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Mechanism of delayed hypersensitivity reactions : in vitro and in vivo studies of the possible role of… Wong, Fook Chuen 1977

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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<H 20 40 60 Tube number I 80 0.6 04 0.2 -< Figure 7 Chromatographic separation of rabbit plasma globulins on DEAE-Sephadex A-50. The pH was maintained at 8.0 and a gradient of NaCl was used to elute the protein. P r e k a l l i k r e i n eluted with i n i t i a l peak of protein. Hageman factor (Pre-PKA) eluted with the t h i r d peak of protein. 68b T u b e n u m b e r Figure 8 Chromatographic separation on DEAE-sephadex A-50 of fractions containing Hageman factor (pre-PKA) The fractions were obtained from the f i r s t DEAE-Sephadex column as shown i n Figure 7. 68c Tube number Figure 9 Chromatography on DEAE-Sephadex A-50 of prekal-l i k r e i n pooled from the f i r s t DEAE-Sephadex column as shown i n Figure 7. After a c t i v a t i o n , k a l l i -k r e i n was quantitated by BAEe hydrolysis. 68d Tube Number Figure 10 Chromatography on CM-Sephadex C-50 of prekal-l i k r e i n pooled from the second DEAE-Sephadex column as shown i n Figure 9. After a c t i v a t i o n , k a l l i k r e i n was quantitated by BAEe hydro l y s i s . 68e M O 0-5T 0-4H 0.3H 0-2H 0-1-HF + Kao l i n H F H F -I-Control super-nate h 7-5 5.0 2.5 H F +-SRF-r ich super-nate Figure 11 A c t i v a t i o n of Hageman factor by k a o l i n and lymphokine. The a c t i v i t y of Hageman factor was assessed by i t s a b i l i t y to 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 which i n turn hydrolyzes BAEe to BA. SRF-rich supernatant (lymphokine) d i d not activate Hageman fa c t o r . 69 3. Bioassay for Kinin Release from Fresh Plasma Exposed to Lymphokines Fresh plasma kinin-forming system contains three components i n precursor form: inactive Hageman factor, prekal-l i k r e i n and kininogen. When negatively charged p a r t i c l e s such as k a o l i n were added, HF was activated so as to generate the conversion of p r e k a l l i k r e i n which, i n turn activated the produc-t i o n of k i n i n from kininogen. K i n i n release from plasma was detected by using the i s o l a t e d guinea p i g ilieum. Figure 12 shows that k i n i n release was obtained from plasma incubated with k a o l i n for three minutes. When fresh plasma was incubated with 10-fold concentrated lymphokine or "control supernatant" for two or ten minutes, k i n i n release could not be detected. V EFFECT OF LYMPHOKINES ON MAST CELLS The preliminary tests showed that lymphokine preparations and two histamine l i b e r a t o r s , ATP and l e c i t h i n a s e A were without e f f e c t on the mesenteric mast c e l l s of the guinea pig. It has been reported that the guinea p i g mast c e l l s were r e s i s t a n t to compound 48/80 (Mota and Vugman, 1956) and l e c i t h i n a s e A (Boreus, 1960). Rat mast c e l l s were then used as a target c e l l for the experiment. Figure 13 showed that r a t mast c e l l s were s e n s i t i v e to l e c i t h i n a s e A. Only small amounts of histamine were released by sal i n e c o n t r o l , lymphokine or control supernatant. 69a The release of k i n i n from fresh plasma exposed to lymphokine and k a o l i n . K i n i n was not released from fresh plasma a f t e r incubation with lympho-kine. These tracings represent the contraction of the guinea p i g ileum i n the Schultz-Dale apparatus after exposure to each substance. 69b Figure 13 The release of histamine from suspension of is o l a t e d mast c e l l s of the r a t s . Small amount of histamine was released spontaneously from the mast c e l l s a f t e r incubation with s a l i n e , control super-natant and lymphokine. Lymphokine did not cause s p e c i f i c a l l y the release of histamine from the rat mast c e l l s in v i t r o . Tracing are guinea pig ileum contractions i n the Schultz-Dale apparatus. 69c LYMPHOKINE OR 20 25 TIME (MIN) Figure 14 E f f e c t of adding lymphokine and ADP on the o p t i c a l g density of p l a t e l e t - r i c h plasma (5 x 10 lymphokine ;-p l a t e l e t s / m l ) . control supernatant. Lymphokine did not induce p l a t e l e t aggregation and the p l a t e l e t character-i s t i c when ADP was added to p l a t e l e t - r i c h plasma containing lymphokine was also not affected. 70 VI EFFECT OF LYMPHOKINES ON PLATELETS Figure 14 showed that lymphokines did not induce p l a t e l e t aggregation. When ADP was added to p l a t e l e t - r i c h plasma containing lymphokine or control supernatant, the o p t i c a l density decreased immediately. Af t e r two minutes, the o p t i c a l density began to increase again. VII ENZYME TREATMENT OF LYMPHOKINES 1. E f f e c t of Soluble Neuraminidase on Lymphokine A c t i v i t y Before concluding that soluble neuraminidase acted d i r e c t l y on MIF and SRF, i t was necessary to determine whether or not neuraminidase i t s e l f i n t e r f e r e s with the migration of macrophage and whether or not i t induces skin inflammatory reactions. In preliminary t e s t s , neuraminidase at a concentra-ti o n of 0.1-0.2 U/ml would i n h i b i t the migration of macrophages (Figure 15) and induce hemorrhagic skin lesions (Figure 16 and Tables 4). Heating to 56°C for 15 minutes resulted i n loss of enzymatic a c t i v i t i e s (Figure 17) and skin r e a c t i v i t y (Figure 16). However, the heat-inactivated enzyme i t s e l f i n t e r f e r e s with the migration of macrophages (Figure 15). Thus, soluble neuraminidase was not able to be used for the study of MIF a c t i v i t y . In order to study the SRF a c t i v i t y , lymphokine may f i r s t be treated with neuraminidase (0.166 U/ml) p r i o r to i n a c t i v a t i o n of the enzyme at 56°C for 15 minutes. 70a Table 4 E f f e c t of b i o l o g i c a l products on lymphokine assays. B i o l o g i c a l products I n h i b i t i o n of macrophage migration Skin r e a c t i v i t y Concananavlin A, 10 ug/ml Fe t a l c a l f serum, 15% Soluble neuraminidase 0.2 units/ml Soluble neuraminidase 56°C, 15 min Supernatant from neuraminidase-agarose Supernatant from chymotrypsin-agarose Normal guinea pig serum p o s i t i v e p o s i t i v e p o s i t i v e p o s i t i v e negative negative negative po s i t i v e p o s i t i v e (hemorrhagic) po s i t i v e negative negative negative 70b Neuraminidase Heated (0.2 u/ml) Neuraminidase (0.2 u/ml) Insoluble 15% guinea p i g neuraminidase- serum i n RPMI-agarose 1640 medium Figure 15 Ef 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. Both soluble neuraminidase and heated neura-minidase i n h i b i t e d the migration of macrophage while insoluble neuraminidase-agarose and normal guinea pig serum did not. 70c Figure 16 Hemorrhagic skin inflammatory reaction at 4 hours af t e r the intradermal i n j e c t i o n of CI. perfringens neuraminidase. Ea, at concentration of 0.1 U/ml; Eb, at concentration of 0.2 U/ml; HE, neuramini-dase (0.2 U/ml) which had been heated at 56°C for 15 minutes. Neuraminidase induced hemorrhagic inflammatory reactions while heated enzyme did not. 70d 10 20 30 Time (Min) Figure 17 Heat s t a b i l i t y of soluble and immobilized neuraminidase of CI. perfringens at 56°C. o o , enzyme at room temperature for p o s i t i v e control; ;A /A , soluble enzyme; ——A A > 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<C0.01 when compared with untreated lymphokine (Appendix 3). 71b /jg Chymotrypsin Figure 18 Standard curve for the hydrolysis of alpha-casein by chymotrypsin. Ordinate: Absorbance of the hydrolysis products l i b e r a t e d from alpha-casein i n 20 minutes by the action of chymotrypsin. Abscissa: pg chymotrypsin i n 2 ml incubation mixture. The arrows indicate the method of c a l c u l a t i n g the a c t i v i t y of chymotrypsin-agarose. 71c 10 20 30 40 50 mil Neuraminidase Figure 19 Standard curve for the hydrolysis of NAN-lactose by the neuraminidase of CI. perfringens. Ordinate: Absorbance of N-acetyl neuraminic acid (NANA) li b e r a t e d from NAN-lactose i n the t h i o b a r b i t u r i c acid assay. Abscissa: mU neuraminidase i n 0.25 ml incubation mixture. The arrows indicate the method of c a l c u l a t i n g the a c t i v i t y of neuraminidase-agarose. 72 Experiments showed that both MIF and SRF a c t i v i t i e s were completely abolished by incubation of lymphokine with chymotrypsin-agarose for two hours at 37°c (Table 5; Figures 20, 21 and 22). The average i n t e n s i t y of skin blueing (pg of Evans blue per 2.25 sq cm of punch) of an untreated lymphokine supernatant was 23.1 pg Evans blue/punch while the i n t e n s i t y of skin blueing produced by lymphokine supernatants af t e r treatment with 0.3 ml of wet chymotrypsin-agarose f e l l to 5.1 pg Evans blue/punch. In the MIF assay, 67.9% i n h i b i t i o n of migration was reduced to 3%. Table 5 and Figure 20 show that the mean i n t e n s i t y of skin blueing caused by lymphokine incubated with neuraminidase-agarose was reduced s i g n i f i c a n t l y from 23.1 pg Evans blue/punch to 18.1 ug Evans blue/punch. In the MIF assay, 68% i n h i b i t i o n of migration was s i g n i f i c a n t l y reduced to 15%. It i s important to note that less than 20% i n h i b i t i o n of migration is considered as negative. These experiments demonstrated that incubation of lymphokine with neuraminidase-agarose could abolish the MIF a c t i v i t y a f t e r three hours at 37°C. Although t h i s enzyme caused s i g n i f i c a n t p a r t i a l destruction of the skin inflammatory a c t i v i t y , the lymphokine retained most of this a c t i v i t y (Figure 20). 7 2 a C O -Q _C C O o O) 1 J| % Migration Inhibition EE SRF Activity Untreated Chymotrypsin- Neuraminidase- Control Lymphokine treated treated Supernatant Lymphokine Lymphokine Figure 20 MIF and SRF a c t i v i t i e s a f t e r incubation with immobilized agarose-bound enzymes. 72b Figure 21 Skin inflammatory reactions at 4 hours after 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. Skin bluing induced by intravenous i n j e c t i o n of Evans blue demonstrates the increased vascular permeability i n inflammation s i t e . 11C, control supernatant; HA, lymphokine; 11AE, lymphokine treated with immobile neuraminidase for 3 hours at 37°C; Chy lh and 2h, lymphokine treated with immobile chymotrypsin for 1 and 2 hours at 37 ° c . Three injections were made for each test. 72c C o n t r o l Lymphokine supernatant supernatant Lymphokine t r e a t e d Lymphokine t r e a t e d with neuraminidase- with chymotrypsin-agarose agarose Figure 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 . Both immobilized neuraminidase and chymotrypsin destroyed MIF a c t i v i t y . 73 Microscopically, the patterns of the four-hour skin reactions induced by untreated and neuraminidase-treated lymphokines were s i m i l a r (Figure 23). The h i s t o l o g i c a l sections of the skin lesions were characterized by a mixed PMN-mononuclear i n f i l t r a t e i n the deeper dermis. 73a Figure 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. a, lymphokine; b, neuraminidase-treated lymphokine; c, control supernatant. The patterns of the skin reactions induced by lympho-kine and enzyme-treated lymphokine were s i m i l a r . The histology of the ski n lesions were characterized by a mixed PMN-mononuclear i n f i l t r a t e i n the deeper dermis. No inflammatory c e l l s were found i n the dermis f o l -lowing the intradermal i n j e c t i o n of control supernatant. 