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The role of prostaglandins in the control of the immune response to an autologous red blood cell antigen (Hb)
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
8,42w29
( 1977)
The Role of Prostaglandins in the Control of the Immune Response to an Autologous Red Blood Cell Antigen (Hb) MIGHAL
ZIMECKI’,”
AND
DAVID
R.
WEBB,
Department of Cell Biology. Roche Institute 41‘ Molrc~rlrr New lersev 07110 Received November 13. 1976
JR.~ Biology. Nutle~.
When spleen cell or peritoneal exudate cell cultures are exposed to inhibitors of prostaglandin synthetase, the number of spontaneous, anti-autologous erythrocyte antigen (Hb)antibody-forming cells is increased. This result is obtained both in C57B l/65 mice as well as in NZB mice. The cell cultures require 24-48 hr exposure to the prostaglandin synthetase inhibitors before their effects are observed. Treatment of spleen cell or peritoneal exudate cell cultures with mitomycin C blocks DNA synthesis but has no effect on the enhancement of the anti-Hb response induced by the prostaglandin synthetase inhibitors. This suggests that the prostaglandins play a role in repressing or controlling the expression of certain autoimmune clones.
INTRODUCTION
In recent years, increased attention has been directed toward understanding the nature of cellular control mechanisms which function in immunocompetent cells (1, 2). It has become clear that immunocompetent cells possess a large array of receptors other than immunoglobulins which may control the way these cells function (3,4). Some of these control functions have been shown to operate when the immune system is challenged by “non-self’ antigens (5-7). With the report by Linder and Edgington (8) that both NZB mouse strains as well as others possess the capacity to spontaneously respond to both exposed and/or unexposed autologous erythrocyte antigens, it has become possible to investigate the nature of the controls which operate in “anti-self’ immune responses. The two antigens in question have been termed X and Hb. The X antigen is found on the surface of mouse erythrocytes and only NZB mice appear to have anti-X-antibody-forming cells (8). The Hb antigen is not exposed and is only revealed by treating mouse erythrocytes with the proteolytic enzyme bromylase (8). Virtually all strains of mice tested have been shown to possess spontaneous anti-Hb-antibody-forming cells in the spleen and in the peritoneum. In culture, the number of anti-Hb-antibody-forming cells increases in the absence of any added antigen (9, 10). More recent investigations by De Heer and Edgington have suggested that the development of the anti-Hb response is a function of altered immunoregulatory influences (11). In addition,
1 Recipient of a Fulbright-Hays Fellowship. * Dr. Michal Zimecki is on leave from the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. 3 To whom all correspondence should be addressed. 420 Copynghr 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
PROSTAGLANDIN
CONTROL
OF ANTI-Hb
RESPONSE
421
Cunningham has reported that animals treated with antilymphocyte serum had increased numbers of splenic PFC to the Hb antigen (12). It has recently been found in our laboratory that prostaglandins play a role in the nonspecific regulation of lymphocyte effector-cell function at both the T- and B-cell level (13, 14, 15). Therefore, it was of interest to investigate whether or not prostaglandins might also be involved in the control of autoimmune responses. The experiments presented in this report suggest that prostaglandins do play a role in regulating the autoimmune response to Hb antigen. MATERIALS
AND METHODS
Animals. C57B1/6J male mice, 6-10 weeks old, were purchased from Jackson Laboratories (Bar Harbor, Maine). The NZB mice were obtained from Dr. Norman Talal, University of California. In each experiment, pools of spleens or peritoneal exudates from at least 10 mice were used to prepare the cultures. Antigens. Mouse erythrocytes (MRBC) were obtained by orbital bleeding under ether anesthesia and collected in 0.1 M sodium citrate solution. Prostaglandins and PG synthesis inhibitors. Prostaglandins E, and Fzo,(PGE, and PGF& were obtained from Hoffmann-LaRoche Inc. and were stored at -20°C in 95% ethanol. Immediately prior to use, they were diluted with medium to the appropriate concentrations. Control cultures received medium plus ethanol at the appropriate concentrations. The concentrations used (1 O-5 M) had previously been shown to be optimal for inhibiting the appearance of antibody-forming cells to sheep erythrocytes and polyvinyl pyrolidone (13, 15). At these concentrations, PGE, gives optimal elevation of cyclic AMP in whole spleen cells and thymocytes, whereas PGFzo, has no effect on cyclic AMP or cyclic GMP levels (D. Webb, unpublished observations). Indomethacin, an irreversible inhibitor of PG synthesis was purchased from Sigma (St. Louis, MO.). ru-6-Chloro-cY-methyl-carbazole-2acetic acid (Ro-20-5720), a competitive reversible inhibitor of prostaglandin synthesis, and octadeca-9-12, diynoic acid (Ro-3-1314), an irreversible inhibitor of prostaglandin synthesis, were obtained from Hoffmann-LaRoche Inc. (Nutley, N.J.). The action of the Ro compounds is limited to the inhibition of prostaglandin synthesis so far as is known (16). All inhibitors were prepared in 7.5% carbonate buffer (GIBCO) and diluted with complete medium before addition. Control cultures received appropriately diluted medium without inhibitors. The dilutions of synthetase inhibitors used in these experiments had previously been shown to be optimal for the inhibition of prostaglandin synthesis (13, 14). Bromelain-RBC preparation. MRBC were washed three times with Hank’s balanced salt solution (HBSS); equal volumes of packed erythrocytes were incubated with bromelase (Bromelain, Dade Reagents, Inc., Miami, Fla.) at 37°C for 30 min. After incubation, MRBC were washed three times with HBSS (8). Preparation of spleen cells. Whole spleen cell suspensions were prepared from spleens which were minced, filtered through a stainless-steel screen, and washed two times with HBSS. Preparation of peritoneal exudate cells and cell purification. Peritoneal exudate cells (PEC) were collected by washing the peritoneal cavity of mice with 2 ml of precooled HBSS using a sterile Pasteur pipette. Cells were washed three times with HBSS before suspending them in culture medium.
