Morley, et al., 1973). 2. Nature of Lymphokines Lymphokines are c l a s s i f i e d according to ef f e c t s on the target c e l l s i n b i o l o g i c a l assays i n v i t r o or in vivo (David and David, 1972). The i n t e r a c t i o n of lymphocytes with s p e c i f i c antigen produces factors causing a v a r i e t y of b i o l o g i c a l e f f e c t s , including i n h i b i t i o n of macrophage migration, aggregation of macrophages, chemotactic a t t r a c t i o n of c e l l s , d i r e c t or i n d i r e c t k i l l i n g of c e l l s , stimulation or i n h i b i t i o n of c e l l d i v i s i o n , and ski n reaction. Up to now, over a dozen lymphokine a c t i v i t i e s have been 15 i d e n t i f i e d . Migration i n h i b i t o r y factor (MIF) is one of the most extensively studied. Lymphokine(s) af f e c t s various c e l l s , such as macrophages, lymphocytes, neutrophils, eosinophils and basophils. However, the macrophage as target c e l l is one of the most thoroughly investigated. I t i s believed that macrophages serve as the main ef f e c t o r c e l l s of DH reactions. Lymphokines can a l t e r many physical and b i o l o g i c a l functions of macrophage as shown i n the following l i s t : (1) I n h i b i t i o n of migration (David et al., 1964) (2) Aggregation (Lolekha, Dray and Gotaff, 1970) (3) Chemotaxis i n v i t r o . (Ward et a l . , 1970; Wahl et a l . , 1974) (4) Decrease i n macrophage volume (Poulter and Turk, 1975a) (5) Reduction of electrophoretic m o b i l i t y of macro-phages (Caspary, 1972; Preece and Light, 1974) (6) Reduction i n the i n t e r f a c i a l energy of the macro-phage c e l l surface (Thrasher _ t al., 1973) (7) Macrophage disappearance reaction (Sonozaki and Cohen, 1971) (8) I n h i b i t i o n of spreading (Dekaris e _ a l . , 1969) 16 (9) P r o l i f e r a t i o n (Hadden et a l . , 1975) (10) Macrophage a c t i v a t i o n (David, 1975; Poulter and Turk, 1975b, c) (11) Macrophage arming factor (Dy et 1976) 3. C h a r a c t e r i s t i c s of the Lymphokines It i s not the intent here to review the c h a r a c t e r i s t i c s of a l l the lymphokines. The factors which are relevant to the present study are those which have been p a r t i a l l y characterized and have a c t i v i t i e s which suggest that they play a role i n the DH reactions. I t should be remembered that few lymphokine a c t i v i t i e s have been i s o l a t e d from in v i v o h y p e r s e n s i t i v i t y reactions and the precise r o l e which lymphokines have i n these reactions has never been demonstrated by d i r e c t observations. I t i s assumed that the in v i t r o a c t i v i t i e s w i l l occur in vivo and may be used to explain the mechanisms of DH. In the following paragraphs the c h a r a c t e r i s t i c s of MIF, Chemotactic Factor (CF), Lymphotoxin (LT), Mitogenic Factor (MF), Macrophage A c t i v a t i n g Factor (MAF) and Skin Reactive Factor (SRF) w i l l be reviewed. ( i ) Migration I n h i b i t o r y Factor (MIF) As described previously, MIF was one of the f i r s t lymphokine a c t i v i t i e s to be i d e n t i f i e d and has been extensively studied. The assay procedure i s r e l a t i v e l y simple (Bloom and 17 Glade, 1971). In b r i e f , peritoneal exudate c e l l s from normal guinea pigs are packed into c a p i l l a r y tubes by centrifugation and then placed i n chambers containing tissue culture medium with or without MIF. The peritoneal exudate c e l l s which consists mostly of macrophages, s p i l l out of the ends of the c a p i l l a r y tube and migrate away from the tube on the f l o o r of the chamber. After a period of time the area of migration i n test and control chambers i s measured and MIF a c t i v i t y i s expressed as a percentage of i n h i b i t i o n . In almost a l l cases, the apparent molecular weight of MIF has been determined by f i l t r a t i o n through Sephadex. Molecular weights of about 25,000 (Bloom and Jimenez, 1970), 35-55,000 (Remold e t a l . , 1970) and 60-70,000 (Bennett and Bloom, 1968; Dumonde et a l . , 1972) have been reported. However, upon sucrose density gradient centrifugation (Remold _ t al., 1970), the molecular weight appeared close to 30-60,000. In any case, molecules of MIF are smaller than any known class of immunoglobulin. MIF obtained from guinea pig lymphocytes i s r e s i s t a n t to heating at 56°C for 30 min but i s destroyed by heating at 80°C for 30 min (David and David, 1972). MIF i s soluble i n 40-50% saturated ammonium s u l f a t e , but p r e c i p i t a t e s i n 80-90% saturated s o l u t i o n (Dumonde et a l . , 1972; Morley et al., 1973). Its i s o e l e c t r i c point i s approximately 5.3 (Sorg and Bloom, 1973). 18 However, a recent report by Remold and Mednis (1977) showed that supernatants from Con A-activated guinea pig LN lymphocytes contained two distant molecular MIF species, based on d i f f e r e n t i s o e l e c t r i c points and d i f f e r e n t e l u t i o n patterns from a Sephadex G-75 column. One, pH 3-MIF had an i s o e l e c t r i c point of 3.0-4.5 and a M.W. of 65,000 and the other pH 5-MIF had an i s o e l e c t r i c point of 5.0 to 5.5 and a M.W. of between 25,000-43,000. MIF i s r e s i s t a n t to both DNase and RNase (David and David, 1972) but is destroyed completely by digestion with chymotrypsin and p a r t i a l l y by digestion with t r y p s i n (Remold and David, 1971). These findings suggest that MIF i s a protein. Further, Remold and David (1971) suggest that guinea pig MIF may be an acid glycoprotein which has a s i a l i c acid moiety. This i s based on the following findings: (a) MIF i s sensitive to neuraminidase (Remold and David, 1971); (b) Upon electrophoresis at pH 9.1, MIF a c t i v i t y migrates anodally more r a p i d l y than serum albumen, that i s , i n the prealbumin f r a c t i o n (Remold et a l . , 1970); (c) The density on cesium chloride gradients i s p ^ ^ = 1.45-1.41 which i s greater than that of protein (p^,_ = 1.34) (Remold and David, 1971). 19 L i t t l e i s known concerning the i n t e r a c t i o n of MIF with the macrophage membrane. Leu et: al., (1972) reported that macrophages possess receptors which bind guinea pig MIF and the receptor i s destroyed by t r y p s i n treatment. Later, Remold (1973) found that the action of MIF i s blocked by L-fucose and could be prevented by trea t i n g the macrophage with fucosidase. He concluded that L-fucose may be a part of the receptor. Studies by Fox and MacSween (1974) using a f f i n i t y chromatography have indicated that guinea pig MIF can bind to fucosamine-agarose and can be subsequently eluted from the column with buffers containing high concentrations of L-fucose. I t has been shown that the response of macrophages to MIF is enhanced when the c e l l s were pretreated with a v a r i e t y of esterase i n h i b i t o r s such as antithrombin I I I , alpha-2-antitrypsin, Cl i n h i b i t o r , d i i s o p r o p y l fluorophosphate and soybean t r y p s i n i n h i b i t o r (Remold, 1975; Remold et a l . , 1975). These findings suggest the presence of serine esterase on the macrophage membrane which counteracts the action of MIF. This serine esterase may play a r o l e i n control of macrophage response to MIF (David, 1975). On the other hand, plasma esterase i n h i b i t o r s such an antithrombin III may i n t e r a c t jLn vivo with the macrophage and may play a ro l e i n enhancing the response of macrophage to this lymphokine i n the inta c t host (Piessens et a l . , 1977). 20 ( i i ) Chemotactic Factor (CF) Ward _ t al., (1969) f i r s t found that MIF-rich supernatant also contained a chemotactic factor f or macrophages. Furthermore, Ward (1970) demonstrated that t h i s factor elutes from Sephadex G-100 i n fract i o n s containing albumin and s l i g h t l y smaller molecules and i s distinguishable from the MIF by electrophoresis. Mononuclear chemotactic factor i s found i n the gel albumin f r a c t i o n whereas MIF i s found i n the gel prealbumin f r a c t i o n . Wahl et a l . , (1974) reported that the molecular weight of chemotactic factor i s 12,500 and suggested that the factor obtained by Ward et a l . , (1970) may be i n an aggregated form. Moreover, t h i s factor i s r e s i s t a n t to neuraminidase and heat (Remold, 1972; Wahl e t a l . , 1974). Studies by Cohen et. al., (1973) with extractions obtained from skin s i t e of DH reactions indicated that the extractions showed chemotactic a c t i v i t y for monocytes and lymphocytes but not neutrophils. An i n t e r e s t i n g f i n d i n g was that no detectable MIF a c t i v i t y was found i n the extracts. Intradermal i n j e c t i o n of these extracts into normal guinea pig induced mononuclear i n f i l t r a t i o n . Kambara et a l . , (1977) confirmed these findings and further demonstrated that two mononuclear chemotactic factors extracted from DH s k i n lesions were obtained and were found i n the pseudoglobulin f r a c t i o n . The f i r s t one which had a s i m i l a r molecular weight to IgG was h e a t - l a b i l e and the other with a molecular weight of 12,000 was 21 heat-stable. These chemotactic factors may explain why a majority of nonsensitized host c e l l s accumulate at the s i t e of DH reaction i n response to s p e c i f i c antigenic signals. ( i i i ) Lymphotoxin (LT) In general, LT i s produced by mitogen-stimulated lymphocytes for i n v i t r o studies (reviewed by Rosenau and Tsoukas, 1976). LT i s n o n s p e c i f i c a l l y cytotoxic to a great v a r i e t y of target c e l l s (Williams and Granger, 1973). The mouse L - f i b r o b l a s t c e l l l i n e i s the most sensitive indicator c e l l of LT a c t i v i t y (Granger, 1971). Human, guinea pig and mouse LT have been extensively investigated. Kolb and Granger (1968, 1970) have estimated the molecular weight of LT produced by PHA-stimulated human and mouse lymphoid c e l l s to be within the ranges of 