74 DISCUSSION I PRODUCTION OF LYMPHOKINES The present study confirmed that the incubation of guinea pig LN lymphoid c e l l s with Con A or s p e c i f i c antigen, r e s u l t s i n the release of factors into the supernatant which are able to cause skin inflammation and i n h i b i t i o n of macrophage migration. The release of lymphokines from the stimulated lymphocytes is dependent on the following technical considerations: (1) number of lymphocytes i n the culture medium; (2) concentration of mitogen and antigen; (3) time of incubation; and (4) type of c e l l s . In the tissue culture system of the present experiments, 1-I.5xl0 7 v i a b l e c e l l s / m l i n medium RPMI-1640 with Con A or antigen was incubated for 20 hours at 37°C i n the presence of 5% C0 2 i n a i r . Preliminary studies showed that high concentrations of c e l l s i n the culture medium such as 5 x l 0 7 c e l l s / m l (used by Pick et a l . 1969) would change the pH of the culture medium even with Earle's buffer. Hank's buffer i n the culture medium was not suitable due to i t s weak buf f e r i n g power. Con A at a concentration of 10-20 pg/ml i n the culture medium was found to be optimal and the optimum for DNP-BGG was 50 pg/ml. The present results were s i m i l a r to the findings of Ford and Ashworth (1976) showing that an optimal output of MIF was achieved at between 20-100 pg DNP-BGG/ml and Con A 5-10 pg/ml. It 75 was i n t e r e s t i n g that DNP-BGG at concentrations of 200 ug/ml could not induce the release of lymphokines from the s e n s i t i z e d lymphocytes. The reason i s obscure. I t i s also important that unpurified LN lymphocytes were found to be much more potent i n re l e a s i n g lymphokine materials than p u r i f i e d lymphocytes (Table I ) . P u r i f i e d lymphocytes, presumably T lymphocytes, were obtained by passage of LN c e l l s through a glass bead column at 37°C to which macrophages and B lymphocytes adhere (Rosenthal et a l . , 1972). The present re s u l t s indicated that lymphokine production by LN c e l l s of guinea pig required macrophages and confirmed the most recent findings by many inv e s t i g a t o r s . There is 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 factor (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; Waldron et a l . , 1973; Nelson and Leu, 1975; Yoshinaga etal., 1975; Rosenstreich et a l . , 1976). Complete removal of macrophages by one step passage through a glass bead column i n the present experiments i s impossible. I f a high c e l l concentration i n tissue culture i s used, even though the contaminating c e l l s are a small percentage of the t o t a l , t h e i r 76 numbers i n the tissue culture medium can be s u f f i c i e n t to exert an influence on the lymphocyte a c t i v a t i o n . The high c e l l number of 5 x 10' c e l l s / m l has been employed for the production of SRF and MIF (Pick et a l . , 1969; 1970). In t h e i r tissue culture system for lymphokine production, the p o s s i b i l i t y of contaminating macrophages i s very r e a l . II SEPARATION OF LYMPHOKINES 1. Gel F i l t r a t i o n The present experiments showed that by employing a Sephadex G-100 column, the maximal MIF a c t i v i t y was found i n f r a c t i o n I II whose molecular weight was s l i g h t l y smaller than that of peroxidase (M.W. 44,000). The estimates of other authors from gel f i l t r a t i o n of MIF have ranged from 33,000-55,000 (Remold et a l . , 1970) to 56,000-82,000 (Dumonde et a l . 1972). Similar amounts of MIF a c t i v i t y was present i n f r a c t i o n II (the area of bovine serum albumin, MW 67,000) and f r a c t i o n IV (the area of chymotrypsinogen A, MW 25,000). The r e s u l t s confirm the fin d i n g of Remold et a l . , (1972) that MIF from Con A stimulated lymphocytes i s heterogenous. The d i s t r i b u t i o n of ski n r e a c t i v i t y i n f r a c t i o n s III, IV, and V was si m i l a r to the findings of M a i l l a r d et a l . , (1972) that v a s o a c t i v i t y was present i n a single peak l y i n g between human albumin (M.W. 77 67,000) and cytochrome (M.W. 12,500) with average molecular weight of 39,000. It was concluded that Sephadex G-100 column chroma-tography could not separate MIF and SRF. 2. Polyacrylamide Gel Electrophoresis It would be of in t e r e s t to determine whether skin r e a c t i v i t y i s separable from MIF aft e r electrophoresis on acrylamide gels. The method was according to that of Remold et a l . , (1970) with s l i g h t modification. A preparative gel electrophoresis apparatus was used. The gel was polymerized without tetramethylethylene diamine (TEMED) and i n the presence of only 0.017% ammonium persulphate by using long wavelength u l t r a v i o l e t l i g h t . This was due to the fact that TEMED and higher concentrations of ammonium persulphate are toxic to c e l l s i n culture (Remold et a l . , 1970). UV l i g h t was used to polymerized the gel because i t could accelerate polymerization while fluorescent l i g h t could not i f the gels contained no TEMED and only a small amount of persulphate. Both maximal MIF and SRF a c t i v i t i e s were found i n E-3 (Figure 4 and Table 2), which correspond to the albumin region. This f i n d i n g contradicts the studies of Remold et a l . , (1970) who demonstrated that MIF a c t i v i t y was recovered from the pre-albumin. In conclusion, i t is obvious that MIF and SRF are substances which 78 have very similar physical properties and may, i n f a c t , be due to c l o s e l y r e l a t e d or si m i l a r molecules. Most recently, by the use of i s o - e l e c t r i c focusing technique, separation and p a r t i a l p u r i f i -cation of MIF, colony stimulating factor and i n t e r f e r o n from mouse lymphokine has been achieved (Trudgett, 1976). It would be i n t e r e s t i n g to know whether i s o e l e c t r i c focusing i s applicable i n the separation of guinea pig lymphokines. I l l EFFECTS OF BIOLOGICAL PRODUCTS ON LYMPHOKINE ASSAYS Many b i o l o g i c a l products are involved i n the preparation, treatment and assays of lymphokines. When Con A, for example, i s used as a mitogen to produce lymphokines i n tissue culture, i t may contaminate the lymphokine preparations. Preliminary tests were performed i n an attempt to determine whether neuraminidase, Con A and f e t a l c a l f serum i n t e r f e r e with the lymphokine assays. The r e s u l t s i n Table 4 show that 0.1-0.2 U/ml of neuraminidase, which was usually used for the treatment of lymphokine, could cause a skin inflammatory reaction (Figure 16) and i n h i b i t migration of macro-phages (Figure 15). Heating to 56°C for 15 minutes resulted i n loss of enzymatic a c t i v i t y (Figure 17) and skin r e a c t i v i t y (Figure 16). The heated neuraminidase i t s e l f i n t e r f e r e d with macrophage migration. Obviously, lymphokines treated with soluble neuraminidase were not su i t a b l e for MIF assay. This finding was i n 79 agreement with the c r i t i c i s m of Papagoriou et a l . , (1974) who showed that neuraminidase i n t e r f e r e d with the migration of human lymphoid c e l l s and guinea pig peritoneal macrophages. Thus, complete removal of the neuraminidase from the treated samples appears to be an absolute requirement. During the i n v e s t i g a t i o n of MIF i n the supernatants of c e l l c ultures, i t was discovered that f e t a l c a l f serum (FCS) i n h i b i t e d the ^n v i t r o migration of macrophages i n control supernatants. On the other hand, migration of macrophages was not i n h i b i t e d by normal guinea p i g serum. This fi n d i n g confirms the report of Fox et a l . , (1974), who demonstrated that FCS contained MIF-like material. Moreover, MIF-like material from FCS could be p a r t i a l l y or com-p l e t e l y inactivated by either d i a l y s i s , repeated freezing and thawing, heating to 56°C for 30 minutes, or changes of pH beyond the range of 6.0 to 7.7. As guinea pig serum did not i n t e r f e r e with the MIF assay, i t was an excellent serum supplement i n MIF assays. It was found that Con A alone at a concentration of 10 pg/ml or higher would produce skin lesions (Figure 24) and would i n h i b i t markedly the migration of peritoneal macrophages (Taylor et a l . , 1975). Similar skin lesions induced by Con A were reported by Kind and Peterson (1976); Schwartz et a l . , (1970); P e l l e y and Schwartz (1972) and Schier et a l . , (1974). Taylor et a l . , (1975) demonstrated that ConA at as low concentrations as 1 ug/ml would 79a Figure 24 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 Con A solution at d i f f e r e n t concentrations 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 i n inflammation s i t e . C, i n j e c i o n of s a l i n e ; Con A, i n j e c t i o n of Con A at a concen-t r a t i o n of 10, 20 and 40 pg/ml. Low dose (10 pg/ml) of Con A s t i l l induced inflammation. 80 a f f e c t MIF assay r e s u l t s . Therefore, removal of Con A from the incubation supernatant i s compulsory p r i o r to MIF assays. (To avoid this a r t i f a c t caused by ConA, antigen-induced production of lympho-kines was then used for subsequent experiments). There are two methods which can be used to remove Con A from Con-A-activated lymphocyte culture supernatants. The f i r s t method involves Sephadex f r a c t i o n a t i o n of the culture and supernatant since Con A is absorbed by Sephadex (Agrowal and Goldstein, 1965). However, Schwartz et. a l . , (1970) and Pe l l e y and Schwartz (1972) reported that Con A could only be completely removed by absorption to the Sephadex gel i n the absence of serum supplement. In the presence of serum, Con A i s eluted i n the fractions prior to albumin and thus w i l l i n t e r f e r e with lymphokine assay. The second method is the incubation of lymphocytes with insoluble Con A i n serum-free medium for 24 hours which can y i e l d sizeable amounts of MIF and mitogenic factor (Geczy et _al., 1975; F r i e d r i c h et a l . , 1975). This makes i t easy to remove the mitogen from the culture supernatant by simple cen t r i f u g a t i o n . 81 IV PROTEASE ACTIVITY OF LYMPHOKINES The p o s s i b i l i t y was considered that lymphokines may possess non-specific inflammatory materials which are unrelated to mediators associated with delayed h y p e r s e n s i t i v i t y and which are released from the stimulated lymphocytes. As an example, Janoff and Zeligs (1968) demonstrated that PMN lysosomal enzymes could e l i c i t a skin inflam-matory reaction. Houck et. a l . , (1973) demonstrated that LNPF and lymphokines from mouse lymphocytes had SRF a c t i v i t y which was due to the presence of acid protease. The r e s u l t s shown i n Table 3 re-vealed that lymphokine preparations did not contain neutral protease a c t i v i t y and that there were only small amounts of acid protease a c t i v i t y i n two batches of lymphokine preparations. Addition of pepstatin (a s p e c i f i c i n h i b i t o r of acid protease) to the lymphokine preparation did not i n h i b i t the skin reaction and suggested that the skin reaction induced by lymphokine was not due to acid protease. Furthermore, macrophage lysosomal preparations with acid protease a c t i v i t y , but with no neutral protease a c t i v i t y , did not e l i c i t any s k i n reaction. Thus i t i s doubtful that the permeability increasing a c t i v i t y of SRF and LNPF is due to the presence of cathepsin D-like enzyme (acid protease). A possible reason for the difference between the present results and those reported by Houck et^ a l . , (1973) may stem from the species of animal used. The l a t t e r used rats as the animal model. A comparison of the a c t i v i t i e s of lympho-82 kines obtained from cultures of guinea pig and rat lymphocytes i s necessary. Furthermore, human PMN lysosomal preparations containing neutral protease could cause skin reaction (Figure 6 and Table 3). Addition of pepstatin to the PMN lysosomal preparation could not i n h i b i t the skin reaction, suggesting that PMN lysosomal enzymes e l i c i t i n g the skin reaction was not due to acid protease, but could be due to neutral protease or other inflammatory materials. Recently, Movat et^ a l . , (1976) demonstrated that neutral protease with kininogenase a c t i v i t y was i s o l a t e d from human PMN leukocyte by cation exchange chromatography and g e l - f i l t r a t i o n . However, the p o s s i b i l i t y that lymphokine might have s p e c i f i c neutral protease such as kininogenase, collagenase, elastase and SH-dependent protease could not be completely excluded from these experiments. Assays for these s p e c i f i c enzymes were not done. Grayzel et a l . , (1975) showed that p r o t e o l y t i c a c t i v i t y could be determined by measuring the TCA soluble radioactive peptides released from 3 H-acetylated casein or haemoglobin. They found that human peripheral blood lymphocytes contained a number of proteases, including cathepsin D, a neutral serine protease(s) and t h i o l protease(s). Furthermore, the neutral protease was bound to the surface of the lymphocytes but not secreted into the medium. Therefore, a more sens i t i v e assay for measuring p r o t e o l y t i c a c t i v i t y i n the lymphokine preparation may be necessary. 