Protease inhibitor regulation of B-cell differentiation

CELLULAR

IMMUNOLOGY

Protease

60,

155-167 (1981)

Inhibitor

Regulation

of B-Cell Differentiation’

P. K. ARORA,’ H. C. MILLER,~ AND L. D. ARONSON Departments of Microbiology

and Public Health and Medicine. Michigan State University. East Lansing, Michigan 48824

Received September 9. 1980; accepted October 8. 1980 Trasylol (aprotinin), also referred to as kallikrein inactivator, is known to bind and inactivate a variety of proteases, such as trypsin, chymotrypsin, cathepsin, etc., and was used in the present study as a probe for studying protease regulation of lymphoid differentiation. At concentrations of 100 to 2000 kallikrein inactivating units (KIU) per culture, this inhibitor suppressed both the primary and secondary plaque-forming cell (PFC) response of mouse spleen cells to SRBC in vitro. This suppression was not antigen specific and blocked T-independent responses as well. Suppression by Trasylol was not due to depletion effect on the antigen and its inhibitory capacity was reversible. The degree of suppression was dependent on the time of addition of Trasylol to the cultures; i.e., Trasylol added to antigen-stimulated cultures up to 48 hr after initiation of cultures was immunosuppressive whereas at 72 hr after initiation or later it did not suppress. Pretreatment of spleen cells with this inhibitor for approximately 6 hr before exposure to the antigen did not affect the immune response. When preincubated with trypsin, the suppressive activity of Trasylol was abrogated. Trasylol did not affect T helper cells or adherent cells, but it selectively suppressed the B-cell differentiation. These results suggest that protease inhibitors play an important role in immunoregulation.

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 (1, 2, 3). Inhibitors of proteolytic enzymes have been shown to affect such activities of cells as growth (4, 5) and the response to plant lectins (6). Protease inhibitors are abundant in normal serum and one of their roles is to provide immunologic stasis (7). We have proposed that a,-antitrypsin (LU,-AT)~, one of the major protein constituents of normal human serum, has an immunoregulatory function. A noncytotoxic immunosuppressive effect by this protease inhibitor was demonstrated on the primary antibody response both in vitro and in vivo. Furthermore, we observed q-AT suppression of antigen-dependent B-cell differentiation without ’ This work was supported by grants from the National Institutes of Health (CA-13396) the American Cancer Society (CD-SlA), and Children’s Leukemia Foundation of Michigan (ORD-21195). 2 Present address: Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205. 3 Recipient of an American Cancer Society Faculty Research Award (FRA-147). 4 Abbreviations used: PFC, plaque-forming cells; PI, protease inhibitor; at-AT, or-antitrypsin; SRBC, sheep erythrocytes; FCS, fetal calf serum; MEM, minimal essential medium; PBS, phosphate-buffered saline; HSA, human serum albumin; KIU, kallikrein inactivating unit. 155 0008-8749/81/070155-13$02.00/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.

