CELLULAR I1VI1VIUNOLOGY28, 59-68
(1977)
Effects of C-Reactive Protein on Lymphocyte Functions III. Inhibition of Antigen-Induced Lymphocyte Stimulation and Lymphokine Production 1 R. F. IV~oRTENSEN,2 D. BRAUN, AND I-I. GEWURZ ~ Department of Immunology, Rush Medical School, and Department of Microbiology, University of Illinois at the Medical Center, Chicago, Illinois 60612 Received May 23, 1976 The effect of purified human C-reactive protein (CRP) on antigen-induced lymphOcyte proliferation and lymphokine production by human lymphocytes was assessed since it was previously shown that CRP binds primarily to T cells and inhibits the mixed lymphocyte response. Peripheral blood lymphocytes from antigen-sensitive donors displayed a diminished proliferative response to both purified protein derivative (PPD) and Candida antigen in the presence of CRP, a response which was proportional to the concentration of CRP. Addition of CRP to human lymphocytes in culture markedly inhibited the production of migration inhibitory factor (MIF) not only initiated by antigen but also triggered by the lectins PHA and Con A; however, CRP did not interfere with the action of preformed MIF on the indicator migrating cells. CRP also greatly reduced the antigen-triggered production of another lymphokine, the ehemotactic factor for monocytes. These findings suggest that elevated levels of CRP may modulate cell-mediated immune responses and may contribute to the transient anergy associated with inflammation. INTRODUCTION C-reactive protein ( C R P ) 4 is a serum constituent which rapidly increases during the acute stage of the inflammatory response (1, 2) and was originally defined by its calcium-dependent precipitation of the pneumococcal cell wall teichoic acid, C-polysaccharide ( C P S ) (3, 4). Serum concentrations of this protein are used clinically as an indicator of febrile illnesses and inflammation (5, 6). Like antibody, purified C R P has recently been demonstrated to activate the classical complement pathway efficiently when reacting with both C P S (7, 8) and certain polycationic substances (9, 10) in the absence of specific antibody. Purified human C R P has z This work was supported by Grant A1 12870-01 from the National Institutes of Health, a grant from the Leukemia Research Foundation, Inc., and Grant IM-101 from the American Cancer Society. -~Recipient of U.S. Public Health Service Postdoctoral Fellowship CA 03626 IMB. Present address and address for correspondence: Michigan Cancer Foundation, 110 East Warren, Detroit, Michigan 48201. s H. G. holds the Thomas J. Coogan, Sr., Chair of Immunology established by Marjorie Lindheimer Everett. ~Abbreviations used in this paper: CRP, C-reactive protein; CPS, C-polysaccharide; CTX, lymphocyte-derived chemotactic factor for monocytes; Con A, concanavalin A; MIF, migration inhibitory factor; MLR, mixed lymphocyte response; PHA, phytohemagglutinin; PPD, purified protein derivative (of tuberculin) ; PE, peritoneal exudate. 59 Copyright © 1977 by Academic Pres~, Inc. All rights of reproduction in any form reserved.
ISSN 0008-8749
60
MORTENSEN, BRAUN AND GEWuRZ
also been shown to bind primarily to human T lymphocytes and to the T cells of extrathymic lymphoid organs of mice (11, 12). This binding led to the inhibition of the mixed lymphocyte response ( M L R ) and the generation of cytolytic T cells without inhibiting proliferation in response to the lectins phytohemagglutinin ( P H A ) and concanavalin A (Con A) (11, 12). Other investigators have described both direct stimulation of lymphocytes by CRP (13, 14) and inhibition of the P H A stimulation of leukocytes by CRP (15). The functional diversity of T lymphocytes is thought to reflect most likely the activities of T-cell subpopulations. For example, a dissociation of the effector T cells mediating the specific cytolysis of allogeneic cells from the T cells producing macrophage migration inhibitory factor ( M I F ) in response to specific antigen has been observed using different experimental approaches (16-18). Since we had already shown that CRP inhibits the proliferation of those T cells responding to allogeneic cells (11), this study was undertaken to determine the effect of CRP on antigen-induced lymphokine production and lymphocyte proliferation. Another reason for examining the effect of CRP on these correlates of cellular immunity (19) is the association between anergy and depressed in vitro lymphocyte responsiveness in a diverse group of diseases in which nonantibody serum proteins are thought to contribute to the lowered responsiveness [reviewed in Refs. (20-22)]. Thereforel we have determined the effect of CRP on MIF induction by P H A and Con A (23-25) as well as its effect on the antigen-induced MIF and ehemotactic factor for monocytes (CTX) (26). The present report shows that purified human CRP inhibits antigen-induced lymphocyte proliferation and chemotactic factor production and both antigen- and mitogen-induced MIF formation. These findings are consistent with the interpretation that CRP may modulate cell-mediated immune responses during the acute stages of various inflammatory reactions. M A T E R I A L S AND M E T H O D S Isolation of CRP. CRP was purified from pooled human ascites and pleural fluids by affinity chromatography on CPS covalently bound to Bio-Gell A-50m (BioRad Laboratories, Richmond, Calif.) exactly as described in detail elsewhere (8). The basis for this purification method is the calcium-dependent binding of CRP to CPS (4); therefore, CRP was eluted from the CPS-agarose beads with 0.02 M citrate in 0.01 M Tris-buffered (pH 7.5) saline and further purified by filtration on Bio-Gel A-0.5m to remove aggregates and subunits of CRP. The documentation of the purity of CRP prepared in this manner has been outlined previously (8). Purified CRP (1-2 mg/ml) was dialyzed against 0.15 M saline, sterilized by Millipore filtration, and stored at 4°C for less than 1 month before use. A monospecific antiserum raised in goats was used to determine CRP concentrations by radial diffusion. Lymphocyte cultures. Lymphocytes were separated from heparinized blood obtained from individuals displaying delayed cutaneous reactivity to either 5 tuberculin units of purified protein derivative ( P P D ) or 25/xg of Candida antigen (Dermatophytin 0, Hollister-Stier, Spokane, Wash.) by centrifugation on Ficoll-Hypaque. For measurements of antigen-induced lymphocyte stimulation, peripheral lymphocytes were cultured in R P M I 1640 containing 10% heat-inactivated fetal calf serum with various concentrations of either P P D (Connaught Labs, Toronto, Canada) or Candida in microcultures (Microtest II, Falcon Plastics, Oxnard, Calif.) with 2 × 105 cells/well. CRP was added at the time the cultures were ini-
CRP iNtItBITION OF T-CELL ACTIVATION
61
tiated. The cells were cultured for 144 hr and then labeled for 18 hr with 0.05 ~Ci of [2-Cl~]thymidine (55 mCi/mmol, SchwarzMann, Division of Becton Dickinson, Orangeburg, N.Y.). The lymphocytes were harvested with a multiple automatic sample harvester (27), and the incorporated thymidine was counted by liquid scintillation. Cultures for generating MIF consisted of 4 × 106 peripheral lymphocytes from sensitive donors in 1.0-ml cultures (13 × 100-ram Falcon tubes) containing either 25/zg/ml of P P D or 250 ~g/ml of Candida. Mitogen-induced M I F was obtained from similar cultures of peripheral lymphocytes (2 x 106/ml) pulsed with either P H A at 1 ~l/ml (PHA-P, Difco Labs, Detroit, Mich.) or Con A at 2 ~g/ml (Nutritional Biochemicals Corp., Cleveland, Ohio) for 1 hr at 37°C, after which the cells were washed three times with medium and then cultured in the presence or absence of CRP. Supernatants from both mitogen- and antigen-stimulated cell cultures were collected at 48 hr and examined for M I F activity. Assay for MIF. The supernatants from the human lymphocyte cultures were tested in an indirect M I F assay utilizing nonsensitized guinea pig peritoneal cells as indicators (54). The assay used was the microdroplet method of Harrington and Statsny (28). 