83 V LYMPHOKINES EXPOSED TO FRESH PLASMA, HAGEMAN FACTOR AND PREKALLIKREIN In order to assess the p o s s i b i l i t y that lymphokine might activate the kinin-forming system of plasma, two experiments were set up: (1) lymphokine preparation was exposed to 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 , (2) lymphokine preparation was exposed d i r e c t l y to fresh plasma. The r e s u l t s demonstrated that k i n i n was not released when fresh plasma was incubated with lymphokine for two and ten minutes and assayed on the guinea pig ileum (Figure 12). Furthermore, lympho-kine did not activ a t e HF (Figure 11) and p r e k a l l i k r e i n . Similar r e s u l t s have been reported by M a i l l a r d e_t a l . , (1972). They demon-strated that heated serum with SRF supernatant would not contract the guinea p i g ileum. These experiments indicate that lymphokine preparations contained no activator for kinin-forming systems of fresh plasma. This r e s u l t i s not s u r p r i s i n g because the previous experi-ment showed that there was no p r o t e o l y t i c a c t i v i t y at pH 3.5 and 7.0 i n the lymphokine preparations. M a i l l a r d et a l . , (1972) reported 84 that e s t e r o l y t i c a c t i v i t y for ATEe or BAEe was not detected i n SRF supernatant. Moreover, Cochrane et a l . , (1973) demonstrated that a c t i v a t i o n of HF i n so l u t i o n was obtained with t r y p s i n , k a l l i -k r e i n , plasmin, and Factor XI (plasma thromboplastin antecedent). Kinin can be released from kininogen by the action of k a l l i k r e i n (Wuepper and Cochrane, 1972), venom enzyme (Suzuki et: s i l . , 1965; Hamberg and Rocha e S i l v a , 1957) and PMN lysosomal neutral protease (Movat et a l . , 1973, 1976). The enzymes which are l i k e l y candidates for activators are not present i n lymphokine preparations. M a i l l a r d et a l . , (1972) demonstrated that i n an iji v i vo system, polybrene and protamine sulphate which protect the a c t i v a -tors of HF would i n h i b i t SRF a c t i v i t y , and that sodium d i e t h y l -dithiocarbamate which i s an antagonist of kininase would enhance the a c t i v i t y . Therefore, these authors suggested that the SRF a c t i v i t y may be re l a t e d to the a c t i v a t i o n of kinin-forming system. In the present in v i t r o system, the f a i l u r e of d i r e c t a c t i v a t i o n of kinin-forming system implied that lymphokine i s not a d i r e c t media-tor of skin inflammatory reaction. I t i s most l i k e l y that the skin reaction induced by lymphokine may be a secondary action and i s complicated. (See Discussion Section X) 85 VI EFFECTS OF LYMPHOKINES ON MAST CELLS Preliminary tests showed that antihistamine (mepyramine) did not reduce the increased vascular permeability produced by the intradermal i n j e c t i o n of lymphokines. This suggests that mast c e l l s are not involved i n this reaction. This r e s u l t confirms the f i n d i n g of M a i l l a r d et al., (1972). Moreover, i n v i t r o tests indicate that lymphokine preparations and two histamine l i b e r a t o r s , ATP and l e c i t h i n a s e A, did not stimulate mast-cell responses i n the guinea pig. This confirms the findings of Boreus (1960) who demonstrated that guinea pig mast c e l l s did not show the t y p i c a l d i s r u p t i o n following the incubation with compound 40/80 and l e c i t h i n a s e A, which caused the disruption of the rat and hamster mast c e l l s . Rat mast c e l l s were therefore used as a target c e l l i n subsequent experiments. Figure 13 shows that the rat mast c e l l s were s e n s i t i v e to l e c i t h i n a s e A. A small amount of histamine was present i n control group (s a l i n e only), lymphokine and "control supernatant". This was due to the spontaneous release from the rat mast c e l l s (Johnson and Moran, 1966). It seems l i k e l y that lymphokine does not cause the release of histamine from mast c e l l s of rats i n v i t r o and that mast c e l l s are not involved i n the s k i n inflammatory reaction produced by lymphokines. 86 VII EFFECT OF LYMPHOKINES ON PLATELETS The involvement of p l a t e l e t s i n inflammation has been suggested by some investigators (Redei and Kelemen, 1969; Gorg and Kovacs, 1969). Due to the multiple b i o l o g i c a l a c t i v i t i e s of lympho-kines, i t seemed reasonable to assume there may be in t e r a c t i o n s between lymphokine and p l a t e l e t s . The experiments demonstrated that lymphokine produced from s e n s i t i v e LN lymphoid c e l l s did not induced p l a t e l e t aggregation. This r e s u l t contradicts the findings of Lavelle et a l . , (1975) who demonstrated that s e n s i t i z e d mononuclear c e l l s from the spleen of ne p h r i t i c rabbits upon re-exposure to the s e n s i t i z i n g antigen, produced a substance which was capable of inducing p l a t e l e t aggregation. The important difference between the present experiment and that of Lavelle et a l . , (1975) i s the source of lymphoid c e l l s . The spleen c e l l s used by these authors probably contained macrophages (Shortman et a l . 1971; Mosier, 1967). Several investigators indicate that lymphokines would induce the release of enzymes from macrophages into the culture supernatant (Pantalone and Page, 1975; Wahl et al. 1974). Therefore, a p o s s i b i l i t y i s that lymphokines released from lymphocytes which were stimulated by s p e c i f i c antigens w i l l subsequently activ a t e macrophages to release p l a t e l e t aggregation factor (PAF). This factor released from macrophages i s probably a h y d r o l y t i c enzyme. It has been reported that p r o t e o l y t i c enzymes such as t r y p s i n , pronase, papain and 87 thrombin would cause p l a t e l e t aggregation (Mustard and Packham, 1970). Furthermore, Lavalle et a l . , (1975) demonstrated that the properties of PAF produced by the spleen c e l l s were sim i l a r to that of thombin. Thus, for further studies, i t would be i n t e r e s t i n g to determine whether or not macrophages are required i n the production of PAF by guinea pig spleen c e l l s . VIII ENZYME TREATMENT OF LYMPHOKINES 1. Chymotrypsin-agarose In these experiments, the b i o l o g i c a l a c t i v i t i e s of lymphokine and enzyme-treated lymphokine as measured by macro-phage migration i n h i b i t i o n and by skin reaction were compared. The data i n Table 5 and Figure 20 c l e a r l y demonstated that MIF and SRF a c t i v i t e s were completely destroyed by incubation with chymotrypsin-agarose for two hours and i s consistent with other evidence that MIF and SRF are proteins. The re s u l t s confirm that findings of Remold and David (1971) who demonstrated that MIF a c t i v i t y could be completely abolished by dige s t i o n for 16 hours with chymotrypsin which was coupled to a copolymer of ethylene and maleic anhydride. Furthermore, Pick et a l . , (1969) indicated that skin active substances could be completely destroyed by pepsin digestion and p a r t i a l l y destroyed by tryp s i n and papain. Other b i o l o g i c a l a c t i v i t i e s 88 of lymphokines such as lymphotoxin, chemotactic fa c t o r , and macrophage a c t i v a t i o n factor were not tested following treatment with chymotrypsin-agarose. It was believed that the p r o t e i n - l i k e materials of lymphokine would be destroyed by chymotrypsin. Therefore, i t was d i f f i c u l t to assess the close c o r r e l a t i o n between MIF and SRF by using immobilized chymotrypsin. MIF and SRF are r e s i s t a n t to DNase and RNase (David and David, 1972; Pick et a l . , 1969) which makes i t u n l i k e l y that a c t i v i t y i s due to a nu c l e i c acid moiety. The insoluble gel-enzymes were chosen because they could be completely removed by centrifugation and f i l t r a t i o n . This insured that the enzymes would not be present i n the treated lymphokines which were subsequently assayed for MIF and SRF a c t i v i t i e s . 2. Neuraminidase-agarose The experiments shown i n Table 4 and Figure 15 indicated that incubation of lymphokine with neuraminidase-agarose could abolish MIF a c t i v i t y a f t e r three hours at 37°C. This r e s u l t confirms the findings of Remold and David (1971) that MIF i s a glycoprotein, the a c t i v i t y of which was destroyed by neuraminidase. These authors used soluble enzyme i n the incubation with lymphokine. A f t e r incubation, the mixture was chromatographed separately on a Sephadex G-100 column and the fractions e l u t i n g just 89 aft e r albumin were assayed for MIF a c t i v i t y . T h e o r e t i c a l l y , separation of MIF from neuraminidase with g e l - f i l t r a t i o n chromatography i s d i f f i c u l t due to t h e i r s i m i l a r molecular weight (neuraminidase MW 56,000 and MIF MW 33,000-55,000) (Drzeniek, 1972; Remold and David, 1971). A b o l i t i o n of MIF a c t i v i t y by neuraminidase indicated that terminal s i a l i c a c i d residues are necessary for i t s b i o l o g i c a l a c t i v i t y . However, human ,MIF from se n s i t i z e d human lymphocytes stimulated by streptokinase-streptodornase was r e s i s t a n t to neuraminidase and had a molecular weight of 25,000 (Rocklin et. a l . 1972 ; Remold, 1972) . Probably the most i n t e r e s t i n g observation was that neuraminidase completely destroyed MIF a c t i v i t y while causing only a s l i g h t drop of SRF a c t i v i t y (Table 5 and Figure 20). MIF i s therefore more s e n s i t i v e to neuraminidase but there is s t i l l a s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t of the enzyme on SRF. In addition i t was noted that microscopic studies of the skin showed that the mononuclear and n e u t r o p h i l i c i n f i l t r a t i o n s induced by either the treated or untreated lymphokines were s i m i l a r . These findings suggest that: (1) MIF may contribute i n part to the ski n inflammatory reaction but is not mainly responsible for i t within 4 hours of i n j e c t i o n of a lymphokine preparation. 