85,000-100,000 and 90,000-150,000 res p e c t i v e l y . However, recent studies showed that the molecular weight of mouse LT produced by PHA-stimulated spleen c e l l s i s 41,000 and i t s i s o e l e c t r i c point i s between 4.4-4.8. Human LT can be separated by gel f i l t r a t i o n into two basic f r a c t i o n s ; those are alpha-LT with M.W. 90,000 and beta-LT with M.W. 45,000 (Walker e t a l . , 1976). Alpha-LT i s heat-stable. Beta-LT i s r e l a t i v e l y unstable i n medium containing serum and appears to predominate early a f t e r mitogenic stimulation. Further, Hiserodt e_t a l (1976) demonstrated that beta-LT can be resolved into at l e a s t 2 22 components (termed beta-LT^ and beta-LT 2) by chromatography on DEAE-cellulose or electrophoresis on polyacrylamide g e l . Beta-LT^ i s r e l a t i v e heat-stable and more p o s i t i v e l y charged. Guinea pig LT causes a high degree of thymidine release from prelabeled L - c e l l s , but no release from guinea pig lung f i b r o b l a s t s (Bloom, 1971). Guinea pig LT appears to be d i s t i n c t from MIF and chemotactic factor (Coyne, 1973). It i s s e n s i t i v e to heating at 60°C for 30 min and to chymotrypsin but i s r e s i s t a n t to neuraminidase. Further, anti-guinea pig LT serum i n h i b i t s the action of LT but f a i l s to i n h i b i t the MIF and mitogenic a c t i v i t i e s of lymphokines (Gately et. a l . , 1975) The mechanism by which LT exerts i t s cytotoxic e f f e c t on the target c e l l s i s unknown. William and Granger (1973) reported that human LT r a p i d l y binds to d i f f e r e n t target c e l l s . Moreover, recent studies with labeled LT have shown that only LT-sensitive c e l l s contain s p e c i f i c h i g h - a f f i n i t y , limited capacity receptors capable of binding about 600 molecules of LT per c e l l ; whereas a non-specific l o w - a f f i n i t y , high-capacity component i s present i n both LT-sensitive and r e s i s t a n t c e l l s (Tsoukas et a l . , 1976). Further, observation with immuno-electron microscopy shows that LT binds to the c e l l u l a r surface i n patches and a filamentous web underlies the plasma membrane binding s i t e s (Friend and Rosenau, 1977). 23 (i v ) Macrophage a c t i v a t i n g factor (MAF) MAF l i k e MIF i s sens i t i v e to neuraminidase (Nathan, 1973). However, macrophage a c t i v a t i o n by incubation of lymphokines requires at least 48 hours (Nath et al., 1973; Nathan et a l . , 1973; Poulter and Turk, 1975c; Sober et a l . , 1976). MAF a c t i v i t y includes increased adherence of macrophage to the substrate (Nathan, et a l . , 1971), phagocytic a c t i v i t y (Barnet et a l . , 1968), spreading, (Nath et ail., 1973), glucose oxidation through the hexose monophosphate shunt (Nathan et a l . , 1971; Nath et a l . , 1973; Poulter and Turk, 1975c), b a c t e r i c i d a l - a c t i v i t y (David, 1975), glucosamine incorporation (Hammond et a l . , 1975; Sober et a l . , 1976), increased h y d r o l y t i c enzymatic a c t i v i t y (Pantalone and Page, 1975; Poulter and Turk, 1975c) and enchanced tumoricidal a c t i v i t y (David, 1975). Enhancement of MAF, l i k e that of MIF, was obtained when macrophages were pretreated with the plasma esterase i n h i b i t o r , antithrombin III or the surface reactant, di a z o t i s e d s u l f a n i l i c a c i d (Piessens et a l . , 1977). I t seems l i k e l y that MAF i s important f o r the enhancement of DH reaction. (v) Mitogenic factor (MF) Mitogenic factor causes a s t r i k i n g increase i n thymidine incorporation by nonsensitized lymphocytes (Hart et a l . , 1973; Gately et a l . , 1975; M i l l s , 1975) and macrophages (Hadden et a l . , 24 1975). Wolstencraft and Dumonde (1970) found that mitogenic a c t i v i t y was also found i n MIF-rich supernatant from antigen-stimulated guinea pig lymphocytes. The ro l e which mitogenic factor plays i s s t i l l obscure. It may help to amplify the DH reaction in vivo by stimulatory p r o l i f e r a t i o n of uncommitted lymphocytes. ( v i ) Skin reactive factor (SRF) Bennet and Bloom (1968) f i r s t found that MIF-rich supernatant from stimulated guinea pig lymphocytes i n serum-free medium would cause delayed-type skin reactions when injec t e d into the skin of normal guinea pigs. The reaction began at 3 hours, and reached a peak at 6-12 hours. At 4 hours, h i s t o l o g i c a l examination revealed a mononuclear c e l l i n f i l t r a t i o n with r e l a t i v e l y few neutro-p h i l s . Subsequently, Pick et a l . (1969) and Dumonde et a l . (1969) confirmed the production of SRF by antigen s e n s i t i z e d lymph node lymphocytes. Further, stimulation of nonsensitized lymphocytes by Con A also induces the production of SRF (Pick et al., 1970a, b; Schwartz et a l . , 1970). Fra c t i o n a t i o n studies of SRF indicated that t h i s a c t i v i t y i s found i n the same Sephadex f r a c t i o n as MIF, e l u t i n g from Sephadex G-100 and G-200 i n the f r a c t i o n containing molecules of the s i z e of serum albumin and of molecules of s l i g h t l y smaller molecular weight (Bennett and Bloom, 1968; Pick et a l . , 1969, 1970b). 25 Pick et al., (1969) demonstrated that SRF is soluble i n 40% ammonium su l f a t e but i s p r e c i p i t a t e d by 66% ammonium s u l f a t e . SRF retains most of i t s a c t i v i t y following heating at 56°C for 30 min but i s destroyed by heating at 100°C for 30 minutes (Pick et a l . , 1970). The SRF a c t i v i t y i s completely destroyed by pepsin and p a r t i a l l y destroyed by t r y p s i n and papain (Pick e__a_.., 1969). It is r e s i s t a n t to DNase and RNase. However, the s t a b i l i t y of SRF to neuraminidase and chymotrypsin has not yet been reported. Up to now, highly p u r i f i e d forms of lymphokines have not yet been prepared. It i s not yet known whether a l l these lymphokine a c t i v i t i e s are due to separate factors or are due to one substance which possess several a c t i v i t i e s . However, evidence i s accumulating that several of the a c t i v i t i e s can be distinguished on the basis of t h e i r physiochemical nature and t h e i r s t a b i l i t y to enzymes. It has been shown that chemotatic factor for macrophages and mitogenic factor are r e s i s t a n t to neuraminidase (Wahl et a l . , 1974; David and David, 1972), whereas both MIF and MAF a c t i v i t i e s are destroyed by neuraminidase. This suggests that the l a t t e r two may be the same or c l o s e l y r e l a t e d substances (Nathan et a l . , 1973; David, 1975). On the other hand, i t has been shown that MIF and MF d i f f e r i n molecular weight and other physical properties (Ashworth et a l . , 1975). 26 4. Lymphokine Production In Vivo U n t i l now, there i s no clear proof of a p h y s i o l o g i c a l r o l e for any of the lymphokines. Some evidence has been presented i n support of the notion that lymphokines are a c t u a l l y l i b e r a t e d i n vivo i n DH reactions. Hays et. ajL., (1973) found that considerable amounts of MIF and MF were detected i n the lymph of sheep which were immunized against BCG and subsequently challenged with PPD. In addition, an increase i n lymphocyte output through both the l e s i o n and the lymph node was measured. Sa l v i n _ t _ l . , (1973, 1974) reported that MIF and type II i n t e r f e r o n were released into the c i r c u l a t i o n of mice with DH a f t e r stimulation with s p e c i f i c antigen. Kaufman et a l . , (1975) found that the serum and central lymph from mice immunized with l i v e L i s t e r i a monocytogenes s i x days previously and boosted four hours p r i o r to c o l l e c t i o n , contained MIF as assayed by MIF a c t i v i t y i n v i t r o . More recently, Postlethwaite and Snyderman (1975) devised a system i n the guinea pig which allows the sampling of peritoneal f l u i d p e r i o d i c a l l y before and a f t e r i n t r a p e r i t o n e a l antigen challenge with horseradish peroxidase. Within 24 hours of administration of the antigen into a previously s e n s i t i z e d guinea pig, the peritoneal f l u i d contained a chemotactic substance which was s i m i l a r to that from s e n s i t i z e d lymphocytes stimulated with s p e c i f i c antigen in v i t r o . After 48 hours, the chemotactic a c t i v i t y present i n the peritoneal f l u i d returned to the 27 pre-challenge l e v e l . Furthermore, using the same system, Postlethwaite and Kang (1976) found that the MIF a c t i v i t y of the peritoneal f l u i d was maximal at 48 hours after antigen challenge while no MIF a c t i v i t y was detected at 96 hours. It i s of i n t e r e s t to note that i n these studies, chemotactic a c t i v i t y was found p r i o r to MIF a c t i v i t y . In a l l , these findings substantiate the b e l i e f that lymphokines are produced i n DH reactions. I l l PROBLEMS IN CORRELATION OF IN VITRO LYMPHOKINE ACTIVITY WITH DELAYED-TYPE HYPERSENSITIVITY IN VIVO 1. MIF and SRF As described previously MIF i s one of the most thoroughly investigated lymphokines. It has been shown to correlate with DH i n viv o . However, the precise r o l e of MIF i n DH i s a question of great i n t e r e s t . Bennet and Bloom (1968) have shown that i n j e c t i o n of MIF-rich fractions into the skin of normal guinea pigs i s followed by the development of an erythematous and indurated reaction which appears within 3-4 hours. In f a c t , some physical and chemical properties of SRF and MIF are s i m i l a r . These are: (1) s i m i l a r molecular weight, (2) resistance to heat, (3) s e n s i t i v i t y to some p r o t e o l y t i c enzymes and resistance to DNase and RNase. However, the s t a b i l i t y of SRF to neuraminidase and chymotrypsin has not yet 28 been reported. It would be i n t e r e s t i n g to see i f SRF i s s e n s i t i v e to neuraminidase and chymotrypsin, both of which destroy MIF a c t i v i t y , and also to determine where SRF a c t i v i t y i s found a f t e r gel electrophoresis. 