13,riefly, mineral oil-induced peritoneal exuate cells from healthy female guinea pigs were washed three times in Hank's balanced salt solution and placed in 2 ml of R P M I with 20% fetal calf serum. A pellet of these cells was resuspended in a mixture of equal parts of medium 199 ( 2 × ) and 0.4% Sea Plaque agarose to obtain a 25% (v/v) suspension of the migrating cells. Droplets (2 /xl) of this suspension were dispensed into Lab-Tek tissue culture chamber slides (eight wells/slide) which had been precoated with a droplet of 0.8% Sea Plaque agarose. The agarose droplets with cells were refrigerated (10 rain) and then covered with the test supernatant (0.2 ml/well). After 48 hr of incubation, the migration areas were projected on a Nikon Model 6C overhead profile comparator at 20X magnification; the areas of migration traced on paper were measured by planimetry. The values of all eight migration areas from one slide were used to calculate a mean migration index on a calculator using a program described earlier by Paque et al. (29). All of the supernatants were coded so that MIF assays were performed in a single-blind manner. Induction and assay of monocyte chemotactic factor ( CTX). Leukocytes from PPD- and Candida-sensitive donors were isolated from heparinized blood by dextran (1: 10, 5 ~ dextran-250, Pharmacia) sedimentation. The cells were adjusted to 2.0 × 106 lymphocytes (approximately 4 × 106 leukocytes/ml) in R P M I 1640 with 0.5% human AB serum and incubated in 1.0-ml cultures with either P P D (25 /~g/ml) or Candida antigen (100 ~g/ml) for 36 hr. The culture supernatants were assayed for chemotactic activity against nonimmune guinea pig peritoneal exudate (PE) cells by Dr. Sharon Wahl (Laboratory of Microbiology and Immunology, National Institute of Dental Research, N I H ) exactly as described previously (30). Chemotactic activity was expressed as the mean number of mononuclear cells per oil immersion field which have migrated through a 5-ram Nucleopore polycarbonate filter on each of 20 oil immersion fields selected from triplicate filters. RESULTS
Effect of CRP on Antigen-Induced Lymphocyte Stimulation To determine if CRP had an effect on specific antigen-induced D N A synthesis of lymphocytes, purified human CRP was added directly to cultures of sensitized
62
IVIORTENSEN, BRAUN AND GEWURZ TABLE 1 Etfect of Purified Human CRP on the Stimulation of Lymphocytes from Sensitized Donors by PPD ~ PPD (~g/ml) 125 50 25 12.5 0
FC~4-]thymidine incorporation (cpm 4- SD) 0 ~g of CRP/ml
25 ~g of CRP/ml
100 ~g of CRP/ml
28674-270 51624-446 46604-327 30324-410 4824- 57
16634-178 24874-244 26364-319 12854-215 4144- 87
14824-222 17604-190 11964-152 11824-110 3774- 65
Peripheral blood lymphocytes obtained from donors showing a delayed cutaneous response with induration of 10 mm or more to 5 tuberculin units of PPD. Data from one of four similar experiments are shown; cultures were performed in triplicate. lymphocytes in the presence of the appropriate antigen. The lymphocytes were obtained from individuals displaying delayed cutaneous reactivity to either tuberculin P P D or Candida antigen. Significant inhibition of the blastogenic response to P P D occurred with as little as 25/xg/ml of CRP and was more pronounced at 100 ~g/ml of CRP (Table 1). This inhibition was observed not only at the P P D concentration giving optimal stimulation (50 ~g/ml) but also at concentrations lower and higher than this, indicating that the antigen dose-response relationship was not altered by the presence of CRP. The reduction of the proliferative response was proportional to the C R P concentration in the cultures. These concentrations of C R P have previously been documented by us to be nontoxic for human lymphocytes (11), and they were observed to be nontoxic throughout the incubation period in these experiments when examined by trypan blue dye exclusion. Further evidence for the noncytotoxicity of C R P is seen by the unaltered background D N A synthesis in cultures without antigen but with C R P (Table 1). C R P apparently does not interfere with the uptake of labeled thymidine since it did not inhibit P H A - or Con A-induced D N A synthesis (11). In similar experiments using lymphocytes from subjects with delayed sensitivity to Candida, we observed that C R P inhibited the mitogenic response induced by TABLE 2 Effect of Purified Human CRP on the Stimulation of Sensitized Lymphocytes by Candida Antigen~ ]-C14]thymidine incorporation (cpm 4- SD)
Candida Antigen (#g/ml)
0/~g of CRP/ml
50 ~g of CRP/mI
100 gg of CRP/mI
250 125 62 31 0
61204-252 6426:t=550 43904-302 37304-607 9654- 67
40664-416 28744-408 22524-270 20834-280 10504- 54
35354-295 26314-210 18324-312 20424-357 10144- 80
Peripheral blood lymphocytes from donors displaying delayed cutaneous reactivity to 25 /~g of Candida antigen of at least 10 mm in diameter with induration. Data are from one of five similar experiments; cultures were performed in triplicate.
CRP INIIiBiTION
OF
T - C E L L
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FIG. 1. The effect of purified CRP on the production of MIF by sensitized human peripheral lymphocytes in response to antigen. Lymphocytes from sensitive donors were incubated with either 25 /xg/ml of PPD or 250 gg/ml of Candida antigen in the presence and absence of 50 ~g/ml of purified human CRP. Culture supernatants (48 br) were tested for MIF activity against guinea pig peritoneal exudate cells. Mean migration index (-+SD) is shown for four experiments with each of the two antigens.
Candida antigen (Table 2). Again, the CRP-mediated inhibition was observed over a range of concentrations of Candida antigen and was proportional to the amount of CRP present. When tested by double diffusion on Ouchterlony plates, there was no indication of a reaction between CRP and either of the antigens, suggesting that CRP does not lower the antigenic concentration to suboptimal stimulating levels in the cultures. These experiments examining the response to two different recall antigens demonstrate that CRP markedly inhibits, but does not completely abrogate, the proliferative response of sensitized lymphocytes to specific antigens. Effect of CRP on the Generation of M I F Delayed hypersensitivity reactions depend on specific antigen-committed lymphocytes which in the presence of that antigen secrete soluble lymphokines that are in vitro correlates of the delayed skin reactivity; the most widely studied of these mediators is M I F (19, 31). Since D N A synthesis and cell division are not required for the elaboration of M I F (32, 33) and since mediator production and lymphocyte stimulation do not always correlate (31), it is possible that CRP might inhibit lymphocyte blastogenesis without affecting mediator production. Therefore, we examined the effect on the production of M I F by antigen-sensitive lymphocytes at a single concentration of CRP (50 ffg/ml) which inhibited proliferation. CRP inhibited the production of M I F in response to both P P D and Candida antigen, as shown by indirect assays for M I F with guinea pig P E cells as indicators (Fig. 1). Concentrations of CRP of less than 25 /~g/ml failed to inhibit M I F generation significantly (data not shown). A migration index of 0.60 or less in the indirect assay as used here was considered indicative of a significant amount of MIF. The inhibition by CRP was reproducible with cells from different donors. The controls for these experiments consisted of supernatants from cultures of the same lymphocytes exposed to CRP (50 ffg/ml) in the absence of antigens (Fig. 1) or supernatants
64
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FIG. 2. The effect of CRP on the induction of MIF by PHA and Con A. Peripheral human lymphocytes were exposed to PHA (1 /zl/ml) or Con A (2 #g/mi) for 60 min, washed, and then placed in culture with or without 50/~g/ml of CRP. Supernatants collected at 48 hr were tested for MIF activity with guinea pig peritoneal exudate cells. Mean migration index (-+SD) for three separate experiments is shown. from the control cultures reconstituted with either P P D (25 ffg/ml) or Candida antigen (250 ffg/ml); none of these supernatants caused significant migration inhibition. Since the release of M I F and other lymphokines can be triggered nonspecifically by the T-cell mitogens P H A and Con A (23-25), we also tested the effect of CRP on M I F formation induced by each of these substances. In order to minimize the migration inhibitory effect of the mitogens themselves on the indicator cells, the lymphocytes were exposed to P H A or Con A for 1 hr, which has been shown to be sufficient for the optimal binding of these mitogens (34), and then incubated for 48 hr in the presence or absence of CRP (50 ffg/ml). As seen in Fig. 2, CRP inhibited the production of M I F under these conditions. Thus, CRP significantly inhibited the triggering of M I F by both the antigens and the mitogens tested here.
