.—--.<..‘ LIBRARY ' Michigan State University This is to certify that the .. thesis entitled REGULATION OF THE IMMUNE RESPONSE B¥—PROTEASE—$NH{BL$0RS presented by PRINCE KUMAR ARORA has been accepted towards fulfillment of the requirements for _m&_degree in Y AND PUBLIC HEALTH "gay/4 pay/N HAROLD c. MILLER, Ph.D. Major professor Date b 11 1 78 0-7639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. mu m7 » .qtf‘é 300 ALJb . ROLE OF PROTEASE INHIBITORS IN THE REGULATION OF THE IMMUNE RESPONSE By Prince Kumar Arora A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1978 ABSTRACT ROLE OF PROTEASE INHIBITORS IN THE REGULATION OF THE IMMUNE RESPONSE By Prince Kumar Arora For a long time cell biologists have proposed that proteolytic enzymes play a role in cell proliferation and lymphocyte differentiation. Protease inhibitors [P.I.] on the other hand inhibit these proteolytic enzymes thereby abrogating their functions. Two protease inhibitors alpha-l-antitrypsin [oi-AT] and Trasylol were examined for their role in the regulation of the immune response. An in xi££g_modified plaque forming cell [PFC] assay was used to measure the immune response. al-AT was found to have a regulatory effect on the immune response. In concentrations of 100 to 1000 ug/ml of murine spleen cell culture, al-AT significantly suppressed the anti-SRBC PFC response. In_yigg. studies confirmed these findings. Results indicated that this protein does not bind to antigen, and it does not lose its biologic activity under normal cell culture conditions. -AT was found to be homogeneous, 0‘1 when examined by isoelectric focusing and by disc gel electr0phoresis. al-AT suppressed antigen-dependent B-lymphocyte differentiation without affecting adherent or T-cells. Since one of the unique properties of Trasylol is its protease- inhibiting capacity similar to that of a -AT, this kallikrein inactivator l Prince Kumar Arora was tested as a model of protease inhibitor for further determining various parameters of immuno-regulation brought about by protease inhibitor. In concentrations of 100 to 2000 kellikrein inactivating units [KIU] per m1 of culture, this inhibitor suppressed both the pri- mary and secondary PFC response. Suppression by Trasylol was not antigen-specific. This was further verified by the demonstration that Trasylol suppressed in gitrg_PFC response of spleen cells against dextran. Suppression by Trasylol was not due to depletion effect on the antigen. Inhibitory capacity of Trasylol was reversible and the degree of suppression was dependent on the time at which trasylol was added to the cultures. Trasylol added to antigen-stimulated cultures up to 48 h after initiation of cultures was immuno-suppressive whereas at 72 h after initiation or later it did not suppress. Pretreatment of spleen cells with this inhibitor, for 6 h before exposure to the antigen did not affect the immune response. When pre-incubated with trypsin, the suppressive activity of Trasylol was abrogated. Similar to our previous observations with al-AT, Trasylol did not appear to affect T-cells or adherent cells, but it suppressed the B-lymphocyte differen- tiation suggesting that Trasylol has an immunoregulatory function. By the use of incorporation of radiolabeled thymidine ([BHJ- thymidine) into mouse spleen cells and plasmacytoma [MOPC-Zl], the inhibitory effect of Trasylol on mitogen-induced lymphocyte triggering was also studied in 31359. DNA synthesis was effectively inhibited by 250 to 1000 KIU of Trasylol when response was induced by lipopolysac- charide [LPS] of Escherichia coli. The inhibitory effect of Trasylol Prince Kumar Arora was reversible. 0n the contrary, DNA synthesis of spleen cells was not inhibited by the inhibitor when the cells were stimulated with 1 ug of concanavalin-A [Con-A] per culture. DNA synthesis and growth rate of plasmacytoma, a B-cell tumor, was also reversibly inhibited by Trasylol. Taken together these results suggest that the target of inhibitory action of protease inhibitors such as al-AT and Trasylol was the B-lymphocyte. Fluorescent labeling studies have also supported these findings since B-lymphocytes and plasmacytoma display intense binding of labeled protease inhibitor [both Trasylol and al-AT] compared to low levels found on T cells or lymphoblastoid cell line [S.49.l, T-cell tumor]. The functional significance of these protease inhibitors as regulators of lymphocyte differentiation and tumor growth is discussed. DEDICATED TO MY PARENTS ii ACKNOWLEDGEMENTS I wish to express my sincere appreciation to my adviser, Dr. Harold Miller, for his help and guidance in my research throughout my graduate program. Thanks are also due to my other committee members, Drs. Tobi Jones, Walter Esselman and Lawrence Aronson, for their helpful suggestions throughout my research project. My sincere gratitude goes to my friends and colleagues, Bill Freimuth, P. Narayanan, Bill Eschenfeldt, Barbara Laughter, Warren Coon, and others, whose help, counseling and encouragement made my studies as a graduate student an enjoyable experience. Finally, but most impor- tantly, I would like to acknowledge the patience, help and encouragement of K. Barbara Chin, throughout my graduate studies. iii TABLE OF CONTENTS Page LIST OF TABLES ................. . ............. . ..... . ....... ..... vi LIST OF FIGURES ............ ..... ............................... . vii INTRODUCTION.... ............ . ....................... .. .......... 1 LITERATURE REVIEWOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOO O ............ 3 I. Protease Inhibitors.. ..... .............. ...... ........... 4 A. Alphal-Antitrypsin........ ............ ...... .......... 6 Biochenical Characterization.......................... 7 Determination and Isolation of al-AT....... ........ ... 7 Genetic Aspects of al-AT............ ..... ...... ....... 8 Mechanism of Inhibition by al-AT............. ......... 9 Physiologic Function of 01-AT......................... 10 Pathogenesis of al-AT Deficiency ...... . ..... . ....... .. 11 B. Trasylol................. ................... . ....... .. 13 Historical Perspective................................ 13 ISOIationOOOO......OOCOOOOOOOO ...... 0 OOOOOOOOOOOOOO .0. 15 Biochemical Characterization............... ........... 15 Protease Binding Site.............................. 17 Three-dimensional Structure........................ 17 Shape........................................ ...... 17 Isoelectric Point and Stability.... ..... . .......... 18 Enzyme-Inhibitor Interaction......... ........... ...... 18 Inhibition Spectrum of Trasylol.......... ........... .. 19 TrYPSinIOOOOOOOOOOOOO......OOOOOOOOOOOOOOOOOOOOOOOO 19 Chymotrypsin......... ....... . .......... . ....... .... 20 Kallikrein............................... .......... 20 PlasminOOO.........OOOOOOOOOOOOOO0...... ........... 21 Distribution and Execretion of the Inhibitor.. ........ 21 Pharmacology of Trasylol.............................. 22 iv TABLE OF CONTENTS--continued Trasylol as Therapeutic Agent ......................... Trasylol and the Immune Response ..... . ................ II. Proteolytic Enzymes ......... ... .......................... Serine Proteases........ .................... .......... Mechanism of Protease Action... ...... .. ..... ... ....... BIBLIOGMPHYOOOOOOO0.0.0.0... ...... 0...... ..... ......OOCOOOOOOOO ARTICLE 1 - al-ANTI-TRYPSIN IS AN EFFECTOR 0F IMMUNOLOGIC STASIS ARTICLE 2 - PROTEASE INHIBITOR REGULATION OF B CELL DIFFERENTIA- TION I. The Effect of Trasylol on the Primary and Secondary Antibody Response............ .......... ARTICLE 3 - PROTEASE INHIBITOR (TRASYLOL) INTERACTION WITH LYMPHOID CELLS............. ..... .... ..... . .......... Page 23 24 25 26 27 30 40 51 90 LIST OF TABLES TABLE Page 1. Protease Inhibitors in Human Serum ....... . ................. 5 Article 1 1. Effect of al-AT on the Anti-SRBC Responses of Mice ......... 46 2. Kinetics of al-AT on In Vitro Anti-SRBC Responses... ....... 47 3. a -AT Effect on Adherent and Non-Adherent Spleen Cells FOIIOWing Antigen StimUIationooococo-00.000000000000000.coo 48 4. The Immunobiological Effects of al-AT on T-Cells, B-Cells and Adherent Cells of Spleen .............. . ................ 49 Article 2 1. Dose-Response Effect of Trasylol on the.lB Vitro Primary Antibody Response........ ................ ........ .......... 68 2. Trasylol Suppression of Memory Response.. ....... . .......... 70 3. Trasylol Regulation of the Anti-Dextran PFC Response of Normal and B-Cell SpleeDOOOOO......OOOOOOOO ..... 0.0.0.0.... 72 4. Trypsin Neutralization of Trasylol Immuno-suppression...... 73 5. Trasylol Effect on Adherent and Non-adherent Spleen Cells Following Antigen Stimulation.............................. 74 6. The Immunobiological Effects of Trasylol on T-Cells, B-Cells and MacrOphages of Spleen.............. ...... . ..... 75 vi LIST OF FIGURES FIGURE 1. 2. Article 2 Attempt to override Trasylol suppression by increasing the antigen doseIOOOOO ..... 000...... ....... ......OOOOOOOOOOOO Kinetics of Trasylol suppression on the immune response... Article 3 . Suppression of LPS-stimulation of spleen cells by various concentrations of Trasylol.......................... ...... Suppression of mouse plasmacytoma MOPC-Zl growth by Trasy10100000000.0.0000.........OOOOOOO...000...... 0000000 . Suppression of mouse plasmacytoma MOPC—21 growth by Trasy101 0000000000000 ......IOOOOOOOOOOOOOOO ........ O ...... . Fixed normal spleen cells (a) and Trasylol treated spleen cells (b) stained with FITC-Trasylol. Enriched population of B-cells (c) and T-cells (d) stained with FITC-Trasylol. Plasmacytoma [MOPC-Zl] cells (e) and lymphoblastoid [8.49.1] cells (f) stained with FITC-Trasylol. .......... .. vii Page 69 71 106 108 109 110 INTRODUCTION The role of proteolytic enzymes in cellular functions has been expanded in recent years. Proteases have been found on the surface and in the medium of cultured cells and it has been suggested that a proteo- lytic mechanism may be of importance in cell proliferation and lympho- cyte differentiation. Protease inhibitors, on the other hand, are known to inhibit or inactivate proteolytic enzymes thereby abrogating their function. Furthermore they also inhibit proteases isolated from lympho- cytes. The major objective of this research was to investigate the role of alpha—l-antitrypsin [cl-AT], a major protease inhibitor present in the normal serum, in the regulation of the immune response and to deter- mine the type of lymphoid cell population regulated by the protease inhibitor. Trasylol is a protease inhibitor similar to 0 -AT, and was 1 used as a model for further determining various parameters of immuno- regulation brought about by protease inhibitor. Literature on the protease inhibitors, a -AT and Trasylol is pre- 1 sented with respect to the biochemistry, genetics, mechanism of protease inhibition and the inhibition spectrum, and their role in pathogenesis and the immune response is also discussed. Finally the role of al-AT and Trasylol in the regulation of the immune response is examined with emphasis on specific