90 (2) Other b i o l o g i c a l materials may be present i n the lymphokine preparation which contribute to SRF a c t i v i t y and are not sens i t i v e to neuraminidase. This is not s u r p r i s i n g since lymphokine preparations are known to contain chemotactic factor, mitogenic factor and lymphotoxins, a l l of which may contribute to the skin inflammatory reaction. (See Discussion Section X) (3) The neuraminidase preparations may contain contaminating enzymes with other s p e c i f i c i t i e s . The neuraminidase was tested for protease a c t i v i t y at pH 5.0 and 7.0 using a z o c o l l as substrate. A very low a c t i v i t y could be detected at pH 5.0 which i s the same pH for neuraminidase digestion. I t i s possible that t h i s a c i d p r o t e o l y t i c a c t i v i t y caused the loss of SRF a c t i v i t y a f t e r digestion with neuraminidase. (4) It i s possible that both MIF and SRF a c t i v i t i e s are se n s i t i v e to neuraminidase but that SRF i s for some reason much more r e s i s t a n t to digestion with the enzyme. Dose-response experiments were not done to explore this point. It would be necessary to plot log-dose response curves of MIF and SRF versus enzyme concentrations and d i l u t i o n s of lymphokines. S i m i l a r l y , d i f f e r e n t times of digestion should indicate the optimal times for complete destruction of MIF a c t i v i t y and the rate at which SRF a c t i v i t y i s diminished. These 91 experiments would give important information on the mechanisms by which the neuraminidase preparations a f f e c t MIF and SRF lymphokine a c t i v i t i e s . In this regard, preliminary experiments showed that a f t e r digestion of lymphokine with neuraminidase for 1 hour MIF a c t i v i t y decreased from 70% to 38%, and after digestion for 3 hours the a c t i v i t y decreased to 15%. (5) There are two possible interpretations of these data: (a) The MIF and SRF a c t i v i t i e s are present i n two d i f f e r e n t materials, of which MIF i s sensitive to neuraminidase and SRF i s not, as discussed i n Section (4). (b) The MIF and SRF a c t i v i t i e s are properties of the same material and the assays d i f f e r i n s e n s i t i v i t y , the SRF assay being more s e n s i t i v e than the MIF. It may be noted, however, that Morley Wolstencroft and Dumonde (1973) have shown that the two assays have si m i l a r s e n s i t i v i t y , which i f true, would favour the the f i r s t i n t e r p r e t a t i o n of the present data. It was reported that neuraminidase destroyed guinea pig MIF, but did not a f f e c t the a c t i v i t i e s of chemotactic factor and lymphotoxin (David, 1975; Coyne et. a l . 1973). At least two such kinds of mediators could be present i n the neuraminidase-treated lymphokine preparation. The present experiments have shown that MIF and SRF a c t i v i t i e s were completely abolished by chymotrypsin. Other 92 investigators had shown that chymotrypsin would also destroy lymphotoxin (Coyne et a l . 1973) and chemotactic factor (Wahl et a l . 1974). Therefore, i t i s quite l i k e l y that the skin reactive factor may represent the e f f e c t of a combination of a number of factors including at l e a s t chemotactic factor and lymphotoxin. Furthermore, the p a r t i a l loss of skin reaction caused by neuraminidase i s not necessary due to the a b o l i t i o n of MIF a c t i v i t y by neuraminidase. It i s possible that other glycoproteins which have terminal s i a l i c acid residues may also cause skin reactions, or one glycoprotein molecule may possess two active s i t e s , one for MIF and the other for SRF. I t i s emphasized here that although MIF i s not mainly responsible for the skin reaction, i t may be involved i n the enhancement the DH reaction i n vi v o . More recently, Geczy et a l . , (1975 and 1976) reported that antilymphokine antibody t o t a l l y suppressed the delayed skin response of s e n s i t i z e d guinea pigs challenged with PPD. Their in v i t r o studies have shown that MIF could be completely absorbed from whole supernatants by immuno-adsorbent columns prepared with rabbit anti-guinea p i g lymphokine antibody while SRF and MF were not retained. This recent i n v i t r o study supports the present findings that lymphokines treated with neuraminidase to remove MIF would r e t a i n SRF a c t i v i t y . 93 Remold and David (1971) indicated that guinea pig MIF i s an acid molecule migrating i n electrophoresis as a prealbumin. Unfortunately, attempts to separate MIF from chemotactic factor by electrophoresis were unsuccessful. 94 IX PROBLEMS ON THE STUDIES OF THE MECHANISM OF LYMPHOKINE EFFECTS ON MACROPHAGE It has been unequivocably shown that s e n s i t i z e d lymphocytes generate a number of lymphokines with a v a r i e t y of b i o l o g i c a l a c t i v i t i e s which are dependent upon the assay system employed. Whether or not a l l lymphokines are involved i n DH reactions, and whether the a c t i v i t i e s detected are due to one or separate e n t i t i e s , s t i l l remain unresolved. These questions w i l l probably remain unanswered u n t i l chemical separation and p u r i f i c a t i o n of the factors i s achieved. MIF was the f i r s t lymphokine to be described and i s the lymphokine most thoroughly investigated. As described i n the previous section, lymphokines may modify the behaviour of macro-phages. Recently, data was presented that the c e l l surface of macrophages was altered following i n t e r a c t i o n with MIF. Poulter and Turk (1975a and b) found that rapid changes occurred i n macrophages after contact with lymphokines for one hour. These change include the rounding of c e l l s (Nath et a l . , 1973), decreased macrophage volume (Poulter and Turk, 1975a), decreased permeability, decreased biosynthetic p o t e n t i a l (Poulter and Turk, 1975b), and the cross-linkage of sulphydryl groups. Poulter and Turk (1975b) proposed that these c e l l u l a r changes may r e f l e c t the functional e f f e c t of macrophage i n h i b i t i o n . In addition, macrophage aggrega-95 t i o n , reduction i n electrophoretic m o b i l i t y and the reduction i n the i n t e r f a c i a l energy of the c e l l surface may also be rel a t e d to the a l t e r a t i o n of macrophage membrane following contact with lymphokines. It must be emphasized that a l l the changes described occurred following the incubation with lymphokine for a period of one to five hours. Whether or not the changes observed were due s o l e l y to the added lymphokine or may be due, i n part, to " a l t e r e d " macrophages upon contact with lymphokine i s a question of great i n t e r e s t . Remold (1973) found that L-fucose blocked the e f f e c t of guinea pig MIF on macrophages and that macrophages incubated with alpha-L-fucosidase no longer responded to MIF. He proposed that L-fucose i s an important part of the receptor for MIF on the macrophage plasma membrane. It i s possible that one material i n lymphokine acts on a macrophage receptor and causes the many physical and b i o l o g i c a l changes which are observed. In other words, one material i n lymphokine posesses several b i o l o g i c a l a c t i v i t i e s . To support t h i s hypothesis, i t seems important to f i n d out whether or not the immobilized neuraminidase-agarose which has been used i n the present experiment can abolish the various a c t i v i t i e s of lymphokines. 96 X POSSIBLE MECHANISM OF DELAYED HYPERSENSITIVITY REACTIONS - A HYPOTHESIS A possible mechanism of DH reaction i s outlined i n Figure 25. This i s based on in v i t r o and in vivo observations described previously. It i s postulated that DH reactions may be manifested i n two stages, (1) i n i t i a l reaction and (2) enhancement. Lymphokines may be c l a s s i f i e d into two types with respect to the time course of inflammation. Those are (1) f a s t - a c t i n g lymphokines and (2) slow-acting lymphokines. Fast-acting lymphokines are those which cause a skin reaction within 4 hours of i n j e c t i o n s as shown i n Figure 21. These skin reactions do not seem to depend on MIF since they are not greatly affected by treatment of the lymphokine preparations with neuraminidase. Part of the skin reaction at 4 hours may therefore be a t t r i b u t e d to SRF which probably involves chemotactic factor and lymphotoxin. Lymphotoxin has been found to be cytotoxic to many types of c e l l s ^n v i t r o from many d i f f e r e n t animal species (Granger, 1972) and thus may take part i n e l i c i t i n g the increase in vascular permeability in vivo (Wilhelm, 1973). Generally, i n v i t r o studies have shown that chemotactic factor has a rapid action. On the other hand, MIF, MAF and MF have a slow action iji v i t r o , and thus these factors may belong to the category of slow-acting lymphokines. The slow-acting lymphokines may be responsible for the a m p l i f i c a t i o n of the i n i t i a l skin reaction. 96a Figure 25 Possible Mechanism of Delayed H y p e r s e n s i t i v i t y Reaction Lymphocytes activated by s p e c i f i c antigen or mitogens --» Lymphokines «•• Fas t - a c t i n g lymphokines Slow-acting lymphokines Chemotactic f a c t o r s for macrophage PMN eosinophil Accumulation of leukocytes at the r e a c t i o n s i t e I I I Lymphotoxin SRF MIF Macrophage a c t i v a t i n g factor. Cytoxic to y tissue Increase i n vascular permeability Immobilization of macrophage at the rea c t i o n s i t e Macrophage Activated macrophage I n i t i a l manifestation of delayed h y p e r s e n s i t i v i t y Mitogenic f a c t o r f o r lymphocyte macrophage Increase i n DNA synthesis and p r o l i f e r a t i o n A c t i v a t i o n of lymphocyte i J, A c t i v a t i o n of lymphocyte Increase i n phagocytosis and adherance Release of various h y d r o l y t i c enzymes Increase i n glucose o x i d a t i o n through hexose shunt A c t i v a t i o n of kinin-forming system Increase i n vascular permeability A c t i v a t i o n of complement system and other serum p r o t e i n Chemotactic factor Tissue damage Enhancement of delayed h y p e r s e n s i t i v i t y 97 It i s possible that the primary event i n skin inflammation due to delayed h y p e r s e n s i t i v i t y reactions may be due to the e f f e c t of chemotactic factor and lymphotoxin. Chemotactic f a c t o r may a t t r a c t more leukocytes to the inflammation s i t e . Lymphotoxin may exert a d i r e c t cytotoxic action on structures l i k e the vascular endothelium and w i l l mediate changes i n vascular permeability. Cohen et a l . , (1973) found that extracts of skin s i t e s with DH reactions possessed chemotactic a c t i v i t y for monocytes and lympho-cytes and could produce inflammatory reactions i n normal guinea p i g skin. Thus, they suggested that c l a s s i c delayed hypersensitivy may be mainly due to chemotactic phenomena. However, chemotactic substance(s) i n the extract of skin s i t e s of DH may be very compli-cated. Activated serum proteins such as proteins of the k i n i n -forming system and the f i f t h component of complement, which have chemotactic a c t i v i t y (Wilkinson, 1974) may also be present i n the extract and may be involved i n DH reactions. Cohen et a l . , (1973) e s p e c i a l l y pointed out that the extracts of skin had no detectable MIF a c t i v i t y . The present results have shown that neuraminidase-treated lymphokine i n which MIF a c t i v i t y was destroyed s t i l l retained most of i t s skin r e a c t i v i t y . Thus, i t i s suggested that MIF i s not a main cause of inflammation. Chemotactic factor and lymphotoxin are also r e s i s t a n t to neuraminidase (David, 1975) and therefore neura-98 minidase-treated lymphokines w i l l probably r e t a i n the a c t i v i t y of mononuclear c e l l a t t r a c t i o n and cytotoxic action. The second step i n DH reactions i s perhaps an enhancement stage. Slow-acting lymphokines such as macrophage a c t i v a t i n g factor may a c t i v a t e the accumulated macrophages (Mackaness, 1969; Nathan et a l . , 1971; 1973) which i n turn, release active h y d r o l y t i c enzymes (Wahl et. aJL., 1974; Pantalone and Page, 1975). Recent