Lack of Effect of CRP on the Activity of MIF An important consideration in these experiments was whether CRP exerted its inhibitory effect by blocking the expression of M I F activity rather than its production. To test this possibility, varying amounts of CRP were added to supernatants with M I F activity obtained from either P H A - or antigen (PPD)-triggered lymphocytes and then tested for changes in that activity. Concentrations of CRP of 25 and 100 ~g/mi did not significantly alter the migration inhibitory action of the supernatants or the migration of the indicator cells in the presence of control supernatants (Table 3). However, at 200 t~g/ml, CRP slightly increased the migration inhibitory activity of the MIF-containing supernatants. Therefore, CRP most likely exerts its inhibitory effect at the level of the activated lymphocytes elaborating M I F rather than by modifying the activity of M I F already formed.
Effects of CRP on Antigen-Triggered Chemotactic Factor Another lymphokine, the chemotactic factor (CTX) for monocytes, which has been shown to be distinct from M I F (26), was also measured in culture super-
CRP INI-IIBITION OF T-CELL ACTIVATION
65
TABLE 3 Lack of Effect of CRP on the Migration Inhibitory Activity of MIF-Containing Supernatants~ CRP (gg/ml)
Migration index b
-25 100 200
Control
PPD
PHA
1.00 0.90 0.95 0.90
0.63 0.54 0.48 0.37
0.38 0.39 0.30 0.20
a Purified CRP added to supernatants from lymphocyte cultures containing MIF induced by PPD (25 ~g/ml) or PHA (1 ~l/ml). b Mean migration index with a SD of less than 10%. n a t a n t s of P P D - and C a n d i d a - s t i m u l a t e d leukocyte cultures. T h e presence of C R P d u r i n g the incubation p e r i o d significantly l o w e r e d the a m o u n t of chemotactic activity in response to both antigens with leukocytes obtained from different donors ( T a b l e 4 ) . W i t h both antigens, the p e r c e n t a g e of inhibition of C T X activity was g r e a t e r than 7 5 % . T h e presence of C R P alone in the culture fluids did not p r o d u c e a chemotactic response. T h e s e findings taken t o g e t h e r with the d a t a showing inhibition of M I F f o r m a t i o n indicate that C R P m a y significantly influence those in v i t r o p a r a m e t e r s associated with delayed hypersensitivity. DISCUSSION T h e studies described here were u n d e r t a k e n to d e t e r m i n e the effect of purified h u m a n C-reactive protein on both a n t i g e n - i n d u c e d proliferation and m e d i a t o r p r o duction by h u m a n p e r i p h e r a l l y m p h o c y t e s and r e p r e s e n t an extension of our earlier investigations showing that C R P inhibited the response to allogeneic cells (11, 12). T h e results p r e s e n t e d herein clearly d e m o n s t r a t e that the interaction of C R P with sensitized h u m a n l y m p h o c y t e s resulted in a p r o n o u n c e d inhibition of both specific TABLE 4 Effect of CRP on Antigen-Induced Formation of Monocyte Chemotactic Factor Antigen ~
CRP (gg/ml)
Chemotactic activity b
-PPD PPD
--50 50
134-3 30 4- 3 15 4- 3 10 4 - 1
--
--
Candida Candida
--
174-3 51 -4- 6 26 4- 3 16 4 - 2
--
--
50 50
Lymphocytes from sensitive donors incubated with either PPD (25 ~g/ml) or Candida anti~:en (100 #g/ml) ; supernatants collected at 36 hr for testing. b Mean number of migrating guinea pig monocytes per oil immersion field (4-SEM).
66
1VIORTENSEN, BRAUN AND GEWURZ
antigen-induced proliferation and lymphokine production. Although the extent of the inhibition of DNA synthesis was never complete, it was proportional to the amount of CRP and occurred over a range of antigen concentrations. Since there was no indication of a reaction between CRP and the antigens, it can be inferred that CRP exerted its inhibitory effect at the level of the cells required for either proliferation or mediator production, especially since we and others have presented evidence for the binding of CRP with lymphocytes (11, 13, 15). The addition of CRP to the cultures of sensitized lymphocytes with antigen abrogated the production of two distinguishable soluble mediators, MIF and chemotactic factor, which have both been implicated in the genesis of delayed sensitivity reactions (19). Efforts to correlate CRP concentration with the degree of inhibition of MIF formation by antigens were unsuccessful, perhaps because assays for the lymphokines are considerably more qualitative than quantitative. Inasmuch as the addition of CRP to MIF-containing supernatants failed to alter their migration inhibitory activity, it seems most likely that the CRP acts at the level of the MIF- or CTX-producing cells, or the adherent cells necessary for their induction (31). The inhibition of mediator production does not by itself indicate that CRP affects only T-lymphocyte functions since several investigators have demonstrated production of lymphokines by non-T cells (35-38). Nonetheless, CRP probably does affect the T cells in a mixed population since it inhibits proliferation in response to specific antigens, a property of human T cells but not of B cells (35). We also observed inhibition of MIF production induced by Con A and PHA, although we have previously shown that these same CRP preparations did not inhibit DNA synthesis induced by these lectins (11). A dissociation between mediator production and blastogenesis has been noted previously ( 3 9 ~ 1 ) , and it has been suggested that this may reflect the activity of distinct T-lymphocyte subpopulat'ions, with only one of the populations making MIF (35). The selective inhibition of antigen- but not mitogen-induced proliferation may represent a selectivity of CRP for that lymphocyte subpopulation which recognizes antigen. That distinct mitogen- and antigen-reactive T cells exist is supported by studies in which cells dividing in response to antigen were inactivated without altering subsequent proliferation to mitogens (42) and by studies in which antigen- and mitogen-reaetive cells were separated on the basis of density (43). Moreover, we have recently observed that antigen-induced lymphoblasts bound CRP, but mitogen-induced lymphoblasts did not (44). The characteristics of the CRP-dependent inhibitory effects described here would indicate that CRP inhibits events that precede thymidine incorporation into DNA since M I F formation is independent of cell division (32, 33, 35) and occurs as early as 6-8 hr after stimulation (45). The underlying metabolic changes following CRP binding and their relationship to the diminution of T-cell activities remains to be established. CRP may be one factor that contributes to the impaired lymphocyte responsiveness and anergy associated with certain inflammatory diseases (20-22), viral infections (46), and the tumor-bearing state (47), since these conditions are accompanied by elevated serum CRP levels (48, 49). Other serum proteins such as immunoregulatory a-globulin (50), a-fetoprotein (51), low density lipoproteins (52), and at-acid glycoprotein 5 have also been shown to alter T-lymphocyte functions, Chlu, K. 1Vi.,Mortensen, R. F., Osmand, A. P., and Gewurz, H., fmmunology, in press.