cell populations regulated by the protease inhibitor. In these studies we proposed that alpha-l-antitrypsin, a constitu- ent of normal human serum, has an immunoregulatory function based on demonstration of a non-cytotoxic immuno~suppressive effect on the pri- mary antibody response both in gi££g_and lE;XiXQ by this protease inhibitor. Furthermore, it was observed that 0 -AT suppressed antigen- 1 dependent B-lymphocyte differentiation without affect on adherent or T-cells. Results of these experimental approaches are presented in the first of three manuscripts [published in Nature, 214;589 (1978)]. The second article [submitted for publication] extend this concept by examining the effect of polyvalent enzyme inhibitor Trasylol on the primary and secondary immune response to both T-dependent and T-independ- ent antigens. This investigation is the first study in which Trasylol has been examined for its effect on the immune response. Further experi— ments have been carried out to investigate if immunoregulation by Trasylol is due to its protease inhibiting capacity and also to deter- mine its mode of action. Finally, the third article [submitted for publication] focuses on the role of Trasylol in its inhibition of lymphocyte stimulation by mitogens and gives evidence that this action is exerted only on B-lymphocytes of spleen. Fluorescent microscopy was used to verify that protease inhibitor binding was more selective for B—lymphocytes. LITERATURE REVIEW Immuno regulation, a fundamental and ubiquitous mechanism, can be brought about by certain normal serum proteins. Feedback inhibition of the immune response by antibodies represents a specific system of regu- lation (l). Immune-regulatory factors which nonspecifically inhibit an immune response are subjects of great interest but remain poorly under- stood. The bulk of experimental evidence to date suggests that a number of proteins present in normal human serum can serve as immuno-suppressors or as immuno-enhancers. Recently a-globulin rich fraction of Cohn Fraction IV, designated IRA (immune-regulatory alpha globulin) has been implicated in suppressing the in 31539 antibody response of spleen cells to sheep red cells without cytotoxicity (2). Results from the studies of Chase (3) also indicate that az-macroglobulin, one of the protease inhibitors [P.I.] present in normal serum limits the human lymphocyte response to phytohemagglutinin [FHA-P] and Concanavalin-A [Con-A] as measured by 3H-thymidine incorporation. A similar role for al-anti- trypsin, a protease inhibitor and a constituent of the normal serum can be postulated to be involved in the regulation of the immune response. The role of proteolytic enzymes in cellular functions has been expanded in recent years. Proteases have been found on the surface and in the medium of cultured cells (4). They have been implicated in alter— ing the electrOphoretic mobility of lymphocytes (5), in lymphocyte migration in 3139 (6), and even in lymphocyte blast transformation (7). Protease inhibitors [P.I.] on the other hand, have a function to inhibit proteolytic enzymes produced by leukocyte granules (8). Furthermore, they also inhibit proteases produced by certain bacteria during their infection (9). More recently protease inhibitor like N-a—tosyl-L-lysyl chloromethyl ketone [TLCK] has been shown to affect an intracellular protease thought to be responsible for lymphocyte blastogenesis (10). The function of al-antitrypsin [a protease inhibitor] in the serum is unknown, nor is it known why there is an increase in serum of this proteinase inhibitor in certain pathological conditions (11). One intriguing possibility is that a -antitrypsin has immuno-regulatory 1 properties, which are important for the exemption of intracellular pathogens and even tumor cells from immuno-logical attack. Immuno-suppression by protease inhibitor, a -antitrypsin, as has 1 been demonstrated by us [published results] may lead to further under- standing of the severe deficiency diseases of the immune system, an association between protease inhibitor and immune deviation and finally the relationship between immune deviation and disease. I. Protease Inhibitors The earliest observation concerning the anti—tryptic activity of serum date back to the end of the last century (12) and the first attempts to concentrate and isolate protease inhibitors [P.I.] were reported by Landsteiner in 1900 (13). However, it has been only during the last 25 years, that nearly six P.I.'s of human plasma have been isolated and chemically and biologically characterized. Protease inhibitors are found not only in plasma, but also in organs and tissues sensitive to proteolysis, i.e., the lungs, mucous membranes of the nasal respiratory and 0.1. tract (14). Although a number of P.I.'s are already well characterized, only -antitrypsin [cl-AT] and a -macroglobulin [oz-M] have turned out to 0‘1 2 be important with regard to both their concentration in the plasma and specificity; the other inhibitors are only present in trace amounts. Heimburger (14) lists six different protease inhibitors well character- ized in human serum (Table l). * Table l. Protease Inhibitors in Human Serum Normals (mg/d1) 01-Antitrypsin al-AT 180-280 01—Antichymotrypsin ol~X 30-60 Inter-a-trypsin inhibitor IaI 20-70 Antithrombin III AT III 22-39 Cl—Inactivator Cl INA 15-35 azéMacroglobulin aZ-M 150-350 males 175-420 females * Data taken from Table 1 of Reference #14. A. Alpha -Antitrypsin 1 Since the first successful isolation of al-AT by Moll, Sudden and -AT until 1964, when Brown in 1958 (15) nothing much was done on a1 Laurell and Erickson (16) described a -AT—deficiency in man and estab- l lished an association between homozygous al-AT deficiency and obstruc- tive emphysema of lungs. Since then a number of phenotypes of al-AT deficiency have been reported in the literature. It has been observed that the frequency of chronic pulmonary emphysema is significantly higher in subjects with homozygous deficiencies of al-AT than in the general population (11). al-antitrypsin [al-AT] has been identified as an alpha-l-globulin. Enzymes shown to be affected by its inhibitory properties include tryp- sin, chymotrypsin, pancreatic elastiase, skin collagenase, renin, urokinase, Hageman factor cofactor and the neutral proteases of poly- morphonuclear leukocytes (17,22). These enzymes include an elastase, a collagenase and a non—specific protease capable of digesting vascular basement membranes (23,24). al-AT can also inhibit the acid protease present in alveolear macrophages (25). Inhibition of kallikrein is negligible (26). al-Antitrypsin can be found in a number of body fluids such as tears, perilymph, lymph, saliva, colostrum, mother's milk, duodenal fluid, gall bladder bile, synovial fluid, cervical mucus, semen and amniotic fluid (17,27-31). It is also found in platelets and in mega- karyocytes (32). In the serum, exogenous 01-AT has a half life of only about a week, most of the loss being by catabolization in the liver (33). Biochemical Characterization: Although the a1~AT isolated from deficient patients can be distinguished electrophoretically from that of normal persons, immunologically and functionally [i.e., as an inhibitor of trypsin or elastase in gitrg] it is identical. This glyco- protein has a molecular weight ranging from 49,500 to 54,000, consists of a single polypeptide chain [389 amino acids with the predominance of aspartate, glutamate, leucine and lysine]. The carbohydrate content [nearly 12%] consists of four side chains of two different types [containing hexose, glucosamine and sialic acid] (34). In severely deficient persons it has less N-acetyl-glucosamine, mannose, galactose and sialic acid [N-acetyl-neuraminic acid] than that from normal persons (35). The isoelectric point is in the pH range of 4 to 6. 1 estimated by radial immunodiffusion (36) or by measuring the trypsin Determination and Isolation of a -AT: al-Antitrypsin is usually inhibiting capacity of serum. In the latter test the residual activity of trypsin is measured after the enzyme had been incubated for a fixed period of time with a dilution of the serum to be tested (37). More recently Delforge (38) did crossed immunoelectrophoresis of a -AT using 1 thin layer isoelectrifocusing [TLIEF] in polyacrylamide as the first dimension. This system provided increased resolution of the patterns associated with the polymorphic expression of a -antitrypsin. 1 Isolation of al-AT from serum is a difficult procedure and the results are not always satisfactory. Kueppers (36) isolated apparently pure 01-AT by electrophoresis on a Pevikon block.with subsequent filtra- tion on a Sephadex G-200 column, but one of the steps included removal of sialic acid by neuraminidase. Such desialylated a -AT preparation 1 preserved its antigenic properties and after labeling with 1251 behaved ingizg as a native protein (39). Musiani and Tomasi, Jr. (34) used affinity chromatography employing an antiserum which had been depleted of al-AT antibodies. Their final preparation was homogeneous by immuno- logical and physiochemical criteria. Hao_e£nal. (40) fractionated human plasma by using polyethylene glycol and obtained purified [>95Z] al-AT with a yield of nearly 14 percent. Genetic Aspects of al-AT: Functionally and immologically the -AT proteins are identical. Only by electrophoresis on starch gel in O‘1 discontinuous acid buffers and by isoelectric focusing [IEF] can vari- ants be detected with certainty. A system of labeling based on letters of the alphabet has been adopted, the electrophoretically slowest being denominated Z, the usual M, and the faster F. S, the second most common type, falls between M and Z. The system itself is titled "Pi" for protease inhibitor. Although reports of at least 26 different Pi alleles have appeared in the literature since the system of letters was agreed upon, a number of the alleles have apparently been found in only one individual or one family. All available data support the hypothesis of codominant alleles, that is, each allele controls the production of one kind of al-AT mole- cule uninfluenced by the corresponding allele on the other member of the chromosome pair. No more than two sets of bands have ever been found in a single subject's pattern (41,42) although mosiacism is theoretical- ly possible. Presumably, these are at a single locus, but the specific chromosome carrying the Pi genes has yet to be determined. Evidence for linkage with the Gm gene, which codes for the constant part of the heavy chain of IgG, is rather good and favors chromosome 2 (43,44). There does not appear to be good evidence for any other linkage (42,44). PiM is by far the most common allele with a frequency of at least 0.87 (as). Other alleles include P18, P12, PiMZ, P122 and PiSZ (45). A few cases have been discovered in which levels of circulating al—AT are virtually undetectable and no electrophoretic pattern at all is obtainable (46,47). Lack of display of characteristic PAS-positive globules in the liver strongly suggests that deletion of the Pi gene has null or