2. SRF and Plasma Mediator-producing Systems The pharmacological studies on SRF by M a i l l a r d et. a l . , (1972) indicated that SRF a c t i v i t y was i n h i b i t e d by polybrene and protamine s u l f a t e which counteract the a c t i v a t i o n of Hageman factor i n the kinin-forming system and was enhanced by sodium diethyl-dithiocarbamate which i s an i n h i b i t o r of kininases. M a i l l a r d et a l . , (1972) also found that the depletion of test animals of c i r c u l a t i n g C3 or neutrophils did not influence the SRF a c t i v i t y of those animals. Antihistamine and serotonin antagonists did not diminish SRF action. These authors concluded that SRF a c t i v i t y may be rel a t e d to the a c t i v a t i o n of Hageman factor and i s not a d i r e c t mediator of vascular permeability. Hageman factor can be activated by: a) contact with negatively charged surface of glass, k a o l i n and b i o l o g i c a l materials such as collagen and basement membrane, b) a v a r i e t y of proteases including t r y p s i n , k a l l i k r e i n , plasma c l o t t i n g factor XI (reviewed by Ryan and Majno, 1977). 29 However, activated Hageman factor can act on the following systems: a) kinin-forming system, b) c l o t t i n g system, and c) f i b r i n o l y t i c system. In the kinin-forming system, activated Hageman factor activates p r e k a l l i k r e i n to form k a l l i k r e i n , which i n turn, cleaves kininogen to produce k i n i n . (Cochrane and Wuepper, 1971). K i n i n i s a potent mediator of vascular permeability and causes contraction of guinea pig ileum (Rocha e S i l v a , 1970). In order to determine whether or not guinea pig lymphokines can d i r e c t l y activate the k i n i n forming system, p a r t i a l l y p u r i f i e d Hageman factor and p r e k a l l i k r e i n which were i s o l a t e d from rabbit plasma were interacted with guinea p i g lymphokine i n a series of experiments. I t i s well known that lymphokines could a f f e c t the a c t i v i t y of macrophage, lymphocytes, neutrophils, eosinophils and f i b r o b l a s t s . Since p l a t e l e t s and mast c e l l s both contain mediators of the inflammatory response (Bloom, 1974; and Zucker, 1974), studies were undertaken to investigate whether or not lymphokines can a f f e c t the a c t i v i t i e s of p l a t e l e t s and mast c e l l s . 30 MATERIALS AND METHODS I PREPARATION OF LYMPHOKINES 1. Animals Male Hartley s t r a i n guinea pigs, weighing 350-600 g, were used as c e l l donors and test animals. 2. Preparation of Antigen Dinitrophenylated bovine gammaglobulin (DNP-BGG) was used an antigen. Conjugation of BGG to 2,A-dinitrobenzene sulfonate (DNB-SO^) was done according to the methods of L i t t l e and Eisen (1967). 300 mg BGG and 300 mg K 2C0 3 were dissolved i n 15 ml of water, and 300 mg of DNB-SO^ sodium s a l t (Eastman) were added. The s o l u t i o n was s t i r r e d at room temperature for 24 hours i n the dark and then centrifuged at 20,000 g for 15 minutes. It was passed through a Sephadex G-25 column (2.5 x 30 cm, running buffer 0.005 M sodium phosphate, pH 7.5) to separate the DNP-BGG from DNP. The excluded peak was c o l l e c t e d , dialyzed against d i s t i l l e d water, frozen i n dry ice-methanol or l i q u i d nitrogen and then l y o p h i l i z e d . Since DNP-BGG was poorly soluble i n s a l i n e , i t was reconstituted i n s t e r i l e d i s t i l l e d water f i r s t at 10 mg/ml and then 9 mg of NaCl/ml was added. 31 3. Mitogen Concanavalin A (Con A, Calbiochem Lab) was used as lymphocyte a c t i v a t o r . Con A was dissolved i n sal i n e so that 0.1 ml of mitogen s o l u t i o n contained 100 ug of Con A. 4. Preparation of Animals a. S e n s i t i z a t i o n with DNP-BGG Four to eight guinea pigs were in j e c t e d with 100 pg of DNP-BGG i n 0.2 ml of saline emulsified i n an equal volume of complete Freund's Adjuvant (CFA) (Difco, H37Ra). 0.1 ml of the antigen-adjuvant emulsion was injected into each footpad. Lymph node (LN) c e l l s were harvested 14 days af t e r s e n s i t i z a t i o n , as described i n Section 1-6. b. S e n s i t i z a t i o n with Freund's Adjuvant In order to obtain an increased number of lympho-cytes, the guinea pigs were in j e c t e d with Complete Freund's adjuvant. 0.6 ml was injected i n the four foot pads and 0.4 ml was in j e c t e d subcutaneously into the nuchal region. LN c e l l s were obtained 2-3 weeks after the CFA i n j e c t i o n . The LN c e l l s were used for mitogen a c t i v a t i o n . 32 5. Culture Medium RPMI-1640 (GIBCO) was supplemented with 100 U/ml p e n i c i l l i n , 100 ug/ml streptomycin and 2 mM glutamine. 6. Lymphocyte Preparation The enlarged regional lymph nodes from s e n s i t i z e d guinea pigs were, obtained from the a x i l l a r y , p o p l i t e a l , and inguinal regions, and were teased i n RPMI-1640 medium with s p e c i a l forceps which were equipped with 5 27-G needles at the t i p ( F i g . 1). The LN c e l l s were separated from tissue fragments by f i l t e r i n g through a 150 mesh s t e r i l e s t e e l screen. The LN c e l l s were washed twice with RPMI-1640 medium by centrif u g i n g at 220 g for 10 minutes at 4°C. The washed LN c e l l s were resuspended and were centrifuged at 50 g for 1 minute at 4°C to remove the clumps. The lymphocytes were 50-60% v i a b l e by trypan blue dye exclusion. C e l l suspensions were di l u t e d to 1-1.5 x 10 7 v i a b l e cells/ml i n the RPMI-1640 medium. 7. P u r i f i c a t i o n of Lymphocytes from LN C e l l s P u r i f i e d lymphocytes from LN c e l l s were prepared according to the method of Shortman et a l . , (1971) with s l i g h t m o dification. S i l i c o n i z e d glass beads, 60-80 mesh (Applied Science Lab. Inc.) were immersed i n 50% ethyl alcohol i n a 250 ml beaker to lower surface tension. An equal volume of d i s t i l l e d water was added. The glass 32a Figure 1 Instruments for lymphokine studies. Five needle rakes which were used to tease lymph nodes i n RPMI-1640 medium. A p l a s t i c tube with holes i n one end which was introduced into the peritoneal cavity of abdomen to c o l l e c t macrophages. 33 beads i n d i l u t e alcohol were s t e r i l i z e d at 15 lb pressure for 15 minutes. In t h i s way, a i r attached to the glass beads was completely removed. The glass beads were washed with d i s t i l l e d water. A 50 ml p l a s t i c syringe was used as the column. It was f i l l e d with saline and contained a glass wool plug. The suspended beads were introduced with a Pasteur pipette below the surface of the s a l i n e . A f t e r packing was complete, RPMI-1640 medium containing 20% guinea pig serum, which was warmed at 37°C, was used to wash Q the column. 10 ml of c e l l suspension (10 cells/ml) was added and allowed to enter the column. The macrophages and B-lymphocytes were allowed to adhere on the glass beads for 20 minutes at 37°C. Subsequently, the column was washed slowly with warm medium at a flow rate of 2 ml/min, and the f i r s t 50 ml of the eff l u e n t was co l l e c t e d i n centrifuge tubes. C e l l suspensions were adjusted to 1-1.5 x 10^ c e l l s / m l . The percentage of v i a b l e lymphocytes was over 90%, the y i e l d was about 30%. 34 LYMPHOKINE PRODUCTION IN VITRO 1. S p e c i f i c Antigen A c t i v a t i o n The c e l l suspension from non-purified or p u r i f i e d lymphocyte preparations was divided into two parts. To the experimental part, DNP-BGG was added to a f i n a l concentration of 50 ug/ml. (Preliminary experiments indicated that 50 pg/ml of the DNP-BGG was optimal to produce MIF and SRF). To the co n t r o l , an equal volume of s a l i n e was added. The c e l l suspensions, 45-50 ml each, were cultured i n 250 ml tissue culture flasks (Falcon P l a s t i c s ) . The cultures were gassed with 5% CO^ i n 95% a i r and incubated on a rocker platform at 37°C. After 20-24 hours of incubation, the c e l l suspensions were centrifuged for 20 minutes at 1000 g at 4°C. To the control cultures an amount of DNP-BGG equal to the experimental group was added a f t e r incubation. The supernatants were concentrated 10 f o l d and p u r i f i e d p a r t i a l l y by s a l t f r a c t i o n a t i o n as described i n Section III-3. 2. Non-specific A c t i v a t i o n of Lymphocytes Con A mitogen sol u t i o n was added to c e l l suspensions (1-1.5 x 10 7 cells/ml) to give a f i n a l concentration of 10 pg Con A/ml (Remold et a l . , 1972). To 35 control f l a s k s , an equal volume of s a l i n e was added. The c e l l culture procedure was the same as described previously. Con A was added to the supernatant of the control culture within 24 hours of incubation. After centrifugation, both active and control supernatants were dialyzed against PBS and concentrated to 1.5 ml. These concentrated supernatants were used as described i n the following: ( i ) For f r a c t i o n a t i o n by Sephadex G-100 as described i n Section I I I - l . ( i i ) For comparison of MIF a c t i v i t y produced by unpurified and p u r i f i e d LN lymphocytes which were stimulated with Con A. The concentrated active and control supernatants were applied on a small Sephadex G-100 column (height 4 cm, diameter 2.5 cm) which was e q u i l i b r a t e d i n PBS (Horvat et a l . , 1972). Con A was removed by binding to the Sephadex gel (Agrawal and Goldstein, 1965). The eluant was concentrated to one tenth volume of the o r i g i n a l supernatant. 