CRP INI-tlBITION OF T-CELL ACTIVATION
67
and all possess physical characteristics distinctly different from each other and C R P . I n t e r m s of biological activity, C R P is distinguishable from these factors in that it does not inhibit proliferation in response to Con A or P H A (11, 12). C R P has been recently classified with other factors that affect lymphocyte functions but which are produced by nonlymphoid cells, i.e., a cytokine (53). Although the significance of the modification of the response of lymphocytes to various immunological stimuli by C R P or other acute phase proteins is not yet clear, one proposed role for such interactions is to prevent autosensitization following tissue damage and inflammation (53). ACKNOWLEDGMENTS The authors wish to thank Linda Mauser for her careful technical assistance and Dr. Sharon Wahl ( N I H ) for her measurements of the chemotactic factor. REFERENCES 1. 2. 3. 4. 5. 6.
Anderson, H. C., and McCarty, M., Amer. Y. Med. 8, 445, 1950. Claus, D., Osmand, A. P., and Gewurz, H., f. Lab. Cli~. Med. 86, 375, 1975. Tiller, W. S., and Francis, T., Jr., J. E.rp. Med. 52, 561, 1930. Abernethy, T. J., and Avery, O. T, Y. E.vp. Med. 73, 173, 1941. Hedlund, P., Acta Med. Sca~d. Suppl. 361, 1, 196t. Good, R. A., In "Rheumatic Fever" (L. Thomas, Ed.), p. 115. University of Minnesota Press, Minneapolis, 1952. 7. Kaplan, M. H., and Volanakis, J. E., Immm~ology 112, 2135, 1974. 8. Osmand, A. P., Mortensen, R. F., Siegel, J., and Gewurz, H., J-. Exp. Med. 142, 1065, 1975. 9. Siegel, J., Rent, R., and Gewurz, H., J. Exp. Med. 140, 631, 1974. 10. Siegel, J., Osmand, A. P., Wilson, M. F., and Gewurz, It., J. Exp. Med. 142, 709, 1975. 11. Mortensen, R. F., Osmand, A. P., and Gewurz, H., f. Exp. _]/fed. 141,821, 1975. t2. Mortensen, R. F., and Gewurz, H., J. Imm*tnology 116, 1244, 1976. 13. Hornung, M., and Fritchi, S., Nature Nezv Biol. 230, 84, 1971. 14. Hornung, M. 0., Proe. Soe. Exp. Biol. Meal. 139, 1166, 1972. 15. Hokama, Y., Paik, Y. P., Yanagihara, E., and Kimura, L., J. ReticuIoendothel. Soc. 13, 111, 1973. 16. Henney, C. S., Gaffney, J., and Bloom, B. R., Y. Exp. Med. 140, 837, 1974. 17. Tigelaar, R. E., and Gorczynski, IR. M., Y. Exp. Med. 140, 267, 1974. 18. Harrington, J. T., Cell. Immunol. 24, 195, 1976. 19. Bloom, B. R., Adva~. lmmunoI. 13; 101, 1971. 20. Gatti, R. A., La~,cet 1, 1351, 1971. 21. Bryceson, A., l~ "Progress in Immunology--II" (L. Brent and J. Holborow, Eds.), pp. 61-68. North-Holland, Amsterdam, 1974. 22. Kantor, F. S., N. Engl. J. Med. :292, 629, 1975. 23. Lamellin, J. P., and Vassali, P., Nature (London) 229, 426, 1971. 24. Pick, E., Brostoff, J., Krejci, and Turk, J. L., Cell Immunol. 1, 92, 1970. 25. Pelley, R., and Schwartz, H. J., Proc. Soc. E.~'p. Biol. Med. 141,373, 1972. 26. Ward, P. A., Remold, H. G., and David, J. R., Cell. Immm, ol. 1, 162, 1970. 27. Hartzman, R. J., SegalI, M., Bach, M. L., and Bach, F. H., Tra~*splantation 11,268, 1971. 28. t-Iarrington, J. T., and Stastny, P., J. Immmzol. 110, 752, 1973. 29. Paqne, R. E., Kniskern, P. J., Dray, S., and Baram, P., .f. Imm:u~wI. 103, 1014, 1969. 30. Wahl, S. M., Altman, L. C., Oppenheim, J. J., and Mergenhagen, S. E., Int. Arch. Allergy Appl. Immunol. 46, 768, 1974. 31. David, J. R., and David, R. A., Progr. Allercjy 16, 300, 1972. 32. Rocklin, R. E.,/. Imm,tnol. 110, 674, 1973. 33. Bloom, B. R., Gaffney, J., and Jimenez, L., 7. hmmtnol. 109, 1395, 1972, 34. Jones, G., Cell. Immnnol. 9, 393, 1973.
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35. Rocklin, R. E., ~ a c Dermott, R. P., Chess, L., Schlossman, S., and David, J. R., J. Exp. Med. 140, 1303, 1974. 36. Yoshida, T., Sonozaki, H., and Cohen, S., f. Exp. Med. 138, 784, 1973. 37. Mackler, B. F., Altman, L. C., Rosenstreich, D. L., and Oppenheim, J. J., Nature (London) 243, 834, 1974. 38. Epstein, L. B., Kreth, W., and Herzenberg, L. A., Cell. Immunol. 12, 407, 1974. 39. Levin, A. S., Spitler, L. E., Stites, D. P., and Fudenberg, H. H., Proc. Nat. Acad. Sci. USA 67, 821, 1970. 40. Chaparas, S. D., Thor, D. E., Godfrey, H. P., Baer, H., and Hedrick, S. R., Science 170, 637, 1970. 41. Rocklin, R. E., Chilgren, R., Hong, R., and David, J. R., Cell. Immuno[. 1, 290, 1970. 42. Lazda, V. A., and Baram, P., J. Immunol. 112, 1705, 1974. 43. Durkin, H. G., and Waksman, B. H., Proc. Nat. Acad. Sci. USA 72, 5090, 1975. 44. Croft, S., Mortensen, R. F., and Gewurz, H., Science 192, 685, 1976. 45. Bennett, B., and Bloom, B. R., Transplantation 5, 996, 1967. 46. Notkins, A. L., Mergenhagen, S. E., and Howard, R. J., Amlu. Rev. M icrobiol. 24, 525, 1970. 47. Harris, J., and Bagai, R. C.. Med. Clin. N. Amer. 56, 501, 1972. 48. Sprunt, D. H., Hale, W. M., Chang, F. C., Richmond, S. G., and Erickson, C. C., Science 122, 273, 1955. 49. Riley, R. F., Hokama, ¥., Colver, V., Coleman. M. K., and Dowdy, A. I~., Cancer Res. 18, 833, 1958. 50. Monzoian, J. O., Glasgow, A. H., Nimberg, R. D., Cooperband, S. R., Schmid, I(., Saporoschetz, I., and Mannick, J. A., J. Immunol. 113, 266, 1974. 51. Murgita, R., and Tomasi, T., J. Exp. Med. 141, 440, 1975. 52. Curtiss, L. K., and Edgington, T. S., f. Immunol. 116, 1452, 1976. 53. Waksman, B. H., and Namba, Y, Cell. Immunol. 21, 161, 1976. 54. Thor, E. D., Jureziz, R. E., Veach, $. R., Miller, E., and Dray, S. Nature (London) 219, 755, 1968.