Pi-. occurred, and the allele is denominated Pi M allele has been further divided into a number of subtypes. Johnson (48) reported Mlamb and MBaldwin subtypes with acid starch gel electrOphoresis. Kueppers (49) reported a heterozygous subtype Pi MMI' Others (50,51) reported MllM2 subtypes. Genz 33 a1. (52) further divided the subtypes into the homozygous types Ma, Mb, and Mc and the hetero- zygotes into Mab, Mac, and Mbc. There is considerable overlap in the terminologies of these subtypes and a standard nomenclature has yet to be determined by workers in this field. Mechanism of Inhibition by -AT: The serine proteases that are 0‘1 inhibited by 01-AT appear to cleave peptides by attaching the active catalytic site of the enzyme to one of two particular locations in the amino acid chain of the peptide (18). They probably combine with the 01-AT molecule in the same fashion (18). Since chemical modification of the lysine residues of the molecule renders it incapable of inhibiting 10 trypsin and chymotrypsin, whereas other modifications affecting other amino acids leave its function intact, the lysyl bond is believed to be the reactive inhibitor site, at least for these two proteases (53). Cohen 33.31. (54) pr0posed that a Lys-Thr bond is cleaved during base catalyzed disruption of the complex and that a new carboxyl terminal lysyl residue is formed. These workers disrupted the complex using 180B labeled base. The new carboxyl terminal lysyl residue became labeled with 180. This conforms to the known distribution of oxygens which occurs during the base catalyzed hydrolysis of acyl esters, which would occur if the al-AT-trypsin complex were an acyl ester or if an acyl intermediate formed during the base catalyzed hydrolysis of a tetrahedral adduct. This led them to hypothesize that trypsin binds to a Lys-Thr bond near the amino terminal end of the al-AT. The bond is probably analogous to that usually formed between trypsin and its sub- strates. Since there are no intrachain disulfide bonds at the amino terminal end in al-AT, a tetrahedral complex is most likely. With an excess of elastase the al-AT molecule itself may be cleaved and inacti- vated; however, the peptide fragment remaining with the elastase mole- cole appears to inactivate it as well (55). Physiologic Function of a1-AT: The physiologic function of al-AT J. in the serum is unknown. It is not clear why there is an increase of this protease inhibitor in certain physiologic and pathologic conditions. The level of 01-AT considerably increases in variety of conditions such as local inflammation, surgery, malignant tumors, injection of bacterial endotoxin, nephritis, pregnancy, and even during administration of ll contraceptive hormones (56-60). The rise of al-AT concentration follow- ing trauma is quite steep and maximum values [twice the normal level] are reached in human subjects on the third day after gastrectomy (57) or after injection of typhoid vaccine (60). Heterozygotes for the deficiency gene have an elevation of 0 -AT but it reaches barely 50 per- 1 cent of the response in normal individuals (56). Recent work by Arora gt 31. (61) suggests that 0 -AT play an 1 immuno-regulatory role in the suppression of antigen-dependent B-lympho- cyte differentiation without affecting adherent or T-cells. Pathogenesis of 0 -AT Deficiency: Severely deficient subjects of l phenotype PiZ and probably PiSZ are much more susceptible to the develop— ment of emphysema or chronic bronchitis [or both] than the general popu— lation, the large majority of whom are of phenotype PiM (62,63). Patients of PiMZ phenotype are more likely to have symptomatic chronic obstructive lung disease than those of phenotype PiM (64). Larsson .E£.21' (62) investigating aspects of lung function in smokers noted that abnormalities of elastic recoil, residual volume and closing capacity were significantly more prevalent in the PiMZ smokers than in the PiM smokers. The experimental animal model to imitate natural emphysema closely was created by the intratracheal instillation of papain. Recently Fisk and Kuhn (65) observed emphysema-like changes in the lungs of the Blotchy mouse [Blotchy allele is one of several mutations at the Mottled locus on the mouse X-chromosome] and suggested Blotchy strain to be a useful model to investigate how abnormalities of connective tissue 12 proteins influence pulmonary structure and function. The discovery that emphysema often deve10ps prematurely in persons who have virtually no ol-globulin, a protein known to inhibit at least one proteolytic enzyme in gitgg, directed investigators toward a search for a responsi- ble in 3132 enzyme. Proteases capable of producing emphysematous lesions in animal lungs were found in purulent sputum (66) and also extracted from the granules of polymorphonuclear leukocytes (23). Leukocyte elastase when instilled into the trachea of hamsters could produce the lesions of emphysema (67). Alveolar macrophages also released an elastase, but it was much less readily inhibited by al-AT than the elastase from leukocytes (68). Characteristically, the emphy— sema found in the lungs of PiZ cases is of panlobular type, although some patients do have the more common centrilobular variety (69). In 1968, Sharp noted a serum electrophoretic pattern without an al-globulin peak and found it belonged to a child with cirrhosis (1?). Microscopic examination of biopsy material showed that the globules in the hepatocytes readily combine with fluorescent-labeled antibody to human 01-AT (70-72). Those who have studied the frequency of 0 -AT deficiency in hepatic 1 cancer have found that 10 to 50 percent of patients with hepato-cellular carcinoma have the characteristic globular bodies in their livers, although usually not in the tumor cells themselves (73-75). Although proteolytic enzymes have access to several other tissues and could conceivably cause disease in them also if inhibition were ineffective, convincing data relating al-AT deficiency to any organ 13 other than the lung and the liver are sparse. Chronic pancreatitis or pancreatic fibrosis is only suggested by a few reports (76-78). Data in rheumatoid arthritis are conflicting (79,80). Sporadic cases of glomerulonephritis in a -AT deficiency with 1 liver disease have been reported (17), and the deposits containing al-AT found along the basement membrane have led to speculation that leakage of Z protein from dying liver cells may act as an antigen in the formation of immune complexes (81). B. Trasylol Historical Perspective: In 1925, Frey (82) discovered in mice a blood-pressure lowering substance of high molecular weight which was later termed Kallikrein from the greek name "kallikreas" for pancreas (83) and which today is included among what are known as the kinino- genases. The first inhibitor for kallikrein was detected in the blood plasma by Frey and Kraut and was termed kallikrein inactivator (84,85). In 1930, the existence of a further kallikrein inhibitor in the bovine parotid, spleen, liver, and lymph nodes was verified by Kraut £5 31. (85). Astrup (87) found an inhibitor of fibrinolysis in extracts of the bovine lung. Werle gg_al. (88) recognized that a connection exists between the inhibitors from the mentioned animal organs. He found that the kalli- krein inhibitor he had described was also an inhibitor for trypsin and chymotrypsin, that the pancreatic trypsin inhibitor of Kunitz is also capable of inhibiting kallikrein (89), and that the inhibitor from the bovine lung has the same inhibitor specificity as the kallikrein-trypsin l4 inhibitor of the other bovine organs (90). Werle together with Marx was able to show that all these inhibitors also inhibit plasmin (90). The inhibitor known as the kallikrein-trypsin inhibitor occurs only in some ruminants; in particular some bovine organs contain large quantities of it. It differs from the selective trypsin inhibitor which has been verified in the human, canine, porcine and bovine pancreas and which probably occurs in all mammals. It also differs from the inhibi- tor detected in the canine submandibuloris for trypsin and chymotrypsin and in the mammalian accessory genital glands for trypsin (91). A significant distinguishing feature is that the inhibitory spectrum of the protease inhibitors of the serum (92) is different from that of the inhibitor from bovine organs (92). The inhibitors from the bovine parotid and lung are chemically identical (93). Many characteristics indicate that the kallikrein-trypsin inhibitors from other bovine organs, especially the liver, are no different from this inhibitor (92). The kallikrein-trypsin inhibitor from the bovine lung is industrially gained on a large scale in an almost pure form and is commercially available as Trasylol ® [Bayer, Germany]. In 1936, Kunitz (86) isolated a basic trypsin inhibitor from bovine pancreas which--as we know now from the work of many different laboratories-is identical with Trasylol. The basic trypsin inhibitor Trasylol is not identical with the strongly monospecific trypsin inhibi- tor which occurs in the pancreas of all mammals. Besides the specific trypsin inhibitor bovine pancreas also contains the polyvalent inhibitor Trasylol. Furthermore, in contrast to the specific trypsin inhibitor, 15 Trasylol is secreted neither into the pancreatic juice nor saliva. It has been functionally recognized as an intracellular effector, the specific target of which is hitherto unknown (94). Isolation: A process for isolating the kallikrein-trypsin inhibi- tor consists of alcohol-fractionation of the tissue extracts containing the inhibitor and subsequent paper-electrophoretic purification (95). From dealbuminized tissue extracts the inhibitor can be bound and frac- tionated to cation exchanges (96). Precipitation and selective adsorp- tion methods, in conjunction with exchanges chromatography, are likewise applied for its isolation on a fairly large scale. The inhibitor can be brought to crystallization by salting it out (95). In this form, and also after the chromatographic purification, the action of l KIU [Kallikrein Inhibitor Unit] is bound to 0.14-0.15 pg of organic sub- stance. Biochemical Characterization: Biochemists have focused on the polypeptide Trasylol structure, its amino acid composition and sequence, its inhibitory spectrum, and on the mechanism of its inhibitory action. Common features of the enzymes inhibited by Trasylol are a serine residue in their active center. This is also true for kallikrein from pig pancreas, the active center of which, according to the studies of Fiedler 35 a1. (97), is the sequence Asp-Ser-Gly. The condition for the elucidation of the amino acid composition of a polypaptide like Trasylol is its isolation in sufficient amounts in a pure state. l6 Kunitz (86) prepared this inhibitor in a relatively pure form elegantly by binding it to pure trypsin and splitting the complex by trichloro—acetic acid. After further purification the amino acid compo- sition and sequence of this preparation were examined by Laskowaski and Laskowaski (94,98). He found 58 amino acids and 4 amide groups. The molecular weight calculated from these results was 6513. The amino acid sequence and the position of the 8-8 bridges (Figure l) were V 10 _15 _ k Tyr —OThr—OGly —‘Pro —°Cys 4LysF'tlf:-.Ayg \ \ l f to TNT ‘ a ”' Pro v 1 1 A19 Lyst Alo‘—Arg¢— Cy5°—Gly¢- Ohm Ile Glu (L1 1"” —' PM 5+ Us {‘5 Set —-. A10\ 71' 35 AF 20 Lgu 55 3\ Glu ‘71] 77' 5 Cys ECy5‘—Thr0—-:r9*—Mel0-Cys¢—Asn‘/ ‘ I?!” 1 L L BOlllfl PM 1 PM .4 G! G! Al 1 ._. H ° ......nlmy Asp 58 1 g—j. LCCU _.