36 III SEPARATION OF LYMPHOKINES 1. Fr a c t i o n a t i o n of Sephadex G-100 The active supernatants (60-120 ml) from Con A-activated lymphocytes were dialysed extensively against PBS for 24 hours at 4°C with three changes. After d i a l y z i n g , the supernatants were concentrated to 1.5 ml with an Amicon u l t r a f i l t r a t i o n c e l l using the UM-10 membranes i n ice-bath. The concentrated supernatants were c l a r i f i e d by c e n t r i f u g a t i o n for 5 minutes at 2000 g. They were then applied to 1.6 x 100 cm column of Sephadex G-100 (Pharmacia) i n PBS. The column was eluted with the same buffer at 4°C at a flow rate of 0.3 ml/min and 2.0 ml f r a c t i o n s were c o l l e c t e d . The Sephadex column was precalibrated with 4-5 mg of the following molecular weight markers: blue dextran, BSA, peroxidase, chymotrypsinogen A and RNase. The protein contents of the eluted fractions were determined (Lowry e_t a l . , 1951) using BSA as the protein standard. A f r a c t i o n c o l l e c t o r (LKB Unicord II) was used i n a cold box. Eluant fract i o n s near the void volume of the columns contained protein of high molecular weight were pooled and referred to as F - l . Albumin equivalent f r a c t i o n s (M.W. 67,000) were pooled and r e f e r r e d to as F-II. Peroxidase 37 equivalent f r a c t i o n s (M.W. 44,000) were referred to as F-III. Chymotrypsinogen A and RNase equivalent fractions were referred to as F-IV and F-V respectively. Each f r a c t i o n was concentrated to 3 ml with Amicon u l t r a f i l t r a t i o n c e l l s using UM-10 membranes and dialyzed against RPMI-1640 medium. The MIF and SRF a c t i v i t i e s of each f r a c t i o n were determined (See Section IV). The control supernatants were separated from Con A by passing through a small Sephadex' G-100 column as described i n section II-2. 2. Polyacrylamide Gel Electrophoresis Fractions I I , III and IV, separated on the Sephadex G-100 column as described previously was further separated by polyacrylamide gel electrophoresis. The electrophoretic separations were based on the method of Remold ejt £__., (1970) with s l i g h t modifications. The lower separation gel was composed of the following solutions: 2.5 mg% r i b o f l a v i n i n H^ O 2.0 ml 1.5 M T r i s HC1 pH 9.1 2.0 ml 7.5% acrylamide and 0.467% .methyl bisacrylamide i n IL^ O 4.0 ml 0.017% ammonium persulfate 2.0 ml 38 The upper stacking gel was composed of the following solutions: 2.5 mg% r i b o f l a v i n i n IL^ O 1.0 ml 0.28 M Tris-phosphate at pH 6 . 7 1.0 ml 10% acrylamide and 2.5% bisacrylamide 1.5 ml 0.017% ammonium persulphate 1.0 ml Sucrose 0.6 gm Running gels and stacking gels were prepared by u l t r a v i o l e t polymerization i n 120 x 16 mm tubes for preparative electrophoresis and i n 105 x 6 mm tubes for a n a l y t i c a l e l e c t r o -phoresis. The upper chamber buffer (cathode) was 0.043 M T r i s glycine buffer pH 9.1. The buffer i n the anode chamber was 0.12 M T r i s HCl at pH 8.1. Fractions I I , III and IV, eluted from Sephadex G-100 column, were concentrated to 1 ml, and the density was increased by adding g l y c e r i n (10% v/v). This lymphokine sample was then c a r e f u l l y layered onto the surface of the spacer gel. A current of 6 mA at 80 V was applied u n t i l the sample had completely entered the spacer gel. Then the current was adjusted to 9 mA at 150 V for the remainder of the run which took approximately 4 hours. After the run, the gel was removed from the glass column and was cut with 39 razor blades into f i v e fractions (1.5 cm each) s t a r t i n g from the bromophenol blue front. Fraction E2 was the pre-albumin f r a c t i o n while E3 was the albumin f r a c t i o n when compared with guinea pig serum run i n the same process. A l l fiv e fractions were placed i n d i v i d u a l l y i n glass columns (140 x 16 mm). The lower ends of the glass tubes were attached to d i a l y s i s sacs. The material from each gel f r a c t i o n was eluted by electrophoresis by using 0.043 t r i s - g l y c i n e buffer at pH 8.9. E l u t i o n of E l , 2 and 3 took one hour; E4 and 5 took three hours. The eluent of each f r a c t i o n (about 4 ml) was thoroughly dialyzed against 400 ml of PBS for 24 hours with a change of PBS and subsequently with a change of RPMI-1640. The f r a c t i o n s were then concentrated to 3 ml with Amicon "Minicon" concentration k i t . The MIF and SRF a c t i v i t i e s of each f r a c t i o n were determined (See Section IV). 3. Salt F r a c t i o n a t i o n of Lymphokines P a r t i a l p u r i f i c a t i o n of lymphokines was based on the fact that lymphokine i s soluble at 40% saturation of ammonium sulphate and p r e c i p i t a t e s at 90% (Dumonde et a l . , 1972). The supernatants were l y o p h i l i z e d and redissolved i n one tenth t h e i r volume i n 0.01 M HCl. Following the removal of insoluble material by c e n t r i f u g a t i o n , the ten f o l d concentrates were p r e c i p i t a t e d by slowly adding ammonium sulphate to 40% saturation according to the nomogram of 40 Dixon (1953). A f t e r standing for 20 minutes, the suspensions were centrifuged at 10,000 g for 20 minutes to remove salted-out proteins, e s p e c i a l l y DNP-BGG. The supernatant fract i o n s of 40% saturated ammonium sulphate were p r e c i p i t a t e d by slowly adding ammonium sulphate to 90% SAS and standing for 20 minutes at 4°C. After c e n t r i f u g a t i o n at 50,000 g for 30 minutes, the p r e c i p i t a t e was dissolved i n water and dialyzed against water for 24 hours at 4°C and subsequently l y o p h i l i z e d . IV ASSAYS FOR LYMPHOKINES 1. Skin Inflammatory Test for SRF A c t i v i t y a. By Measuring Erythema The dorsal skin surfaces of male Hartley s t r a i n guinea pigs weighing 350-400 g were clipped. The animals were injec t e d intramuscularly with mepyramin (5 mg/kg) at 30 minutes p r i o r to skin test. 0.1 ml of the 10-fold concen-trated lymphokine test solutions dissolved i n s a l i n e or i n culture medium were injec t e d intradermally with a tuberculin syringe using 26-gauge needles. The diameters of the erythema produced were recorded 1-4 hours a f t e r the i n j e c t i o n s . 41 b. By Measuring the Increase i n Vascular Permeability The procedure i s the same as described i n IV-la, except that 0.1 ml of test solutions were i n j e c t e d i n t r a -dermally followed by the intravenous i n j e c t i o n of Evans Blue (20 mg/kg) (BDH) i n saline v i a the saphenous vein or ear vein . The guinea pigs were s a c r i f i c e d four hours after the i n j e c t i o n of the various lymphokine test solutions. The blue lesions were measured and punched out with a standard punch 1.5 cm i n diameter according to the method of Ukade __t a l . , (1970). 4.0 ml of formamide (Fisher S c i e n t i f i c Co.) was added to each piece of skin containing a blue l e s i o n and was incubated at 56°C for three days. The o p t i c a l density of the f i l t r a t e was measured at 620 nm with a spectrophotometer. The t o t a l amount of dye i n the piece of s k i n was calculated by means of a standard c a l i b r a t i o n curve and was correlated with the SRF a c t i v i t y of the lymphokine test s o l u t i o n . 2. I n h i b i t i o n of Macrophage Migration Test for MIF A c t i v i t y A micro assay for the macrophage migration i n h i b i t i o n was adopted according to the modification by Hughes (1972). The micro-modification, which used narrow bore polyethylene tubing (0.023" I.D.) instead of glass c a p i l l a r y tubes, has the advantages 42 of easy handling and cutting. Furthermore, due to the narrow I.D., 10^ peritoneal c e l l s can y i e l d 10-20 c a p i l l a r i e s . 15 ml of s t e r i l e mineral o i l (B.P.) was injected i n t r a p e r i t o n e a l l y into male guinea pigs (500-600g) under l i g h t ether anaesthesia. Af t e r 5 days, blood from cardiac puncture was allowed to stand for one hour at room temperature, and then centrifuged twice at 220 g for 20 minutes. The serum was inactivated at 56°C for 40 minutes. This guinea pig serum was used to suspend the macrophages i n the MIF assay. After cardiac puncture, the abdominal skin was clipped and swabbed with 70% ethanol and the skin i n c i s e d from diaphragm to pubis, leaving the musculature i n t a c t . 100 ml of s a l i n e containing 10 Units/ml of heparin was i n j e c t e d into the peritoneum. The peritoneum was then vigorously massaged to disperse the peritoneal exudate c e l l s (PEC). An i n c i s i o n was made i n the abdomen and a p l a s t i c tube (0.5 cm O.D.) with holes i n one end was introduced ( F i g . 1). The peritoneal f l u i d was withdrawn completely with a 20 ml p l a s t i c syringe and centrifuged at 220 g for 10 minutes at 20°C. The supernatant was removed by suction, and the PEC were washed three times with cold RPMI-1640. The y i e l d s of PEC were usually 1-2 x 10 c e l l s per guinea pig. After the f i n a l washing, the c e l l s were resuspended i n RPMI-1640 containing 15% homologous serum with 100 U/ml of p e n i c i l l i n and 100 ug/ml of streptomycin to (43 give c e l l concentrations of 5 x 107 c e l l s / m l . Polyethylene tubing (0.023" I.D.) was cut with a razor blade into 6 cm long segments. The c a p i l l a r i e s were loaded using a tube r c u l i n syringe with a 25 gauge needle with 4 cm of PEC suspension leaving at least a 0.5 cm a i r space. To seal the c a p i l l a r y , the end with the a i r space was melted i n a flame and quickly squeezed with forceps. The c a p i l l a r i e s were centrifuged at 1000 g for three minutes i n a 3 ml conical tube. The length of r e s u l t i n g c e l l p e l l e t after centrifugation was 2-3 mm. The c a p i l l a r i e s were cut with a razor blade at the c e l l - f l u i d i n t e r f a c e and transferred immediately to MIF chambers (Cie Mini-Lab Co.). Four c a p i l l a r i e s containing c e l l s were placed inside a disposable MIF chamber and mounted with s i l i c o n e grease. A coverglass was placed over the chamber and sealed with s i l i c o n e grease. The o r i f i c e of each c a p i l l a r y was inward and made contact with the bottom surface to allow PEC to migrate downward. Test solutions (lymphokine, enzyme-treated lymphokine or control supernatant) were i n RPMI-1640 containing 15% guinea pig serum, 100 Units/ml of p e n i c i l l i n and 100 ug/ml of streptomycin was introduced c a r e f u l l y through the side holes which were then sealed with s i l i c o n e grease. Chambers were incubated at 37°c for 18-20 hours. The patterns of macrophage migration were photographed. The r e l a t i v e areas of macrophage migration from the c a p i l l a r y ends were 44 determined by tracing the fan areas of migrated macrophages from the photographs and by weighing the areas which were cut out. The migration i n h i b i t o r y potency of lymphokines or of enzyme-treated lymphokines were expressed as a percentage i n h i b i t i o n of migration, as follows: Area of migration i n supernatant of antigen Percent i n h i b i t i o n = (1 - stimulated lymphocytes ) x 100 of migration Area of migration i n supernatant of non-stimulated lymphocytes Less than 20% of MIF is considered as negative and more than 20% as po s i t i v e (Pick and Turk, 1972). 