422
ZIMECKI
AND
WEBB
Purified “B” cells were prepared from PEC as follows: The PEC were suspended in 20 ml of MEM containing 10% fetal calf serum and poured onto 100 mm petri dishes. Following 30 to 40-min incubations at 37°C the nonadherent cells were decanted and treated with ATS (1:20 dilution) plus guinea pig complement (1:20 dilution). The preparation of the antithymocyte serum (ATS) and its properties have been previously reported (15). After 30-min incubation at 37°C the cells were washed three times in HBSS, counted, and cell viability was measured by use of trypan blue. The presence of macrophages was assayed by spreading on glass coverslips and uptake of colloidal carbon. The number of macrophages never exceeded 1%. Also, this procedure has previously been shown to eliminate all T-cell helper function and T-specific mitogen responses (14). The concentration of purified cells was approximately 1 X lo6 cells/culture. The cultures were prepared according to the method of Mishell and Dutton (17) using a medium described by Click et al. ( 18) which does not require daily supplement. All experiments were performed using at least four cultures per group, and the results are reported as the mean ? standard error. Estimation of statistical significance was performed using a two-tailed t test; values of P > 0.1 are regarded as not significant. The primary in vitro antibody response was measured after 4 days of culture using the hemolytic plaque assay as modified by Mishell and Dutton (17). Mitomycin C ~r~ntmen~. Mitomycin C (Mit. C), (Sigma) was added to PEC or spleen cells at concentrations of 40 &ml for 30 min. The PEC were then washed three times in HBSS and cultured for 96 hr. Such a procedure blocks more than 90% of DNA synthesis after 96 hr of culture. RESULTS
Effect of inhibitors of PG synthesis on the uppearance of anti-Hb-antibodyforming cells in vitro. Previous studies in this laboratory had established that inhibitors of PG synthesis could alter the IgM response to T-dependent and T-independent antigens (14, IS). In Table 1 are shown the results of several experiments in which spleen cells or PEC from two strains of mice (C57B1/6J and C,WHeJ) were exposed to the PG synthesis inhibitor Ro-20-5720 for 4 days in culture and then assayed for anti-Hb PFC. As may be seen, in the presence of the
EFFECT OF Ro-20-5720
TABLE I ON THE APPEARANCE
OF Amr-Hb
PFCi IO6 recovered Mice C57B 1165 Control Ro-20-5720,
IO-”
CsH/HeJ Control Ro-20-5720,
IO-” M
M
PFC cells”
Spleen
Peritoneal
33 -t 1.4 58 2 4.6 P < 0.1
6,052 10,539
exudate
lr 3.50 I 616 P i
cells
0.02
667 2 22 1,157 -t 81 P < 0.05
-
” The data are reported as the means of the number error. Mice used were C57B1/6J males, 6- 10 weeks
in Vitro
of PFC from old.
quadruplicate
cultures
? standard
PROSTAGLANDIN
CONTROL
OF ANTI-Hb
423
RESPONSE
inhibitor, the number of anti-Hb PFC is increased in both strains of mice in both the spleen cell cultures and PEC cultures. No PFC to unmodified MRBC could be detected (data not shown). That this is not an artifact due to the type of inhibitor used is demonstrated by an experiment in which three different inhibitors of PG synthesis were used. As may be seen in Table 2, both Ro-3-1314 and Ro-2@5720 are comparable in their ability to enhance the appearance of anti-Hb PFC. In this particular experiment, indomethatin is even more effective in stimulating the appearance of anti-Hb PFC in peritoneal cell cultures.
EFFECT
OF THREE
DIFFERENT
TABLE 2 PG SYNTHESIS ON PEC CULTURES IN C57Bl16J
INHIBITORY IN
OF
Mice
THE
APPEARANCE
OF
ANTI-Hb PFC
MICE
PFC/ lo6 recovered PEC” 606 1,274 1,319 3,069
Control Ro-3-1314, 1O-6 M Ro-20-5720, lO-6 M Indomethacin, lo-” M UThe data are reported as the mean of quadruplicate
f f 2 f
17 81, P <. 0.1 59, P c 0.05 66, P -c: 0.02
cultures 2 standard error.
Effect of inhibitors of PG synthesis on the appearance of anti-H6 PFC in NZB mice. Since anti-Hb antibody activity was originally reported in NZB mice (8), we determined whether or not the PG synthetase inhibitors exerted any effect in this strain. In the experiment shown in Table 3, NZB mouse PEC were exposed to Ro-2@5720 ( lop6 M) at 0,24,48 or 72 hr after culture initiation and the cultures were assayed after 96 hr. The results show that roughly equal stimulation of anti-Hb PFC occurs when the inhibitor is added 0, 24, or 48 hr after culture initiation. Cultures receiving the inhibitor at 72 hr demonstrated little enhancement. Effect of PG synthetase inhibitor on different subpopulations. Studies with T-dependent and T-independent antigens had suggested that both T cells and B cells
TABLE EFFECT
OF
Ro-20-5720
ON THE
APPEARANCE
CULTURES
Mice Control Ro-20-5720 (lo-” M)
FROM
3 ANTI-Hb PFC J-MONTH-OLD NZB OF
Time of addition (hr) 0 24 48 72
IN PERITONEAL
EXUDATE
CELL
MICE
PFC/106 recovered PE cell@ 766 1,455 1,479 1,851 931
zk 45 (939-574) 2 80 (1940-1272) P < 0.05 -r- 141 (1963-1143) P < 0.05 -t 121 (2500-1323) P < 0.05 -+ 67 (1275-700) P < 0.30
0 The data are reported as the mean from quadruplicate cultures 2 standard error. The values in parentheses represent the range of PFC/106 values obtained. The MRBC were treated with bromelain as outlined in Materials and Methods.
424
ZIMECKI
AND
WEBB
were capable of making and responding to PG (14, 15). In the experiment shown in Table 4, PEC from C57B1 mice were depleted of macrophages and T cells, and the effect of Ro 20-5720 on the appearance of anti-HB PFC was ascertained. The results show that depletion alone enhances the appearance of anti-Hb PFC, and exposure of depleted cultures to PG synthetase inhibitor even further enhances the appearance of anti-Hb PFC.
TABLE EFFECT
OF MACROPHAGE
AND
T-CELL
DEPLE-HON EXPOSED
Mice Control Ro-20-5720 lo-” h4
4 ON THE
TO
Type of cells Whole “B” Whole “B”
A~PEARWCE
OF AN.rr-Hb PFC IN PEC
Ro-20-5720
PEC cells PEC cells
PFCI IO6 PEC? 1064 -c 78 1680 _c 114 2018 t 433, P < 0.05 319.5 -r 305, P < 0.05
RThe data are reported as the mean of triplicate cultures k standard error. C57B116J mice were used.
Effect of mitomycin C and prostaglandin on the appearance of anti-Hb PFC in PEC and spleen cells exposed to indomethacin. The enhancing effect observed with inhibitors of PG synthetase raises the question as to whether the enhancement represents a derepression of preexisting anti-Hb clones or is affecting clonal expansion. In the experiments shown in Table 5, PEC or spleen cell cultures were exposed to mitomycin C (Mit.C) prior to culture with indomethacin. The Mit.C prevents any increased DNA synthesis and, thus, the effect of indomethacin on the appearance of anti-Hb PFC may be measured in the absence of proliferation. The results show that in Mit.C-treated PEC cultures indomethacin still increases the number of anti-Hb PFC cultures when compared to cultures receiving only Mit.C. In the PEC, Mit.C exposure alone increases the number of PFC against Hb as measured per lo6 recovered cells. This is due to a reduction in the number of ceils per culture without a comparable reduction in the number of PFC. However, the number of PFC per culture in Mit.C-treated PEC cultures is less than that observed in the untreated controls. In the spleen cell cultures, Mit.C has no discernible effect on cell recovery while 90% of the DNA synthesis is inhibited. Although the number of PFC is low, these results do support the observations made with reference to the PEC; Mit.C does not block the ability of indomethacin to enhance the number of anti-Hb PFC even though Mit.C alone inhibits the appearance of splenic PFC, or reduces the cell recovery (with less reduction in DNA synthesis) in the PEC. The data presented so far have dealt with effects of inhibitors of PG synthesis. Since these results show that inhibiting PG synthesis increases the number of anti-Hb PFC, an experiment was performed to see whether or not PG added
+
-
+
66.2 69.8 77.6 71.6
IN
PEC
6
69~ 262 117-c 762
3 3 8 8
902 1 322 % 5 195 ? 16
143?