__JAsn \ \Pro *—Arg \Gly ¢- Ala; Lys - Aloo/ / Figure 1. Amino acid sequence and S-S—bridges of the polyvalent pro- lease inhibitor (Trasylol) from bovine pancreas, respectively bovine lung. (Source: Figure 2 from Reference #112.) clarified nearly at the same time by Anderer and Hornle (99) and by Laskowski (100). A special difficulty in these efforts was the fact that at that time no enzyme was known which could attack Trasylol. In the meantime we know from the work of Kassell (101) that thermolysine is able to split Trasylol at 60-80°C into several peptides. l7 Protease Binding Site: The work of Archer (102) and of Laskowski Sr. and Jr. and their groups (100) revealed the importance of the free NH2 group of the lysine 15 residue for the inhibitory activity against trypsin (98). Fritz and co-workers showed that this is true also for the inhibition of chymotrypsin and kallikrein (98). Three-dimensional Structure: The crystallization of Trasylol by Schultz and Kraut (103) Opened up the possibility of elucidation of the 3—dimensional configuration of Trasylol. This was performed by Huber _g£.al. (194), after isomorphone crystallization with primarily aromatic mercury containing compounds. Shape: According to Huber (104) the Trasylol molecule is pear- shaped (Figure 2). Figure 2. Three-dimensional structure of the polyvalent prolease inhibitor (Trasylol) from bovine lung. (Source: Figure 4 from Reference #112.) l8 Isoelectric Point and Stability: The isoelectric point is at pH 10.5 on account of its slight molecular size, the inhibitor is dialys- able and not precipitable by de-albuminizing reagents. The inhibitor is remarkably stable in the neutral and particularly in the acid range, even at fairly high temperatures; and contrary to what was formerly assumed it is also relatively insensitive in the alkaline range (105). The lysine 15 residue is situated at the top of the molecule in an extremely exposed position. Most of the basic posi- tively charged amino acids are found in the upper half of the molecule whereas the acidic negatively charged ones are located at the base of the molecule. Engyme-Inhibitor Interaction: According to Huber (104) the ex- posed situation of the reactive center and the high dipole moment are of importance for the rapid and correct orientation of the inhibitor to the active center of mutual actions of enzymes and this inhibitor, salt bridges and hydrophobic effects are of special importance. They may explain the high affinity between inhibitor and enzymes. The lysine 15 side chain of Trasylol fits into the binding product, e.g., of trypsin (104) like a lysine containing substrate and in this way blocks the activity of the enzyme (Figure 3, on the following page). The inhibition of trypsin and chymotrypsin by Trasylol is perman- ent. There is no splitting of a peptide bond which is the case with many other neutral inhibitors of trypsin. In an acidic medium the inhibitor can be regenerated from the complex without any alterations. l9 Lysine Substrate RN \\\\\\\\ \t \\ ////. Trypsin Substrate Figure 3. Binding pocket of trypsin with a lysine containing substrate for trypsin. (Source: Figure 5 from Reference #112.) Inhibition Spectrum of Trasylol: The polyvalent inhibitor in- hibits several enzymes of different substrate specificity, which however have in common the esterolytic and proteolytic activity and partly also the kinin—liberating activity. The inhibition extends to trypsin, chymotrypsin, the different kinds of kallikrein, plasmin as well as some bacterial proteinases. Trypsin: The reaction of trypsin with Trasylol is pH—dependent above pH 11 and below pH 6, and it is reversible. In each case the proteolytic, esterolytic and kinin-liberating activity is equally strongly blocked. With trypsin the inhibition is linear with increasing inhibitor quantities only up to a limit value which lies between 70 and 20 90% inhibition. In the physiological pH range, 1 KIU inhibits the activity of 0.56 to 0.7 pg of trypsin. The dissociation constant of the trypsin-inhibitor complex is extremely low. For pH 7.8 an inhibitor 11 mol/l. The dissociation of the complex increases constant is 2 x 10- as the pH decreases, i.e., the inhibitor constant becomes larger [at pH 4.0, e.g., the Ki = 2.6 x 10.9 mol/l] (105). Chrmotrypsin: l KIU of Trasylol inhibits the activity of 0.44 ug of chymotrypsin. Expressed in unity of weight, the ratio of the trypsin to the chymotrypsin inhibition is 1:0.68. A dissociation of the chymo- trypsin inhibitor complex occurs likewise in the acid range, the dissoci- ation constant being, however, considerably greater than that for the trypsin-inhibitor complex. In a mixture of trypsin and chymotrypsin, trypsin is preferentially inhibited by Trasylol (105). Kallikrein: The kallikrein-inhibiting capacity of the inhibitor is of special significance, because it was the starting point of the entire development in the field of Trasylol. While the kallikrein—trypsin inhibitor is capable of inhibiting the kinin—liberating [proteolytic] and esterolytic activity of the kallikreins, the trypsin inhibitors from soya beans and ovomucoid inhibit only the serum kallikrein. Kallikrein-inhibitor complex has a relatively high dissociation constant [at pH 7.8 the Ki = 1.2 x 10-8 mol/l]. At pH 4.0 the kallikrein-inhibitor complex is already completely dissociated. Since the inhibitor is more firmly bound to trypsin than to kallikrein, when trypsin is added to the kallikrein-inhibitor complex, it displaces the kallikrein from its bond with the inhibitor. Once the 21 trypsin-inhibitor complex has formed, it is no longer segregated by the addition of kallikrein even when this kallikrein is added in large sur- plus quantities (105). Plasmin: Measured in terms of splitting synthetic substrates, 1 KIU inhibits the activity of 6 ug of plasmin. Pronase-P is a protein- ase mixture from Streptomyces griseus. 3.75 ug of the enzyme is inhibited by l KIU. Papain is inhibited by Trasylol only to a barely measurable extent [1 KIU inhibits 0.002 ug of papain]. Pepsin is neither inhibited by Trasylol nor is Trasylol attacked by pepsin (105). Distribution and Execretion of the Inhibitor: After injecting the rat with 12000 KIU/kg, Trautschold and Werle (89) examined the distribu- tion of the inhibitor over the individual organs. The inhibitor was initially found in slight quantities in all organs, then it selectively concentrated in the liver, with a maximum 30 min after the injection. After a latent period of about 10 min, the inhibitor content in the renal tissue began to rise; after 1 h it was 50% whereas only about 10% were still present in the blood. 4-5 h after the injection, almost the entire quantity of inhibitor administered was found in the kidneys. Tests undertaken with Trasylol using tritium as tracer (90) showed that the inhibitor is excreted in a biologically effective form only to a slight extent in the course of several days; the main quantity appears only as tritium activity in the urine. From this, one may conclude that there is extensive decomposition of the inhibitor in the renal tissues. Trasylol-as polypeptide with a relatively low molecular weight-- is distinguished by a very good toleration even in high dosage. 22 The mouse LD50 is about 2.5 x 106 KIU/kg (106). The dog tolerates i.v. doses up to l x 106 KIU/kg without complications. Rats tolerate high Trasylol doses less well; especially operationally damaged animals may show severe shock-like reactions with high dosage (107). This is one of the reasons why the rat is ill-suited for tests with Trasylol. In rare cases, patients with a history of allergy can after repeated applications show intolerance reactions (106). Pharmacology of Trasylol: Besides its capacity of inhibiting serine enzymes, Trasylol has another kind of activity: it may exert pharmacological actions. Pauschinger ggdal. (108) found that Trasylol accelerates the blood flow in the calf of the leg of man by arterial dilation and removes constriction. This observation was corroborated by Breght and co-workers (109) who studied 22 yiggg the effect of Trasylol on the tonus of peripheral arteries and veins from cattle and of veins from the human leg. They found that in very low concentrations [10.6 to 10.8 g/ml] Trasylol relaxes the arteries and constricts the veins. The mechanisms of these effects is unknown. The inhibiting effect of Trasylol in the development of edema in rat paws according to Kaller (110) could be elicited besides by inhibi- tion of kinin liberation also by a direct permeability reducing action of Trasylol on the proximal capillaries in the inflamed area. Also the distribution of Trasylol in the body after i.v. injections shows some specificity of the affinity of Trasylol to special organs or membranes (111). They could show that Trasylol also stabilizes the membranes of lysosomes of leukocytes and inhibits their degranulation. 23 Trasylol as Therapeutic Agent: Trasylol was employed therapeutic- ally for the first time in 1953 as a trypsin inhibitor on the suggestion of Frey and Werle (112, 113). Werle (112) reported large series of patients having acute pancreatitis who were treated with Trasylol. Thereafter, the favorable therapeutic action of Trasylol was confirmed :hn numerous clinical reports. The first attempt to use Trasylol thera- peutically was based on the assumption of a premature activation of trypsin during acute pancreatitis. Though it was possible to demon- strate active trypsin in pancreatitis, it must be assumed that inter- mediary active trypsin acts as a primary activator of other pancreatic enzymes in cellular micro-areas. There result four points at which Trasylol exerts a therapeutic action: 1. By inhibition of locally activated kininogenases Trasylol prevents the spreading of edema and stasis. This is caused by the action of kinins which increase vascular permeability, and dilate capillaries. 2. There occurs a partial inhibition of the reactions which lead to activation of the proteolytic system. 3. Under the influence of Trasylol a diminution of the secretory rate and secretion of pancreatic enzymes occurs. 4. Generalized liberation of kinins which leads to circulatory shock is prevented. Trasylol should be administered in the very beginning of acute pancreatitis so that it may act on the initial edema. 