3. Histology Biopsy specimens were excised from the intradermal i n j e c t i o n s i t e at 4 hours. The biopsies were fixed i n 10% buffered formalin, dehydrated i n d i l u t i o n s of alcohol and i n dehydrated acetone with four changes, and embedding i n polyg l y c o l methacrylate. Sections 3 um i n thickness were stained with haematoxylin and eosin. 45 V PROTEOLYTIC ACTIVITY IN LYMPHOKINE SUPERNATANTS 1. Acid Protease A c t i v i t y Acid protease a c t i v i t y was estimated according to the method of Mycek (1970). 0.2 ml of 0.5% (W/V) alpha-casein (Worthington) i n 0.5 ml of 1 M c i t r a t e buffer at pH 3.5 and 0.9 ml of water were mixed and held at 38°c for 5 minutes. 0.1 ml of 10-fold concentrated lymphokine, "control group supernatant", or macrophage lysosomal enzyme (as p o s i t i v e control, prepared according to the method of Werb and Cohn, 1974) was added to the casein mixture and incubated at 38°C for 30 minutes. 2.0 ml of 5% TCA was then added. After 10 minutes, the mixture was centrifuged at 900 g for 10 minutes and f i l t e r e d through Whatman No. 3 f i l t e r paper. Control flasks were i d e n t i c a l with the experimental group with the exception that the various test solutions were added af t e r the addition of TCA. The concentra-ti o n of peptides i n the f i l t r a t e s were determined by the method of Lowry et a l . (1951) against a BSA standard. 2. Neutral Protease A c t i v i t y Neutral protease a c t i v i t y was measured according to the method of Hayashi (1965). 0.1 ml of 2% alpha- casein and 0.2 ml of phosphate buffer (pH 7.2) were mixed and held at 37°C for 5 minutes. 0.1 ml of the test solutions (lymphokine, control 46 supernatant or PMN lysosomal enzyme kindly supplied by Dr. R. T. Abboud, Vancouver General Hospital) and 0.2 ml of 10 mM cysteine i n s a l i n e were added to the substrate s o l u t i o n . The mixture was incubated at 37°C for 1 hour. An equal volume of 6% t r i c h l o r a c e t i c acid was added and allowed to react for 10 minutes. Subsequently the reaction mixture was centrifugated and then f i l t e r e d . The concentration of peptides i n the f i l -trates were determined by the method of Lowry et a l . (1951). VI LYMPHOKINE EXPOSED TO HAGEMAN FACTOR, PREKALLIKREIN OR FRESH PLASMA The methods described i n the following were used to determine whether lymphokines can d i r e c t l y a c t i v a t e Hagman factor or p r e k a l l i k r e i n . 1. Separation of Hageman Factor and P r e k a l l i k r e i n Rabbit Hageman factor and p r e k a l l i k r e i n were p u r i f i e d according to the methods of Cochrane and Wuepper (1971) and Wuepper and Cochrane (1972). Plasma g l o b u l i n f r a c t i o n s soluble at 25% saturation and insoluble at 50% saturation of ammonium sulphate contained the p r e k a l l i k r e i n and Hageman factors which were separated out with DEAE-column chromatography as follows: 47 a. Globulin F r a c t i o n 150-200 ml of blood were c o l l e c t e d from the ear artery of rabbits and dispensed into p l a s t i c tubes containing 1/10 volume of a c i d - c i t r a t e anticoagulant. The blood was centrifuged twice at 2,000 g for 30 minutes at 4°C. Saturated neutral ammonium sulphate was slowly added to the plasma i n a p l a s t i c beaker to 50% SAS at 4°C and s t i r r e d for 30 minutes after which i t was centrifuged at 13,000 g for 10 minutes. The p r e c i p i t a t e was resuspended i n 25% SAS and s t i r r e d for 20 minutes. The suspension was centrifuged at 13,000 g for 10 minutes at 4°C. The supernatant was then dialyzed against the s t a r t i n g buffer of DEAE-Sephadex column. b. Anionic exchange chromatography Chromatographic separation was done at 4°C. DEAE-Sephadex A-50 (Pharmacia) was swollen i n d i s t i l l e d water and e q u i l i b r a t e d with 0.01 M phosphate s t a r t i n g buffer at pH 8.0 and containing 0.001 M EDTA and 50 pg/ml polybrene ( A l d r i c h Chemical Co.). The f i n a l i o n i c strength was 0.02. The DEAE-Sephadex A-50 was packed into a p l a s t i c 3.1 x 75 cm column by gravi t y . The dialyzed g l o b u l i n f r a c t i o n was added to the column and washed with 0.01 M phosphate s t a r t i n g buffer. The e l u t i o n was performed with a l i n e a r s a l t gradient, using 600 ml of s t a r t i n g buffer and 600 ml of 48 terminal buffer, (0.5 M NaCl, 0.01 M phosphate buffer pH 8.0 and 0.001 M EDTA). The eluent was c o l l e c t e d i n the p l a s t i c tubes (10 ml/tube). The amount of p r e k a l l i k r e i n and Hageman factors i n each tube was assayed as described i n the following sections. 2. P r e k a l l i k r e i n Assay The amount of p r e k a l l i k r e i n i n each f r a c t i o n was determined according to the method of Wuepper and Cochrane (1972). In summary, the p r e k a l l i k r e i n was f i r s t activated by t r y p s i n . Subsequently, the activated p r e k a l l i k r e i n was allowed to react with benzoyl arginine ethyl ester (BAEe). In the presence of activated p r e k a l l i k r e i n , BAEe was hydrolyzed to benzoyl-L-arginine. The amount of activated p r e k a l l i k r e i n which was able to produce 1 u mole benzoyl-L-arginine/minute at 37°C was taken as 1 unit. Trypsin (2 x c r y s t a l l i z e d , Sigma) was dissolved i n 0.001 M HC1 to give 1 mg/ml. P r i o r to use, t r y p s i n was d i l u t e d to 920 ug/ml with 0.1 M T r i s buffer at pH 7.5. Lima bean t r y p s i n i n h i b i t o r (LBTI) (Worthington), 1 mg/ml T r i s buffer saline (TBS) (0.01 M T r i s , 0.15 N NaCl, pH 7.55), was used to i n h i b i t the t r y p s i n . LBTI does not i n h i b i t p r e k a l l i k r e i n a c t i v i t y . The a c t i v a t i o n of p r e k a l l i k r e i n to k a l l i k r e i n was performed as follows: 49 Experimental Control 1 Fractions from 0.1 ml f r a c t i o n c o l l e c t o r Trypsin 0.1 ml 0.1 ml LBTI 0.1 M T r i s buffer, 0.1 ml 0.2 ml pH 7.5 The reaction mixture i n each tube was incubated i n a water bath at 37°C for 15 minutes. It i s evident that i n each of the controls the p r e k a l l i k r e i n should not be activated. Subsequently, the hydrolysis of BAEe to benzoyl-L-arginine by activated p r e k a l l i k r e i n ( k a l l i k r e i n ) was performed as follows: Control Control 2 3 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.2 ml Experimental Control Control Control 1 2 3 LBTI (1 mg/ml) 1 m Mole BAEe i n PBS 0.1 ml 3.0 ml 0.1 ml 3.0 ml 3.0 ml 0.1 ml 3.0 ml A l l the tubes were incubated i n a waterbath at 37°C for 20 minutes, and then r a p i d l y cooled i n i c e . The o p t i c a l density at 253 nm of each tube was read, and the concentration of benzoyl-L-arginine was determined from a standard curve. 50 3. Assay for Hageman Factor The amount of Hageman factor i n each f r a c t i o n was determined by the method of Cochrane and Wuepper (1971). Hageman factor (HF) was activated by Kaolin i n TBS buffer. The amount of activated Hageman factor (bound to kaolin) was measured by adding p r e k a l l i k r e i n . The conversion of p r e k a l l i k r e i n to the active enzyme ( k a l l i k r e i n ) was measured by i t s a b i l i t y to hydrolyze 1 mM BAEe as described previously. The procedure was performed as follows: Experimental Control Control Control 1 2 3 Fractions from 0.2 ml - 0.2 ml f r a c t i o n c o l l e c t o r TBS, pH 7.55 - 0.2 ml 0.2 ml 0.4 ml Kaolin suspension 0.2 ml 0.2 ml (5 mg/ml i n TBS) The suspensions were shaken at room temperature (23°C) for 20 minutes and then centrifuged at 450 g for 5 minutes at 15°C. The supernatants were removed and 0.1 ml of 0.1 M T r i s at pH 7.55 and 0.1 ml of p r e k a l l i k r e i n s o l u t i o n were added to the k a o l i n containing activated HF. The tubes were incubated at 37°C for 40 minutes and then 3.0 ml of 1 mM BAEe was added. Incubation was continued for a further 40 minutes. The tubes were then cooled on i c e , centrifuged at 2,000 g for 5 minutes at 4°C and the o p t i c a l density was measured at 253 nanometers. 51 4. Further P u r i f i c a t i o n of P r e k a l l i k r e i n a. Rechromatography on DEAE-Sephadex (DEAE-II) The fractions having p r e k a l l i k r e i n a c t i v i t y from DEAE c e l l u l o s e chromatography were pooled and concentrated by p r e c i p i t a t i o n with 60% SAS. The p r e c i p i t a t e was dissolved i n 50 ml of s t a r t i n g buffer (0.01 M phosphate, pH 6.9, 0.001 M EDTA) and dialyzed against the s t a r t i n g buffer. After c e n t r i f u g a t i o n , the clear protein s o l u t i o n was chromatographed on DEAE Sephadex A-50 column (3.1 x 50 cm) at pH 6.9. Fractions were eluted by a sodium chloride gradient i n phosphate-EDTA buffer (0.14 M NaH 2 po 4 , 0.2 M NaCl, 0.001 M EDTA). Each f r a c t i o n was tested for p r e k a l l i k r e i n a c t i v i t y . b. CM-Sephadex C-50 cation exchange chromatography CM-Sephadex was swollen with water and e q u i l i b r a t e d with citrate-phosphate s t a r t i n g buffer (0.1 M c i t r i c acid, 0.2 M Na2HP0^, pH 5.8, i o n i c strength u=0.05). The gel was packed i n a p l a s t i c column (2.5 x 40 cm). The f r a c t i o n having p r e k a l l i k r e i n a c t i v i t y from DEAE-II was dialyzed against citrate-phosphate s t a r t i n g buffer. The sample was applied to the column and washed. Proteins were eluted with a li n e a r gradient which u t i l i z e d 0.3M NaCl with citrate-phosphate buffer (pH 7.5). Each f r a c t i o n was tested for p r e k a l l i k r e i n a c t i v i t y . 