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altering nonactivated T cells or adherent cells. Recently an a-globulin rich fraction (Cohn Fraction IV, immune regulatory a-globulin, IRA) has also been implicated in suppressing the in vitro antibody response of mouse spleen cells to sheep red cells without cytotoxicity (8). Chase (9) found that crl-macroglobulin, another protease inhibitor in normal serum, limits the human lymphocyte response to phytohemagglutinin and concanavalin A. N-cr-tosyl+lysl chloromethyl ketone (TLCK), a synthetic protease inhibitor, has been shown to react with an intracellular protease thought to be responsible for lymphocyte blastogenesis (6). A similar role for Trasylol, a polyvalent enzyme inhibitor isolated from bovine pancreas can be postulated to regulate the immune response. Trasylol (aprotinin), originally isolated by Frey (lo), Kraut et al. ( 1 1), and Kunitz and Northrop (12), has been used in the treatment of hemorrhages caused by hyperfibrinolysis and pancreatitis, where it inhibits excessive activation of certain proteases which occur during the development of the disease. Prokopenko and Drobyazgo (13) observed that Trasylol could inhibit the formation of 7 S immunoglobulins in rabbits with experimental atherosclerosis. They proposed that Trasylol was inhibitory to the immunostimulant factor that circulated in the blood of experimental atherosclerotic animals. Nakamura and co-workers (14) observed that Trasylol could abolish both the proteolytic activity and thymocyte-helping potency of supernatant of mouse polymorphonuclear (PMN) leukocytes cultivated in vitro. The studies of Higuchi et al. ( 15) indicate that Trasylol reversibly inhibits antigen, alloantigen, or mitogen-triggered synthesis of DNA, RNA, and protein in lymphocytes. These workers proposed that the Trasylol inhibition of lymphocyte triggering could be due to its interference with the helper action of macrophages on lymphocytes. Reduction in lymphocyte stimulation was also observed for PHA, Con A, LPS, and dextran sulphate responses when Trasylol was added (16). While much is now known about the chemical nature (a small peptide, MW 6512), in vivo synthesis and potential therapeutic importance of Trasylol, the mechanisms involved in its regulation of the immune response and functional significance are not understood. Since it has a protease-inhibiting capacity similar to that of cqantitrypsin (a,-AT), one intriguing possibility is that Trasylol also has immunoregulatory properties. The present studies make use of this polyvalent enzyme inhibitor to probe the effects on primary and secondary immune responses to both T-dependent and Tindependent antigens. Experiments:have been designed to determine the mechanism and target cell of Trasylol-mediated immunologic stasis. MATERIALS

AND

METHODS

Animals C57BL/lO X C3H/He (BCFI) female mice were obtained Views Farms, Clinton, Tennessee. Cells for Culture

from Cumberland

and Assay

Spleen cells were obtained aseptically from 9 to 16-week-old BCF, mice. 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

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cells were washed once and resuspended in medium CMRL 1066 (Grand Island Biological Co., Grand Island, N.Y.) 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/liter gentamicin. Spleen cells were cultured (7) in 35-mm tissue culture dishes (Falcon Plastics, Division of BioQuest, Oxnard, Calif.). Viability of cells was determined by trypan blue exclusion in all experiments. Biologicals

Trasylol (aprotinin), the trypsin and kallikrein inhibitor (FBA-Bayer Pharmaceuticals, New York, N.Y.), obtained in lyophylized form and free of benzyl alcohol, was diluted in spleen cell medium (pH 7.2-7.4) to obtain a desired concentration per culture. Trasylol containing 10,000 KIU/ml was obtained from FBA Pharmaceuticals, Mobay Chemical Corporation, New York, New York. Thin-layer horizontal SDS-gel electrophoresis demonstrated only one zone. In addition, one protein peak was eluted from a Sephadex G-50 column, as determined at OD 280. Trypsin inhibitory capacity (17) demonstrated the material was capable of inhibiting 0.80 mol trypsin per mole of Trasylol. Thus the material was homogenous and 80% active as a proteolytic enzyme inhibitor. Trypsin, 2~ crystallized, dialyzed, and lyophilized (Sigma Chemical Co., St. Louis, MO.) was also dissolved in spleen cell medium to obtain the desired concentration. It was assayed using the method of Chase and Shaw (18). Antigens

Sheep erythrocytes (SRBC) were obtained from a single animal (Lot 8227121, Grand Island Biological Co.) and were stored in Alsever solution. Before use, the SRBC were washed three times in sterile phosphate-buffered saline (PBS) and suspended to 1 X lo9 cells/ml in spleen cell culture medium. Clinical grade dextran with an average molecular weight of 2 X lo6 (Sigma Chemical Co.) 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 of Dextran to SRBC