(1977)
Effects of C-Reactive Protein on Lymphocyte Functions III. Inhibition of Antigen-Induced Lymphocyte Stimulation and Lymphokine Production 1 R. F. IV~oRTENSEN,2 D. BRAUN, AND I-I. GEWURZ ~ Department of Immunology, Rush Medical School, and Department of Microbiology, University of Illinois at the Medical Center, Chicago, Illinois 60612 Received May 23, 1976 The effect of purified human C-reactive protein (CRP) on antigen-induced lymphOcyte proliferation and lymphokine production by human lymphocytes was assessed since it was previously shown that CRP binds primarily to T cells and inhibits the mixed lymphocyte response. Peripheral blood lymphocytes from antigen-sensitive donors displayed a diminished proliferative response to both purified protein derivative (PPD) and Candida antigen in the presence of CRP, a response which was proportional to the concentration of CRP. Addition of CRP to human lymphocytes in culture markedly inhibited the production of migration inhibitory factor (MIF) not only initiated by antigen but also triggered by the lectins PHA and Con A; however, CRP did not interfere with the action of preformed MIF on the indicator migrating cells. CRP also greatly reduced the antigen-triggered production of another lymphokine, the ehemotactic factor for monocytes. These findings suggest that elevated levels of CRP may modulate cell-mediated immune responses and may contribute to the transient anergy associated with inflammation. INTRODUCTION C-reactive protein ( C R P ) 4 is a serum constituent which rapidly increases during the acute stage of the inflammatory response (1, 2) and was originally defined by its calcium-dependent precipitation of the pneumococcal cell wall teichoic acid, C-polysaccharide ( C P S ) (3, 4). Serum concentrations of this protein are used clinically as an indicator of febrile illnesses and inflammation (5, 6). Like antibody, purified C R P has recently been demonstrated to activate the classical complement pathway efficiently when reacting with both C P S (7, 8) and certain polycationic substances (9, 10) in the absence of specific antibody. Purified human C R P has z This work was supported by Grant A1 12870-01 from the National Institutes of Health, a grant from the Leukemia Research Foundation, Inc., and Grant IM-101 from the American Cancer Society. -~Recipient of U.S. Public Health Service Postdoctoral Fellowship CA 03626 IMB. Present address and address for correspondence: Michigan Cancer Foundation, 110 East Warren, Detroit, Michigan 48201. s H. G. holds the Thomas J. Coogan, Sr., Chair of Immunology established by Marjorie Lindheimer Everett. ~Abbreviations used in this paper: CRP, C-reactive protein; CPS, C-polysaccharide; CTX, lymphocyte-derived chemotactic factor for monocytes; Con A, concanavalin A; MIF, migration inhibitory factor; MLR, mixed lymphocyte response; PHA, phytohemagglutinin; PPD, purified protein derivative (of tuberculin) ; PE, peritoneal exudate. 59 Copyright © 1977 by Academic Pres~, Inc. All rights of reproduction in any form reserved.
ISSN 0008-8749
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MORTENSEN, BRAUN AND GEWuRZ
also been shown to bind primarily to human T lymphocytes and to the T cells of extrathymic lymphoid organs of mice (11, 12). This binding led to the inhibition of the mixed lymphocyte response ( M L R ) and the generation of cytolytic T cells without inhibiting proliferation in response to the lectins phytohemagglutinin ( P H A ) and concanavalin A (Con A) (11, 12). Other investigators have described both direct stimulation of lymphocytes by CRP (13, 14) and inhibition of the P H A stimulation of leukocytes by CRP (15). The functional diversity of T lymphocytes is thought to reflect most likely the activities of T-cell subpopulations. For example, a dissociation of the effector T cells mediating the specific cytolysis of allogeneic cells from the T cells producing macrophage migration inhibitory factor ( M I F ) in response to specific antigen has been observed using different experimental approaches (16-18). Since we had already shown that CRP inhibits the proliferation of those T cells responding to allogeneic cells (11), this study was undertaken to determine the effect of CRP on antigen-induced lymphokine production and lymphocyte proliferation. Another reason for examining the effect of CRP on these correlates of cellular immunity (19) is the association between anergy and depressed in vitro lymphocyte responsiveness in a diverse group of diseases in which nonantibody serum proteins are thought to contribute to the lowered responsiveness [reviewed in Refs. (20-22)]. Thereforel we have determined the effect of CRP on MIF induction by P H A and Con A (23-25) as well as its effect on the antigen-induced MIF and ehemotactic factor for monocytes (CTX) (26). The present report shows that purified human CRP inhibits antigen-induced lymphocyte proliferation and chemotactic factor production and both antigen- and mitogen-induced MIF formation. These findings are consistent with the interpretation that CRP may modulate cell-mediated immune responses during the acute stages of various inflammatory reactions. M A T E R I A L S AND M E T H O D S Isolation of CRP. CRP was purified from pooled human ascites and pleural fluids by affinity chromatography on CPS covalently bound to Bio-Gell A-50m (BioRad Laboratories, Richmond, Calif.) exactly as described in detail elsewhere (8). The basis for this purification method is the calcium-dependent binding of CRP to CPS (4); therefore, CRP was eluted from the CPS-agarose beads with 0.02 M citrate in 0.01 M Tris-buffered (pH 7.5) saline and further purified by filtration on Bio-Gel A-0.5m to remove aggregates and subunits of CRP. The documentation of the purity of CRP prepared in this manner has been outlined previously (8). Purified CRP (1-2 mg/ml) was dialyzed against 0.15 M saline, sterilized by Millipore filtration, and stored at 4°C for less than 1 month before use. A monospecific antiserum raised in goats was used to determine CRP concentrations by radial diffusion. Lymphocyte cultures. Lymphocytes were separated from heparinized blood obtained from individuals displaying delayed cutaneous reactivity to either 5 tuberculin units of purified protein derivative ( P P D ) or 25/xg of Candida antigen (Dermatophytin 0, Hollister-Stier, Spokane, Wash.) by centrifugation on Ficoll-Hypaque. For measurements of antigen-induced lymphocyte stimulation, peripheral lymphocytes were cultured in R P M I 1640 containing 10% heat-inactivated fetal calf serum with various concentrations of either P P D (Connaught Labs, Toronto, Canada) or Candida in microcultures (Microtest II, Falcon Plastics, Oxnard, Calif.) with 2 × 105 cells/well. CRP was added at the time the cultures were ini-
CRP iNtItBITION OF T-CELL ACTIVATION
61
tiated. The cells were cultured for 144 hr and then labeled for 18 hr with 0.05 ~Ci of [2-Cl~]thymidine (55 mCi/mmol, SchwarzMann, Division of Becton Dickinson, Orangeburg, N.Y.). The lymphocytes were harvested with a multiple automatic sample harvester (27), and the incorporated thymidine was counted by liquid scintillation. Cultures for generating MIF consisted of 4 × 106 peripheral lymphocytes from sensitive donors in 1.0-ml cultures (13 × 100-ram Falcon tubes) containing either 25/zg/ml of P P D or 250 ~g/ml of Candida. Mitogen-induced M I F was obtained from similar cultures of peripheral lymphocytes (2 x 106/ml) pulsed with either P H A at 1 ~l/ml (PHA-P, Difco Labs, Detroit, Mich.) or Con A at 2 ~g/ml (Nutritional Biochemicals Corp., Cleveland, Ohio) for 1 hr at 37°C, after which the cells were washed three times with medium and then cultured in the presence or absence of CRP. Supernatants from both mitogen- and antigen-stimulated cell cultures were collected at 48 hr and examined for M I F activity. Assay for MIF. The supernatants from the human lymphocyte cultures were tested in an indirect M I F assay utilizing nonsensitized guinea pig peritoneal cells as indicators (54). The assay used was the microdroplet method of Harrington and Statsny (28). 