52 5. Further P u r i f i c a t i o n of Hageman Factor The second chromatographic p u r i f i c a t i o n for HF employed DEAE-Sephadex A-50 i n a p l a s t i c column (2.5 x 25 cm). The s t a r t i n g buffer contained 0.01 M phosphate and 0.001 M EDTA at pH 8.0 and the terminal buffer contained 0.14 M NaH2PO^H205 with 0.2 M NaCl and 0.001 M EDTA. 6. Lymphokine Exposed to HF and P r e k a l l i k r e i n The procedures were the same as the assays for p r e k a l l i k r e i n and HF except that lymphokine or " c o n t r o l group supernatant" replaced t r y p s i n i n the p r e k a l l i k r e i n assay and k a o l i n i n the HF assay. 7. Bioassay for K i n i n Release from Fresh Plasma Exposed to the Lymphokines This experiment attempted to detect any substance i n the lymphokine supernatant which would activate the k i n i n system as detected i n the Schultz-Dale apparatus. K i n i n release was measured on the ileum of guinea pigs (180-200 gm). The guinea pigs were s a c r i f i c e d by a blow on the head and bled. The ileum was cut into 2 cm segments and put into Tyrode solution containing atrophine s u l f a t e (1 ug/ml) and mepyramine maleate (0.1 ug/ml) i n a 10 ml glass Schultz-Dale bath aerated with a i r at 37°C. 53 Fresh c i t r a t e plasma (1 part of 0.38% c i t r a t e i n PBS to 9 parts of blood) was the substrate. As a ru l e , 0.1 ml of sample (lymphokine, control supernatant or k a o l i n i n 5 mg/ml) was incubated with 0.2 ml of plasma, 0.05 ml of 10 mM o-phenanthroline.HCl (kininase i n h i b i t o r ) and 0.1 ml of PBS, pH 7.4 for 2-10 minutes at 37°C (Girey et a l . , 1972). The mixture was then bo i l e d for 15 minutes i n the steam bath. 20-200 u l of mixture was added into the glass chamber which contained 5 ml of Tyrode solution and the suspended ileum. The k i n i n present i n the test solution w i l l stimulate the ileum and cause contraction. The ileum was standardized with synthetic bradykinin (Sandoz). The amplitude of contractions was recorded, and t r i p l i c a t e measurements were made on a l l samples. 54 VII EFFECT OF LYMPHOKINES ON MAST CELLS 1. E f f e c t of Lymphokine on Mesenteric Mast C e l l s of Guinea Pigs Degranulation of mast c e l l s of guinea pig mesentery was estimated according to the method of Boreus (1960). Guinea pigs weighing 250 gra were k i l l e d by a blow and bled. Pieces of mesentery were incubated at 37°C i n 0.4 ml of Tyrode s o l u t i o n (pH 7.4) with 0.1 ml of concentrated lymphokine or control supernatant added to the suspension for 15, 30 or 60 minutes. Lecithinase A (Boreus, 1960) and ATP (Kiernan, 1972) at the f i n a l concentration of 30 ug/ml and 5 mM res p e c t i v e l y were used as p o s i t i v e controls of histamine l i b e r a t o r s . After incubation, the mesentery was fixed i n 50% (v/v) ethanol with 1% (v/v) a c e t i c acid and 4% (v/v) lead subacetate for at least 20 minutes and stained for 30-40 seconds i n 0.5% t o l u i d i n e blue s o l u t i o n at pH 4.0. Mast c e l l degranulation was determined by microscopic observation. 2. E f f e c t of Lymphokine on Isolated Mast C e l l s of the Rat Mast c e l l s were obtained by lavage of peritoneal c a v i t i e s of male Wistar rats (230-250 gm) with heparinized (10 U/ml) Tyrode sol u t i o n . The peritoneal c e l l suspension was centrifuged at 300 g for 5 minutes. Rat peritoneal c e l l s 55 (10-15% mast c e l l ) were resuspended i n 4 ml of Tyrode solution and layered onto 4 ml of 30% F i c o l l (Johnson and Moran 1966). The tube was centrifuged at 450 g for 20 minutes at room temperature. The mast c e l l s were located i n the upper t h i r d of the 30% F i c o l l s o lution. The mast c e l l s were washed twice i n Tyrode sol u t i o n . The r e s u l t i n g c e l l suspension contained between 85-95% mast c e l l s , as determined by counting c e l l s stained with tolui d i n e blue i n a haemocytometer. The mast c e l l s were resuspended i n the incubation medium at 1.6 x lo**/ml. The incubation medium (pH 7.2) contained 150 mM NaCl, 2.7 mM KC1, 1 mM CaCl 2 , 4 mM Na 2HP0 4, 5.6 mM Dextrose and 0.1% bovine serum albumin. 0.4 ml of mast c e l l s were incubated with ei t h e r 0.1 ml of histamine-releasing agent ( l e c i t h i n a s e A, 100 ug/ml), lymphokine, or "control supernatant", at 37°C for 30 minutes. After incubation, the c e l l s were centrifuged at 900 g for 5 minutes. Histamine present i n the supernatant was detected by stimulation of guinea pig ileum contraction i n the Schultz-Dale apparatus using histamine standards. 56 VIII EFFECT OF LYMPHOKINES ON PLATELETS The test for p l a t e l e t aggregation was according to the method of Born (1962). S i l i c o n i z e d glassware was used throughout i n this procedure. Guinea pig blood was obtained by heart puncture. 18 ml of guinea pig blood was mixed with 2 ml of 3.8% trisodium c i t r a t e (pH 7.4). P l a t e l e t - r i c h plasma (PRP) was obtained by the c e n t r i f u g a t i o n of the blood mixture at 300 g for 15 minutes at room temperature. Platelet-poor plasma (PPP) were obtained by c e n t r i f u g a t i o n at 2,000 g for 20 minutes at 4°C. The concentration of p l a t e l e t s i n PRP was estimated by counting i n a hemocytometer chamber, and then o adjusted to 5 x 10 /ml by adding PPP. a 2.5 ml aliquots of PRP (5 x 10 /ml) were pipetted into 5 ml Coleman cuvettes and incubated with constant s t i r r i n g at 37°C. The o p t i c a l density of the PRP was read at a wavelength of 600 nm i n a Beckman spectrophotometer. Subsequently, the o p t i c a l density of PRP was read at one-half to one minute i n t e r v a l s following addition of either 100 u l s a l i n e , 40 u l of 10~4M ADP, 100 u l of lymphokine (concentrated 10-20 fold) or 100 u l of "control supernatant" (concentrated 10-20 f o l d ) . P l a t e l e t aggregation was indicated by the decrease i n o p t i c a l density following the addition of these substances. 57 IX ENZYME TREATMENT OF LYMPHOKINE PREPARATION Neuraminidase of C l . perfringens (Type V, Sigma) and chymotrypsin (Sigma) were immobilized by conjugation to CNBr-activated Sepharose 4B (Pharmacia) according to the method described i n A f f i n i t y Chromatography (Pharmacia) and were used to int e r a c t with lymphokines. 1. Preparation of Immobilized Enzymes a. Preparation of Chymotrypsin-agarose 0.8 gm of CNBr-activated Sepharose 4B was re-swollen and washed twice with 1 mM HC1 for 5 minutes i n a 40 ml centrifuge tube to remove the dextran and lactose which were added to the activated gel i n order to preserve i t s a c t i v i t y under freeze-drying. After centrifugation, the 2.5 ml of swollen gel was resuspended i n 2 ml of 1 mM HC1 andtransferred to a 5 ml p l a s t i c tube. This suspension was centrifuged at 900 g for 3 minutes. Following the removal of the supernatant, 2.5 ml of coupling buffer (0.1 M NaCO^ and 0.5 M NaCl at pH 8.0) containing 10 mg of chymotrypsin was added to the gel and mixed thoroughly. The suspension was placed i n an end-over-end mixer for 2.5 hours at room temperature. After centrifugation to remove excess protein and coupling buffer, 2.5 ml of 1 M ethanolamine (adjusted to 58 pH 7.75 with 1 N HCl) was added to i n a c t i v a t e the remaining groups i n the CNBr-activated Sepharose for two hours at room temperature. The gel was transferred to a 5 ml syringe containing a M i l l i p o r e coarse p r e f i l t e r on the bottom. The gel was washed a l t e r n a t e l y with high pH borate buffer (0.1 M, pH 8.0) and low pH acetate buffers (0.1 M, pH 4) f i v e times to remove any trace of non-covaltently bound chymotrypsin and blocking reagent. Chymotrypsin-agarose was stored i n 2 M (NH 4) 2so 4 at pH 7.0. The a c t i v i t y of the enzyme-gel was determined by incubating aliquots of the preparation with 1% of casein as substrate i n 0.1 M borate buffer at pH 8.0. (see section IX-ld) b. Preparation of Clostridium Perfringens Neuraminidase-agarose 0.6 g of CNBr-activated Sepharose 4 B was reswollen and washed as previously described. 2 mg of neuraminidase i n 2.5 ml coupling buffer at pH 7.7 was added into the p l a s t i c tube containing the swollen Sepharose. The suspensions were placed i n an end-over-end mixer for 16 hours i n a cold room (4°c). The remaining active groups were inactivated, and the enzyme-gel washed, also as previously described. The a c t i v i t y of neuraminidase-agarose was determined by incubating aliquots of the preparation with NAN-lactose (Sigma) as substrate i n 0.05 M acetate buffer at pH 5.0 (See Section IX-ld). The neuraminidase-agarose was stored i n 2 M ammonium sul f a t e at pH 7.0. 59 c. Preparation of Control Neuraminidase-agarose The procedure was the same as the preparation of neuraminidase-agarose except the enzymatic a c t i v i t y was destroyed at 56°C for 15 minutes before coupling to agarose. d. Determination of enzymatic a c t i v i t i e s ( i ) Chymotrypsin-agarose. The a c t i v i t y of the Chymotrypsin-agarose was determined according to the modified method of Axen and Ernback (1971). The reaction mixture contained 1 ml of chymotrypsin-agarose suspension (0.1 ml wet chymotrypsin-agarose/ml) or chymotrypsin s o l u t i o n (1-20 ug/ml) and 1 ml of 1% alpha-casein s o l u t i o n i n 0.1 M borate buffer (pH 8.0). The mixture containing soluble enzyme was incubated at 37°C for 20 min with constant shaking. The mixture containing insoluble enzyme suspension was s t i r r e d with a small magnetic s t i r r i n g bar. Aft e r 20 min of incubation, 2 ml of 6% TCA was added and allowed to stand for 30 min. The blank was prepared by adding 2 ml of 6% TCA into 1 ml of substrate, followed by the addition of 1 ml of enzyme so l u t i o n . The o p t i c a l density of the f i l t r a t e s were measured at 280 nm and plotted against standard amounts of chymotrypsin to give a standard curve. The a c t i v i t y of the chymotrypsin-agarose was measured by comparison with the standard curve. 