24 Further indications for therapeutical use of Trasylol are chronic relapsing pancreatitis, postoperative pancreatitis, various forms of shock, e.g., during peritonitis, and large tissue damage. Activation of proteolytic systems in blood as it occurs in hyperfibrinolysis, and with activation of the thrombin system may also be influenced by Trasylol. Trasylol and the Immune Response: Few studies have investigated the regulation of immune response by Trasylol. Prokopenko and Drobyazgo (114) studied the effect of Trasylol on antibody formation under normal conditions and in experimental atherosclerosis. They observed that Trasylol sharply inhibits the formation of specific immunoglobulins [especially 78] in rabbits with experimental atherosclerosis but does not affect antibody formation in healthy animals. These authors pro- posed that some form of equilibrium arises in rabbits with experimental atherosclerosis between the immuno—depressant effects of cholestrol or its metabolis products and the immunostimulant action of a serum factor which is probably a proteolytic enzyme or its activator. Trasylol disturbs this equilibrium and thus causes inhibition of antibody bio- synthesis in animals with experimental atherosclerosis. While studying the interaction between lymphocytes and inflamma- tory exudate cells, Nakamura and his coeworkers (115) found that the supernate of Ephziggg cultivated polymorphonuclear leukocytes [PMN-SUP] contain a factor which has an enhancing effect on the thymocyte-response. Thymocyte-helping potency of the SUP was abolished by adding Trasylol in soluble form or by passing SUP through a Trasylol affinity column. These authors thus assumed that the enhancing effect of PMN on 25 thymocyte-proliferative response was associated with the function of a neutral protease released from the cells, because the hemoglobinolytic activity of lymphocyte helping fraction was also inhibited when pre- incubated with Trasylol. They termed this protease, a lymphocyte help- ing protease [LHP]. More recently, Higuchi gg'al. (116) have described the inhibitory action of Trasylol on antigen or mitogen-induced lymphocyte triggering. By the use of incorporation of radiolabeled thymidine, uridine, and leucine into mouse lymphocytes, these workers observed that DNA synthe- sis, as well as RNA and protein syntheses are effectively inhibited by 0.3-2.5 x 10—7 mol of Trasylol when responses are induced by homologous antigen, allogeneic cells, phytohemagglutinin [FHA] of Escherichia coli. DNA synthesis by splenic cells is not inhibited by Trasylol when the cells are stimulated with a relatively large amount of Concanavalin-A [Con-A]. Furthermore, the antigen-induced DNA synthesis by nonadherent lymph node cells is enhanced by the culture supernatant of macrophages, and this helping effect of macrophage supernatant is effectively inhibited either by soluble or insoluble Trasylol. Their observations lead them to the proposal that the inhibitory action of Trasylol on lymphocyte triggering operates indirectly interfering with the helping action of macrophages on lymphocytes. 11. Proteolytic Enzymes The role of proteolytic enzymes in cellular functions has been expanded in recent years. Proteases have been found on the surface and 26 in the medium of cultured cells (4). There are also extensive reports that proteases produce a wide variety of effects on lymphocytes. Among these effects are: an alteration of electrophoretic mobility (5), a change in the pattern of homing, i.e., the migration of lymphocytes 33 3139 (6), an unmasking of certain alloantigens or an increase in antigenicity (117), and an alteration in the binding of antilymphocyte serum [ALS] (5). Recently, Hatcher g£_§l, (118) have isolated a cyto— toxic protease from human lymphocytes. The work of Hirschhorn ggugl. (10) suggests that an intracellular protease is involved in the blasto- genic response of lymphocytes to PHA. The same authors also demonstrated that protease inhibitors like epsilon-amino caprioc acid and TLCK could suppress the lymphocyte response to PHA. Saito g£.al. (119) also reported that another protease inhibitor leupeptin suppress lymphocyte transformation and agreed with the previous authors that the inhibition of lymphocyte transformation was due to the inhibition of an intracellu- lar protease. Serine Proteases: Most of the proteases inactivated by protease inhibitors, come under the trypsin family. This class of serine proteases is defined by the presence of a uniquely reactive serine side chain which makes a covalent ester bond to the carbonyl carbon atom of the susceptible bond in substrates to form an acyl-enzyme, and which reacts similarly with a number of covalent inhibitors (120). Two families of trypsin and subtilin have been well studied. Members of the trypsin family include: trypsin, chymotrypsin, elastase, plasmin, thrombin, collagenase and proteolytic enzymes of the complement system. 27 Mechanism of Protease Action: The mechanism of action of serine proteases on their specific substrates is generally formulated according to the following scheme: E + I e===> [El] e———* [EI]* g—e- [EI]** Fe [EI']* 6% E + 1' Present evidence (121,122) suggests that protease inhibitors function in a role analogous to that of a substrate. [EI]* represents an inactive complex in the form of a tetrahedral adduct which in the case of STI and trypsin involved arginine-63 of STI and active site serine residue of trypsin (123,124). This complex can be dissociated, however, by kinetic control (125) to yield primarily the virgin inhibitor 1. The existence of this postulated acyl-enzyme intermediate was resolved by Huang and Liener (126). They presented the evidence indicating that a small but detectable fraction of the STI-trypsin com- plex does in fact exist in the form of acyl intermediate [ester bond involving the carboxyl group of arginine-63 and the active serine residue of trypsin] in equilibrium with the predominent tetrahedral Species. This demonstration was facilitated through the use of 125I-labeled STI and the subsequent "trapping" of the acyl-enzyme inter- mediate. Bode_g£.al. (127) using X—ray crystallography compared the struc- tures of the free components with the molecules in the complex and observed the structures of stable intermediates of proteolysis. Their studies indicated that complex forms a tetrahedral adduct with a long bond of 2.6OA between C of lys 15 [inhibitor] and 0Y of serine 195. 28 C of lys 15 [inhibitor] is strongly tetrahedrally deformed with the carbonyl oxygen —340 out of the plane formed by N, C, Ca3. This agrees with NMR-studies on the complex, and suggests that a considerable degree of proton transfer from Ser 195 to His 57 occurs in the complex (127). In view of the functional similarities of all protein protease inhibitors, Bode_g£_al. (127) believed that their association with trypsin follows the same stereo-chemical principles. These workers also noted that complexes with other members of the trypsin protease family resemble each other closely due to the strongly conserved active site and substrate contact segments. Kaplan and Bona (128) have shown the mitogenic effects of pro— teases such as pronase and trypsin. It can be postulated that mitogens and antigens cause the release and activation of an endogenous protease at the lymphocyte surface whose proteolytic activity is essential for the subsequent events of blast transformation. Neutral proteases such as trypsin, chymotrypsin, pronase and pancreatic elastase have been shown to induce lymphocyte transformation and proliferation (7). Grayzel g5 31. (129) even have found a neutral protease bound to lymphocyte cell membrane. The extent to which proteases exist bound to the surface of cells or secreted by them remains to be determined. The function of a surface protease is a matter of conjecture but its presence on the lympho- cyte, a cell whose surface is the site of considerable cellular activity including the migration and shedding of receptors and the secretion of 29 immunoglobulins, suggests that proteases may play a role in the dynamic processes of the lymphocyte surface membrane. It is quite conceivable that cell surface or exogenous proteases may also be mitogenic and thus have a role in the initial differentiation of lymphoid cells, since they short-circuit the endogenous proteolytic step supposed to be common to all blast transformation. Observations that both exogenous and endogenous proteases have a role in the lymphocyte blast transformation, and also the studies that protease inhibitors like TLCK and leupeptin can suppress the action of such proteases, lead to the hypothesis that protease inhibitor present in the normal serum, may have its immunoregulatory effect on lymphocytes in two ways: 1. 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The induction of pulmonary emphysema with human leukocyte elastase. Am. Rev. Respir. Dis. 116:469. Janoff, A., R. Rosenberg, and M. Galdston. 1971. Elastase-like, esteroprotease activity in human and rabbit alveolar macrophage granules. Proc. Soc. Exp. Biol. Med. 136:1054. Greenberg, S. D., D. E. Jenkins, and P. M. Stevens, SE.El' 1973. The lungs in homozygous alphal antitrypsin deficiency. Am. J. Clin. Pathol. 66:581. Altman, R. P., and R. Chandra. 1976. Biliary hypoplasia consequent to al-antitrypsin deficiency. Surg, Forum 22:377. Aagenaes, 0., A. Matlary, and K. Elgjo, EE.§l° 1972. Neonatal cholestasis in alpha-l-antitrypsin deficient children: Clinical, genetic, histological, and immunohistochemical findings. Acta. Paediatr. Scand. 61:632. Bhan, A. K., R. J. Grand, and H. R. Colten, E£.§lf 1976. Liver in -antitrypsin deficiency: Morphologic observations and 16_vitro synthesis of al-antitrypsin. Pediatr. Res. 16:35. Lieberman, J. 1974. Emphysema, cirrhosis, and hepatoma with alpha-l-antitrypsin deficiency. Ann. Intern. Med. ‘61z850. Palmer, P. E., and H. J. Wolfe. 1976. a -antitrypsin deposition in primary hepatic carcinomas. Arch. Pathol. Lab. Med. 100:232. Blenkinsopp, W. K., and G. P. Haffenden. 1977. Alpha-l-antitrypsin bodies in the liver. J. Clin. Pathol. 66:132. Novis, B. H., G. 0. Young, and S. Bank, 66_§1. 1975. Chronic pan- creatitis and alpha-l-antitrypsin. Lancet 2:748. Mihas, A. A., and B. I. Hirschowitz. 1976. Alpha chronic pancreatitis. Lancet 2:1032. l-antitrypsin and Freeman, H. J., W. M. Weinstein, and T. K. Shnitka, et a1. 1976. Alpha -antitrypsin deficiency and pancreatic fibrosis. Ann. Intern. 1 Med. 85:73. Arnaud, P., R: M. Galbraith, and W. P. Faulk. 1977. Increased frequency of the M2 phenotype of alpha—l-protease inhibitor in juvenile chronic polyarthritis. J. Clin. Invest. .99i1442- Brackertz, D., and F. Kueppers. 1977. Alpha-l-antitrypsin pheno- types in rheumatoid arthritis. Lancet 2:934. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 36 Moroz, S. P., E. Cutz, and J. W. Balfe, ££.él° 1976. Membranopro- 1iferative glomerulonephritis in childhood cirrhosis associated with alphal-antitrypsin deficiency. Pediatrics 62:232 Frey, E. K. 1926. Zusammenhange zwischenherzarbeit und niereu— tatigkeit. Arch. Klin. Chir. 142:663 Frey, E. K., H. Kraut, and E. Werle. 1950. Kallikrein - Pedutin, Enke Verlag, Stuttgart. Frey, E. K., and H. Kraut. 1928. Ein neues kreislanfhormon und seine wirkung nannynschmiedebergs. Arch. Exp. Path. Pharma. 133:1 Kraut, H., E. K. Frey, and E. Werle. 1930. Uber die inaktivierung des kallikreins. Z. Physiol. Chem. 192:1. Kunitz, M., and J. H. Northrop. 