studies have shown that peritoneal macrophages activated by t h i o g l y c o l l a t e i n culture were able to secrete plasminogen a c t i v a t o r , collagenase, elastase (Unkeless et a l . , 1974; Webb and Gordon, 1975) and probably other p r o t e o l y t i c enzymes. Most of these products are active at neutral pH and are released as newly synthesized material, not storage products. These enzymes are d i r e c t l y discharged i n t o phagocytic vacuoles. The neutral protease may contribute a great deal to the breakdown of connective tissue protein and to the i n j u r y of blood vessels. For instance, plasminogen activator released from the stimulated macrophages is capable of converting plasminogen to plasmin which i s involved i n l y s i s of f i b r i n and a c t i v a t i o n of the t h i r d component of complement (Spragg, 1974). Further, Schorlemmer et a l . , (1976) have found that macrophages possess a C3b receptor. Most recent observation has shown that p u r i f i e d adherent monocytes produced more when exposed to lymphokine-rich supernatant (Littman and Ruddy, 1977). It was suggested that enzymes released 99 from stimulated macrophages could cleave C3, generating more C3b, which would bind with C3b receptor of macrophage and then, i n turn, induce further enzyme release. Furthermore, i t has been reported that a material which causes an inflammatory reaction i n the skin could also be obtained by incubating normal macrophages with a lymphokine preparation (Pick et al. 1971) and with PPD (Heise and Weiser, 1969). An important problem for future studies is to determine whether neutral protease can be released by lymphokine-activated macrophages and whether these proteases can activate the known serum systems such as kininforming and complement systems which may also contribute to the enhancement of DH reaction. For instance, a new h e a t - l a b i l e chemotactic factor which d i f f e r e d from the lymphocyte-derived chemotactic factor was i s o l a t e d from the extracts of skin s i t e with DH reaction. (Kambara et. a l . 1977). It i s d i f f e r e n t to correlate MIF demonstrated i n v i t r o with the DH reaction in vivo. David (1975) demonstrated that MIF i s in d i s t i n g u i s h a b l e from MAF. It i s possible that macrophage a c t i -vating factor and not MIF may be p a r t l y responsible for enhancement of DH reaction. These questions cannot be answered u n t i l the factors responsible for the various lymphokine a c t i v i t i e s can be better separated, characterized and p u r i f i e d . 100 Mitogenic factor w i l l cause an increase i n DNA synthesis i n non-immune lymphocytes (Hart et a l . , 1973; Gately et a l . , 1975; M i l l s , 1975) and macrophages (Hadden et. a l . , 1975). The r o l e which mitogenic factor plays i n DH is s t i l l obscure. The most recent report demonstrated that mitogenic factor from LN c e l l s could be separated from lymphotoxin and the p u r i f i e d mitogenic factor was capable of inducing production of lymphotoxin by lymphoid c e l l s (Gately et a l . , 1976). It would seem l i k e l y that mitogenic factor may amplify DH reactions by stimulating lymphoid c e l l s to produce lymphokines and induce macrophage a c t i v a t i o n . Lymphocyte-macrophage and lymphocyte-lymphocyte interactions may occur at various stages during the development of DH reaction; macrophages probably serve as the main eff e c t o r c e l l s amplifying the reaction which leads to v i s i b l e skin inflammation. 101 SUMMARY AND CONCLUSION The main theme of this study was to determine the bio-l o g i c a l action of lymphokines during the course of the delayed hyper- s e n s i t i v i t y reaction. Four groups of experiments were conducted to investigate: (1) the production and the separation of lymphokines, ( 2 ) the protease a c t i v i t y of lymphokines 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 response and MIF. Guinea pig lymph node lymphocytes, stimulated by either s p e c i f i c antigen, DNP-BGG, or concanavalin A, were able 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 macrophage migration i n h i b i t i o n (MIF) and skin r e a c t i v i t y (SRF). The supernatants generated from unpurified lymphokines possessed higher MIF a c t i v i t y which suggested that macrophages may be involved i n the production of lymphokines. The separation of MIF and SRF by the techniques of g e l - f i l t r a t i o n , electrophoresis and 102 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 s u l f a t e was unsuccessful, i n d i c a t i n g that the physical properties of MIF and SRF were s i m i l a r . Lymphokine preparations did not contain neutral protease a c t i v i t y and there was only a small amount of acid protease a c t i v i t y i n some batches of lymphokine preparations. Addition of pepstatin (a s p e c i f i c i n h i b i t o r of acid protease) to the lymphokine prepara-t i o n did not i n h i b i t the skin reaction, which suggested that the skin reaction induced by lymphokines was not due to acid protease. Further, i t was demonstrated that lymphokines did not activate Hageman factor and p r e k a l l i k r e i n . Lymphokines did not contain a idetectable activator of the kinin-forming system. Lymphokine preparations did not cause the release of histamine from mast c e l l s i n v i t r o . S i m i l a r l y , lymphokines did not induce p l a t e l e t aggregation. These findings suggested that mast c e l l s and p l a t e l e t s were not involved i n the delayed hyper-s e n s i t i v i t y reaction. 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 . This ensured that the enzymes would not be present i n the treated lymphokines which were subsequently assayed for MIF and SRF a c t i v i t i e s . It was found that MIF and SRF a c t i -v i t i e s were completely destroyed by incubation with chymotrypsin-103 agarose for two hours. This confirmed that MIF and SRF are proteins. Incubation of lymphokines with neuraminidase-agarose for 3 hours at 37°C could reduce markly the MIF a c t i v i t y , which indicated that terminal s i a l i c acid residues were probably necessary for MIF a c t i v i t y and that MIF i s a glycoprotein. However, using the skin blueing t e s t , neuraminidase-agarose treated lymphokines retained most of skin r e a c t i v i t y although s i g n i f i c a n t p a r t i a l destruction of SRF a c t i v i t y occurred. When lymphokine was treated with soluble neuraminidase and then heated at 56°C for 15 min. to inacti v a t e enzyme a c t i v i t y , most of i t s SRF a c t i v i t y was also retained. The ^n v i t r o MIF assay i s widely employed as an i n v i t r o c orrelate of delayed h y p e r s e n s i t i v i t y reactions. The present findings showed that neuraminidase-treated lymphokines l o s t MIF a c t i v i t y while most of the SRF a c t i v i t y was retained. I t i s sug-gested that the sk i n inflammatory reaction induced by lymphokines i s not mainly due to MIF. A possible mechanism of the delayed h y p e r s e n s i t i v i t y reaction is postulated as follows, and can be divided into two stages which include (1) i n i t i a l reaction and (2) enhancement. Lymphokines may be c l a s s i f i e d into f a s t - a c t i n g and slow-acting lymphokines. Fast-acting lymphokines are those which cause a skin r e a c t i o n within 4 hours of i n j e c t i o n . These skin reactions do not 104 seem to depend on MIF since they are not affected by treatment of the lymphokine preparations with neuraminidase. Part of the skin reaction at 4 hours may therefore be attributed to SRF which pro-bably involves chemotactic factor and lymphotoxin. Lymphotoxin has been found to be cytotoxic to many c e l l types from many d i f f e r e n t animal species and may take part i n e l i c i t a t i o n of increased vas-cular permeability. Chemotactic factor has been shown i n v i t r o to have a rapid action. On the other hand, MIF, macrophage a c t i v a t i o n factor and mitogenic factor have a slow action i n v i t r o and there-fore these lymphokines belong to the category of slow-acting lymphokines. The slow-acting lymphokines may be responsible for the am p l i f i c a t i o n of the i n i t i a l skin reaction. MIF i n h i b i t s the migration of macrophages away from the reaction s i t e . MAF activates the accumulation of macrophages into the inflammatory s i t e . Hydrolytic enzymes w i l l probably be released by the the activated macrophages. MF stimulates the lymphocytes which i n turn w i l l produce more lymphokines to amplify the e x i s t i n g reaction. During the course of the delayed h y p e r s e n s i t i v i t y reaction, lympho-cyte-macrophage and lymphocyte-lymphocyte interactions occur. The lymphocytes may serve as the i n i t i a l producer of lymphokines while macrophages serve as the main e f f e c t o r c e l l s which i n turn amplifies the previous reactions, thus leading to v i s i b l e skin inflammation. 105 BIBLIOGRAPHY Agrowal, B. B. and Goldstein, I. J., 1965. S p e c i f i c binding of concannavalin A to cross-linked dextran gels. Biochem. J . 96: 23C-25C. Altman, L. C. and Kirchner, H., 1972. The production of a monocyte chemotactic factor by agammaglobulinemic chicken spleen c e l l s . J. Immunol. 109: 1149-1151. Ashworth, L. A. E., Eckersley, B.J. and Ford, W. H., 1975. Com-parison of the properties of two antigen-induced guinea pig lymphokines. Int. Arch. A l l e r g y Appl. Immunol. 48: 143-155. Ashworth, L. A. E. and Ford, W. H., 1976. Mitogenic factor as an i n  v i t r o correlate delayed h y p e r s e n s i t i v i t y i n the guinea pig. Int. Arch. Al l e r g y Appl. Immunol. 50: 583-592. Axen, R. and Ernback, S., 1971. Chemical f i x a t i o n of enzymes to cyanogen halide activated polysaccharide c a r r i e r s . Eur. J. Biochem. 18: 351-360. Barnet, K., Pekarek, J. and Johanovsky, J., 1968. Demonstration of s p e c i f i c induction of erythrocyte phagocytosis by macro-phages from normal, non-sensitized rabbits by a factor released from lymph node c e l l s of immunized rabb i t s . Experientia 24: 948-949. Bennett, B. and Bloom, B. R., 1967. Studies on the migration i n h i -b i t o r y factor associated with delayed-type hypersensi-t i v i t y : Cytodoynamics and s p e c i f i c i t y . Transplantation 5: 996-1000. Bennett, B. and Bloom, B. R., 1968. Reactions i n vivo produced by a soluble substance associate with delayed-type hypersensi-t i v i t y . Proc. Nat. Acad. S c i . U.S.A. 59: 756-762. Blaese, R. M., Oppenheim, J. J., Seager, R. C. and Waldmann, T. A. ,1972. Lymphocyte-macrophage i n t e r a c t i o n i n antigen induced i n v i t r o lymphocyte transformation i n patients with the Wiskoff-Aldrich syndrome and other diseases with anergy. C e l l . Immunol. 4: 228-242. 106 Bloom, B. R., 1971. In v i t r o approaches to the mechanism of c e l l -mediated immune reactions. Adv. Immunol. 13: 101-208. Bloom, G. D., 1974. Structural and biochemical c h a r a c t e r i s t i c s of mast c e l l s . In the Inflammatory Process. Edited by B. W. Zweifach, L. Grant and R. T. McCluskey. Academic Press, N. Y. pp. 544-599. Bloom, B.R. and Bennett, B., 1968. Migration i n h i b i t o r y factor associated with delayed-type h y p e r s e n s i t i v i t y . Federation Proceedings 27: 13-15. Bloom, B. R. and Chase, M. W., 1967. Transfer of delayed-type h y p e r s e n s i t i v i t y . A c r i t i c a l review and experimental study i n the guinea pig. Progr. A l l e r g y 10: 151-245. Bloom, B. R. and Glade, P. R., 1971. In V i t r o methods i n c e l l -medicated immunity. Academic Press, New York. Bloom, B. R. and Jimenez, L., 1970. Migration i n h i b i t o r y factor and the c e l l u l a r basis of delayed-type h y p e r s e n s i t i v i t y reac-tions. Amer. J. Pathol. 