PFCYculture
5 PFC AND
EXPOSED
0.1 .OOl
14973 656 14320 593
36
108
82 43
It 703 '55 +- 1479 2 115
1884 * 1255 f
< 0.1
2162 '1100"
CPM/culture”
TO INDOMETHACIN
< 0.001
2 0.7 3, P < 2, P <
f 60 k 16 2 17,P f 233, P
PFC/lO” cellsb
CULTURES
232 7 f 31 + 20 f
652 921 1282 2047
CELL
LIExpressed as cells/culture x 10m5. PEC cultures were initiated at a concentration of 1 x lo6 PEUculture; spleen ceII cultures were initiated at a concentration of 15 x IOVculture. Mice used were C57B1165 males, 6- 10 weeks old. Indomethacin was added at the time of culture initiation. b The data are reported as the mean of triplicate cultures * standard error. ’ Cultures were pulsed with [3H]Tdr for 4 hr and then precipitated in cold 10% trichloroacetic acid (TCA). The TCA precipitates were washed in TCA and then resuspended in 0.5 N NaOH and assayed for radioactivity. The data are reported as the mean counts per minute per culture f standard error.
Indomethacin ( 1O-6 M)
Spleen cells Control
+
0.99 2.43 0.99
Indomethacin ( 1O-6 M)
2.31
+
TABLE OF AivrI-Hb
Cell recovery”
APPEARANCE
-
ON THE
PEC Control
C (Mit.C)
Mit.C
OF MITOMYCIN
Cultures
EFFECT
B g 2. z
5
?
$
i
3 g
2
f! E
$ 0 3 : F
426
ZIMECKI
AND
WEBB
exogenously will block the appearance of anti-Hb PFC. In Table 6, the results show that the addition of PGE, reduces by 50% the number of PFC. The addition of PGF,, also reduces the number of PFC but to a lesser degree (37%). This reduction does not appear to be due to an alteration in cell number since cell recovery is comparable to that obtained in the control cultures. It should be noted that in experiments reported elsewhere (15) the exogenous addition of PG in the presence of a prostaglandin synthetase inhibitor has little effect on the ability of PG to suppress the immune response. TABLE THE EFFECT OF PGE,
Culture Control PGF,, (IO-‘M) PGE, (IO-” M)
AND PGF,,
6
ON THE APPEARANCE
Cell recover9
OF
ANTI-Hb PFC IN PEC CUI.I,UKES”
PFCiculture
2.31 2.30 2.39
143 5 6 93 + 9 75 '- 5
PFU lo6 PEC 652 f 60 411 t 42,P
a Cultures were prepared in triplicate from C57BMJ mice, 6- 10 weeks old. at a concentration of 1 x 106 PEG/culture. The data are reported as the mean 2 standard error. PC additions were made at the time of culture initiation. b Expressed as cells per culture x 105.
DISCUSSION
These data show that, when the synthesis of prostaglandin is inhibited in cultured spleen cells or PEC, the number of anti-Hb PFC is increased. Furthermore, at least one of the controlling cell populations appears to consist of B cells. Finally, the prostaglandins appear to exert their action by controlling the expression of anti-Hb clones. Tolerance to self-antigens is one of the basic requirements for any immune system. How tolerance is achieved has been a topic of considerable controversy (19, 20). That immunocompetent cells exist, in otherwise normal mice, which are capable of responding to self-antigens has been established (a), at least concerning autologous erythrocyte antigens. The existence of such clones requires that there be a means for controlling their expression. One possible mechanism would be some kind of a suppressor cell-mediated control. Indeed, the evidence of Cunningham (12), employing ALS to enhance the appearance of anti-Hb PFC, is consistent with such a hypothesis. However, the evidence presented by Lord and Dutton (9, 10) suggested that a direct effect of the antigen itself on the autoantibody-forming cells could not be ruled out. Thus, the question remains as to the nature of the controlling mechanism(s) which operate to regulate the expression of anti-Hb responses. Recently, we have described evidence suggesting that PG plays a large role in the nonspecific regulation of immune response for both T-dependent and T-independent antigens (13,15). In the case ofthe T-independent antigen, polyvinyl pyrolidone, it was possible to demonstrate two kinds of control; one involving the autoregulation of B cells via prostaglandin, and a second controlling mechanism
PROSTAGLANDIN
CONTROL
OF ANTI-Hb
RESPONSE
427
involving T cells which was not dependent on PG (15). The data presented in this report are compatible with the idea that a similar kind of control system is operative in the anti-Hb response. Clearly, the removal of T cells from PEC enhances the appearance of anti-Hb PFC. This may be the result of simple enrichment of responding B cells, rather than the removal of a specific T suppressor cell. And, in fact, the data of Lord and Dutton (10) support the notion that T cells are not required for the induction of anti-Hb clones. Even more enhancement occurs if, in addition to the removal of T cells, PG synthesis inhibitors are added. This implies that 0 antigen-bearing T cells may not be required in order for PG to regulate anti-Hh responses and suggests that the B cells may regulate themselves. This is, in fact, what was found in studies involving the T-independent antigen polyvinyl prolidone (15). Furthermore, in experiments to be reported (Zimecki and Webb, submitted for publication) it is clear than anti-8 treatment of glass-wool-purified B cells removes all suppressor T-cell activity. Thus, it is unlikely that suppressor T cells are present in either our PEC or B-cell preparations. These results do not conflict with the evidence presented by Cunningham (12), but rather suggest that there exist several levels of control in both B-cell and T-B-cell interactions. In fact, the antilymphocyte serum experiments reported by Cunningham do not exlude the possible autoregulatory influence of B cells. . In addition to identifying the nature of at least one of the controlling substances, the question remained concerning the mechanism by which the control operates. The E series PG has been reported by several workers (3, 22, 23) both to have antiproliferative properties and also to block effector-cell functions such as the release of antibody, possibly via an alteration of cyclic AMP levels (3,2 1,24). The results presented in this report are compatible with these observations in that by blocking proliferation with Mit.C in either PEC or spleen cell cultures it is still possible to enhance the anti-Hb responses by exposure to PG synthetase blockers. This suggests that by blocking the synthesis of PG we may be affecting the ability of anti-Hb B cells to make antibody. In this regard, it should be noted that in cultures exposed to prostaglandin synthetase inhibitors, the inhibition of PG synthesis persists for 96 hr at a level of 20-50% of untreated control cultures (Webb and Nowowiejski, submitted for publication). It should also be noted that Mit.C treatment alone