1936. Isolation from beef pancreas of crystalline trypsinogen, trypsin, a trypsin inhibitor, and an inhibitor-trypsin compound. J. Gen. Physiol. 16:991. Astrup, T., and O. K. Albrechtssen. 1957. Estimation of the plas- minogen activator and the inhibitor in animal and human tissue. Scand. J. Clin. Lab. Invest. .6:233. Werle, E., L. Maier, and E. Ringelmann. 1952. Hemmung von protein- asen durch kallikrein - inaktivatoren. Naturwiss. 66:328. Trautschold, I., and E. Werle. 1961. Spektrophotometrische bestim- mung des kallikreins und seiner inaktivatoren. Z. Physiol. Chem. 325:48. Werle, E. 1964. Uber einen hemmkbrper ffir kallikrein und trypsin in der rinderlunge. Z. Physiol. Chem. 338:228. Haendle, H., H. Fritz, I. Trautschold, and E. Werle. 1965. Uber einen hormonabhangigen inhibitor ffir proteolytische enzyme in mannlichen accessorischen geschluhts drfisen und in sperma. Z. Physiol. Chem. 666:185. Trautschold, 1., E. Werle, H. Haendle, and H. Sebening. 1964. 16 Pathogenese, Diagnostik, Klinik und Therapie der erkrankungen des exokrinen pankreas. N. Henning, K. Heinkel, and H. Schon, eds. Schattauer—Verlag, Stuttgart. p. 289. Anderer, F. A. 1965. Z. Naturforsch. 206:499. Vogel, R., I. Trautschold, and E. Werle. 1968. 16 Natural Protein- ase Inhibitors, Academic Press, New York, London. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 37 Kraut, H., W. Korbel, W. Sholtan, and F. Schultz. 1960. Versuche zur isolierung des kallikrein-inaktivators. III. Elektrophoretische reinigung und molekulargeweichtsbestimmung in des ultrazentrifuge. Z. Physiol. Chem. 621:90. Trautschold, I. 1965. Habilitationsschrift. Med. Fakultat, Munchen. Fiedler, F., B. Muller, and E. Werle. 1971. Die aminosafiresequen im bereich des serins inaktiven zentrum des kallikreins aus schweinipankreas. Z. physiol. Chem. 352:1463. Fritz, H. 1971. 16_Proceedings of the International Research Conference on Proteinase Inhibitors, Munich. H. Fritz and H. Tscheche, eds. De Gruyter, Berlin, New York. p. l. Anderer, F. A., and S. Hornle. 1965. Z. Naturforsch. 20b:457. Laskowski, Sr. M., and B. Kassell. 1965. The basic trypsin inhibi- tor of bovine pancreas. V. The disulfide linkages. Biochem. Biophys. Res. Commun. 26:463. Kassell, B., M. Radicevic, M. J. Ansfield, and M. Laskowski, Sr. 1965. The basic trypsin inhibitor of bovine pancreas. IV. The linear sequence of 58 amino acids. Biochem. Biophys. Res. Commun. 18:255. Chauvet, J., and R. Acher. 1967. The reactive site of the basic teypsin inhibitor of pancreas. Role of Lys 15. J. Biol. Chem. 242:4274. Kraut, H., N. Bhargara, F. Schultz, and H. Zimmermann. 1963. Versuche zur isolierung des kallikrein-inaktivators. IV. Kristal- lisation und aminosaurezusammensetzung vergleich unit des trypsin- inhibitor von kunitz und Northrop. Z. Physiol. Chem. .2233230° Huber, R., A. Ruhlmann, and W. Steigemann. 1971. 16 Proceedings of the International Research Conference on Proteinase Inhibitors, Munich. De Gruyter, Berlin, New York. p. 50. Trautschold, I., E. Werle, and G. Zickgraf—Rfidel. 1965. Concerning the kallikrein-trypsin inhibitor. p. 3. Benchelt, H. 1963. Isolierung eines kallikrein-inaktivators aus rinderlunge und seine identifizier unit dem inaktivator aus rinder- parotis. Medizin. U. Chemie. 2:763. Creutzfeldt, W., and W. F. Caspary. 1972. Intestinal absorption of pancreatic kallikrein and some aspects of its physiological role. Kiniogenases: 16 Synposium on Physiological Properties and Pharma— cological Rationale. Haberland, G. L., and J. W. Rohen, eds. Schattaueg-Verlag, Stuttgart - New York. p. 67. 108. 109. 110. 111 O 112. 113. 114. 115 O 116. 117 O 118. 119. 38 Pauschinger, P., P. Matis, and H. Rieckert. 1969. 16_Neue Aspekte der Trasylol-Therapie 3, Schattauer, Stuttgart, New York. p. 81. Brecht, K., H. Nguyen-Duong, R. Fisher, F. Hillenbrand, P. Matis, H. J. Schneider, and W. Schuizer. 1971. Versuche zur isolierung des kallikrein-inaktivators. Arztl. Forsch. 26:86. Kaller, H. 1966. .lB Neue Aspekte des Trasylol Therapie, Schattauer, Stuttgart-New York. p. 152. Arndts, D., K. 0. Ralker, P. T5r3k, and E. Haberman. 1970. Studien zur verteilung und elimination eines proteinasen-inhibitors unit isotopentechniken. Arztl. Forsch. .26:667. Werle, E. 1972. Trasylol: A short survey on its history, bio- chemistry and activities. ‘16_New aspects of Trasylol therapy. Vol. 5. Protease Inhibition in Shock Therapy. Brendel, W., and G. L. Haberland, eds. Schattauer, Stuttgart-New York. p. 9. Frey, E. K. 1953-54. Uber eine neue innersekretorische funktion des pankreas. Therapieworsche. 6:323. Prokopenko, L. C., and L. D. Drobyazgo. 1975. Effect of the poly- valent proteinase inhibitor Trasylol on antibody formation under normal conditions and in experimental atherosclerosis. Bull. Exp. Biol. Med. .26:558. Nakamura, S., M. Yoshinaga, and H. Hayashi. 1976. Interaction between lumphocytes andinflammat ory exudate cells. 11. A proteo- lytic enzyme released by polymorphonuclear leukocytes as a possible mediator for enhancement of thymocyte response. J. Immunol. 117:1. Higuchi, S., S. Ohkawara, S. Nakamura, and M. Yoshinaga. 1977. The polyvalent protease inhibitor, Trasylol, inhibits DNA synthesis of mouse lymphocytes by an indirect mechanism. Cell. Immunol. 34:395. Schlessinger, M., and D. B. Amos. 1971. Effect of neuranmindase on serological prOperties of murine lymphoid cells. Transpl. Proc. 3:895. Hatcher, V. B., G. S. Lazarus, and A. I. Grayzel. 1977. A cyto— toxic protease isolated from human lymphocytes. Fed. Proc. Saito, M., T. Yoshizawa, T. Aoyagi, and Y. Magai. 1973. Involve— ment of proteolytic activity in early events in lymphocyte trans— formation by PHA. Biochem. Biophys. Res. Commun. .62:569. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 39 Kraut, J. 1977. Serine proteases: Structure and mechanism of catalysis. Am. Rev. Biochem. 46:331. Laskowski, M., Jr., and R. W. Sealock. 1971. Protein proteinase inhibitors - molecular aspects. Enzymes,_3rd ed. 3:375. Fritz, H., H. Tschesche, L. J. Greene, and E. Truscheit, eds. 1974. Proteinase Inhibitors, Boveyer Symposium V, Berlin, Springer— Verlag. Blow, D. M., J. Janin, and R, M. Sweet. 1974. Mode of action of soybean trypsin inhibitor [Kunitz] as a model for specific protein- protein interactions. Nature [London] 249:54. Sweet, R. M., H. T. Wright, J. Janin, and D. M. Blow. 1974. Crystal structure of the complex of porcine trypsin with soybean trypsin inhibitor [Kunitz] at 2.62 resolution. Biochemistry 13:4212. Sealock, R. W., and M. Laskowski, Jr. 1969. Enzymatic replacement of the original by a lysyl residue in the reactive site of soybean trypsin inhibitor. Biochemistry §:3703. Huang, J. S., and I. E. Liener. 1977. Interaction of the Kunitz soybean trypsin inhibitor with bovine trypsin. Evidence for an acyl-enzyme intermediate during complexation. Biochemistry 16:2474. Bode, W., P. Schwager, and R. Huber. 1974. Structural studies on the pancreatic trypsin inhibitor-trypsin complex and its free components: Structure and function relationship in serine protease inhibition and catalysis. Structure and Function of Plasma Pro- teins. Allison, Ed. p. 43. Kaplan, J. C., and C. Bona. 1974. Proteases as mitogens: The effect of trypsin and pronase on mouse and human lymphocytes. Exp. Cell Res. §§:388. Grayzel, A. I., V. B. Hatcher, and G. S. Lazarus. 1975. Protease activity of normal and PHA stimulated human lymphocytes. Cellular Immunol. l§:210. ARTICLE 1 ALPHAl-ANTITRYPSIN IS AN EFFECTOR OF IMMUNOLOGIC STASIS 40 ALPHAl—ANTITRYPSIN IS AN EFFECTOR OF IMMUNOLOGIC STASIS A number of proteins present in normal serum can serve as suppres- sors or enhancers of the immune response. Recently an a-globulin rich fraction of Cohn Fraction IV, designated IRA (immune-regulatory arglobulin), has been implicated in suppressing the in yitrg_antibody response of mouse spleen cells to sheep red cells without cytotoxicity.l Studies by Chase2 also indicate that 02-macroglobulin, one of the protease inhibitors present in normal serum, limits the human lymphocyte response to phytohemagglutinin, and Concanavalin-A as measured by 3H-thymidine incorporation. A similar role for al-antitrypsin (ml-AT), the major protease inhibitor in normal serum, can be postulated to be involved in the regulation of the immune response. Proteases have been found on both the surface and in the medium of cultured cells3; they have also been implicated in altering the electrophoretic mobility,4 in yigg_migration5 and blast transformation6 of lymphocytes. The physiologic function of al-AT in the serum is unknown. It is not clear why there is an increase of this protease inhibitor in certain physiologic and pathologic conditions.7 Results of the present communi- cation suggest that al-AT plays an immunoregulatory function by having a suppressive effect on the in 3139 and in xi££g_immune response of mouse spleen cells against sheep red cells (SRBC). For these studies spleens from 9-16 week old hybrid C57BL/10XC3H (BC3F1) female mice were dissected free of the surrounding fascia and cells were expressed from 41 42 the capsules. Single cell suspensions were gently aspirated and expelled by syringe through needles of 21 and 27 gauge. The cells were washed once then resuspended in spleen cell medium.8 Viability was checked by the Trypan blue exclusion method. al-AT isolated from normal human plasma was obtained from Dr. Y. L. Hao (American National Red Cross, Blood Research Center, Bethesda, Maryland 20014) and from Dr. Charles B. Glazer (The Institute of Medical Sciences, San Francisco, California 94115). al-AT activity was determined by measurement of its trypsin inhibitory capacity9 and quanti- tatively confirmed by radial immunodiffusion.10 It was also found to have a normal M1 phenotype pattern by isoelectric focusing11 and by disc gel electrophoresis.12 Immunoregulatory effects of protease inhibitors were studied in_!izg by injecting various concentrations of al-AT into groups of BCFl mice. 01-AT ranging from 250 ug to 1000 pg was given intravenously into mice along with 107 SRBC. The control groups received either human serum albumin (HSA), or human gamma globulin (HGG). Results presented in Table 1 indicate that these concentrations of al-AT significantly suppressed the plaque response. Furthermore, as the dose of al-AT was increased, greater suppression of PFC response resulted. Next al-AT effects on the immune response were studies in gigrg by adding various amounts of this protease inhibitor to spleen cell cultures. 