60: 453-465. Bloom, B. R. and Shevach, E., 1975. Requirement for T c e l l s i n the production of migration i n h i b i t o r y factor. J. Exp. Med. 142: 1306- 1311. Bloom, B. R., Stoner, G., Gaffney, J., Shevach, E. and Green, I., 1975. Production of migration i n h i b i t o r y factor and lympho-toxin by non-T c e l l s . Eur. J. Immunol. 5: 218-220. Boreus, L. 0., 1960. A comparison of mast-cell reactions i n the r a t s , hamster and guinea pig. Acta Physiol. Scand. 49: 251-260. Born, G. V. R., 1962. Aggregation of blood p l a t e l e t s by adenosine diphosphate and i t s r e v e r s a l . Nature (London) 194: 927-929. Bray, M. A., Dumonde, D.C, Hanson, J. M., Morley, J., Wolstern-c r o f t , R. A. and Smart, J. V., 1976. Heterogeneity of guinea pig lymphokines revealed by p a r a l l e l bioassay. C l i n . Exp. Immunol. 23: 333-346. Calderon, J., K e i l y , J. A., Lefko, J. L., and Unanue, E. R., 1975. The modulation of lymphocyte function by molecules secreted by macrophages. J. Exp. Med. 142: 151-164. 107 Caspary, E. A., 1972. The mechanism of antigen-induced e l e c t r o -phoretic mobility reduction of guinea pig macrophages. C l i n . Exp. Immunol. 11: 305-309. Cassidy, J. T., Jourdian, G. W. and Roseman, S., 1965. The s i a l i c acids. VI. P u r i f i c a t i o n and properties of s i a l i d a s e from Clostridium perfringens. J. B i o l . Chem. 240: 3501-3512. Chase, M. W., 1945. The c e l l u l a r transfer of cutaneous hyper-s e n s i t i v i t y to tuberculin. Proc. Sco. Exp. B i o l . Med. 59: 134-135. Chen, C , and Hirsch, J., 1972. The e f f e c t s of mercaptoethanol and peritoneal macrophages on the antibody-forming capacity of nonadherent mouse spleen c e l l s i n v i t r o . J. Exp. Med. 136: 604-617. Cochrane, C. G., Revak, S. D., and Wuepper, K. D., 1973. A c t i v a t i o n of Hageman factor i n s o l i d and f l u i d phases. J. Exp. Med. 138: 1564-1583. Cochrane, C. G. and Wuepper, K. D., 1971. The f i r s t component of the kinin-forming system i n human and rabbit plasma. I t s r e l a t i o n s h i p to c l o t t i n g factor XII (Hageman f a c t o r ) . J. Exp. Med. 134: 986-1004. Cohen, S., 1976. Cell-mediated immunity and the inflammatory sys-tem. Human Pathology 7: 249-264. Cohen, S., 1977. The r o l e of cell-mediated immunity i n the induction of inflammatory responses. Amer. J. Pathology 88: 502-528. Cohen, S., and McCluskey, R. T., 1973. Delayed h y p e r s e n s i t i v i t y . In Rose, N.R., Milgrom, F., and Van Oss, C. J. ( E d i t o r s ) : P r i n c i p l e s of Immunology. New York, The MacMillan Co., pp. 189-204. Cohen, S., McCluskey, R. T. and Benacerraf, B., 1967. Studies on the s p e c i f i c i t y of the c e l l u l a r i n f i l t r a t e of delayed hyper-s e n s i t i v i t y reactions. J. Immunol. 98: 269-273. Cohen, S. Ward, P. A., Yoshida, T. and Burek, C. L., 1973. Bio-l o g i c a l a c t i v i t y of delayed h y p e r s e n s i t i v i t y skin reaction s i t e s . C e l l . Immunol. 9: 363-376. 108 Cohen, S. and Yoshida, T. 1976. I n h i b i t i o n by T c e l l products of MIF production by B c e l l s . Fed. Proc. 35: 389. Coyne, J. A., Remold, H. G., Rosenberg, S. A., and David, J. R., 1973. Guinea pig lymphotoxin (LT). I I . Physiochemical properties of LT produced by lymphocytes stimulated with antigen or concanavalin A: Its d i f f e r e n t i a t i o n from migra-ti o n i n h i b i t o r y factor (MIF). J. Immunol. 110: 1630-1637. Cummings, M. M., Hoyt, M. and G o t t s h a l l , R. Y., 1947. Passive transfer of tuberculin s e n s i t i v i t y i n the guinea pig. Publ. Health Resp. 62: 994-997. David, J. R., Al-Askari, S., Lawrence, H.S. and Thomas, L., 1964. Delayed h y p e r s e n s i t i v i t y i n v i t r o . I. The s p e c i f i c i t y of i n h i b i t i o n of c e l l migration by antigens. J. Immunol. 93: 264-273. David, J. R., 1975. Macrophage a c t i v a t i o n by lymphocyte mediators. Fed. Proc. 34: 1730-1736. David, J. R., and David, R. R., 1972. C e l l u l a r h y p e r s e n s i t i v i t y and Immunity. Progr. A l l e r g y . 16: 300-449. David, J. R., and Becker, E. L., 1974. I n a b i l i t y to block the a c t i -v i t y of guinea pig migration i n h i b i t o r y factor with d i - i s o p r o p y l phosphorofluoridate. Eur. J. Immunol,, 4: 287-289. Dekaris, D., Fauve, R. M., and Raynaud, M., 1969. Delayed hyper-s e n s i t i v i t y and i n h i b i t i o n of macrophage spreading i n vivo and i n v i t r o studies of tuberculin and streptococcal hyper-s e n s i t i v i t y i n guinea pigs. J. Immunol. 103: 1-5. Diamantstein, T., and Ulmer, A., 1976. Two d i s t i n c t lymphocyte stimulating soluble factors (LAF) released from murine peritoneal c e l l s . I. The c e l l u l a r source and the e f f e c t of cGMP on t h e i r release. Immunology 30: 741-747. Dixon, M., 1953. A nomogram for ammonium sulphate s o l u t i o n . Biochem. J. 54: 457-458. Drzeniek, R., 1972. V i r a l and b a c t e r i a l neuraminidases. Current Topics i n Microbiology and Immunology. 59: 35-74. Heidelberg, Springer-Verlag. 109 Dumonde, D. C , Page, D. A., Mathew, M., and Wolstencroft, R. A., 1972. Role of lymphocyte a c t i v a t i o n products (LAP) i n cell-mediated immunity. I. Preparation and p a r t i a l p u r i -f i c a t i o n of guinea pig LAP. C l i n . Exp. Immunol. 10: 25-47. Dumonde, D. C , Wolstencroft, R. A., Panayi, G. S., Matthew, M., Morley, J., and Howson, W. T., 1969. "Lymphokines." Non-antibody mediators of c e l l u l a r immunity generated by lymphocyte a c t i v a t i o n . Nature (London) 224: 338-342. Dvorak, H. F., 1974. Delayed h y p e r s e n s i t i v i t y . The inflammatory Process. Second e d i t i o n . Vol. 3. Edited by B. W. Zweifach, L. Grant, R. T. McCluskey, New York, Academic Press, pp. 291-345. Dy, M., Kamoun, P., D i m i t r i u , A., and Hamburger, J., 1976. Studies on mouse macrophage arming factor and i t s molecular weight. Transplantation 21: 273-275. Folch, H., Yoshinaga, M., and Waksman, B. H., 1973. Regulation of lymphocyte responses i n v i t r o . I I I . I n h i b i t i o n by adherent c e l l s of the T-lymphocyte response to phytohemagglutinin. J. Immunol. 110: 835-839. Ford, W. H., Ashworth, L. A. E., and Inder, S., 1976. E f f e c t of the concentration inducing agent on the output of lymphokines in the guinea p i g . Eur. J. Immunol. 6: 135-138. Fox, R. A., Gregory, D. S., and Feldman, J. D., 1974. Migration i n h i b i t i o n factor (MIF) and migration stimulation factor (MSF) i n f e t a l c a l f serum. J. Immunol. 112: 1861-1866. Fox, R. A., and MacSween, J. M., 1974. The i s o l a t i o n of migration i n h i b i t o r y f a c t o r . Immunol. Comm. 3: 375-390. F r i e d r i c h , W., Lazary, S., Geczy, C , and DeWeck, A. L., 1975. Lymphokines. I. Use of insoluble concanavalin A for the production of migration i n h i b i t o r y factor i n guinea pig lymphocyte cultures. Int. Arch. A l l e r g y Appl. Immunol. 49: 504-518. Friend, D. A., and Rosenau, W., 1977. T a r g e t - c e l l membrane a l t e r a -tions induced by lymphotoxin. Amer. J. Pathol. 86: 149-155. 110 Gately, M. K., Gately, C. L., Henney, C. S., and Mayer, M. M. 1975. Studies on lymphokines: The production of antibody to guinea pig lymphotoxin and i t s use to d i s t i n g u i s h lympho-toxin from MIF and mitogenic factor. J. Immunol. 115: 817-826. Gately, C. L., Gately, M. K., and Mayer, M. M., 1975. The molecular dimensions of mitogenic factor from gyinea pig lymph node c e l l s . J. Immunol. 114: 10-16. Gately, C. L. Gately, M. K. and Mayer, M. M., 1976. Separation of lymphocyte mitogen from lymphotoxin by lymphoid c e l l s stimu-lated with the p a r t i a l l y p u r i f i e d mitogen: A possible a m p l i f i c a t i o n mechanism of c e l l u l a r immunity and a l l e r g y . J. Immunol. 116: 669-675. Gately, M. K., and Mayer, M. M., 1972. The e f f e c t of antibodies to complement components C2, C3 and C5 on the production and action of LT. J. Immunol. 109: 728-734. Gately, M. K., and Mayer, M. M., 1974. The molecular dimensions of guinea pig lymphotoxin. J. Immunol. 112: 168-177. Geczy, C. L., F r i e d r i c h , W., and DeWeck, A. L., 1975. Production and i n vivo e f f e c t of antibodies against guinea pig lymphokines. C e l l Immunol. 19: 65-77. Geczy, C. L., Geczy, A. F., and DeWeck, A. L., 1976. Antibodies to guinea pig lymphokine. II. Supression of delayed hyper-s e n s i t i v i t y reactions by a "second generation" goat antibody against guinea pig lymphokines. J. Immunology 117: 66-72. Gery, I., and Waskman, B. H., 1972. Potentiation of the T-lympho-cyte response to mitogens. II. The c e l l u l a r source of potentiating mediator(s). J. Exp. Med. 136: 143-155. Girey, G. J. L., Talamo, R. C , and Coleman, R. W., 1972. The kine-t i c s of the release of bradykinin by K a l l i k r e i n i n normal human plasma. J. Lab. C l i n . Med. 80: 496-505. Gorg, P., and Kovacs, I. B., 1969. The a l t e r a t i o n of p l a t e l e t be-haviour during various conditions and the e f f e c t s of a n t i -inflammatory agents on p l a t e l e t s aggregation and thrombus formation. In: Inflammation Biochemistry and Drug Inter-action. (Eds. A. B e r t e l l i and J . C. Houck; Excerpta Medical Foundation, Amsterdam, 1969). pp. 197-203. I l l Granger, G. A., 1971., In "In v i t r o methods i n cell-mediated immu-n i t y " . Edited by B. R. Bloom and P. Glade. Academic Pres., New York. pp. 38-40. Granger, G. A., 1972. Lymphokines — The mediators of C e l l u l a r Immunity. Ser. Haemat. 4: 8-40. Grayzel, A. I., Hatcher, V. B., and Lazards, G. S., 1975. Protease a c t i v i t y of normal and PHA stimulated human lymphocytes. C e l l . Immunol. 18: 210-219. Hadden, J. W., Sadlik, J. R., and Hadden, E. M., 1975. Macrophage p r o l i f e r a t i o n induced i n v i t r o by a lymphocyte factor. Nature (London) 257: 483-485. Hamberg, U., and Rocha e S i l v a , M., 1957. On the release of brady-k i n i n by try p s i n and snake venoms. Arch. Int. Pharmacodyn. 110: 222-238. Hammond, M. E., Selvaggio, S., and Dvorak, H. F., 1975. Antigen-enhanced glycosamine incorporation by peritoneal macrophages in cell-mediated h y p e r s e n s i t i v i t y . I. Studies on Biology and mechanism. J. Immunol. 115: 914-921. Hart, D. A., Jones, J. M. and Nisonoff, A., 1973. Mitogenic factor from inbred guinea pigs. C e l l Immunol. 9: 173-185. Havermann, H., Horvat, M., Sodomann, C. P., Havemann, K., and Burger, S., 1972. Protease a c t i v i t y as a possible mechanism of migration i n h i b i t o r y factor. Eur. J. Immunol. 2: 97-99. Hay, J. B., Lachmann, P. J . , and Truka, Z., 1973. The appearance of migration i n h i b i t o r y factor and a mitogen i n lymph draining tuberculin reactions. Eur. J. Immunol. 3: 127-131. Hayashi, H., Udaka, K., Miyoshi, H., and Kudo, S., 1965. Further study of c o r r e l a t i v e behaviour between s p e c i f i c protease and i t s i n h i b i t o r i n cutaneous arthus reactions. Lab. Invest. 14: 665-673. Heise, E. R., and Weiser, R. S., 1969. Factors i n delayed hyper-s e n s i t i v i t y : Lymphocyte and macrophage cytotoxins i n the tuberculin reaction. J. Immunol. 103: 570-576. 112 Hiserodt, J. C , F a i r , D. S., and Granger, G. A., 1976. I d e n t i f i c a -t i o n of multiple c y t o l y t i c components associated with the beta-LT class of lymphotoxins released by mitogen-activated human lymphocytes i n v i t r o . J. Immunol. 117: 1503-1506. Horval, M., Havemann, K., Sodoman, C. P., and Burger, S., 1972. Mitogenic factor and migration i n h i b i t o r y factor i n super-natants of serum free human lymphocyte culture stimulated with concanavalin A. Int. Arch. A l l e r g y 43: 446-456. Horton, J. E., Oppenheim, J. J., Mergenhagen, S. E., and Raisz, L. G., 1974. The requirement for macrophage-lymphocyte i n t e r -action for the production of osteoclast a c t i v a t i o n factor by lymphocytes. J. Immunol. 