For dextran coupling, SRBC were washed three times in PBS, pH 7.4, and resuspended to a 50% cell suspension in PBS. Two milliliters of dextran solution containing 10 mg/ml were added to 2 ml of 50% SRBC as reported by Ghanta et al. (19). After incubation during stirring at 37°C the dextran-coupled SRBC (Dex-SRBC) were washed three times with PBS to remove any unreacted dextran. The cells were resuspended in Eagle’s minimal essential medium (MEM) with Hanks’ salts to a final concentration of 1 X 109/ml, and stored at 4°C. DextranSRBC were always prepared fresh a few hours before their use. Thymectomy

Four-week-old mice were thymectomized, according to the methods of Miller (20). They were allowed to rest for at least 1 month prior to irradiation. When

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thymectomized mice were sacrificed, the mediastinum was examined macroscopically for the presence of thymus remnants. No such remnants were found. Irradiation

Ten- to twelve-week-old mice received 900 rad of whole body irradiation the “Co y-irradiation source in the Department of Food Science at Michigan University. The animals were rested for at least 4 hr before transplantation.

from State

Cell Enrichment Bone marrow-derived B cells transplantation. Bone marrow cells from tibias and femurs of lo- to 12-week-old normal BCF, mice were gently aspirated in MEM 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 in MEM and was then diluted to the appropriate concentration to be injected. Each mouse received 5 X lo6 bone marrow cells that were pretreated with a-Thy-l.2 antiserum and fresh guinea pig serum to kill residual T cells. Splenic macrophages. Spleen cells of normal mice were placed in tissue culture petri dishes (Falcon Plastics). After 24, 48, or 72 hr of incubation at 37”C, the nonadherent cells were separated from the adherent cells by repeated washing with fresh spleen cell medium. The adherent cells were used as a source of macrophages. Splenic T-cells and B-cells. A nylon wool column (soaked in MEM + FCS, 37”C, 1 hr) was washed with 20 ml MEM + FCS. One column was used for every 2 X 10’ nonadherent cells. Resuspended nonadherent cells were poured over the column, and then incubated for 45 min at 37°C. T cells from the column were recovered by dropwise additions of 25 ml MEM + FCS [details have been published elsewhere (21)]. 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 was repeated three to five times to recover maximum yields of the B cells. The Bcell preparation was further purified by treatment with mouse anti-Thy-l.2 serum and complement. Detection of the cell type regulated by Trasylol was studied using individual cell populations preincubated with predetermined amounts of Trasylol. An assay system was set up containing cell populations (untreated with Trasylol) which were reconstituted with the respective cell population which was preexposed to Trasylol. The effects were monitored as changes in the plaque response compared to the untreated control group. Homolytic

Plaque-Forming

Cell (PFC) Assay

Primary antibody responses to sheep erythrocytes (SRBC) were measured using the method described by Mishell and Dutton (22). Viable spleen cells (2 X 10’) in 1 ml of culture medium and 0.05 ml of SRBC (1.5%) were cultured for 4 days in 35-mm tissue culture dishes (Falcon Plastics) with rocking. Trasylol was tested for immunosuppressive activity by adding 0.1 ml when setting up cell cultures unless otherwise stated. The spleen cell cultures were incubated for 4 days at 37°C in a humid 8% CO2 atmosphere. No difference in viability or concentration could be detected with spleen cells cultured in the presence or absence of Trasylol. Sus-

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pensions of the spleen cell cultures (0.1 ml) were assayed using the Jerne hemolytic plaque method modified for use with agarose gel on glass microscopic slides (23). The antibody responses have been expressed as mean PFC per culture f SE (standard error of the mean). Each determination represents the mean of at least five cultures per group. Secondary synthesis to SRBC was studied using groups of mice injected via tail vein with 10’ SRBC/mouse on Day 0. After 14, 21, and 35 days, the primed spleen cells were cultured in the presence or absence of antigen as described in the previous section. Trasylol was added in 0.1 ml at the beginning of culture unless otherwise stated. After 4 days of incubation in an atmosphere of 8% CO*, at 37”C, suspensions from these spleen cell cultures (0.1 ml) were assayed using the method described in the previous section. The antibody responses are expressed as mean PFC per culture -t SE. Each preparation was cultured as five cultures per group. RESULTS Suppression