appears to enhance the number of PFC per 10” in PEC, although the number of PFC per culture is actually reduced. This raises the possibility that Mit.C may be toxic for certain PEC but not for the majority of the antibody-forming cells since a good response is still present. The data provided by the spleen cell culture experiment are consistent with the data obtained using PEC, although in spleen cells, Mit.C treatment causes a reduction in the number of PFC without a corresponding reduction in cell number. Two explanations are possible. The first, that Mit.C may be toxic for antibody-forming cells, seems unlikely since in PEC cultures antibody-forming cells are not affected. The second possibility is that some proliferation of anti-Hb clones is necessary in the spleen and is blocked by Mit.C exposure. That some proliferation could be involved in the development of anti-Hb clones is suggested by the data obtained using PGE, and PGF,,. The PGs are biologically active for only a short time and must, therefore, exert their effects very soon after addition. The inhibition observed by adding PG at the time of culture
428
ZIMECKI
AND
WEBB
initiation suggests an effect on a very early stage in development of anti-Hb clones. This might involve the metabolic events which precede and are required for proliferation, although other steps in the differentiation pathway, which do not involve DNA synthesis, may also be involved. The time course experiment using NZB mice is important in this regard. Lord and Dutton (9) have shown that 48 hr are required for the increases in anti-Hb PFC to reach minimum levels. Our data show that adding inhibitors of PG synthesis after 48 hr has less effect when the response is measured at 96 hr. This suggests that whatever metabolic event is sensitive to a block of PG synthesis, at least 48 hr must pass before clear-cut enhancement is evident. Thus, the PG synthetase inhibitors could be exerting effects on metabolic processes necessary for both expression, and possibly proliferation, of the relevant effector cells. It is also possible that anti-Hb cells in the spleen are blocked at a different stage of differentiation than the anti-Hb PEC. Thus, in both cases PG could exert an influence but on different stages of development, e.g., on both differentiation and expression of effector-cell function. The data we have presented here and elsewhere (13-15) imply a role for the prostaglandins in the regulation of a variety of immunocompetent cell functions. Others (23) have also suggested that the prostaglandins play a role in the modulation of inflammatory events. The regulation of cellular events via prostaglandin is of course not unique to the immune system since prostaglandins have been shown to act in a number of tissues (25). The fact that these compounds appear so ubiquitously but act on specific cells or at specific times may be attributed to their short biological and chemical half-life which makes them ideal as intra- tissue or -organ messengers. The specificity of their effects may be related to the modulation of receptor activity or the activity of intermediate enzymes activated by prostaglandins. The precise relationship between the prostaglandins and other regulators of lymphocyte function remains to be determined. Clearly, as we have alluded to earlier, the prostaglandins do not constitute the sole regulatory influence on the spontaneous anti-Hb response. DeHeer and Edington (26) have shown that T cells also possess anti-HB receptors, and Cunningham (12) has provided evidence that T cells may exert control on the appearance of anti-Hb antibody-forming cells. Also, the possibility of direct antigen feedback control has been demonstrated by Lord and Dutton (10). Thus, several regulatory mechanisms may act in concert to control the expression of all potentially autoimmune immunocompetent cells. REFERENCES I. Waksman, B. H., Namba. Y.. Cell. I~nmunol. 21. 161. 1976. 2. Geshon. R., In “Contemporary Topics in Immunobiology” (M. D. Cooper and N. L. Warner, Eds.), Vol. 3. pp. I-35. Plenum Press, New York. 1973. 3. Bomne, H. R., Lichtenstein. L. M.. Melmon, K. L.. Henney . C. S., Weinstein. Y.. and Shearer, G. M., Science 184, 19, 1974. 4. Melmon, K. L.. Weinstein. Y.. Shearer, G. M., and Bourne, H. R., In “Cyclic AMP, Cell Growth. and the Immune Response” (W. Braun, L. M. Lichtenstein, and C. W. Parker, Eds.), pp. 114-134. Springer-Verlag, New York. 1973. 5. Osheroff, P. L., Webb, D. R., and Paulsrud, J., Biochrm. Biophys. Rrs. Commun. 66,425, 1975. 6. Munro. A. J., and Taussig, M. J.. Nature (London) 256, 103, 1975. 7. Plate. J. M. D., Nature (London) 260, 329. 1976. 8. Linder, E.. and Edgington, T. S., J. Immunol. 108, 1615, 1972. 9. Lord, E. M., and Dutton, R. W.. J. Immunol. 115, 1199. 1975.
PROSTAGLANDIN
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
CONTROL
OF
ANTI-Hb
RESPONSE:
429
Lord, E., and Dutton, R. W.,.l. Immunol. 115, 1631, 1975. DeHeer, D., and Edington, T. S., J. Immunol. 113, 1184, 1974. Cunningham, A. J., Nature (London) 254, 143, 1975. Webb, D. R., and Osheroff, P. L., Proc. Nat. Acad. Sci. USA 73, 1300, 1975. Webb, D. R., Jamieson, A. T., and Nowowiejski, I., Cell Zrnmunol. 24, 45, 1976. Zimecki, M., and Webb, D. R., J. Zmmunol. 117, 2158, 1976. Grant, V. N., Baruth, H., Randall, L. 0.. Ashley, C., and Paulrud, J. R., Prostaglandins 10, 59, 1975. Mishell, R. I., and Dutton, R., J. Exp. Med. 126, 423, 1967. Click, R. E., Benck, L., and Alter, B. J., Cell. Zmmunol. 3, 264, 1972. Burnet, M., In “The Clonal Selection Theory of Acquired Immunity.” Vanderbilt Univ. Press, Nashville, Tennessee, 1959. Katz, D. H., and Benacerraf, B., In “Immunological Tolerance, Mechanisms and Potential Therapeutic Applications” (D. H. Katz and B. Benacerraf, Eds.). Academic Press, New York, 1974. Smith, J. N., Steiner, A. L., Newberry, W. M., and Parker, C. W., J. C/in. Invest. JO, 432, 1971. DeRubertis, F. R., Zenser, T. V., Adler, W. H., and Hudson, T., J. Zmmunol. 113, 151, 1974. Bach, M. A., J. Clin. Invest. 5.5, 1074, 1975. Boume, H. R., Lichtenstein, L. M., Melmon, K. L., Henney, C. S., Weinstein, Y., and Shearer, G. M., Science 184, 19, 1974. Samuelson, B, Granstrom, E., Green, K., Hamberg, M., and Hammarstroem, S., In “Annual Reviews of Biochemistry” (E. E. Snell, Ed.), Vol. 44, pp. 669. Annual Reviews Inc., Palo Alto, California. DeHeer, D. H., Edgington, T. S., J. Zmmunol. 116, 1051, 1976.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
8,42w29
( 1977)
The Role of Prostaglandins in the Control of the Immune Response to an Autologous Red Blood Cell Antigen (Hb) MIGHAL
ZIMECKI’,”
AND
DAVID
R.