2 x 107 spleen cells ml“1 with 0.05 ml of 1.5 percent SRBC were cultured in Marbrook vessels at 37°C for 5 days at 8% CO The control group 2. received either HSA, ECG, or an equivalent amount of medium. Hemolytic 43 plaque assays for anti-SRBC producing cells (PFC) were conducted using a modified slide method of Mishell and Dutton.13 At a concentration of 100 ug/culture, 0 -AT significantly reduced the plaque response to 49% 1 of the control. Increasing concentrations of 01-AT from 100 ug/culture to 1000 ug/culture resulted in even greater suppression of PFC responses (data not presented). -AT had no effect on plaque response in concen- o‘1 trations ranging from 1-50 ug/culture. The following are summarized control results (data not presented here). HSA, HGG or acid glycoprotein fraction (prepared according to the method of Hao gt él') served as controls and had no effect upon spleen cell culture responses to SRBC. Also, al-AT was not demonstrated to bind to antigen, and it did not lose biologic activity under described culture conditions. To exclude the possibility that a -AT suppression l was due to an effect on the antigen, SRBC were pretreated with a -AT, 1 washed and then added to the cultures. Results indicated that suppres- sion by a -AT was not due to an effect on the antigen. 1 Kinetic studies (Table 2) indicate that 48 hours exposure to 01-AT was sufficient time to produce a reduction in plaque number which decreased even further when spleen cells are exposed to a -AT for up to 1 5 days. To investigate whether immuno—suppression by al-AT was a result of interaction with the adherent cell population (phagocytic cells) of spleen, groups of cultures were incubated with either 0 -AT or an equi- 1 valent amount of culture medium. After 72 hours, non-adherent cells were separated from adherent cells by repeated washing of the monolayers. Combinations of treated and untreated adherent and non-adherent cell populations were arranged as shown in Table 3. Results of anti-SRBC 44 responses by these cultures indicate that immune-regulation by al-AT was not due to an effect on the adherent cells. To further determine the lymphoid cell type regulated by a -AT, groups of spleen cell cultures 1 were set up as before. After separating the non-adherent cells by repeated washings of the monolayer, the non-adherent cells were further separated into T-cells and B-cells, by passage over nylon-wool columns. The combination of T—cells, B-cells and adherent cells was made as described in Table 4. These studies indicate that al-AT does not affect adherent cells or Tbcells. It does, however, result in a regulation of antigen dependent B-cells responses. In conclusion, with no outward effect on cell viability, al-AT significantly lowers the immune response both in gitrg and in 2129 as measured by the number of plaques produced by spleen cells against SRBC. Immunosuppression by a -AT is not due to an effect on the antigen. 1 Furthermore, in these studies, al-AT did suppress antigen dependent B—cell differentiation without affecting adherent or T-cells. Work is presently in progress to further determine the mechanism of action of 01-AT. Of particular interest is its regulatory effect on cellular proteases. The information obtained through such studies will contribute to our understanding of the molecular event in lymphocyte maturation. In addition to providing basic insight to early events of cell differentiation, these studies may further elucidate the associa— tion between proteinase inhibitor and immune deviation and the relation- ship to various disease states. 45 We thank Dr. Y. L. Hao (Blood Research Laboratory, Methesda, MD) and Dr. Charles B. Glazer (The Institutes of Medical Sciences, San Francisco, CA) for supplying pure samples of a -AT, and Dr. Charles F. 1 Pelzer for his valuable suggestions and critical review of this manu- script. This work was supported by grant CA.13396 from the National Cancer Institute, National Institutes of Health and by an American Cancer Society Faculty Research Award (FRA-147) to HCM. 46 TABLE 1 EFFECT OF al-AT ON THE ANTI-SRBC RESPONSES OF MICE Treatment* Dose Anti-SRBC Responses** ug/mouse (PFC/spleen x 103)+ Direct Indirect ol-AT 1000 75': 4 2 i 0.28 al-AT 500 85.: 11 3‘: 0.8 ol-AT 250 96.: 5.2 9': 2.6 BSA 1000 139 i 7.4 17 :_2.4 --- --- 156 i 16 19‘: 5.5 * Each mouse also received 107 SRBC by intravenous route. **A 0.05 percent solution of agarose in minimal essential medium with Hanks base was distributed in 0.4 ml aliquots into prewarmed Wassermann tubes in 53°C. 0.05 ml of one-fourth (v/v) SRBC suspension and spleen cells (0.1 ml) were added and the mixture then spread on a slide precoated with 0.1 percent agarose. After one hour of incubation at 37°C in humid 8% C0 atmosphere, the slides were treated with 1:10dilution of guinea pig serum as a source of complement. The slides were incubated for another 2-3 hours and PFC counted. + Mean :_standard error of 4 mice. 47 TABLE 2 KINETICS OF 01-AT ON.£§ VITRO ANTI-SRBC RESPONSES* Addition Exposure Time Anti-SRBC of Culture ReSponses to al-AT (# days) (PFC/culture)** 01-AT 5 320 i 43 al-AT 4 395 i 57 al-AT 3 633 i 41 al-AT 2 841 i 71 ol—AT 1 1251 i 120 --- -—— 1229 i 96 * Spleen cell cultures of 2 x 107 cells with SRBC ** Mean :;standard error of 5 cultures 48 TABLE 3 0. -AT EFFECT ON ADHERENT AND NON-ADHERENT 1 SPLEEN CELLS FOLLOWING ANTIGEN STIMULATION Groups Anti-SRBC Responses (PFC/culture)** A* + NA 564 i 36 A + NA" 161 i 11 A* + NA* 33 i 5 A + NA 599 _+_ 50 * al-AT treatment ** Mean :_standard error of 5 cultures 49 TABLE 4 THE IMMUNOBIOLOCICAL EFFECTS OF al-AT 0N T-CELLS, B-CELLS AND ADHERENT CELLS 0F SPLEEN Groups Anti-SRBC Responses (PFC/culture)** T* + B + A 515 i 13 T + B* + A 238 i 19 T* + B + A* 380 i 28 T+B*+A* 165:9 T* + B* + A* 69': 18 T+B+A 552151 * al-AT treatment ** Mean 1 standard error of 5 cultures. Cultures in the7various groups consisted of 10 T-cells and 10 B-cells. 10. 11. 12. 13. 50 REFERENCES . Badger, A. M., Merluzzi, V. J. Mannick, J. A. and Cooperband, S. R., Immunology, 118, 1228-1231 (1977). . Chase, P. S., Cellular Immunology,_§, 544—554 (1972). . Bosmann, H. B., Nature, 249, 144-145 (1974). . Bona, C., Anteunis, A., Robineaux, R. and Halpern, B., Clin. Exptl. Immuno., 12, 377-390 (1972). . Berney, S. N. and Gesner, B. M., Immunology, 18, 681-691 (1970). . Vischer, T. L. and Bertrand, L., Agents Actions,_§, 180-182 (1976). . Koh, A., in Structure and Function of Plasma Proteins (ed. Allison, A. C.) 97-98 (Plenum Press, London and New York, 1974). Schmiege, S. K. and Miller, H. C., J. Immun., 113, 110-119 (1974). . Homer, G. M., Ketchman, B. J. and Zipf, R. E., Clin. Chem., 9, 428- 437 (1963). Mancini, M., Carbonara, A. O. and Heremans, J. F., Immunochem., 2, 235-254 (1965). Kueppers, F., J. Lab. Clin. Med., 88) 151-155 (1976). Davis, B. J., Ann. N. Y. Acad. Sci.,_lgl, 404-427 (1964). Mishell, R. I. and Dutton, R. W., J. Exp. Med., 126, 423—442 (1967). ARTICLE 2 PROTEASE INHIBITOR REGULATION OF B CELL DIFFERENTIATION1 I. The Effect of Trasylol on the Primary and Secondary Antibody Response Running head: Trasylol Suppression of B Cells 51 Prince K. Arora and Harold C. Miller2 Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 52 FOOTNOTES 1. This work was supported by grants from the National Institutes of Health (CA-13396 and AI-12549), and the American Cancer Society (IN-158). 2. H. C. M. is the recipient of an American Cancer Society Faculty Research Award (ERA-147). 3. Abbreviations used in this paper: PFC, plaque-forming cells; P.I., protease inhibitor; a -AT, 0 -Anti-trypsin; SRBC, sheep erythrocytes; l 1 FCS, fetal calf serum; MEM, minimal essential medium; PBS, phosphate- buffered saline; HSA, human serum albumin; KIU, kallikrein inactivat- ing unit. 53 INTRODUCTION Protease inhibitors (P.I.) are known to have a function to inhibit proteolytic enzymes (e.g., elastase; cathepsin—G) produced by leukocyte granules (1). Furthermore, they also inhibit proteases produced by certain bacteria during their infection (2). More recently N-a-tosyl-L- lysyl chloromethyl ketone (TLCK), a protease inhibitor, has been shown to affect an intracellular protease thought to be responsible for lympho- cyte blastogenesis (3). A similar role for Trasylol, a polyvalent enzyme inhibitor isolated from bovine lung, can be postulated to be involved in the regulation of the immune response. Trasylol (aprotinin) originally isolated by Frey (4), Kraut_gt_al. (5), and Kunitz and Northrop (6), has been widely used in the treatment of hemorrhages caused by hyperfibrinolysis and pancreatitis, where it inhibits excessive activation of certain proteinases which occur during the development of the disease. Pharmacologically, this kallikrein inactivator has been shown to dilate arteries and constrict veins (7), prevent edematous development in the inflamed area of rat paws (7) and when injected intravenously, shows binding for special organs or mem- branes (8,9). Prokopenko and Drobyazgo (10) observed that Trasylol could inhibit the formation of 7S immunoglobulins in rabbits with experimental athero— sclerosis. They proposed that Trasylol was inhibitory to the 54 55 immunostimulant factor that circulated in the blood of experimental atherosclerotic animals. Nakamura and co-workers (11) observed that Trasylol could abolish both the proteolytic activity and thymocyte helping potency of SIP [supernatent of mouse polymorphonuclear (PMN) leukocytes cultivated 3513312]. The studies of Higuchi 25 a1. (12) indicate that Trasylol, revers- ibly inhibits DNA, RNA as well as protein synthesis of lymphocytes triggered by homologous antigen, allogeneic cells or mitogens. These workers proposed that the Trasylol inhibition of lymphocyte triggering could be due to its interference with the helper action of macrophages on lymphocytes. The role of proteolytic enzymes in cellular functions has been expanded in recent years. Proteases have been found on the surface and in the medium of cultured cells (13,14,15) and inhibitors of proteolytic enzymes have been shown to affect such activities of cells as growth (16,17) and the response to plant lectins (3). While much is now known about its chemical nature (a small peptide, MW 6512), ganglia synthesis and potential therapeutic importance, the functional significance of Trasylol and the mechanisms involved in its regulation of the immune response is not understood. Since one of the major unique properties of Trasylol is its protease-inhibiting capacity, similar to that of al-antitrypsin (cl-AT) one intriguing possibility is that Trasylol also has immunoregulatory properties. During the course of our previous study (18) we proposed that al-antitrypsin, a constituent of normal human serum, has an 56 immunoregulatory junction based on demonstration of a non-cytotoxic immunosuppressive effect on the primary antibody response b0th.l£;!l££9 and in 3132 by this protease inhibitor. Furthermore, we observed al-AT suppression of antigen-dependent B-cell differentiation without affect on T-cells or adherent cells. The present study extend this concept by examining the effect of a polyvalent enzyme inhibitor Trasylol on the primary and secondary immune response to both T-dependent and T-independent antigens. Further experiments have been done to investigate if immunoregulation by Trasylol is due to its protease inhibiting capacity, and finally to determine its mode of action. MATERIALS AND METHODS Animals: C57BL/10 x C3H/He (BCFl) female mice were from Cumberland View Farms, Clinton, Tennessee. CELLS FOR CULTURE AND ASSAY: Spleen cells were obtained aseptical- ly from 9 to l6-weekrold C3H x C57BL F female mice (Cumberland View 1 Farms, Tenn.). Cell suspensions were prepared by gentle aspiration with a syringe and needles of progressively increasing gauge (21 to 27) to obtain a single cell suspension. Spleen cells were washed once and resuspended in medium CMRL 1066 (Grand Island Biological Co., Grand Island, NY) supplemented with 15% fetal calf serum (FCS) (Grand Island Biological Co.), 0.15 mM L-asparagine, 2 mM L-glutamine, 1 mM sodium pyruvate and 50 mg/l gentamycin. Spleen cells were cultured in 35 mm tissue culture dishes (Falcon Plastics, Division of BioQuest, Oxnard,CA). 