of the in Vitro 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 concentrations. Plaque responses of spleen cells against SRBC were examined after 4 days of incubation. Data shown in Table 1 indicate that when Trasylol was added at a concentration of 100 kallikrein inactivating units (KIU)/culture, the plaque response was reduced to nearly 50% when compared with controls consisting of cultures to which normal human serum albumin (HSA) was added in similar concentrations. The dose response to Trasylol shown in Table 1 indicates that increasing the concentration of Trasylol from 250 to 2000 KIU/culture reduced the PFC number to a significant degree when compared with the control group. Effect of Preincubation Antibody Response

of Spleen Cells with Trasylol

on the Primary

Spleen cells (2 X 10’) in l-ml aliquots were preincubated with 500 and 1000 KIU of Trasylol for 3 and 6 hr. Cultures were then given repeated washings before TABLE

1

Dose-Response Effect of Trasylol on the In Vitro Primary Antibody Response Trasylol (KIU/culture)” 2000 1000 500 250 100 HSA (1000 pg) -

Anti-SRBC responses (PFC/culture)b 20* 19+ 191 + 166 + 274 + 607 f 579 f

y Spleen cell cultures of 2 X 10’ cells/ml of medium with of 1.5% SRBC. b Mean + SE of five cultures per group.

4 4 10 57 12 55 46

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exposure to the antigen. The primary PFC response was measured 4 days after addition of antigen. There was no detectable change in viability of control and treated cultures and no suppression evident in any of the controls (data not shown) indicating that preincubation of spleen cells with Trasylol did not result in suppression of the primary PFC response. To determine if suppression of PFC response by Trasylol was due to some indirect effect on the antigen, the concentration of the antigen was increased from 1.5% to nearly 2.5% SRBC. Any increase in the antigen concentration did not effect the suppressive ability of Trasylol (data not shown), thus indicating that immunosuppression by Trasylol observed in the system was not due to interaction with the antigen. Dose-Response

Effect of Trasylol

on Memory

Experiments were devised to determine whether Trasylol would exert suppressive effects on the secondary antibody response. Groups of mice were primed with 10’ SRBC/mouse on Day 0. After 14, 21, and 35 days spleen cells were collected and cultured with SRBC in the presence or absence of Trasylol. When SRBC-primed spleen cells were exposed to the sensitizing antigen (Table 2), there was an elevated antibody-forming cell response (~8000 PFC/culture). Addition of Trasylol (500 KIU/culture) decreased the secondary response to about 5000 PFC/culture. Suppression of PFC response was even more profound if concentration of Trasylol/ culture was raised to either 1000 or 2000 KIU. Thus, Trasylol significantly reduced (P < 0.05) the secondary plaque response in a dose-dependent manner. Indirect plaque responses (not shown here) were suppressed in similar fashion. Kinetics of the Effect of Trasylol

on Spleen Cells following

Exposure to Antigen

The suppressive effect of Trasylol was examined during early as well as late stages of the immune response. The culture system (i.e., 2 X 10’ spleen cells + SRBC) was incubated with Trasylol in a final concentration of 1000 KIU/culture and Trasylol was removed at various intervals ranging from 6 to 96 hr later. The results of one of these kinetics experiments are presented in Fig. 1. The data indicate TABLE

2

Trasylol Suppression of Memory Response Anti-SRBC Trasylol (KIU/culture) 2000

1000 500

No antigen

14 Days 2666 5890 6234 8228 671

+ + & + f

504 457 846 561

77

responsesb (PFC/culture)’ 21 Days 2232 5186 5920 8753 521

k zk f + f

289 133 204 530 54

35 2421 4439 6300 8166 443

Days + + k f 2

372 163 139 437 667

a Spleen cell cultures of 2 X 10’ cells/ml of medium with 0.05 ml of 1.5% SRBC. b Spleen cells from mice primed intravenously with 1O’SRBC per mouse 14,21, and 35 days previously. ‘Mean + SE of five cultures per group.