WEBB,
Department of Cell Biology. Roche Institute 41‘ Molrc~rlrr New lersev 07110 Received November 13. 1976
JR.~ Biology. Nutle~.
When spleen cell or peritoneal exudate cell cultures are exposed to inhibitors of prostaglandin synthetase, the number of spontaneous, anti-autologous erythrocyte antigen (Hb)antibody-forming cells is increased. This result is obtained both in C57B l/65 mice as well as in NZB mice. The cell cultures require 24-48 hr exposure to the prostaglandin synthetase inhibitors before their effects are observed. Treatment of spleen cell or peritoneal exudate cell cultures with mitomycin C blocks DNA synthesis but has no effect on the enhancement of the anti-Hb response induced by the prostaglandin synthetase inhibitors. This suggests that the prostaglandins play a role in repressing or controlling the expression of certain autoimmune clones.
INTRODUCTION
In recent years, increased attention has been directed toward understanding the nature of cellular control mechanisms which function in immunocompetent cells (1, 2). It has become clear that immunocompetent cells possess a large array of receptors other than immunoglobulins which may control the way these cells function (3,4). Some of these control functions have been shown to operate when the immune system is challenged by “non-self’ antigens (5-7). With the report by Linder and Edgington (8) that both NZB mouse strains as well as others possess the capacity to spontaneously respond to both exposed and/or unexposed autologous erythrocyte antigens, it has become possible to investigate the nature of the controls which operate in “anti-self’ immune responses. The two antigens in question have been termed X and Hb. The X antigen is found on the surface of mouse erythrocytes and only NZB mice appear to have anti-X-antibody-forming cells (8). The Hb antigen is not exposed and is only revealed by treating mouse erythrocytes with the proteolytic enzyme bromylase (8). Virtually all strains of mice tested have been shown to possess spontaneous anti-Hb-antibody-forming cells in the spleen and in the peritoneum. In culture, the number of anti-Hb-antibody-forming cells increases in the absence of any added antigen (9, 10). More recent investigations by De Heer and Edgington have suggested that the development of the anti-Hb response is a function of altered immunoregulatory influences (11). In addition,
1 Recipient of a Fulbright-Hays Fellowship. * Dr. Michal Zimecki is on leave from the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. 3 To whom all correspondence should be addressed. 420 Copynghr 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
PROSTAGLANDIN
CONTROL
OF ANTI-Hb
RESPONSE
421
Cunningham has reported that animals treated with antilymphocyte serum had increased numbers of splenic PFC to the Hb antigen (12). It has recently been found in our laboratory that prostaglandins play a role in the nonspecific regulation of lymphocyte effector-cell function at both the T- and B-cell level (13, 14, 15). Therefore, it was of interest to investigate whether or not prostaglandins might also be involved in the control of autoimmune responses. The experiments presented in this report suggest that prostaglandins do play a role in regulating the autoimmune response to Hb antigen. MATERIALS
AND METHODS
Animals. C57B1/6J male mice, 6-10 weeks old, were purchased from Jackson Laboratories (Bar Harbor, Maine). The NZB mice were obtained from Dr. Norman Talal, University of California. In each experiment, pools of spleens or peritoneal exudates from at least 10 mice were used to prepare the cultures. Antigens. Mouse erythrocytes (MRBC) were obtained by orbital bleeding under ether anesthesia and collected in 0.1 M sodium citrate solution. Prostaglandins and PG synthesis inhibitors. Prostaglandins E, and Fzo,(PGE, and PGF& were obtained from Hoffmann-LaRoche Inc. and were stored at -20°C in 95% ethanol. Immediately prior to use, they were diluted with medium to the appropriate concentrations. Control cultures received medium plus ethanol at the appropriate concentrations. The concentrations used (1 O-5 M) had previously been shown to be optimal for inhibiting the appearance of antibody-forming cells to sheep erythrocytes and polyvinyl pyrolidone (13, 15). At these concentrations, PGE, gives optimal elevation of cyclic AMP in whole spleen cells and thymocytes, whereas PGFzo, has no effect on cyclic AMP or cyclic GMP levels (D. Webb, unpublished observations). Indomethacin, an irreversible inhibitor of PG synthesis was purchased from Sigma (St. Louis, MO.). ru-6-Chloro-cY-methyl-carbazole-2acetic acid (Ro-20-5720), a competitive reversible inhibitor of prostaglandin synthesis, and octadeca-9-12, diynoic acid (Ro-3-1314), an irreversible inhibitor of prostaglandin synthesis, were obtained from Hoffmann-LaRoche Inc. (Nutley, N.J.). The action of the Ro compounds is limited to the inhibition of prostaglandin synthesis so far as is known (16). All inhibitors were prepared in 7.5% carbonate buffer (GIBCO) and diluted with complete medium before addition. Control cultures received appropriately diluted medium without inhibitors. The dilutions of synthetase inhibitors used in these experiments had previously been shown to be optimal for the inhibition of prostaglandin synthesis (13, 14). Bromelain-RBC preparation. MRBC were washed three times with Hank’s balanced salt solution (HBSS); equal volumes of packed erythrocytes were incubated with bromelase (Bromelain, Dade Reagents, Inc., Miami, Fla.) at 37°C for 30 min. After incubation, MRBC were washed three times with HBSS (8). Preparation of spleen cells. Whole spleen cell suspensions were prepared from spleens which were minced, filtered through a stainless-steel screen, and washed two times with HBSS. Preparation of peritoneal exudate cells and cell purification. Peritoneal exudate cells (PEC) were collected by washing the peritoneal cavity of mice with 2 ml of precooled HBSS using a sterile Pasteur pipette. Cells were washed three times with HBSS before suspending them in culture medium.