57 Viability of cells was determined by trypan blue exclusion in all experiments. TRASYLOL: Trypsin-Kallikrein Inhibitor (aprotinin) (FBA Pharma- ceuticals, New York, NY) was diluted in spleen cell medium (pH 7.2-7.4) to obtain a desired concentration/culture. TRYPSIN: 2x crystallized, dialyzed and lyOphilized (Sigma Chemi- cal Co., St. Louis, MO 63178) was also dissolved in spleen cell medium to obtain desired concentration. ANTIGENS: Sheep erythrocytes (SRBC) were obtained from a single animal (Lot #8227121, Grand Island Biological Co., Grand Island, NY) and were stored in Alsever solution. Before use, the SRBC were washed three times in sterile phosphate buffered saline (PBS) and suspended to l x 109 cells/ml in spleen cell culture medium. Clinical grade dextran with an average molecular weight of 2 x 106 (Sigma Chemical Co., St. Louis, MO 63178) was dissolved in pyrogen-free PBS at 20 mg/ml, filtered (Millipore 0.22 mM), stored at -20°C, and thereafter thawed for dilution in spleen cell medium before use. Dextran was added to the cultures in concentrations and at times shown in tables. Dextran, in a wide range of doses, was neither mitogenic nor cytotoxic as evaluated by viable cell counts. COUPLING 0F DEXTRAN T0 SRBC: For dextran coupling SRBC were washed three times in PBS, pH 7.4, and resuspended to a 50% cell suspen- sion in the phosphate buffered saline. Two mililiters of dextran solu- tion containing 10 mg/ml were added to 2 ml of 50% SRBC as reported by Ghanta gt a1. (19). After incubation with stirring at 37°C the dextran- coupled SRBC (Dex-SRBC) were washed three times with PBS to remove any 58 unreacted dextran. The cells were resuSpended in MEM to a final concen- tration of 1 x 109/ml, and stored at 4°C. Dextran-SRBC were prepared fresh a few hours before their use. THEMECTOMY: Fourdweek-old mice were thymectomized, according to the methods of Miller (20). They were allowed to rest for at least one month prior to irradiation. When thymectomized mice were sacrificed, the mediastinum.was examined microscopically for the presence of thymus remnants. No such remnants were found. IRRADIAIYON: Ten- to 12-weekrold mice received 900 Rads of whole body irradiation from the 6oGOV-irradiation source in the Department of Food Science at Michigan State University. The animals were rested for at least 4 hr before transplantation. CeZZ suspensions: Bone marrow cells from tibias and femurs of 10- to lZ-week—old normal BCFl mice were gently aspirated in Eagles minimum essential medium (MEM) in Hanks' salts with a syringe and a 25 gauge needle. Cells were then passed through a 27 gauge needle to produce a dispersed cell suspension. The cell suspension was washed once by centrifugation (170 g) in MEM and was then diluted to the appropriate 6 concentration to be injected. Each mouse received 1 x 10 bone marrow cells. Steps for Separation of‘CeZZs Macrophages: Spleen cells of normal mice were placed in tissue culture petri dishes (Falcon Plastics, Division of BioQuest, Oxnard,CA). After 24, 48 or 72 hours of incubation at 37°C, the non-adherent cells were separated from the adherent cells by repeated washing with fresh 59 spleen cell medium. The adherent cells were used as a source of macro- phages. T—Cells: One column was used for every 2 x 108 non-adherent cells. Before use, the conditioned (soaked in MEM + FCS, 37°C, 1 hr) nylon wool column was washed with 20 ml MEM + FCS. Resuspended non- adherent cells were poured over the nylon wool column, and then incu- bated for 45 min at 37°C. T-cells from the column were recovered by dropwise adding of 25 m1 MEM + FCS. B-Cells: After recovery of T-cells, the above column was washed with 50 ml of MEM + FCS and then pressed and squeezed to recover B-cells. This process of washing and squeezing the nylondwool column was repeated a number of times to recover most of the B-cells. The B-cell preparation was further purified by treatment with mouse anti-theta serum and complement. In order to determine which cell type is regulated by Trasylol, individual cell populations were pre-incubated with known amounts of Trasylol. An assay system containing no Trasylol was reconstituted with each cell population pre-exposed to Trasylol. Any change in the plaque response in comparison to the control group was recorded. HOMCLYTIC PLAQUE FORMING CELL (PFC) ASSAY: a. Primary_Ig.yi£rg_Antibody Synthesis: Primary antibody synthesis to sheep erythrocytes (SRBC) was measured using the method described by Mishell-Dutton (21). Spleen cell suspensions of 2 x 107 viable cells (viabilities greater than 85%) in one ml of spleen cell culture medium and 0.05 ml of SRBC (1.5%) were 60 cultured for 4 days in 35 mm tissue culture dishes (Falcon Plastics, Division of BioQuest, Oxnard, CA) with rocking. Cultures were set up in a total volume of 1.15 ml in the following order: a. 0.05 ml of anti- gen, b. 2 x 107 spleen cells in 1.0 ml of medium, and c. Trasylol to be tested for immunosuppressive activity was added in 0.1 m1 at the begin- ning of culture unless otherwise stated. After the spleen cell cultures were incubated for 4 days at 37°C in a humid 8% C0 atmosphere, the 2 cells from the tissue culture dishes were removed by gentle aspiration with a Pasteur pipette. At the time of assay, viabilities and cell concentrations in each experimental group were measured. There were an average of 2-3 x 106 viable spleen cells remaining in each group after 4 days of cultivation. No discernible difference in viability or con- centration was detected when spleen cells were cultured with or without Trasylol. Suspensions from these spleen cell cultures (0.1 ml) were assayed by the Jerne hemolytic plaque method as modified for use with agarose gel on glass microscopic slides. Details of this procedure are described elsewhere (22). The localized hemolysis-in-gel technique used was to detect and enumerate cellular synthesis of YM antibody [direct plaque forming cells (PFC)]. The antibody responses are expressed as mean PFC per culture i;SE (standard error of the mean). Each preparation was cultured as five cultures per group. b. Secondary In Vigrg Antibody Synthesis: Secondary synthesis to SRBC was measured also by an adaptation of the Mishell-Dutton culture system (21). Groups of mice were injected 61 with 107 SRBC/mouse through their tail vein on day zero. After 14, 21 and 35 days, the primed spleen cells were cultured with and without the antigen as described in the previous section. Trasylol to be tested for immuno-suppressive activity was added in 0.1 ml at the beginning of culture unless otherwise stated. After the 4 day culture period in an atmosphere of 8% 002, at 37°C, the cells were removed from the tissue culture dishes by gentle aspiration with a Pasteur pipette. At the time of assay, viabilities and cell concentrations in each experimental group were measured. No discernible difference in viability or concen- tration was detected when spleen cells were cultured with or without Trasylol. Suspensions from these spleen cell cultures (0.1 ml) were assayed using the method described in the previous section. The anti- body responses are expressed as mean PFC per culture : S.E. Each prep- aration was cultured as five cultures per group. RESULTS Suppression of'the IE_ZZE§9 Antibody Response by Trasylol: In order to study the effect of Trasylol on the immune response, mouse spleen cells were cultured in tissue culture dishes with and without the antigen. Trasylol was added to spleen cell cultures at several concen- trations. The plaque response of spleen cells against SRBC was examined after 4 days of incubation. Data shown in Table 1 indicates that when Trasylol was added at a concentration of 100 KIU/culture, the plaque response was reduced to nearly 50% compared with controls consisting of cultures to which normal human serum albumin (HSA) was added in similar 62 concentrations. The dose-response to Trasylol shown in Table l demon- strates that increasing the concentration of Trasylol from 250 to 2,000 KIU/culture reduced the PFC number to a significant degree when compared with the control group. Effect of'Pre—incubation of'SpZeen Cells with Trasylol on the Primary Antibody Response: 2 x 107 spleen cells in a volume of 1 ml were pre-incubated with 0.1 ml of 500 and 1,000 KIU of Trasylol for 3 and 6 hr. Cultures were then given repeated washings before exposure to the antigen. The primary PFC response was measured 4 days after addi- tion of antigen. There was no measurable change in viability of control and treated cultures and no suppression evident in any of the cultures (data not shown) indicating that pre-incubation of spleen cells with Trasylol in cultures does not result in suppression of the primary PFC response. Effect of'Eacess Antigen on suppression of'Primary Antibody Response by Trasylol: To determine if suppression of PFC response by Trasylol is due to some indirect effect on the antigen, the concentration of the antigen was increased from 1.5% to nearly 2.5% of SRBC. As noted in Figure 1, increase in concentration of the antigen (from 1.5 to 2.5%) in the culture system did not have any effect on the suppressive ability of Trasylol (63-180 PFC/culture), thus indicating that immune-suppression by Trasylol observed (69 i 14 PFC/culture) in the system is not due to its effect on the antigen. Dose-response Effect of Trasylol on Memory: Experiments were done to determine.whether Trasylol would exert suppressive effects on the 63 secondary antibody response. For this purpose, groups of mice were primed with 107 SRBC/mouse on day zero. After 14, 21 and 35 days of priming spleen cells were collected and cultured with SRBC and in the presence or absence of Trasylol (final concentrations ranging from 500 to 2,000 KIU/culture). As seen in Table 2 when SRBC-primed spleen cells were exposed to the same sensitizing antigen, there was a high response of plaque forming cells (>8,000 PFC/culture). Addition of Trasylol (500 KIU/culture) decreased the secondary plaque response to about 6,000 PFC/culture. Suppression of PFC response was even more profound if concentration of Trasylol/culture was raised to either 1,000 or 2,000 KIU. Thus, Trasylol significantly reduced (P cums : m you pmumnaocfiuona Hofizmmuy an .UmMm Nm.~ «0 AB no.0 Magoo mod x N mo mou3uH30 Haoo comaam m A m: ma H sees me A mmwa mNH H Kama ems N H ms o: A mmw mm A ommfi ems A Esau OOH H A m as H Hm” mm H mmsfi ”ma A oaks om NH H as NH A_Nm om A Kmm mos A mass - OOON coca com - ma=t3=c\snv zama>me 4AMEDEJDU\um¢v Ammskssu\=_¥v mmmzoammm ummmuipz< 4o4>mm3. q mam