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OF B CELLS

TRASYLOL

(-)TRASYLOL

t

6

11 TIME

IL !4 46

II 72

OF EXPOSURE

% (HRS)

FIG. 1. Kinetics of Trasylol suppression on the immune response. Spleen cell cultures of 2 X 10’ cells/ ml and 0.05 ml of 1.5% SRBC. Trasylol was removed from the cultures at varying intervals during the incubation period. Cells were washed three times with fresh medium before being added back into the cultures. Data are expressed as means + standard error of five cultures per group. This represents data from one of three similar experiments.

a sharp decrease in PFC response between 18 and 24 hr after antigenic stimulation. The number of PFC/culture up to 18 hr of exposure to Trasylol was approximately 500 and comparable to that of the control group (600 PFC/culture). At 24 hr, however, there was a sharp decline (112 PFC/culture, P < 0.05). Suppression of PFC numbers was more pronounced when spleen cell cultures were exposed to Trasylol for 2 to 4 days (P < 0.01). In a similar fashion, Trasylol (1000 KIU/ culture) was added at various intervals ranging from 0 to 96 hr after initiation of spleen cell cultures. Results indicated a marked suppression in the PFC response if Trasylol was added any time before 72 hr prior to initiation of cultures (data not presented). However, no suppression of PFC response occurred when Trasylol was added between 72 and 96 hr. In another study following 24 hr of incubation, Trasylol was washed from the culture system and PFC response of pretreated cultures was determined on 5, 6, and 7 days (more-prolonged incubation periods than usual). Once Trasylol was removed from the system, immunosuppression was reversed after 24 hr and the response was completely restored to normal levels after another 72 hr (data not presented). Effect of Trasylol

on the Primary

Anti-Dextran

Antibody

Response in Vitro

When the T-independent antigen dextran (MW 2 X lo6 daltons) was tested, Trasylol, at a concentration below 100 KIU/culture, did not produce any change in the primary anti-dextran PFC response. However, at a concentration of 250 KIU/culture Trasylol suppressed the PFC response to nearly 50% of the control

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cultures. With 500 KIU of Trasylol/culture, there were approximately 650 antidextran PFC/culture, compared to the control group with 1606 f 121 PFC/culture. This suppression of primary anti-dextran PFC response was even greater (410 k 48 PFC/culture) when higher concentrations of Trasylol(lOO0 KIU) were used per culture (Table 3). We determined that the effect of Trasylol was directed primarily to those cells synthesizing anti-dextran antibodies by exposing spleen cells (from thymectomized, X-irradiated, and bone marrow-reconstituted mice) to various concentrations of Trasylol in vitro. As presented in Table 3, an enriched B-cell population devoid of Thy-l antigen-bearing cells, responded less well to dextran, than did whole spleen cells. All concentrations greater than 100 KIU of Trasylol culture suppressed the PFC response of spleen cells to dextran. Kinetics of the Trypsin Neutralization

of Immunosuppression

by Trasylol

Trasylol (500, 1000 and 2000 KIU) was preincubated with different concentrations of Trypsin for 3 hr. After this time, the Trasylol-trypsin mixture was added to spleen cell cultures. Immunosuppression of PFC response by 500 and 1000 KIU of Trasylol was abrogated partially by 50 and 100 pg of trypsin and completely by 150 pg of trypsin (data not shown). Even 150 pg (6 mM) of trypsin did not neutralize the effect of 2000 KIU (42 mM) of Trasylol. Trypsin alone in the cultures did not alter PFC responses. Effect of Trasylol

on Select Ceil Populations

It was next important to determine the cell population influenced by the protease inhibitor. Adherent or nonadherent cells were studied by incubation of groups of cultures with either Trasylol (1000 KIU/culture) or an equivalent amount of culture medium. After 24, 48, or 72 hr of incubation, the nonadherent cells were separated from the adherent cells by repeated washing of the monolayers. Combinations of Trasylol-treated and untreated adherent and nonadherent cell popuTABLE Trasylol Regulation of the Anti-Dextran