422
ZIMECKI
AND
WEBB
Purified “B” cells were prepared from PEC as follows: The PEC were suspended in 20 ml of MEM containing 10% fetal calf serum and poured onto 100 mm petri dishes. Following 30 to 40-min incubations at 37°C the nonadherent cells were decanted and treated with ATS (1:20 dilution) plus guinea pig complement (1:20 dilution). The preparation of the antithymocyte serum (ATS) and its properties have been previously reported (15). After 30-min incubation at 37°C the cells were washed three times in HBSS, counted, and cell viability was measured by use of trypan blue. The presence of macrophages was assayed by spreading on glass coverslips and uptake of colloidal carbon. The number of macrophages never exceeded 1%. Also, this procedure has previously been shown to eliminate all T-cell helper function and T-specific mitogen responses (14). The concentration of purified cells was approximately 1 X lo6 cells/culture. The cultures were prepared according to the method of Mishell and Dutton (17) using a medium described by Click et al. ( 18) which does not require daily supplement. All experiments were performed using at least four cultures per group, and the results are reported as the mean ? standard error. Estimation of statistical significance was performed using a two-tailed t test; values of P > 0.1 are regarded as not significant. The primary in vitro antibody response was measured after 4 days of culture using the hemolytic plaque assay as modified by Mishell and Dutton (17). Mitomycin C ~r~ntmen~. Mitomycin C (Mit. C), (Sigma) was added to PEC or spleen cells at concentrations of 40 &ml for 30 min. The PEC were then washed three times in HBSS and cultured for 96 hr. Such a procedure blocks more than 90% of DNA synthesis after 96 hr of culture. RESULTS
Effect of inhibitors of PG synthesis on the uppearance of anti-Hb-antibodyforming cells in vitro. Previous studies in this laboratory had established that inhibitors of PG synthesis could alter the IgM response to T-dependent and T-independent antigens (14, IS). In Table 1 are shown the results of several experiments in which spleen cells or PEC from two strains of mice (C57B1/6J and C,WHeJ) were exposed to the PG synthesis inhibitor Ro-20-5720 for 4 days in culture and then assayed for anti-Hb PFC. As may be seen, in the presence of the
EFFECT OF Ro-20-5720
TABLE I ON THE APPEARANCE
OF Amr-Hb
PFCi IO6 recovered Mice C57B 1165 Control Ro-20-5720,
IO-”
CsH/HeJ Control Ro-20-5720,
IO-” M
M
PFC cells”
Spleen
Peritoneal
33 -t 1.4 58 2 4.6 P < 0.1
6,052 10,539
exudate
lr 3.50 I 616 P i
cells
0.02
667 2 22 1,157 -t 81 P < 0.05
-
” The data are reported as the means of the number error. Mice used were C57B1/6J males, 6- 10 weeks
in Vitro
of PFC from old.
quadruplicate
cultures
? standard
PROSTAGLANDIN
CONTROL
OF ANTI-Hb
423
RESPONSE
inhibitor, the number of anti-Hb PFC is increased in both strains of mice in both the spleen cell cultures and PEC cultures. No PFC to unmodified MRBC could be detected (data not shown). That this is not an artifact due to the type of inhibitor used is demonstrated by an experiment in which three different inhibitors of PG synthesis were used. As may be seen in Table 2, both Ro-3-1314 and Ro-2@5720 are comparable in their ability to enhance the appearance of anti-Hb PFC. In this particular experiment, indomethatin is even more effective in stimulating the appearance of anti-Hb PFC in peritoneal cell cultures.
EFFECT
OF THREE
DIFFERENT
TABLE 2 PG SYNTHESIS ON PEC CULTURES IN C57Bl16J
INHIBITORY IN
OF
Mice
THE
APPEARANCE
OF
ANTI-Hb PFC
MICE
PFC/ lo6 recovered PEC” 606 1,274 1,319 3,069
Control Ro-3-1314, 1O-6 M Ro-20-5720, lO-6 M Indomethacin, lo-” M UThe data are reported as the mean of quadruplicate
f f 2 f
17 81, P <. 0.1 59, P c 0.05 66, P -c: 0.02
cultures 2 standard error.
Effect of inhibitors of PG synthesis on the appearance of anti-H6 PFC in NZB mice. Since anti-Hb antibody activity was originally reported in NZB mice (8), we determined whether or not the PG synthetase inhibitors exerted any effect in this strain. In the experiment shown in Table 3, NZB mouse PEC were exposed to Ro-2@5720 ( lop6 M) at 0,24,48 or 72 hr after culture initiation and the cultures were assayed after 96 hr. The results show that roughly equal stimulation of anti-Hb PFC occurs when the inhibitor is added 0, 24, or 48 hr after culture initiation. Cultures receiving the inhibitor at 72 hr demonstrated little enhancement. Effect of PG synthetase inhibitor on different subpopulations. Studies with T-dependent and T-independent antigens had suggested that both T cells and B cells
TABLE EFFECT
OF
Ro-20-5720
ON THE
APPEARANCE
CULTURES
Mice Control Ro-20-5720 (lo-” M)
FROM
3 ANTI-Hb PFC J-MONTH-OLD NZB OF
Time of addition (hr) 0 24 48 72
IN PERITONEAL
EXUDATE
CELL
MICE
PFC/106 recovered PE cell@ 766 1,455 1,479 1,851 931
zk 45 (939-574) 2 80 (1940-1272) P < 0.05 -r- 141 (1963-1143) P < 0.05 -t 121 (2500-1323) P < 0.05 -+ 67 (1275-700) P < 0.30
0 The data are reported as the mean from quadruplicate cultures 2 standard error. The values in parentheses represent the range of PFC/106 values obtained. The MRBC were treated with bromelain as outlined in Materials and Methods.
424
ZIMECKI
AND
WEBB
were capable of making and responding to PG (14, 15). In the experiment shown in Table 4, PEC from C57B1 mice were depleted of macrophages and T cells, and the effect of Ro 20-5720 on the appearance of anti-HB PFC was ascertained. The results show that depletion alone enhances the appearance of anti-Hb PFC, and exposure of depleted cultures to PG synthetase inhibitor even further enhances the appearance of anti-Hb PFC.
TABLE EFFECT
OF MACROPHAGE
AND
T-CELL
DEPLE-HON EXPOSED
Mice Control Ro-20-5720 lo-” h4
4 ON THE
TO
Type of cells Whole “B” Whole “B”
A~PEARWCE
OF AN.rr-Hb PFC IN PEC
Ro-20-5720
PEC cells PEC cells
PFCI IO6 PEC? 1064 -c 78 1680 _c 114 2018 t 433, P < 0.05 319.5 -r 305, P < 0.05
RThe data are reported as the mean of triplicate cultures k standard error. C57B116J mice were used.
Effect of mitomycin C and prostaglandin on the appearance of anti-Hb PFC in PEC and spleen cells exposed to indomethacin. The enhancing effect observed with inhibitors of PG synthetase raises the question as to whether the enhancement represents a derepression of preexisting anti-Hb clones or is affecting clonal expansion. In the experiments shown in Table 5, PEC or spleen cell cultures were exposed to mitomycin C (Mit.C) prior to culture with indomethacin. The Mit.C prevents any increased DNA synthesis and, thus, the effect of indomethacin on the appearance of anti-Hb PFC may be measured in the absence of proliferation. The results show that in Mit.C-treated PEC cultures indomethacin still increases the number of anti-Hb PFC cultures when compared to cultures receiving only Mit.C. In the PEC, Mit.C exposure alone increases the number of PFC against Hb as measured per lo6 recovered cells. This is due to a reduction in the number of ceils per culture without a comparable reduction in the number of PFC. However, the number of PFC per culture in Mit.C-treated PEC cultures is less than that observed in the untreated controls. In the spleen cell cultures, Mit.C has no discernible effect on cell recovery while 90% of the DNA synthesis is inhibited. Although the number of PFC is low, these results do support the observations made with reference to the PEC; Mit.C does not block the ability of indomethacin to enhance the number of anti-Hb PFC even though Mit.C alone inhibits the appearance of splenic PFC, or reduces the cell recovery (with less reduction in DNA synthesis) in the PEC. The data presented so far have dealt with effects of inhibitors of PG synthesis. Since these results show that inhibiting PG synthesis increases the number of anti-Hb PFC, an experiment was performed to see whether or not PG added
+
-
+
66.2 69.8 77.6 71.6
IN
PEC
6
69~ 262 117-c 762
3 3 8 8
902 1 322 % 5 195 ? 16
143?