60 ( i i ) Neuraminidase-agarose. The a c t i v i t y of the neuraminidase-agarose was determined according to the modified method of Cassidy et a l . , (1965). The reaction mixture consisted of 0.05 M acetate buffer at pH 5.0, 27.5 ug NAN-lactose as substrate, and enzyme solution (5-50 mU) or 0.1 ml of wet enzyme-agarose. The f i n a l volume of the reaction mixture was adjusted to 0.25 ml with acetate buffer. The mixtures containing soluble enzyme were shaken during incubation at 37°c for 15 min. The assay for neuraminidase-agarose was conducted with constant s t i r r i n g . The reactions were terminated by placing the mixtures into an ice bath. Free NANA released from NAN-lactose was determined immediately by the th i o b a r b i t u r i c acid method of Warren (1959). A standard curve for the release of NANA from NAN-lactose was plotted against the concentration of neuraminidase as a measure of the a c t i v i t y of neuraminidase-agarose. 2. The Treatment of Lymphokines with Enzymes a. Lymphokine Treated with Soluble Neuraminidase 2 ml of 20-fold concentrated lymphokine super-natant buffered with 0.2 ml of 1 M acetate at pH 5.0 was divided into two aliqu o t s . In the experimental part, 0.2 U of neuraminidase (0.65 U/mg, Sigma) i n 0.1 ml of acetate buffer was 61 added. After one hour of incubation at 37 C, the solu t i o n was heated at 56°C for 15 minutes to in a c t i v a t e the neuraminidase. In the controls, the lymphokine was heated at 56°C for 15 minutes and then mixed with 0.1 ml of heat-inactivated (56°C, 15 min) neuraminidase. Both were dialyzed overnight against 100 volumes of RPMI-1640 with two changes. These enzyme-treated lymphokine preparations were then tested for t h e i r MIF and SRF a c t i v i t i e s . Some of the dialysed supernatants prepared as described above were reconstituted with 15% homologous guinea pig serum and tested for MIF a c t i v i t y . SRF a c t i v i t y was determined by measuring the increase i n vascular permeability following the intradermal i n j e c t i o n of 0.1 ml of the dialyzed supernatant (See Section I II b). "Control Group Supernatants" (Lymphocyte cultures to which DNP-BGG was added af t e r incubation) was treated and tested for MIF and SRF a c t i v i t i e s i n a si m i l a r manner. In order to determine whether neuraminidase and heated neuraminidase i n t e r f e r e s with MIF or SRF a c t i v i t y , 1 ml of soluble neuraminidase or heated neuraminidase (0.2 U/ml) i n 0.1 M acetate buffer pH 5.0 were dialysed with RPMI-1640 i n a s i m i l a r fashion and used concurrently i n MIF or SRF tests with the neura-minidase-treated lymphokine preparations. 62 b. Lymphokine Treated with Chymotrypsin-Agarose Lymphokine p r e c i p i t a t e from 60 ml of lymphokine supernatants from lymphocytes stimulated by DNP-BGG (See Section III-3) was dissolved i n 2.7 ml of d i s t i l l e d water and 0.3 ml of 1 M borate buffer at pH 8.0. These were compared with similar aliquots of the 20-fold concentrated control group supernatants (lymphocyte cultures to which DNP-BGG was added a f t e r incubation). The samples were then divided into 1.5 ml aliqu o t s . An aliquot of lymphokine supernatant was treated with 0.3 ml of chymotrypsin-agarose. This was incubated for two hours at 37°C with constant s t i r r i n g . Other aliquots of both lymphokine and control were treated with 0.3 ml control agarose (conjugated to inactivated enzyme). A f t e r incubation, the insoluble enzymes were removed by cen t r i f u g a t i o n at 900 g for fi v e minutes at 4°C. The supernatants were then s t e r i l i z e d by M i l l i p o r e f i l t r a t i o n (0.45 um pore s i z e ) , and dialysed against RPMI-1640 for 24 hours. The MIF and SRF a c t i v i t i e s of these enzyme-treated lymphokines and enzyme-treated control group supernatants were determined as described i n Section IX-2a. c. Lymphokine Treated with Neuraminidase-agarose Lymphokine p r e c i p i t a t e from 80 ml of lymphokine or control supernatant (See Section III-3) were dissolved i n 3.6 ml of 63 d i s t i l l e d water and 0.4 ml of 1 M acetate buffer at pH 5.0. The samples were then divided i n 2 ml a l i q u o t s . Aliquots of lymphokine or control group supernatants were treated with 0.4 ml of neuraminidase-agarose, or control heat inactivated neuraminidase-agarose, and incubated for one or three hours at 37°C with constant s t i r r i n g . The MIF and SRF a c t i v i t i e s of these enzyme-treated preparations were determined as described i n Section IX-2a. 64 RESULTS I PRODUCTION OF LYMPHOKINES The a b i l i t y of LN lymphocytes to generate lymphokines before and af t e r removal of macrophages was compared. Parameters for t e s t i n g lymphokines were the MIF and SRF a c t i v i t i e s . As shown i n Table 1, 10-fold concentrated supernatants generated from unpuri-f i e d lymphocytes possessed s i g n i f i c a n t l y higher MIF a c t i v i t y than from p u r i f i e d lymphocytes. The supernatants from macrophage-depleted LN lymphocytes contained no s i g n i f i c a n t MIF a c t i v i t y when stimulated by DNP-BGG and lower lymphokine a c t i v i t y when activated by Con-A. II SEPARATION OF LYMPHOKINES 1. Gel F i l t r a t i o n The concentrated lymphokine obtained from the Con A-stimulated lymphocytes was applied to a Sephadex G-100 column. Each of the five eluate frac t i o n s were pooled, concentrated and tested f or MIF and SRF a c t i v i t i e s (Figure 2 and 3). The MIF and SRF a c t i v i t i e s of each f r a c t i o n were compared with control supernatants. The maximal a c t i v i t i e s of both MIF and SRF were mainly found i n f r a c t i o n III whose molecular weight was s l i g h t l y smaller than that of peroxidase (MW 44,000). Smaller amounts of 64 a Table 1 Comparison of MIF a c t i v i t y of lymphokine produced by unpurified and p u r i f i e d LN lymphocytes stimulated with Con A and DNP-BGG antigen MIF A c t i v i t y ( % ) Supernatant from Con A-activated" lymphocyte-*-Supernatant from DNP-BGG activated lymphocyte Unpurified LN lymphocytes Mean—S.D. n = 3 60 - 14 65 - 10 Macrophage-; depleted LN lymphocytes Mean-S.D. n = 3 3 4 - 9 12 - 11 Significance of difference between unpurified and macrophage-depleted LN lymphocytes P < 1 0 - 6 P< 10~ 6 1. The lymphokine preparations from Con A-activated lymphocytes were prepared by passing through small Sephadex G-100 column. 2. The crude lymphokine preparations were prepared by using ammonium s u l f a t e f r a c t i o n a t i o n for measuring lymphokine a c t i v i t y . 3. See analysis of variance of variance i n Appendix 1. 64b Chymotryp-BSA Peroxidase sinogen A Ribonucleaee Fractions , I , , II [ , 111 | f IV [ | y Intensity of _ + erythema + + + Figure 2 E l u t i o n pattern of lymphokine supernatant on c a l i b r a t e d Sephadex G-100 column. 2 ml of 30 times reconcentrated supernatant of lymphocyte culture stimulated with Con A was applied i n the column. E l u t i o n peaks of marker protein are indicated with arrows. The s o l i d bar represents the percentage of i n h i b i t i o n of migration of peritoneal exudate c e l l s i n the various f r a c -tions . Skin reaction i s indicated by the diameter and i n t e n s i t y of erythema at 6 hours after the intradermal i n j e c t i o n . Both maximal MIF and SRF a c t i v i t i e s were found i n F r a c t i o n I I I . 64c Fraction I Frac t i o n II Fraction III Fraction IV Frac t i o n V Control supernatant Figure 3 I n h i b i t i o n of Macrophage migration by each f r a c -tion from Sephadex G-100. Five eluted f r a c t i o n s (shown i n Figure 2) were concentrated to 10-fold and tested for MIF a c t i v i t y . The maximal MIF a c t i -v i t y was found i n Fract i o n III whose molecular weight was s l i g h t l y smaller to that of peroxidase (M.W. 44j000). The control represents unfrac-tionated control supernatant. 65 MIF a c t i v i t y were present i n f r a c t i o n II (the area of bovine serum albumin) and f r a c t i o n IV (the area of chymotrypsin). The skin r e a c t i v i t y also occurred i n f r a c t i o n II and V. 2. Polyacrylamide Gel Electrophoresis The Sephadex G-100 f r a c t i o n s II, III and IV which contained MIF and SRF a c t i v i t i e s were further p u r i f i e d on polyacrylamide gels. After electrophoresis the gel was cut into f i v e pieces. Both maximal MIF and SRF were found i n E-3 (Figure 4 and Table 2) which corresponded to the albumin region. 3. F r a c t i o n a l P r e c i p i t a t i o n with Ammonium Sulphate This method was designed to concentrate the lymphokine and to remove the antigen DNP-BGG. F r a c t i o n a l p r e c i p i t a t i o n of lymphokine supernatant from DNP-BGG-stimulated lymphocytes was based on the fact that lymphokine i s soluble at 40% saturation of ammonium sulphate (SAS) and p r e c i p i t a t e d at 90% SAS (Dumonde et a l . , 1972; Monley e_t a l . 1973; Bray et a l . 