3

PFC Response of Normal and B-Cell Spleen Anti-dextran

Trasylol (KIU/culture)” 2000 1000 500 250 100 No dextran

responses (PFC/culture)b

Normal spleen

B-Cell spleen’

I* 4 410 + 48 649 f 35 951 2 51 1329 + 86 1606 f I21 9 23+

18f 9 109 2 16 194 f II 534 * 40 744 + 25 824 51 47 ot0

’ Spleen cell cultures of 2 X 10’ cells/ml of medium with 0.05 ml of 1.5% SRBC. b Mean + SE of five cultures per group. ’ Spleen cells from mice previously thymectomized, X irradiated, and reconstituted with 5 X IO6 bone marrow cells.

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lations were arranged as shown in Table 4. These results indicate that when adherent cells were exposed to Trasylol for 24 to 72 hr, there was no suppression of PFC response (approximately 700 PFC/culture) compared with the control groups which went through the similar technical steps of cell separation. When only the nonadherent cells in the culture system were exposed to Trasylol for 24, 48, or 72 hr, the PFC response was suppressed to a significant degree (P < 0.01) compared to the control group (684 * PFC/culture). Suppression of plaque numbers was likewise observed ( 124 + 19 PFC/culture) when both cell populations were exposed to Trasylol and processed through similar steps of cell separation. Effect of Trasylol on T Cells, B Cells, and Adherent Antigen Stimulation

Cells of Spleen following

The lymphoid cell type affected by Trasylol was determined with purified T- and B-cell populations. Groups of spleen cell cultures were selected as adherent and nonadherent cells as before. The nonadherent cells were further separated into T cells and B cells by passage over glass and nylon-wool columns. The combinations of T cells and B cells and adherent cells (A cells) were made as outlined in Table 5. Data suggest that when T cells alone, or T cells + A cells (in the culture system T cells + B cells + A cells) were exposed to Trasylol (1000 KIU/culture) for 24, 48, or 72 hr there was no suppression of the PFC response. When both B cells and A cells in the culture system were exposed to Trasylol for 24, 48, or 72 hr, the plaque number/culture was significantly reduced (P < 0.01). Furthermore, when only B cells were exposed for either 24,48, or 72 hr, there was significant suppression of the PFC response (P < 0.01). These results clearly demonstrate that suppression, brought about by Trasylol, was focused on regulation of antigen-dependent B-cell responses. DISCUSSION Our earlier observations (7) indicated that a,-antitrypsin ((r,-AT) a major protease inhibitor present in the normal serum, played an important role in the regulation of the immune response. Due to difficulties in preparing large amounts of TABLE

4

Trasylol Effect on Adherent and Nonadherent Spleen Cells following Antigen Stimulation Anti-SRBC Groups A* A A* A

+ NAb +NA* + NA* +NA

responses (PFC/cultures)”

24 hr

48 hr

712 + 73 120 + 19 ND ND

702 k 20 167 f 18 ND ND

12 hr 649 125 124 684

+ f f +

40 31 19 38

Note. *, Trasylol treatment. a Mean f SE of five cultures per group. bNonadherent cells were separated from adherent cells by repeated washing of cultures in tissue culture dishes with fresh medium.