PFCYculture
5 PFC AND
EXPOSED
0.1 .OOl
14973 656 14320 593
36
108
82 43
It 703 '55 +- 1479 2 115
1884 * 1255 f
< 0.1
2162 '1100"
CPM/culture”
TO INDOMETHACIN
< 0.001
2 0.7 3, P < 2, P <
f 60 k 16 2 17,P f 233, P
PFC/lO” cellsb
CULTURES
232 7 f 31 + 20 f
652 921 1282 2047
CELL
LIExpressed as cells/culture x 10m5. PEC cultures were initiated at a concentration of 1 x lo6 PEUculture; spleen ceII cultures were initiated at a concentration of 15 x IOVculture. Mice used were C57B1165 males, 6- 10 weeks old. Indomethacin was added at the time of culture initiation. b The data are reported as the mean of triplicate cultures * standard error. ’ Cultures were pulsed with [3H]Tdr for 4 hr and then precipitated in cold 10% trichloroacetic acid (TCA). The TCA precipitates were washed in TCA and then resuspended in 0.5 N NaOH and assayed for radioactivity. The data are reported as the mean counts per minute per culture f standard error.
Indomethacin ( 1O-6 M)
Spleen cells Control
+
0.99 2.43 0.99
Indomethacin ( 1O-6 M)
2.31
+
TABLE OF AivrI-Hb
Cell recovery”
APPEARANCE
-
ON THE
PEC Control
C (Mit.C)
Mit.C
OF MITOMYCIN
Cultures
EFFECT
B g 2. z
5
?
$
i
3 g
2
f! E
$ 0 3 : F
426
ZIMECKI
AND
WEBB
exogenously will block the appearance of anti-Hb PFC. In Table 6, the results show that the addition of PGE, reduces by 50% the number of PFC. The addition of PGF,, also reduces the number of PFC but to a lesser degree (37%). This reduction does not appear to be due to an alteration in cell number since cell recovery is comparable to that obtained in the control cultures. It should be noted that in experiments reported elsewhere (15) the exogenous addition of PG in the presence of a prostaglandin synthetase inhibitor has little effect on the ability of PG to suppress the immune response. TABLE THE EFFECT OF PGE,
Culture Control PGF,, (IO-‘M) PGE, (IO-” M)
AND PGF,,
6
ON THE APPEARANCE
Cell recover9
OF
ANTI-Hb PFC IN PEC CUI.I,UKES”
PFCiculture
2.31 2.30 2.39
143 5 6 93 + 9 75 '- 5
PFU lo6 PEC 652 f 60 411 t 42,P
a Cultures were prepared in triplicate from C57BMJ mice, 6- 10 weeks old. at a concentration of 1 x 106 PEG/culture. The data are reported as the mean 2 standard error. PC additions were made at the time of culture initiation. b Expressed as cells per culture x 105.
DISCUSSION
These data show that, when the synthesis of prostaglandin is inhibited in cultured spleen cells or PEC, the number of anti-Hb PFC is increased. Furthermore, at least one of the controlling cell populations appears to consist of B cells. Finally, the prostaglandins appear to exert their action by controlling the expression of anti-Hb clones. Tolerance to self-antigens is one of the basic requirements for any immune system. How tolerance is achieved has been a topic of considerable controversy (19, 20). That immunocompetent cells exist, in otherwise normal mice, which are capable of responding to self-antigens has been established (a), at least concerning autologous erythrocyte antigens. The existence of such clones requires that there be a means for controlling their expression. One possible mechanism would be some kind of a suppressor cell-mediated control. Indeed, the evidence of Cunningham (12), employing ALS to enhance the appearance of anti-Hb PFC, is consistent with such a hypothesis. However, the evidence presented by Lord and Dutton (9, 10) suggested that a direct effect of the antigen itself on the autoantibody-forming cells could not be ruled out. Thus, the question remains as to the nature of the controlling mechanism(s) which operate to regulate the expression of anti-Hb responses. Recently, we have described evidence suggesting that PG plays a large role in the nonspecific regulation of immune response for both T-dependent and T-independent antigens (13,15). In the case ofthe T-independent antigen, polyvinyl pyrolidone, it was possible to demonstrate two kinds of control; one involving the autoregulation of B cells via prostaglandin, and a second controlling mechanism
PROSTAGLANDIN
CONTROL
OF ANTI-Hb
RESPONSE
427
involving T cells which was not dependent on PG (15). The data presented in this report are compatible with the idea that a similar kind of control system is operative in the anti-Hb response. Clearly, the removal of T cells from PEC enhances the appearance of anti-Hb PFC. This may be the result of simple enrichment of responding B cells, rather than the removal of a specific T suppressor cell. And, in fact, the data of Lord and Dutton (10) support the notion that T cells are not required for the induction of anti-Hb clones. Even more enhancement occurs if, in addition to the removal of T cells, PG synthesis inhibitors are added. This implies that 0 antigen-bearing T cells may not be required in order for PG to regulate anti-Hh responses and suggests that the B cells may regulate themselves. This is, in fact, what was found in studies involving the T-independent antigen polyvinyl prolidone (15). Furthermore, in experiments to be reported (Zimecki and Webb, submitted for publication) it is clear than anti-8 treatment of glass-wool-purified B cells removes all suppressor T-cell activity. Thus, it is unlikely that suppressor T cells are present in either our PEC or B-cell preparations. These results do not conflict with the evidence presented by Cunningham (12), but rather suggest that there exist several levels of control in both B-cell and T-B-cell interactions. In fact, the antilymphocyte serum experiments reported by Cunningham do not exlude the possible autoregulatory influence of B cells. . In addition to identifying the nature of at least one of the controlling substances, the question remained concerning the mechanism by which the control operates. The E series PG has been reported by several workers (3, 22, 23) both to have antiproliferative properties and also to block effector-cell functions such as the release of antibody, possibly via an alteration of cyclic AMP levels (3,2 1,24). The results presented in this report are compatible with these observations in that by blocking proliferation with Mit.C in either PEC or spleen cell cultures it is still possible to enhance the anti-Hb responses by exposure to PG synthetase blockers. This suggests that by blocking the synthesis of PG we may be affecting the ability of anti-Hb B cells to make antibody. In this regard, it should be noted that in cultures exposed to prostaglandin synthetase inhibitors, the inhibition of PG synthesis persists for 96 hr at a level of 20-50% of untreated control cultures (Webb and Nowowiejski, submitted for publication). It should also be noted that Mit.C treatment alone appears to enhance the number of PFC per 10” in PEC, although the number of PFC per culture is actually reduced. This raises the possibility that Mit.C may be toxic for certain PEC but not for the majority of the antibody-forming cells since a good response is still present. The data provided by the spleen cell culture experiment are consistent with the data obtained using PEC, although in spleen cells, Mit.C treatment causes a reduction in the number of PFC without a corresponding reduction in cell number. Two explanations are possible. The first, that Mit.C may be toxic for antibody-forming cells, seems unlikely since in PEC cultures antibody-forming cells are not affected. The second possibility is that some proliferation of anti-Hb clones is necessary in the spleen and is blocked by Mit.C exposure. That some proliferation could be involved in the development of anti-Hb clones is suggested by the data obtained using PGE, and PGF,,. The PGs are biologically active for only a short time and must, therefore, exert their effects very soon after addition. The inhibition observed by adding PG at the time of culture
428
ZIMECKI
AND
WEBB
initiation suggests an effect on a very early stage in development of anti-Hb clones. This might involve the metabolic events which precede and are required for proliferation, although other steps in the differentiation pathway, which do not involve DNA synthesis, may also be involved. The time course experiment using NZB mice is important in this regard. Lord and Dutton (9) have shown that 48 hr are required for the increases in anti-Hb PFC to reach minimum levels. Our data show that adding inhibitors of PG synthesis after 48 hr has less effect when the response is measured at 96 hr. This suggests that whatever metabolic event is sensitive to a block of PG synthesis, at least 48 hr must pass before clear-cut enhancement is evident. Thus, the PG synthetase inhibitors could be exerting effects on metabolic processes necessary for both expression, and possibly proliferation, of the relevant effector cells. It is also possible that anti-Hb cells in the spleen are blocked at a different stage of differentiation than the anti-Hb PEC. Thus, in both cases PG could exert an influence but on different stages of development, e.g., on both differentiation and expression of effector-cell function. The data we have presented here and elsewhere (13-15) imply a role for the prostaglandins in the regulation of a variety of immunocompetent cell functions. Others (23) have also suggested that the prostaglandins play a role in the modulation of inflammatory events. The regulation of cellular events via prostaglandin is of course not unique to the immune system since prostaglandins have been shown to act in a number of tissues (25). The fact that these compounds appear so ubiquitously but act on specific cells or at specific times may be attributed to their short biological and chemical half-life which makes them ideal as intra- tissue or -organ messengers. The specificity of their effects may be related to the modulation of receptor activity or the activity of intermediate enzymes activated by prostaglandins. The precise relationship between the prostaglandins and other regulators of lymphocyte function remains to be determined. Clearly, as we have alluded to earlier, the prostaglandins do not constitute the sole regulatory influence on the spontaneous anti-Hb response. DeHeer and Edington (26) have shown that T cells also possess anti-HB receptors, and Cunningham (12) has provided evidence that T cells may exert control on the appearance of anti-Hb antibody-forming cells. Also, the possibility of direct antigen feedback control has been demonstrated by Lord and Dutton (10). Thus, several regulatory mechanisms may act in concert to control the expression of all potentially autoimmune immunocompetent cells. REFERENCES I. Waksman, B. H., Namba. Y.. Cell. I~nmunol. 21. 161. 1976. 2. Geshon. R., In “Contemporary Topics in Immunobiology” (M. D. Cooper and N. L. Warner, Eds.), Vol. 3. pp. I-35. Plenum Press, New York. 1973. 3. Bomne, H. R., Lichtenstein. L. M.. Melmon, K. L.. Henney . C. S., Weinstein. Y.. and Shearer, G. M., Science 184, 19, 1974. 4. Melmon, K. L.. Weinstein. Y.. Shearer, G. M., and Bourne, H. R., In “Cyclic AMP, Cell Growth. and the Immune Response” (W. Braun, L. M. Lichtenstein, and C. W. Parker, Eds.), pp. 114-134. Springer-Verlag, New York. 1973. 5. Osheroff, P. L., Webb, D. R., and Paulsrud, J., Biochrm. Biophys. Rrs. Commun. 66,425, 1975. 6. Munro. A. J., and Taussig, M. J.. Nature (London) 256, 103, 1975. 7. Plate. J. M. D., Nature (London) 260, 329. 1976. 8. Linder, E.. and Edgington, T. S., J. Immunol. 108, 1615, 1972. 9. Lord, E. M., and Dutton, R. W.. J. Immunol. 115, 1199. 1975.
PROSTAGLANDIN
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
CONTROL
OF
ANTI-Hb
RESPONSE:
429
Lord, E., and Dutton, R. W.,.l. Immunol. 115, 1631, 1975. DeHeer, D., and Edington, T. S., J. Immunol. 113, 1184, 1974. Cunningham, A. J., Nature (London) 254, 143, 1975. Webb, D. R., and Osheroff, P. L., Proc. Nat. Acad. Sci. USA 73, 1300, 1975. Webb, D. R., Jamieson, A. T., and Nowowiejski, I., Cell Zrnmunol. 24, 45, 1976. Zimecki, M., and Webb, D. R., J. Zmmunol. 117, 2158, 1976. Grant, V. N., Baruth, H., Randall, L. 0.. Ashley, C., and Paulrud, J. R., Prostaglandins 10, 59, 1975. Mishell, R. I., and Dutton, R., J. Exp. Med. 126, 423, 1967. Click, R. E., Benck, L., and Alter, B. J., Cell. Zmmunol. 3, 264, 1972. Burnet, M., In “The Clonal Selection Theory of Acquired Immunity.” Vanderbilt Univ. Press, Nashville, Tennessee, 1959. Katz, D. H., and Benacerraf, B., In “Immunological Tolerance, Mechanisms and Potential Therapeutic Applications” (D. H. Katz and B. Benacerraf, Eds.). Academic Press, New York, 1974. Smith, J. N., Steiner, A. L., Newberry, W. M., and Parker, C. W., J. C/in. Invest. JO, 432, 1971. DeRubertis, F. R., Zenser, T. V., Adler, W. H., and Hudson, T., J. Zmmunol. 113, 151, 1974. Bach, M. A., J. Clin. Invest. 5.5, 1074, 1975. Boume, H. R., Lichtenstein, L. M., Melmon, K. L., Henney, C. S., Weinstein, Y., and Shearer, G. M., Science 184, 19, 1974. Samuelson, B, Granstrom, E., Green, K., Hamberg, M., and Hammarstroem, S., In “Annual Reviews of Biochemistry” (E. E. Snell, Ed.), Vol. 44, pp. 669. Annual Reviews Inc., Palo Alto, California. DeHeer, D. H., Edgington, T. S., J. Zmmunol. 116, 1051, 1976.