13,riefly, mineral oil-induced peritoneal exuate cells from healthy female guinea pigs were washed three times in Hank's balanced salt solution and placed in 2 ml of R P M I with 20% fetal calf serum. A pellet of these cells was resuspended in a mixture of equal parts of medium 199 ( 2 × ) and 0.4% Sea Plaque agarose to obtain a 25% (v/v) suspension of the migrating cells. Droplets (2 /xl) of this suspension were dispensed into Lab-Tek tissue culture chamber slides (eight wells/slide) which had been precoated with a droplet of 0.8% Sea Plaque agarose. The agarose droplets with cells were refrigerated (10 rain) and then covered with the test supernatant (0.2 ml/well). After 48 hr of incubation, the migration areas were projected on a Nikon Model 6C overhead profile comparator at 20X magnification; the areas of migration traced on paper were measured by planimetry. The values of all eight migration areas from one slide were used to calculate a mean migration index on a calculator using a program described earlier by Paque et al. (29). All of the supernatants were coded so that MIF assays were performed in a single-blind manner. Induction and assay of monocyte chemotactic factor ( CTX). Leukocytes from PPD- and Candida-sensitive donors were isolated from heparinized blood by dextran (1: 10, 5 ~ dextran-250, Pharmacia) sedimentation. The cells were adjusted to 2.0 × 106 lymphocytes (approximately 4 × 106 leukocytes/ml) in R P M I 1640 with 0.5% human AB serum and incubated in 1.0-ml cultures with either P P D (25 /~g/ml) or Candida antigen (100 ~g/ml) for 36 hr. The culture supernatants were assayed for chemotactic activity against nonimmune guinea pig peritoneal exudate (PE) cells by Dr. Sharon Wahl (Laboratory of Microbiology and Immunology, National Institute of Dental Research, N I H ) exactly as described previously (30). Chemotactic activity was expressed as the mean number of mononuclear cells per oil immersion field which have migrated through a 5-ram Nucleopore polycarbonate filter on each of 20 oil immersion fields selected from triplicate filters. RESULTS
Effect of CRP on Antigen-Induced Lymphocyte Stimulation To determine if CRP had an effect on specific antigen-induced D N A synthesis of lymphocytes, purified human CRP was added directly to cultures of sensitized
62
IVIORTENSEN, BRAUN AND GEWURZ TABLE 1 Etfect of Purified Human CRP on the Stimulation of Lymphocytes from Sensitized Donors by PPD ~ PPD (~g/ml) 125 50 25 12.5 0
FC~4-]thymidine incorporation (cpm 4- SD) 0 ~g of CRP/ml
25 ~g of CRP/ml
100 ~g of CRP/ml
28674-270 51624-446 46604-327 30324-410 4824- 57
16634-178 24874-244 26364-319 12854-215 4144- 87
14824-222 17604-190 11964-152 11824-110 3774- 65
Peripheral blood lymphocytes obtained from donors showing a delayed cutaneous response with induration of 10 mm or more to 5 tuberculin units of PPD. Data from one of four similar experiments are shown; cultures were performed in triplicate. lymphocytes in the presence of the appropriate antigen. The lymphocytes were obtained from individuals displaying delayed cutaneous reactivity to either tuberculin P P D or Candida antigen. Significant inhibition of the blastogenic response to P P D occurred with as little as 25/xg/ml of CRP and was more pronounced at 100 ~g/ml of CRP (Table 1). This inhibition was observed not only at the P P D concentration giving optimal stimulation (50 ~g/ml) but also at concentrations lower and higher than this, indicating that the antigen dose-response relationship was not altered by the presence of CRP. The reduction of the proliferative response was proportional to the C R P concentration in the cultures. These concentrations of C R P have previously been documented by us to be nontoxic for human lymphocytes (11), and they were observed to be nontoxic throughout the incubation period in these experiments when examined by trypan blue dye exclusion. Further evidence for the noncytotoxicity of C R P is seen by the unaltered background D N A synthesis in cultures without antigen but with C R P (Table 1). C R P apparently does not interfere with the uptake of labeled thymidine since it did not inhibit P H A - or Con A-induced D N A synthesis (11). In similar experiments using lymphocytes from subjects with delayed sensitivity to Candida, we observed that C R P inhibited the mitogenic response induced by TABLE 2 Effect of Purified Human CRP on the Stimulation of Sensitized Lymphocytes by Candida Antigen~ ]-C14]thymidine incorporation (cpm 4- SD)
Candida Antigen (#g/ml)
0/~g of CRP/ml
50 ~g of CRP/mI
100 gg of CRP/mI
250 125 62 31 0
61204-252 6426:t=550 43904-302 37304-607 9654- 67
40664-416 28744-408 22524-270 20834-280 10504- 54
35354-295 26314-210 18324-312 20424-357 10144- 80
Peripheral blood lymphocytes from donors displaying delayed cutaneous reactivity to 25 /~g of Candida antigen of at least 10 mm in diameter with induration. Data are from one of five similar experiments; cultures were performed in triplicate.
CRP INIIiBiTION
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T - C E L L
63
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FIG. 1. The effect of purified CRP on the production of MIF by sensitized human peripheral lymphocytes in response to antigen. Lymphocytes from sensitive donors were incubated with either 25 /xg/ml of PPD or 250 gg/ml of Candida antigen in the presence and absence of 50 ~g/ml of purified human CRP. Culture supernatants (48 br) were tested for MIF activity against guinea pig peritoneal exudate cells. Mean migration index (-+SD) is shown for four experiments with each of the two antigens.
Candida antigen (Table 2). Again, the CRP-mediated inhibition was observed over a range of concentrations of Candida antigen and was proportional to the amount of CRP present. When tested by double diffusion on Ouchterlony plates, there was no indication of a reaction between CRP and either of the antigens, suggesting that CRP does not lower the antigenic concentration to suboptimal stimulating levels in the cultures. These experiments examining the response to two different recall antigens demonstrate that CRP markedly inhibits, but does not completely abrogate, the proliferative response of sensitized lymphocytes to specific antigens. Effect of CRP on the Generation of M I F Delayed hypersensitivity reactions depend on specific antigen-committed lymphocytes which in the presence of that antigen secrete soluble lymphokines that are in vitro correlates of the delayed skin reactivity; the most widely studied of these mediators is M I F (19, 31). Since D N A synthesis and cell division are not required for the elaboration of M I F (32, 33) and since mediator production and lymphocyte stimulation do not always correlate (31), it is possible that CRP might inhibit lymphocyte blastogenesis without affecting mediator production. Therefore, we examined the effect on the production of M I F by antigen-sensitive lymphocytes at a single concentration of CRP (50 ffg/ml) which inhibited proliferation. CRP inhibited the production of M I F in response to both P P D and Candida antigen, as shown by indirect assays for M I F with guinea pig P E cells as indicators (Fig. 1). Concentrations of CRP of less than 25 /~g/ml failed to inhibit M I F generation significantly (data not shown). A migration index of 0.60 or less in the indirect assay as used here was considered indicative of a significant amount of MIF. The inhibition by CRP was reproducible with cells from different donors. The controls for these experiments consisted of supernatants from cultures of the same lymphocytes exposed to CRP (50 ffg/ml) in the absence of antigens (Fig. 1) or supernatants
64
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FIG. 2. The effect of CRP on the induction of MIF by PHA and Con A. Peripheral human lymphocytes were exposed to PHA (1 /zl/ml) or Con A (2 #g/mi) for 60 min, washed, and then placed in culture with or without 50/~g/ml of CRP. Supernatants collected at 48 hr were tested for MIF activity with guinea pig peritoneal exudate cells. Mean migration index (-+SD) for three separate experiments is shown. from the control cultures reconstituted with either P P D (25 ffg/ml) or Candida antigen (250 ffg/ml); none of these supernatants caused significant migration inhibition. Since the release of M I F and other lymphokines can be triggered nonspecifically by the T-cell mitogens P H A and Con A (23-25), we also tested the effect of CRP on M I F formation induced by each of these substances. In order to minimize the migration inhibitory effect of the mitogens themselves on the indicator cells, the lymphocytes were exposed to P H A or Con A for 1 hr, which has been shown to be sufficient for the optimal binding of these mitogens (34), and then incubated for 48 hr in the presence or absence of CRP (50 ffg/ml). As seen in Fig. 2, CRP inhibited the production of M I F under these conditions. Thus, CRP significantly inhibited the triggering of M I F by both the antigens and the mitogens tested here.