113: 1278-1287. Houck, J. C., Barrantes, D., and Irausquin, H., 1973. Skin reactive factor and lymph node permeability factor. Agents and Actions 3: 278-283. Hughes, D., 1972. Macrophage migration i n h i b i t i o n t e s t . A c r i t i c a l examination of the technique using a polyethelene c a p i l l a r y tubing micromethod. J. Immunol. Method 1: 403-424. Janoff, A. and Z e l i g s , J. D., 1968. Vascular i n j u r y and l y s i s of basement membrane i n v i t r o by neutral protease of human leukocytes. Science 161: 702-704. Johnson, J. R., and Moran, N. C., 1966. Comparison of several me-thods for i s o l a t i o n of rat peritoneal mast c e l l s . Proc. Soc. Exp. B i o l . Med. 123: 886-889. Kambara, T., Ueda, K., and Maedu, S., 1977. The chemical mediation of delayed h y p e r s e n s i t i v i t y skin reaction. Amer. J. Pathol. 87: 360-370. Kaufmann, S., Weber, L. and Hahn, H., 1975. Macrophage i n h i b i t o r y a c t i v i t y i n serum and central lymph of Listeria-immune mice. Eur. J. Immunol. 5: 799-800. Kiernan, J. A., 1972. The involvement of mast c e l l s i n v a s o d i l a t i o n due to axon reflexes i n injured skin. Quart. J. Exp. Physiol. 57: 311-317. Kind, L. S., and Peterson, W. A., 1968. Concanavalin A in v i v o . Induction of hemorrhagic skin l e s i o n ( A r t h u r - l i k e reactions) i n mice. Science 160: 312-313. 113 Kirchheimer, W. F., and Weiser, R. S., 1947. The tuberculin reac-t i o n . I. Passive transfer of tuberculin s e n s i t i v i t y with c e l l s of tuberculous guinea pigs. Proc. Soc. Exp. B i o l . , 66: 166-170. Kolb, W. P. and Granger, G. A., 1968. Lymphocyte i n v i t r o cyto-t o x i c i t y : Characterization of human lymphotoxin. Proc. Nath. Acad. S c i . 61: 1250-1255. Kolb, W. P. and Granger, G. A., 1970. Lymphocyte i n v i t r o cyto-t o x i c i t y : Characterization of mouse lymphotoxin. C e l l . Immunol. 1: 122-132. L a v e l l e , K. J., R a n s d i l l , B. A. and Trygstad, C. W., 1975. Identi-f i c a t i o n of a new p l a t e l e t aggregation factor released by se n s i t i z e d leukocytes. C l i n . Immunol. Immunopathol. 3: 492-502. Lawrence, H. S., and Landy, M., 1969. Mediators of C e l l u l a r Immunity. Academic Press, New York. Leu, R. W., Eddleston, A. L. W. F., Haddon, J. W. and Good, R. A., 1972. Mechanism of action of migration i n h i b i t o r y factor (MIF). I. Evidence of a receptor for MIF on the peritoneal macrophage, but not on the alveolar macrophages. J. Exp. Med. 136: 589-603. Lipsky, P. E., E l l n e r , J. J. and Rosenthal, A. S., 1976. Phyto-hemagglutinin-induced p r o l i f e r a t i o n of guinea pig thymus-derived lymphocytes. J. Immunol. 116: 868-875. L i t t l e , J. R. and Eisen, H. N., 1967. In: Methods i n Immunology and Immunochemistry. Edited by Williams and Chase. Vol. 1, pp. 130-131. Academic Press, New York. Littman, B. H., and Ruddy, S., 1977. Production of the second com-ponant of complement by human monocytes: Stimulation by antigen-activated lymphocytes or lymphokines. J. Exp. Med. 145: 1344-1352. Lolekha, S., Dray, S., and Gotoff, S. P., 1970. Macrophage aggre-gation i n v i t r o : A correlate of delayed h y p e r s e n s i t i v i t y . J. Immunol. 104: 296-304. 114 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., Randall, R. S., 1951. Protein measurement with the F o l i n phenol reagent. J. B i o l . Chem. 193: 265-275. Mackaness, G. B., 1969. The influence of immunologically committed lymphoid c e l l s on macrophage a c t i v i t y i n v i t r o . J. Exp. Med. 129: 973-992. Mackler, B. F., Altman, L. C , Rosenstreich, D. L., 1974. Induction of lymphokine production by EAC and of blastophenesis by soluble mitogens during human B - c e l l a c t i v a t i o n . Nature (London) 249: 834-837. M a i l l a r d , J. L., Pick, E., and Turk, J. L., 1972. Interaction be-tween "Sensitized lymphocytes" and antigen i n v i t r o . V. Vascular permeability induced by skin-reactive factor. Int. Arch. A l l e r y 42: 50-68. Manheimer, S. and Pick, E., 1973. The mechanism of action of solu-ble lymphocytic mediators. I. A pulse exposure test for the measurement of macrophage migration i n h i b i t o r y factor. Immunology 24: 1027-1034. McAdoo, M. H., Darnenberg, A. M., Hayes, C. J., James, S. P., and Sanner, J. H., 1973. I n h i b i t i o n of cathepsin D-type proteinase of macrophages by pepstatin, a s p e c i f i c pepsin i n h i b i t o r , and other substances. Infection and Immunity 7: 655-665. McCluskey, R. T., Benacacerraf, B. and McCluskey, J. W., 1963. Studies on the s p e c i f i c i t y of the c e l l u l a r i n f i l t r a t e i n delayed h y p e r s e n s i t i v i t y reactions. J. Immunol. 90: 466-477. M i l l s , J. A., 1975. Characterization of guinea pig mitogenic factor. J. Immunol. 114: 45-50. Metaxas, M. N., and Metazas-Buhler, M., 1955. Studies on the c e l l u -l a r transfer of tuberculin s e n s i t i v i t y i n the guinea p ig. J. Immunol. 75: 333-347. Monley, J., Wolstencroft, R. A., and Dumonde, D. C , 1973. The measurement of lymphokines. In: Handbook of Experimental Immunology, second e d i t i o n , 1973. Edited by D. M. Weir, Blackwell S c i e n t i f i c P ublication, London, Chapter 28. 115 Mosier, D. E., 1967. A requirement for two c e l l types for antibody formation i n v i t r o . Science. 158: 1573-1575. Mosier, D. E., and Pierce, C. W., 1972. Functional maturation of thymic lymphocyte population i n v i t r o . J. Exp. Med. 136: 1484-1500. Mota, I., and Vugman, I., 1956. Action of 48/86 on the mast c e l l s and histamine content of guinea pig tissue. B r i t . J. Pharmacol. 11: 304-307. Movat, H. Z., Habal, F. M., and MacMorine, D. R. L., 1976. Neutral protease of human PMN leukocytes with kininogenase a c t i -v i t y . Int. Arch. All e r g y Appl. Immunol. 50: 257-281. Movat, H. Z., Steinberg, S. G., Habal, F. M., and Ranadine, N. S., 1973. Demonstration of a kinin-generating enzyme i n the lysosomes of human polymorphonuclear leukocytes. Lab. Invest. 29: 669-684. Mustard, J. F., and Packham, M. A., 1970. Factors inf l u e n c i n g p l a -t e l e t function: adhesion, release, and aggregation. Pharmacological Reviews. 22: 97-187. Mycek, M. J . , 1970. Cathepsin. In: Methods i n Enzymology. Vol. 19. Edited by G. E. Perlmann and L. Lorand, pp. 285-315. Najarian, J. S. and Feldman, J. D., 1963. S p e c i f i c i t y of passively transferred h y p e r s e n s i t i v i t y . J. Exp. Med. 118:341-352 Nath, I., Poulter, L. W., and Turk, J. L., 1973. E f f e c t of lympho-cyte mediators on macrophages in v i t r o . A c o r r e l a t i o n of morphological and cytochemical changes. C l i n . Exp. Immunol. 13: 455-466. Nathan, C. F., Karnovsky, M. L., and David, J. R., 1971. A l t e r a t i o n of macrophage functions by mediators from lymphocytes. J. Exp. Med. 133: 1356-1376. Nathan, C. F., Remold, H. G. and David, J. R., 1973. Characteri-zation of a lymphocyte factor which a l t e r s macrophage functions. J. Exp. Med. 137: 275-290. Nelson, R. D., and Leu, R. W., 1975. Macrophage reqyirement for production guinea pig migration i n h i b i t o r y factor (MIF) i n v i t r o . J. Immunol. 114: 606-609. 116 Ohishi, M., and Onoue, K., 1975. Functional a c t i v a t i o n of immune lymphocytes by antigenic stimulation i n c e l l mediated immunity. 1. Requirement for macrophages i n antigen-induced MIF production by guinea pig immune lymphocytes i n  v i t r o . C e l l . Immunol. 18: 220-232. Oppenheim, J. J., and Rosenstreich, D. L., 1976. Signals regulating i n v i t r o a c t i v a t i o n of lymphocytes. Prog. All e r g y 20: 65-194. Pantalone, R. M. and Page, R. C , 1975. Lymphokine-induced pro-duction and release of lysosomal enzymes. Proc. Nat. Acad. S c i . U.S.A. 72: 2091-2094. Papageorgiou, P. S., Sorokin, C. F. and Glade, P. R., 1974. Simi-l a r i t y of migration i n h i b i t o r y f a c t o r ( s ) produced by human lymphoid c e l l l i n e and phytohemagglutinin and tuber c u l i n -stimulated human peripheral lymphocytes. J. Immunol. 112: 675-682. Pel l e y , R., and Schwartz, H. J., 1972. The production of migration i n h i b i t o r y factor by nonimmune guinea pig lymphoid c e l l s incubated with concanavalin A. Proc. Soc. Exp. B i o l . Med. 141: 373-378. Pharmacia, 1976. A f f i n i t y Chromatography. Pick, E., Brostoff, J ., K r e j c i , J . and Turk, J. L., 1970. Inter-action between " s e n s i t i z e d lymphocytes" and antigen i n  v i t r o . I I . Mitogen-induced release of skin reactive and macrophage migration i n h i b i t o r y f a c t o r s . C e l l . Immunol. 1: 92-109. Pick, E., K r e j c i , J., Cech, K., and Turk, J. L., 1969. Interaction between " s e n s i t i z e d lymphocytes" and antigen i n v i t r o . I. The release of a skin reactive factor. Immunology 17: 741-767 Pick, E., K r e j c i , J., and Turk, J. L., 1970. Release of skin reac-t i v e factor from guinea pig lymphocytes by mitogens. Nature 225: 236-238. Pick, E., K r e j c i , J. and Turk, J. L., 1971. In vivo action of solu-ble mediators associated with cell-mediated immunity. Int. Arch. A l l e r g y . 41: 18-24. 117 Pick, E., and Turk, J. L., 1972. The b i o l o g i c a l a c t i v a t i o n of solu-ble lymphocyte products. C l i n . Exp. Immunol. 10: 1-23. Piessens, W. Y., Remold, H. G. and David, J. R., 1977. Increased responsiveness to macrophage-activating factor (MAF) after a l t e r n a t i o n of macrophage membranes. J. Immunology 118: 2078-2082. Poulter, L. W., and Turk, J. L., 1975a. Rapid quantitative of changes i n macrophage volume induced by lymphokine i n  v i t r o . C l i n . Exp. Immunol. 19: 193-199. Poulter, L. W., and Turk, J. L., 1975b. Studies on the e f f e c t of soluble lymphocyte products (lymphokines) on macrophage physiology. I. Early changes i n enzyme a c t i v i t y and permeability. C e l l . Immunol. 20: 12-24. Poulter, L. W., and Turk, J. L., 1975C. Studies on the e f f e c t of soluble lymphocyte products (lymphokines) on macrophage physiology. I I . Cytochemical changes associated with a c t i v a t i o n . C e l l . Immunol. 20: 25-32. Postlethwaite, A. E., and Kang, A. H., 1976. K i n e t i c studies on migration i n h i b i t o r y factor (MIF) a c t i v i t y i n a delayed h y p e r s e n s i t i v i t y (DH) reaction i n vivo. Fed. Proc. 35: 390. Postlethwaite, A. E., and Snyderman, R., 1975. Characterization of chemotactic a c t i v i t y produced i n vivo by a cell-mediated immune reaction i n the guinea pig. J. Immunol. 114: 274-278. Preece, A. W., and Light, P. A., 1974. The macrophage electrophor-e t i c m o b i l i t y (MEM) test for malignant disease. Further c l i n i c a l investigations and studies on macrophage slowing f a c t o r s . C l i n . Exp. Immunol. 18: 543-552. Redei, A., and Kelemen, E., 1969. Presence of p l a t e l e t s i n acute experimental inflammatory edema i n h i b i t e d by s a l i c y l a t e or cortisone. In: Inflammation Biochemistry and Drug Interaction. Edited by A. B e r t a l l i and J . C. Houck, Excerpta Medical Foundation, Amsterdam, pp. 261-265. Remold, H. G., 1972. P u r i f i c a t i o n and char a c t e r i z a t i o n of lympho-cyte mediators i n c e l l u l a r immunity: Comparation studies on migration i n h i b i t o r y factor (MIF), chemotactic factor for macrophages and lymphotoxin. Transplant. Rev. 10: 152-176. 118 Remold, H. G., 1973. Requirement for a-L-fucose on the macrophage membrane receptor for MIF. J. Exp. Med. 138: 1065-1076. Remold, H. G., 1974. The enhancement of MIF a c t i v i t y by i n h i b i t a -t i o n of macrophage associated esterases. J. Immunol. 112: 1571-1577. Remold, H. G., and David, J. R., 1971. Further studies of migration i n h i b i t o r y factor (MIF). Evidence for i t s glycoprotein nature. J. Immunol. 