1976). Most of DNP-BGG would be pr e c i p i t a t e d at 40% SAS. The r e s u l t s i n Table 1 show that lymphokine between 40 and 90% saturation not only contained MIF a c t i v i t y , but also caused skin inflammation. Pick et a l . , (1969) demonstrated that the inflammatory material i s soluble at 40% SAS and p r e c i p i t a t e d at 66% SAS. This crude lymphokine preparation 65a Table 2 Gel electrophoresis of the Sephadex G-100 f r a c t i o n s from supernatant of LN lymphocytes cultured with Con A. Three a c t i v i t i e s of lymphokines were found i n the same f r a c t i o n , E3. The control represents unfractionated con-t r o l supernatant. E l = cathode, E5 = anode. Electrophoretic fractions El E2 E3 E4 E5 P o s i t i o n of guinea pig serum protein pre-albumin albumin gamma-gl o b u l i n MIF a c t i v i t y ( i n h i b i t i o n %) -8.9 22.7 66.1 45.5 15 Mononuclear c e l l chemotactic a c t i v i t y 10 f i e l d s , 250xJ 10 81 112 53 34 Skin r e a c t i v i t y Intensity Diameter (cm) + 0.3 + 0.3 +++ 1.2 + 0.9 + 0.8 Assay for chemotactic a c t i v i t y was according to the methods of Wilkinson (1974). Assays for MIF and SRF a c t i v i t i e s were described i n the materials and methods section. 65b Fraction I Fraction II Fraction III Fraction IV F r a c t i o n V Control supernatant Figure 4 Inhibition of macrophage migration by each electrophoretic fraction. After electrophoresis, the gel was cut into 5 fractions starting from the bromophenol blue front and each fraction was tested for MIF activity. The maximal MIF activity was found in E3 Fraction which corresponded to the albumin region. The unfractionated control supernatant represents the control. 66 prepared by using s a l t f r a c t i o n a t i o n can be used f or measuring lymphokine a c t i v i t i e s . The preliminary experiments showed that control supernatant prepared by the same method would not i n t e r f e r e with the migration of macrophage and did not cause skin reaction. The method of preparation of lymphokines by s a l t f r a c t i o n a t i o n i s simple. Therefore, crude lymphokine preparations obtained by s a l t f r a c t i o n a t i o n were always used for studying lymphokine a c t i v i t i e s . I l l PROTEASE ACTIVITY OF LYMPHOKINES The protease a c t i v i t y of lymphokine was determined by using alpha-casein as a substrate at pH 3.5 and 7.0. The r e s u l t s presented i n Table 3 indicate that no s i g n i f i -cant amount of p r o t e o l y t i c a c t i v i t y s i m i l a r to cathepsin-D was found at pH 3.5 i n the lymphokine supernatant. Moreover, lymphokine had no p r o t e o l y t i c a c t i v i t y at pH 7.0 (neutral protease). Homogenates of macrophage and macrophage lysosomal preparation which possessed acid protease a c t i v i t y , were injec t e d intradermally into guinea pigs. An increase i n vascular permeability was not observed one to four hours af t e r the i n j e c t i o n ( F i g . 5). Pepstatin was employed as a s p e c i f i c i n h i b i t o r of acid protease (McAdoo ej_ a l . , 1973). Pepstatin at 1 mM i n h i b i t e d most of 66a Table 3 Protease a c t i v i t y of lymphokine and lysosomal preparations. No. of A c i d i c Neutral Skin batches protease protease r e a c t i v i t y tested a c t i v i t y a c t i v i t y (diameter, cm) O.D.700nm O.D.700nm mean + S.D. mean + S.D. mean + S.D. Control supernatant 10-fold concentrated Lymphokine supernatant 10-fold concentrated Lymphokin^ with pepstatin Macrophage homogenate (10 cells/ml) Macrophage lysosomal enzyme Macrophage lysosomal ^ enzyme with pepstatin 4 Human PMN lysosomal 0 0.02^0.02 2 ND 0.16^0.03 0.14^0.02 0.02^0.01 ND ND ND ND 0.39-0.01 0.2±0 1.6^0.3 1.5^0.2 0.3^ 0.2^0 0.2±0 1.3±0.1 3 1. Pepstatin (a s p e c i f i c i n h i b i t o r of ac i d i c protease) was i n a f i n a l concentration of 1 mM. 2. ND represents not done. 3. Four time d i l u t i o n of o r i g i n a l s o l u t i o n with s a l i n e . 4. Kindly supplied by Dr. R. T. Abbound, Vancouver General Hospital. 66b Figure 5 Skin inflammatory reaction at 4 hours after the intradermal i n j e c t i o n of 0.1 ml of macrophage lysosomal preparation and lymphokine i n the skin of guinea pig. Skin bluing induced by intravenous i n j e c t i o n of Evans blue demonstrates the increased vascular permeability at the inflammation s i t e . LK, lymphokine; LK+E, lymphokine incubated with immobile neuraminidase for 3 hours at 37°C; LK+Pep, lymphokine mixed with pepstatin (1 mM), a s p e c i f i c i n h i b i t o r of acid protease; C, control supernatant; Lys, macrophage lysosomal prepara-tion; Lys+Pep, lysosomal preparation mixed with pepstatin (1 mM); Sal, s a l i n e . 67 the a c t i v i t y of acid protease at pH 3.5 i n v i t r o (Table 3). Lympho-kine was mixed with pepstatin at a f i n a l concentration of 1 mM and was then, i n turn, i n j e c t e d intradermally. The skin r e a c t i v i t y was si m i l a r for lymphokine alone and lymphokine mixed with pepstatin (Figure 5 and Table 3). Human PMN lysosomal preparations, which were used as po s i t i v e controls, had neutral protease a c t i v i t y , and caused strong skin r e a c t i v i t y . Pepstatin i n human PMN lysosomal preparation did not i n h i b i t the skin inflammation (Figure 6). IV LYMPHOKINE EXPOSED TO HAGEMAN FACTOR, PREKALLIKREIN AND FRESH PLASMA In order to measure a c t i v a t i o n of Hageman factor and p r e k a l l i k r e i n , i t was necessary to i s o l a t e and p a r t i a l l y p u r i f y both components. When negatively charged p a r t i c l e s such as k a o l i n i s added, Hageman factor i s bound to the p a r t i c l e s and i s activated so as to generate the conversion of p r e k a l l i k r e i n to k a l l i k r e i n . A quantitative determination of k a l l i k r e i n was performed by the hydrolysis of benzoyl-arginine ethyl ester (BABe) to benzoyl-L-arginine which i s measured at 253 nm at pH 7.6 (Cochrane and Wuepper, 1971). By contrast, when Hageman factor is exposed to enzymes such as tr y p s i n or plasmin, the molecule i s cleaved into three fragments, and these fragments possess enzymatic a c t i v i t y (Cochrane et a l . 1973). This led to the speculation that soluble activators of HF may be released from stimulated lymphocytes. 67a Skin inflammatory reaction at 4 hours after the intradermal i n j e c t i o n of human PMN lysosomal preparation i n the skin of guinea pig. Skin bluing induced by intravenous i n j e c t i o n of Evans blue demonstrates the increased vascular permea-b i l i t y i n inflammation s i t e . C, i n j e c t i o n of sal i n e ; Lys, human PMN lysosomal preparation; Lys+Pep, human lysosomal preparation mixed with pepstatin (1 mM), a s p e c i f i c i n h i b i t o r of acid protease. 68 1. Separation of P r e k a l l i k r e i n and Hageman Factor The g l o b u l i n f r a c t i o n soluble at 25% and insoluble at 55% saturation of ammonium sulphate was applied to a column of DEAE-Sephadex A-50. P r e k a l l i k r e i n eluted i n a sin g l e peak at an io n i c strength of 0.17 as shown i n Figure 7. After e l u t i o n , the HF-rich fractions were passed over a second column of DEAE-Sephadex A-50 as shown i n figure 8. The f r a c t i o n containing the highest HF a c t i v i t y was used as test s o l u t i o n . P a r t i a l l y p u r i f i e d p r e k a l l i k r e i n was prepared by passing the material through two columns of DEAE-Sephadex A-50 (figures 7 and 9) and CM-Sephadex C-50 (Figure 10). The f r a c t i o n containing highest k a l l i k r e i n a c t i v i t y was used as test s o l u t i o n . 2. Lymphokines exposed to Hageman Factor and P r e k a l l i k r e i n A c t i v a t i o n of Hageman factor was accomplished by the addition of lymphokine or k a o l i n suspension at pH 7.5 i n a p l a s t i c tube. The results are shown i n Figure 11. Kaolin suspension at a concentration of 5 mg/ml could activate HF, whereas, HF exposed to equal volume of 10-fold concentration of lymphokines could not be converted to an active form. S i m i l a r l y , lymphokine could not convert p r e k a l l i k r e i n to k a l l i k r e i n . 68a 0-41 r0-8 0 3 H 02H 0.1J I 20 O o f- 1.5 Q O oi enzyme bound to agarose. Neuraminidase a c t i v i t y was completely abolished a f t e r heating at 56°C for 10 minutes. Immobilized enzyme was heat-resistant. 71 As shown i n Table 5, the treatment of lymphokine with neuraminidase followed by heating to 56°C for 15 minutes caused a s i g n i f i c a n t reduction of skin blueing from 55.6 ug Evans blue to 41.3 pg by the c r i t e r i o n of Evans blue extraction compared with untreated lymphokine. However, lymphokine retained most of SRF a c t i v i t y following treatment with soluble neuraminidase. 2. E f f e c t of Insoluble Enzyme-Agarose on Lymphokine A c t i v i t y Water insoluble enzyme, covalently bound to agarose, was chosen to insure complete removal of the enzyme following incubation of lymphokine with enzyme. One ml of wet chymotrypsin-agarose was found to have the same p r o t e o l y t i c a c t i v i t y as 75 pg of soluble chymotrypsin whereas 1 ml of wet neuraminidase-agarose contained 330 mU (NAN-lactose as substrate). (Figure 18 and 19). One ml of lymphokine was digested with 0.2 ml of wet chymotrypsin-agarose or neuraminidase-agarose. However, neuraminidase (Type V, Sigma) contained low protease a c t i v i t y , equivalent to 0.3 ug of trypsin per mg neuraminidase at pH 7.0 and 0.2 pg trypsin/mg at pH 5.0, using Azocoll as a substrate. The p o s s i b i l i t y that lymphokine becomes adsorbed to the agarose gel of the water insoluble enzyme was ruled out by using equal amount of control (inactivated-enzyme) agarose g e l . 71a Table 5 E f f e c t of enzyme on lymphokine a c t i v i t y . r Skin r e a c t i v i t y I n h i b i t i o n of migration (%) Diameter of bluing (cm) Skin bluing (ug Evans blue per 2.25 sq. cm.) A. Soluble enzyme N = 5 mean + S.D. mean + S .D. mean + S.D. Untreated lymphokine ND1 1.5±0.2 55.6-16.5 Neuraminidase-treated lymphokine ND1 1.3^.1 + 2 41.3-9.0 Control supernatant ND1 0.5^.0 10.3-7.6 B. Immobilized enzyme-agarose N = 3 Untreated lymphokine 68^6 1.3^0.1 23.1±4.3 Neuramini dase-treated lymphokine 15±10 3 1.2^0.1 18.1-3.64 Chymotryp s i n -treated lymphokine 3±3 5.1-2.0 Control supernatant 0 0.4^0.1 4.1^0.5 C Neur ami n i da s e-agarose supernatant 4 1. Not done because soluble neuraminidase i n h i b i t e d migration of macrophage. 2. P < 0.035 when compared with untreated lymphokine. 3. P < 0.000001 when compared with untreated lymphokine (Appendix 2). 4. P