Lack of Effect of CRP on the Activity of MIF An important consideration in these experiments was whether CRP exerted its inhibitory effect by blocking the expression of M I F activity rather than its production. To test this possibility, varying amounts of CRP were added to supernatants with M I F activity obtained from either P H A - or antigen (PPD)-triggered lymphocytes and then tested for changes in that activity. Concentrations of CRP of 25 and 100 ~g/mi did not significantly alter the migration inhibitory action of the supernatants or the migration of the indicator cells in the presence of control supernatants (Table 3). However, at 200 t~g/ml, CRP slightly increased the migration inhibitory activity of the MIF-containing supernatants. Therefore, CRP most likely exerts its inhibitory effect at the level of the activated lymphocytes elaborating M I F rather than by modifying the activity of M I F already formed.
Effects of CRP on Antigen-Triggered Chemotactic Factor Another lymphokine, the chemotactic factor (CTX) for monocytes, which has been shown to be distinct from M I F (26), was also measured in culture super-
CRP INI-IIBITION OF T-CELL ACTIVATION
65
TABLE 3 Lack of Effect of CRP on the Migration Inhibitory Activity of MIF-Containing Supernatants~ CRP (gg/ml)
Migration index b
-25 100 200
Control
PPD
PHA
1.00 0.90 0.95 0.90
0.63 0.54 0.48 0.37
0.38 0.39 0.30 0.20
a Purified CRP added to supernatants from lymphocyte cultures containing MIF induced by PPD (25 ~g/ml) or PHA (1 ~l/ml). b Mean migration index with a SD of less than 10%. n a t a n t s of P P D - and C a n d i d a - s t i m u l a t e d leukocyte cultures. T h e presence of C R P d u r i n g the incubation p e r i o d significantly l o w e r e d the a m o u n t of chemotactic activity in response to both antigens with leukocytes obtained from different donors ( T a b l e 4 ) . W i t h both antigens, the p e r c e n t a g e of inhibition of C T X activity was g r e a t e r than 7 5 % . T h e presence of C R P alone in the culture fluids did not p r o d u c e a chemotactic response. T h e s e findings taken t o g e t h e r with the d a t a showing inhibition of M I F f o r m a t i o n indicate that C R P m a y significantly influence those in v i t r o p a r a m e t e r s associated with delayed hypersensitivity. DISCUSSION T h e studies described here were u n d e r t a k e n to d e t e r m i n e the effect of purified h u m a n C-reactive protein on both a n t i g e n - i n d u c e d proliferation and m e d i a t o r p r o duction by h u m a n p e r i p h e r a l l y m p h o c y t e s and r e p r e s e n t an extension of our earlier investigations showing that C R P inhibited the response to allogeneic cells (11, 12). T h e results p r e s e n t e d herein clearly d e m o n s t r a t e that the interaction of C R P with sensitized h u m a n l y m p h o c y t e s resulted in a p r o n o u n c e d inhibition of both specific TABLE 4 Effect of CRP on Antigen-Induced Formation of Monocyte Chemotactic Factor Antigen ~
CRP (gg/ml)
Chemotactic activity b
-PPD PPD
--50 50
134-3 30 4- 3 15 4- 3 10 4 - 1
--
--
Candida Candida
--
174-3 51 -4- 6 26 4- 3 16 4 - 2
--
--
50 50
Lymphocytes from sensitive donors incubated with either PPD (25 ~g/ml) or Candida anti~:en (100 #g/ml) ; supernatants collected at 36 hr for testing. b Mean number of migrating guinea pig monocytes per oil immersion field (4-SEM).
66
1VIORTENSEN, BRAUN AND GEWURZ
antigen-induced proliferation and lymphokine production. Although the extent of the inhibition of DNA synthesis was never complete, it was proportional to the amount of CRP and occurred over a range of antigen concentrations. Since there was no indication of a reaction between CRP and the antigens, it can be inferred that CRP exerted its inhibitory effect at the level of the cells required for either proliferation or mediator production, especially since we and others have presented evidence for the binding of CRP with lymphocytes (11, 13, 15). The addition of CRP to the cultures of sensitized lymphocytes with antigen abrogated the production of two distinguishable soluble mediators, MIF and chemotactic factor, which have both been implicated in the genesis of delayed sensitivity reactions (19). Efforts to correlate CRP concentration with the degree of inhibition of MIF formation by antigens were unsuccessful, perhaps because assays for the lymphokines are considerably more qualitative than quantitative. Inasmuch as the addition of CRP to MIF-containing supernatants failed to alter their migration inhibitory activity, it seems most likely that the CRP acts at the level of the MIF- or CTX-producing cells, or the adherent cells necessary for their induction (31). The inhibition of mediator production does not by itself indicate that CRP affects only T-lymphocyte functions since several investigators have demonstrated production of lymphokines by non-T cells (35-38). Nonetheless, CRP probably does affect the T cells in a mixed population since it inhibits proliferation in response to specific antigens, a property of human T cells but not of B cells (35). We also observed inhibition of MIF production induced by Con A and PHA, although we have previously shown that these same CRP preparations did not inhibit DNA synthesis induced by these lectins (11). A dissociation between mediator production and blastogenesis has been noted previously ( 3 9 ~ 1 ) , and it has been suggested that this may reflect the activity of distinct T-lymphocyte subpopulat'ions, with only one of the populations making MIF (35). The selective inhibition of antigen- but not mitogen-induced proliferation may represent a selectivity of CRP for that lymphocyte subpopulation which recognizes antigen. That distinct mitogen- and antigen-reactive T cells exist is supported by studies in which cells dividing in response to antigen were inactivated without altering subsequent proliferation to mitogens (42) and by studies in which antigen- and mitogen-reaetive cells were separated on the basis of density (43). Moreover, we have recently observed that antigen-induced lymphoblasts bound CRP, but mitogen-induced lymphoblasts did not (44). The characteristics of the CRP-dependent inhibitory effects described here would indicate that CRP inhibits events that precede thymidine incorporation into DNA since M I F formation is independent of cell division (32, 33, 35) and occurs as early as 6-8 hr after stimulation (45). The underlying metabolic changes following CRP binding and their relationship to the diminution of T-cell activities remains to be established. CRP may be one factor that contributes to the impaired lymphocyte responsiveness and anergy associated with certain inflammatory diseases (20-22), viral infections (46), and the tumor-bearing state (47), since these conditions are accompanied by elevated serum CRP levels (48, 49). Other serum proteins such as immunoregulatory a-globulin (50), a-fetoprotein (51), low density lipoproteins (52), and at-acid glycoprotein 5 have also been shown to alter T-lymphocyte functions, Chlu, K. 1Vi.,Mortensen, R. F., Osmand, A. P., and Gewurz, H., fmmunology, in press.