107: 1090-1098. Remold, H. G., David, R. A. and David, J. R., 1972. Characteri-zation o f migration i n h i b i t o r y factor (MIF) from guinea pig lymphocytes stimulated with concanavalin A. J. Immunol. 109: 578-586. Remold, H. G., Katz, A. B., Haber, E., and David, J. R., 1970. Studies on migration i n h i b i t o r y factor (MIF): Recovery of MIF a c t i v i t y after p u r i f i c a t i o n by gel f i l t r a t i o n and dis c electrophoresis. C e l l . Immunol. 1: 133-145. Remold, H. G. and Mednis A., 1977. Two migration i n h i b i t o r y factors with d i f f e r e n t chromatographic behavior and i s o - e l e c t r i c points. J. Immunol. 118: 2015-2019. Remold, H. G., and Rosenberg, R. D., 1975. Enhancement of migration i n h i b i t o r y factor a c t i v i t y by plasma esterase i n h i b i t o r s . J. B i o l . Chem. 250: 6608-6613. Rich, A. R., and Lewis, M. R., 1932. The nature of a l l e r g y i n tu-berculosis as revealed by tissue culture studies. B u l l . Johns Hopkins Hosp. 50: 115-131. Rocha e S i l v a , M., 1970. K i n i n Hormones. S p r i n g f i e l d , 111. Thomos. Rocklin, R. E., Chess, L., MacDermott, R. P., Schlossman, S. F. and David, J. R., 1975. Studies on the production of MIF and mitogenic factor using h i g h l y p u r i f i e d human T and B lymphocytes. Rheumatology 6: 98-105. Rocklin, R. E., MacDermott, R. P., Chess, L., Schlossman, S. F. and David, J. R., 1974. Studies on mediator production by highly p u r i f i e d human T and B lymphocytes. J. Exp. Med. 140: 1303-1316. 119 Rocklin, R. E., and Remold, H. G. and David, J. R., 1972. Charac-terization of human migration inhibitory factor (MIF) from antigen-stimulated lymphocytes. Cell. Immunol. 5: 436-445. Rocklin, R. E., Rosen, F. and David, J. R., 1970. In vitro lympho-cyte response of patients with immunologic deficiency diseases. Correlation of production of macrophage inhibi-tory factor with corelation of production of macrophage inhibitory factor with delayed hypersensitivity. New England J. Med. 282: 1340-1343. Rosenau, W., and Tsoukas, C. D., 1976. Lymphotoxin. Amer. J. Pathol. 84: 580-596. Rosenstreich, D. L., Farrar, J. J. and Dougherty, S., 1976. Abso-lute macrophage dependency of T lymphocyte activation by mitogens. J. Immunol. 116: 131-139. Rosenthal, A. S., Davie, J. M., Rosenstreich, D. L. and Blake, J. T., 1972. Depletion of antibody forming cells and their precursors from complex lymphoid cells and their precursors from complex lymphoid c e l l populations. J. Immunol. 108: 279-281. Ryan, G. B., and Majno, G., 1977. Acute inflammation. Amer. J. Pathology 86: 185-276. Salvin, S. B., and Neta, R., 1975. A possible relationship be-tween delayed hypersensitivity and cell-mediated immunity. Amer. Rev. Resp. Dis. I l l : 373-377. Salvin, S. B., Nishio, J., and Shonnard, J. T., 1974. Two new inhi-bitory activities in blood of mice with delayed hyper-sensitivity, after challenge with specific antigen. Infect. 1mmun. 9: 631-635. Salvin, S. B., Youngner, J. S. and Lederer, W. H., 1973. Migration inhibitory factor and interferon in the circulation of mice with delayed hypersensitivity. Infect. Immun. 7: 68-75. Schorlemmer, H. U., Davies, P., and Allison, A. C , 1976. Ability of activated complement components to induce lysosomal enzyme release from macrophages. Nature 26: 48-49. 120 Schwartz, H. J., Leon, M. A., Pelley, R. P., 1970. Concanavalin A-induced release of skin-reactive factor from lymphoid c e l l s . J. Immunol. 104: 265-268. Schier, W. T., Trotter, J. T. I I I . and Reading, C. L., 1974. In-flammation induced by concanavalin A and other l e c t i n . Proc. Soc. Exp. B i o l . Med. 146: 590-593. Shortman, K., Williams, N., Jackson, H., Russ e l l , P., Byrt, P. and Diener, E., 1971. The separation of d i f f e r e n t c e l l classes from lymphoid organs. IV. The separation of lymphocytes from phagocytes on glass bead columns and i t s e f f e c t on subpopulations of lymphocytes and antibody-forming c e l l s . J. C e l l B i o l . 48: 566-579. Sober, A. J., Haynie, M., Inman, F. P., and David, J. R., 1976. Stimulation by macrophage a c t i v a t i n g factor (MAF) of gluco-samine uptake by guinea pig macrophages i n m i c r o t i t e r plates and c e l l s i n suspension culture. Fed. Proc. 35: 489. Sonozaki, H., and Cohen, S., 1971. The macrophage disappearance reaction: Mediation by a soluble lymphocyte-derived f a c t o r . C e l l . Immunol. 2: 341-352. Sorg, C , and Bloom, B. R., 1973. Products of activated lympho-cytes. I. The use of r a d i o l a b e l l i n g techniques i n the cha r a c t e r i z a t i o n and p a r t i a l p u r i f i c a t i o n of the migration i n h i b i t o r y factor of the guinea pig. J. Exp. Med. 137: 148-170. Spragg, J., 1974. The Plasma Kinin-forming System. In: Mediators of Inflammation, (Edited by G. Weissman, Plenum Press, New York and London), pp. 83-111. Stavitsky, A. B., 1948. Passive c e l l u l a r transfer of the tuberculin type of h y p e r s e n s i t i v i t y . Proc. Soc. Exp. B i o l . 67: 225-227. Suzuki, T., Mizushima, Y., Sato, T., and Iwanaga, S., 1965. P u r i f i -cation of bovine bradykininogen. J. Bichem. (Tokyo) 57: 14-21. Taylor, M. M., Burman, C. J., and Fantes, K. H., 1975. Problems encoutered i n the preparation of migration i n h i b i t o r y factor (MIF) and mitogenic factor (MF) from phytomitogen-stimulated human peripheral lymphocytes and i n the preparation of MIF from human lymphoid c e l l lines (LCL). C e l l . Immunol. 19: 41-57. 121 Thrasher, S. G., Yoshida, T., Van Oss, C. J . , Cohen, S., and Rose, N. R., 1973. Al t e r n a t i o n of macrophage i n t e r f a c i a l tension by supernatants of antigen-activated lymphocyte cultures. J . Immunol. 110: 321-326. Trudgett, A., 1976. P a r t i a l p u r i f i c a t i o n of lymphokine a c t i v i t i e s by i s o e l e c t r i c focussing. J. Immunol. Methods. 10: 1-6. Tsoukas, C. D., Rosenau W. and Baxter, J. D., 1976. C e l l u l a r recep-tors f o r lymphotoxin: C o r r e l a t i o n of binding and c y t o x i c i t y i n s e n s i t i v e and r e s i s t a n t target c e l l . J. Immunol. 116: 184-186. Turk, J. L., 1962. The passive transfer of delayed h y p e r s e n s i t i v i t y i n guinea pigs by the transfusion of i s o t o p i c a l l y l a b e l l e d lymphoid c e l l s . Immunology 5: 478-488. Turk, J. L., and Oort, J ., 1963. A h i s t o l o g i c a l study of the early stages of the development of tuberculin Reaction after passive transfer of c e l l s l a b e l l e d with H-thymidine. Immunology 6: 140-147. Udaka, K., Takeuchi, Y., and Movat, H. Z., 1970. Simple method for quantitation of enhanced vascular permeability. Proc. Soc. Exp. B i o l . Med. 133: 1384-1387. Unanue, E. R., 1972. The regulatory r o l e of macrophages i n a n t i -genic stimulation. Adv. Immunol. 15: 95-165. Unkeless, J . C , Gordon, S., and Reich, E., 1974. Secretion of plasminogen activator by stimulated macrophage. J. Exp. Med. 139: 834-850. Wahl, S. M., Altman, L. C , Oppenheim, J. J. and Merhenhagen, S. E., 1974. J-n v i t r o studies of a chemotactic lymphokine i n the guinea pig. Int. Arch. A l l e r g y apply Immunol. 46: 768-784. Wahl, S. M., Iverson, G. M. and Oppenheim, J. J . , 1974. Induction of guinea pig B c e l l lymphokine synthesis by mitogenic and nonmitogenic signals to Fc, Ig, and C3 receptors. J Exp. Med. 140: 1631-1645. Wahl, L. M., Wahl, S. M., Martin, G. R. and Mergenhagen, S. E., 1974. Production of collagenase by macrophages exposed to lymphocyte products. Fed. Proc. 33: 618. 1 122 Wahl, S. M., Wilton, J . M., Rosenstreich, D. L. and Oppenheim, J. J., 1975. The r o l e of macrophages i n the production of lymphokines by T and B lymphocytes. J. Immunol. 114: 1296-1301. Waldron, J. A., Horn, R. G., and Rosenthal, A. S., 1973. Antigen-induced p r o l i f e r a t i o n of guinea pig lymphocytes in v i t r o : Obligatory r o l e of macrophages i n the recognition of antigen by immune T lymphocytes. J. Immunol. I l l : 58-64. Walker, S. M., Lee, S. C., and Lucas, Z. J., 1976. Cytotoxic a c t i -v i t y of lymphocytes. VI. Heterogeneity of cytotoxins i n supernatants of mitogen-activated lymphocytes. J. Immunol. 116: 807-815. Ward, P. A., Remold, H. G., and David, J. R., 1969. Leukotactic factor produced by s e n s i t i z e d lymphocytes. Science 160: 1079-1081. Ward, P. A., Remold, H. G., and David, J. R., 1970. The production of antigen-stimulated lymphocytes of a leukotactic factor d i s t i n c t from migration i n h i b i t o r y factor. C e l l Immunol. 2: 162-174. Warren, L., 1959. The t h i o b a r b i t a r i c acid assay of s i a l i c acids. J. B i o l . Chem. 234: 1971-1975. Werb, Z. and Cohn, Z. A., 1974. The preparation of macrophage ly s o -somes and phagolysosome. In: Methods i n Enzymology. V o l . 31 Edited by S. Fleischer and L. Packer, Academic Press, pp. 339-345. Wilhelm, D. L., 1973. Chemical Mediators. In: The Inflammatory Process, 2nd Ed., Vol. 2 (Eds. B. W. Zweifach, L. Grant and R. T. McCluskey; Academic Press, New York, 1973) p. 251-301. Wilkinson, P. C , 1974. Chemotaxis and Inflammation. Edinburg, C h u r c h i l l , Livingston. Williams, T. W., and Granger, G. A., 1969. Lymphocyte i n v i t r o c y t o t o x i c i t y : C o r r e l a t i o n of derepression with release of lymphotoxin from human lymphoxytes. J. Immunology 103: 170-178. 123 Williams, T. W., and Granger, G. A., 1973. Lymphocyte i n v i t r o c y t o t o x i c i t y : Mechanism of human lymphotoxin-induced a target c e l l destruction. C e l l . Immunol. 6: 171-185. Wolstencroft, R. A., and Dumonde, D. C , 1970. In v i t r o studies of cell-mediated immunity. I. Induction of lymphocyte trans-formation by a soluble "mitogenic" factor derived from i n t e r a c t i o n of s e n s i t i z e d lymphoid c e l l s with s p e c i f i c antigen. Immunology 18: 599-610. Wuepper, K. D., and Cochrane, C. G., 1972. Plasma p r e k a l l i k r e i n : I s o l a t i o n , c h a r a c t e r i z a t i o n , and mechanism of a c t i v a t i o n . J. Exp. Med. 135: 1-20. Yoshida, T., Sonozaki, H., and Cohen, S., 1973. The production of migration i n h i b i t i o n factor by B and T c e l l s of the guinea p i g . J. Exp. Med. 138: 784-797. Yoshinaga, M., and Waksman, B. H., 1973. Regulation of lymphocyte responses '_in v i t r o . IV. Role of macrophages i n rat lympho-cyte responses and t h e i r i n h i b i t i o n by cytochalasin B. Ann. Immunol. 124: 97-120. Yoshinaga, M., Nakamura, S., Hayashi, H., 1975. Interaction between lymphocytes and inflammatory exudate c e l l s . I. Enhancement of thymocyte response to PHA by products of polymorpho-nuclear leukocytes and macrophages. J. Immunol. 115: 533-538. Zucker, M. B., 1974. P l a t e l e t s . In the Inflammatory Process. Edited by B. W. Zweifach and L. Grant, R. T. McCluskey. Academic Press. New York. pp. 511-543. 124 Appendix 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. ( i ) A c t i v a t i o n with Con A Source DF Mean Square F P Treatment 1 4134.4 0.0 0.000001 Preparation 2 455.3 6.7 0.0069 Interaction 2 630.9 9.2 0.002 Error 18 68.2 Total 23 ( i i ) A c t i v a t i o n with DNP--BGG Source DF Mean Square F P Treatment 1 16433.0 358.7 0.000001 Preparation 2 579.3 12. 7 0.00037 Interaction 2 290.8 6.348 0.0082 Error 18 68.2 Total 23 125 Appendix 2 Analysis of variance of MIF a c t i v i t y of lymphokine treated with neuraminidase-agarose. Source DF Mean Square F P Treatment 1 16643.0 348.7 0.000001 Preparation 2 615.9 12.9 0.0033 Interaction 2 133.3 2.8 0.002 Error 18 47.7 Total 23 Appendix 3 Analysis treated of variance of SRF a c t i v i t y of lymphokine with neuraminidase-agarose. Source DF Mean Square F P Treatment 1 98.4 9.5832 0.01133 Animal 2 106.1 10.3384 0.00368 Interaction 2 0.953 0.0929 0.912 Error 10 10.264 Total 15 

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