164

ARORA, MILLER, TABLE Immunobiological

AND ARONSON 5

Effects of Trasylol on T Cells, B Cells, and Adherent Cells of Spleen Anti-SRBC

Groupsb T* + B +A* T + B* +A* T*+B +A T +B*+A T* + B* + A* T +B +A -

24 hr 162 I? 27 ic 838 f 28+

responses (PFC/culture)” 48 hr

36 IO 15 5

677 + 15 25+ 8 688 f 24 27 + 10

12 hr 692 k 21+ 722 * 26k 32k 809 f 796 +

33 5 41 2 4 26 40

Note. *, Trasylol treatment. ’ Mean + SE of five cultures per group. ’ Cultures in the various groups consisted of IO’ T cells and 10’ B cells.

pure and murine-active al-AT, Trasylol has been used in the present studies as a model for determining various parameters of immunoregulation brought about by protease inhibitors. Trasylol has a defined chemical composition and its mechanism of interaction with proteases is better understood than that for other protease inhibitors. The present investigation represents the first in which this protease inhibitor has been examined for its effect on the immune response. Trasylol has been found to be immunosuppressive on both the primary and the secondary antibody production. Equivalent amounts of medium or normal human serum albumin (HSA), at even higher concentrations, did not suppress the PFC response. Dose-response studies showed that suppression with Trasylol occurred at concentrations greater than 100 KIU/culture which is equivalent to 14 pg/ml or 2.1 PM/liter. This amount represents about fourfold less than that found for cY,-antitrypsin (500 pg/ml or 9.2 PM/liter) (7). Maximal suppression of the 4-day in vitro response to SRBC was achieved by the addition of Trasylol to a final concentration of 2000 KIU/culture. Exposure of the murine splenic cells to suppressive concentrations of Trasylol for nearly 6 hr before addition of the antigen did not alter their responsiveness. Washing the cells two to three times before the addition of antigen was sufficient to remove all suppressive activity (unpublished observation). Presently studies are under way to determine whether prolonged treatment with Trasylol alters surface membrane receptors for antigenic recognition or inhibits intracellular processes required for antibody synthesis. Results presented indicate that the immunosuppressive activity of Trasylol could not be reversed by incubating the spleen cell cultures with surplus of antigen, suggesting that immunosuppression was not due to direct interaction or depletion of the antigen. It is possible that Trasylol may inhibit the binding of antigen either by direct association with the antigen receptor or by association with a receptor of its own that somehow prevents the union of antigen with cell surface antibody receptor. Preincubation of spleen cells or the antigen with suppressive concentrations of Trasylol, before addition into the culture system, has ruled out the above possibility since no suppression resulted in either

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case. Also, due to its small molecular size, it seems unlikely that Trasylol would sterically block the union of antigen with its receptor. Several other findings bear particular importance on understanding the mechanism of Trasylol regulation of the immune response. A sharp decline of the PFC response occurred after 18 to 24 hr when Trasylol was added to spleen cell cultures from 0 to 96 hr. Shorter exposures (6 to 12 hr) resulted in little or no suppression, indicating that the mechanism(s) involved in Trasylol-induced suppression was more complex than simply blocking Ig receptors and prevention of antigenic stimulation. When Trasylol was added to cultures later than 72 hr after immunization, no effect on the immune response occurred. This suggests that the synthetic capacity of the antibody-secreting plasma cell was not affected by Trasylol. However, the early phases of antibody formation such as antigen processing, B-T cell interactions, and clonal proliferation of antigen-sensitive B cells would likely be susceptible to the Trasylol-induced suppression. Furthermore, Trasylol addition to spleen cell cultures at various time periods revealed its ability to interfere with an early event in lymphocyte stimulation (data unpublished). The maximal time for inhibition by Trasylol (18-24 hr) in antigen-stimulated spleen cell cultures, coincides well with findings of Moreau et al. (24) who demonstrated that the blastogenesis of human lymphocytes was restricted by protease inhibitors such as soybean trypsin inhibitor (SBI) and tosyl-L-lysine chloromethyl ketone (TLCK) which had been added only in an early phase of cultivation. We have also demonstrated that this immunosuppression can be progressively reversed by removal of Trasylol from the culture system, completely returning to- normal responsiveness 4 days later (unpublished results). A cytotoxic effect of this agent on lymphocytes was ruled out since antibody synthesis by antigen-stimulated spleen cells was not inhibited by 24 hr pretreatment with Trasylol. Also, cell viabilities were unaltered. When we began this work, controls which we treated with benzyl alcohol in PBS (the preservative and vehicle for commercial Trasylol) were found to be suppressed in PFC or mitogen responses. Viabilities were also 50% lower. It is therefore important that future attempts to use Trasylol be carried out only with the lyophilized material. Development of secondary responses was investigated to determine if this later stage was also affected. Trasylol significantly suppressed immunocompetence of splenic lymphocytes in which memory was induced. When SRBC-primed spleen cells were exposed to the same sensitizing antigen, there was a higher secondary PFC response (15-fold higher) compared to the primary in vitro response. Nearly lo-fold more Trasylol was required to suppress the secondary PFC response when compared to the primary. The inhibition of the in vitro generation of secondary antibody response was in agreement with previous in vitro findings of Glaser et al. (25) who showed that ol-globulin (isolated from normal serum) markedly inhibited plaque and rosette formation both in the primary and secondary response. Trasylol could be neutralized with the protease trypsin. Low concentrations of trypsin had no effect since only trypsin, 100-l 50 kg/ml, preincubated with Trasylol reversed the immunosuppressive effects. The chemical susceptibility of Trasylol paralleled previous work (25-27) in which it was demonstrated that the suppressive activity of normal mouse serum could be neutralized by trypsin. Thus, when saturated with trypsin, Trasylol could not interact with cellular proteases. Pretreatment of T lymphocytes with Trasylol did not result in any suppression