CRP INI-tlBITION OF T-CELL ACTIVATION
67
and all possess physical characteristics distinctly different from each other and C R P . I n t e r m s of biological activity, C R P is distinguishable from these factors in that it does not inhibit proliferation in response to Con A or P H A (11, 12). C R P has been recently classified with other factors that affect lymphocyte functions but which are produced by nonlymphoid cells, i.e., a cytokine (53). Although the significance of the modification of the response of lymphocytes to various immunological stimuli by C R P or other acute phase proteins is not yet clear, one proposed role for such interactions is to prevent autosensitization following tissue damage and inflammation (53). ACKNOWLEDGMENTS The authors wish to thank Linda Mauser for her careful technical assistance and Dr. Sharon Wahl ( N I H ) for her measurements of the chemotactic factor. REFERENCES 1. 2. 3. 4. 5. 6.
Anderson, H. C., and McCarty, M., Amer. Y. Med. 8, 445, 1950. Claus, D., Osmand, A. P., and Gewurz, H., f. Lab. Cli~. Med. 86, 375, 1975. Tiller, W. S., and Francis, T., Jr., J. E.rp. Med. 52, 561, 1930. Abernethy, T. J., and Avery, O. T, Y. E.vp. Med. 73, 173, 1941. Hedlund, P., Acta Med. Sca~d. Suppl. 361, 1, 196t. Good, R. A., In "Rheumatic Fever" (L. Thomas, Ed.), p. 115. University of Minnesota Press, Minneapolis, 1952. 7. Kaplan, M. H., and Volanakis, J. E., Immm~ology 112, 2135, 1974. 8. Osmand, A. P., Mortensen, R. F., Siegel, J., and Gewurz, H., J-. Exp. Med. 142, 1065, 1975. 9. Siegel, J., Rent, R., and Gewurz, H., J. Exp. Med. 140, 631, 1974. 10. Siegel, J., Osmand, A. P., Wilson, M. F., and Gewurz, It., J. Exp. Med. 142, 709, 1975. 11. Mortensen, R. F., Osmand, A. P., and Gewurz, H., f. Exp. _]/fed. 141,821, 1975. t2. Mortensen, R. F., and Gewurz, H., J. Imm*tnology 116, 1244, 1976. 13. Hornung, M., and Fritchi, S., Nature Nezv Biol. 230, 84, 1971. 14. Hornung, M. 0., Proe. Soe. Exp. Biol. Meal. 139, 1166, 1972. 15. Hokama, Y., Paik, Y. P., Yanagihara, E., and Kimura, L., J. ReticuIoendothel. Soc. 13, 111, 1973. 16. Henney, C. S., Gaffney, J., and Bloom, B. R., Y. Exp. Med. 140, 837, 1974. 17. Tigelaar, R. E., and Gorczynski, IR. M., Y. Exp. Med. 140, 267, 1974. 18. Harrington, J. T., Cell. Immunol. 24, 195, 1976. 19. Bloom, B. R., Adva~. lmmunoI. 13; 101, 1971. 20. Gatti, R. A., La~,cet 1, 1351, 1971. 21. Bryceson, A., l~ "Progress in Immunology--II" (L. Brent and J. Holborow, Eds.), pp. 61-68. North-Holland, Amsterdam, 1974. 22. Kantor, F. S., N. Engl. J. Med. :292, 629, 1975. 23. Lamellin, J. P., and Vassali, P., Nature (London) 229, 426, 1971. 24. Pick, E., Brostoff, J., Krejci, and Turk, J. L., Cell Immunol. 1, 92, 1970. 25. Pelley, R., and Schwartz, H. J., Proc. Soc. E.~'p. Biol. Med. 141,373, 1972. 26. Ward, P. A., Remold, H. G., and David, J. R., Cell. Immm, ol. 1, 162, 1970. 27. Hartzman, R. J., SegalI, M., Bach, M. L., and Bach, F. H., Tra~*splantation 11,268, 1971. 28. t-Iarrington, J. T., and Stastny, P., J. Immmzol. 110, 752, 1973. 29. Paqne, R. E., Kniskern, P. J., Dray, S., and Baram, P., .f. Imm:u~wI. 103, 1014, 1969. 30. Wahl, S. M., Altman, L. C., Oppenheim, J. J., and Mergenhagen, S. E., Int. Arch. Allergy Appl. Immunol. 46, 768, 1974. 31. David, J. R., and David, R. A., Progr. Allercjy 16, 300, 1972. 32. Rocklin, R. E.,/. Imm,tnol. 110, 674, 1973. 33. Bloom, B. R., Gaffney, J., and Jimenez, L., 7. hmmtnol. 109, 1395, 1972, 34. Jones, G., Cell. Immnnol. 9, 393, 1973.
68
I~IORTENSEN, BRAUN AND GEWURZ
35. Rocklin, R. E., ~ a c Dermott, R. P., Chess, L., Schlossman, S., and David, J. R., J. Exp. Med. 140, 1303, 1974. 36. Yoshida, T., Sonozaki, H., and Cohen, S., f. Exp. Med. 138, 784, 1973. 37. Mackler, B. F., Altman, L. C., Rosenstreich, D. L., and Oppenheim, J. J., Nature (London) 243, 834, 1974. 38. Epstein, L. B., Kreth, W., and Herzenberg, L. A., Cell. Immunol. 12, 407, 1974. 39. Levin, A. S., Spitler, L. E., Stites, D. P., and Fudenberg, H. H., Proc. Nat. Acad. Sci. USA 67, 821, 1970. 40. Chaparas, S. D., Thor, D. E., Godfrey, H. P., Baer, H., and Hedrick, S. R., Science 170, 637, 1970. 41. Rocklin, R. E., Chilgren, R., Hong, R., and David, J. R., Cell. Immuno[. 1, 290, 1970. 42. Lazda, V. A., and Baram, P., J. Immunol. 112, 1705, 1974. 43. Durkin, H. G., and Waksman, B. H., Proc. Nat. Acad. Sci. USA 72, 5090, 1975. 44. Croft, S., Mortensen, R. F., and Gewurz, H., Science 192, 685, 1976. 45. Bennett, B., and Bloom, B. R., Transplantation 5, 996, 1967. 46. Notkins, A. L., Mergenhagen, S. E., and Howard, R. J., Amlu. Rev. M icrobiol. 24, 525, 1970. 47. Harris, J., and Bagai, R. C.. Med. Clin. N. Amer. 56, 501, 1972. 48. Sprunt, D. H., Hale, W. M., Chang, F. C., Richmond, S. G., and Erickson, C. C., Science 122, 273, 1955. 49. Riley, R. F., Hokama, ¥., Colver, V., Coleman. M. K., and Dowdy, A. I~., Cancer Res. 18, 833, 1958. 50. Monzoian, J. O., Glasgow, A. H., Nimberg, R. D., Cooperband, S. R., Schmid, I(., Saporoschetz, I., and Mannick, J. A., J. Immunol. 113, 266, 1974. 51. Murgita, R., and Tomasi, T., J. Exp. Med. 141, 440, 1975. 52. Curtiss, L. K., and Edgington, T. S., f. Immunol. 116, 1452, 1976. 53. Waksman, B. H., and Namba, Y, Cell. Immunol. 21, 161, 1976. 54. Thor, E. D., Jureziz, R. E., Veach, $. R., Miller, E., and Dray, S. Nature (London) 219, 755, 1968.