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MILLER,

AND ARONSON

of the PFC response of spleen cells to T-dependent antigen SRBC. On the other hand pretreatment of B lymphocytes with Trasylol did result in suppression of the PFC response of spleen cells to both T-dependent and T-independent antigens. These results suggest the target of the Trasylol inhibition to be the B lymphocyte. Further evidence has developed for Trasylol suppression of B-cell dependent mitogen-induced lymphocyte transformation and fluorescent-labeled Trasylol reaction with B cells (unpublished results). There have been reports that B-lymphocyte activity can be enhanced by proteases such as trypsin, pronase, elastase-like protease, and cathepsin (28, 29). One would assume a natural Trasylol-susceptible protease to be involved in the activation of B lymphocytes by antigen. While several laboratories have demonstrated the suppressive actions of protease inhibitors on lymphocyte activation [TLCK (6, 30,31), SBI ( 16, 24) lima bean trypsin inhibitor (34), tosyl+arginine methyl ester (6, 30), EACA (6), trasylol (7, 16, 3 1, 32), TPCK (6, 30), benzoyl+arginine amide (30), leupeptin (33), and antipain (35)]; none of these inhibitors, however, has been reported to have an inhibitory effect selectively directed toward the function of B cells. In fact, only insolubilized SBI was found to have an inhibitory action on a protease associated with the lymphocyte surface (24). The protease inhibitors listed above seem to be heterogeneous with respect to the inhibition profile. They include protease inhibitors of both trypsin and chymotrypsin. It is likely that more than one protease may be involved in lymphocyte triggering and in the future it will be important to elucidate the mechanism and stage of protease inhibitor regulation of lymphocyte responsiveness. A shift in the balance between concentrations of protease inhibitors and proteolytic enzymes may condition the degree of lymphocyte differentiation. Increased concentration of available protease, through experimental manipulation, may drive subsets of B lymphocytes in mitotic cycle and induce the secretion of antibodies of multiple specificities (36, 37) leading to unwanted immunological anomalies. Normal serum protease inhibitors, on the other hand, may be important in the fine regulation of such proteases. In future studies Trasylol will be used as a membrane and cytoplasmic probe to determine the cellular and molecular events of protease regulation of lymphocyte differentiation. ACKNOWLEDGMENT The studies of Mr. Alvin Gabrielsen, in which benzyl alcohol preservative in Trasylol was also found to have inhibitory and cytotoxic capacity, are gratefully acknowledged.

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PROTEASE

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 3 I. 32. 33. 34. 35.

MODULATION

OF B CELLS

167

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