Regulation of the immune response by prostaglandins

CLINICAL

IMMUNOLOGY

AND

lMMUNOPATHOLOGY

15.

106-

122 (1980)

REVIEW Regulation

of the Immune Prostaglandins

JAMES S. GOODWIN

Response

AND DAVID

by

R. WEBB

Received June 15. 1979 This paper reviews work on the regulation of humoral and cellular immunity by prostaglandins. A substantial body of evidence implicates prostaglandins E, and E, as local feedback inhibitors of T-cell activation in vitro and in view. Blockade of prostaglandin synthesis in vitro or in vivn results in an enhanced cellular immune response in a number of different experimental systems. Prostaglandins of the E and F series also modulate humoral immune responses such as B-cell activation or antibody production. Several disease states are discussed where disordered regulation by prostaglandins might have a role in the altered immune responses.

INTRODUCTION

Prostaglandins are produced in every tissue in the body. It was at first difficult to assign a meaningful physiologic role to compounds as ubiquitous as prostaglandins. It has since been realized that prostaglandins act as local hormones; that is, their site of action is the same as their site of production. With the possible exception of parturition, circulating prostaglandins have no systemic actions. The most obvious role for prostaglandins in the immune response is as mediators of inflammation; that is, as proinflammatory compounds. Prostaglandins E, and E, (PGE, and PGE,) cause local vasodilitation and increased vascular permeability when injected intradermally (1). These compounds also potentiate the actions of histamine and bradykinin in causing pain (2) and accumulation of edema fluid (3). The most telling evidence for the predominantly proinflammatory role for prostaglandins is the fact that inhibitors of prostaglandin synthesis are powerful anti-inflammatory agents in view (4). This present review will focus on a less well-recognized action of prostaglandins, what could be considered an anti-inflammatory action-the negative regulation of humoral and cellular immunity. In 1971 Smith er al. reported that PGE, and E,, as well as A, and F1, inhibited [3H]thymidine incorporation into PHAstimulated human lymphocytes (5). Other investigators then showed in various in vitro assay systems that PGE, and E, could depress macrophage inhibitory factor activity (6), leukocyte inhibitory factor production (7), direct cytolysis by activated lymphocytes (8), hemolytic plaque formation (9), and antibody formation (10). One problem with these early studies was the high concentrations of prostaglandins used (1O-6 to lo-” M) to demonstrate inhibition in 13irro. Since the concentration of PGE in inflammatory sites is usually less than 10e8 M (1 l), it was 106 00%1229/80/010106-17$01.00/0 Copyright @ 1980 by Academic Press. Inc. All nghtc of reproduction in any form reserved.

PROSTAGLANDINS

AND

THE

IMMUNE

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RESPONSE

difficult to know whether endogenous prostaglandins actually did inhibit immune function in viva. For example, Berenbaum et al. concluded from their studies that the inhibitory effects of prostaglandins seen in vitro represented a nonspecific toxicity from the high, unphysiologic concentrations of the agents used (12). These doubts were finally dispelled by two sets of experiments performed in different laboratories, both showing a role for endogenous prostaglandins in immune regulation in viva. Webb and Osheroff showed that intravenous injections of sheep red blood cells (sRBC) in mice resulted in an SO-fold increase in splenic PGFzo, within minutes of the injection (13). Prior administration of indomethacin or other Pg synthetase inhibitors to the mice prevented the PGF, increase and also resulted in a significantly increased number of direct plaque-forming cells in the spleen 3 to 5 days after the sRBC injection. Thus prostaglandins exerted a negative regulatory role on formation of plaque-forming cells in viva after antigenie challenge. Plescia and his co-workers found that the immunosuppression caused by certain syngeneic tumors in mice could be reversed in vitro by PG synthetase inhibitors (14). A more dramatic finding was that tumor growth could be slowed in viva by administration of indomethacin to the mice. The experiments in mice by Webb and by Plescia provided strong evidence for a role for endogenous prostaglandins in immunoregulation in certain systems. These results prompted several groups of investigators to reexamine the question of what part prostaglandins might play in the control of immune responses in experimental animals and in humans. PROSTAGLANDIN

REGULATION OF CELLULAR RESPONSES IN VITRO

IMMUNE

Webb and his co-workers have studied the role of prostaglandins in regulating the activation of mouse splenocytes by mitogens. The assay involves the separation of spleen cells on glass-wool columns into two populations: those which do not adhere to glass wool, nonadherent lymphocytes; and those which adhere weakly to glass wool, the glass-adherent lymphocytes. It was first observed by Folch and Waksman (15) that when rat spleen cells were passed over glass-wool columns the nonadherent lymphocytes were more responsive to mitogenic stimulation (i.e., gave a higher degree of [3H]thymidine incorporation). Webb et al. and Webb and Nowowiejski confirmed this observation in mice and were able to show further that a glass-adherent T cell could suppress DNA synthesis when added back to cultures of phytohemagglutinin (PHA)-stimulated nonadherent lymphocytes (16, 17). Inhibitors of PG synthesis such as indomethacin and dl-6-chloroa-methylcarbazole-2-acetic acid (RO-20-5720) could reverse the glass-adherent T-cell suppression of nonadherent lymphocyte DNA synthesis. This was the first indication that the endogenous synthesis of prostaglandins was involved in the regulation of lymphocyte activation. Both nonadherent lymphocytes and glass-adherent T-cells release PGE into the culture medium after PHA stimulation (17). The appearance of PGE in both populations reaches its maximum level by 48 hr in culture. Differences between the two populations relate to the total amount of PGE released. Unstimulated

108

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nonadherent lymphocyte cultures contain roughly 0.5 ng of immunoreactive PGE,. Following exposure to PHA this amount increases to about 5 ng by 48 hr. In contrast, unstimulated glass-adherent T-lymphocyte cultures contain approximately 5 ng of PGE,. Stimulation with PHA induces the glass-adherent T-cells to release 10 ng PGE, by 48 hr. Thus, glass-adherent cells make more tofu/ PGE,; however, the net increase in both nonadherent lymphocytes and glass-adherent T cells is the same. Further, the relative increase is much greater in nonadherent lymphocytes (lo-fold) than in glass-adherent T cells (twofold). These data were of particular interest because they were somewhat unexpected. Earlier studies using prostaglandin synthetase inhibitors had shown that PHA-stimulated nonadherent lymphocytes had no greater DNA synthesis after addition of prostaglandin synthetase inhibitors. In fact, prostaglandin synthetase inhibitors were only effective in enhancing DNA synthesis if nonadherent lymphocytes were first suppressed by glass-adherent T cells. This left a dilemma: how to explain the role PG synthesis in lymphocyte activation when the PG synthesized seemed only to be effective when both cell populations. nonadherent lymphocytes. and glass-adherent T cells were present. It would appear that in this system PGE, does not directly suppress the mitogen-responsive cells. Instead PGE, activates a glass-adherent T-suppressor cell which in turn suppresses the response of the nonadherent lymphocytes to mitogens. When glass-adherent T cells were incubated with various concentrations of PGE, for 24 hr and then washed, they were much more suppressive when added back to mitogen cultures of nonadherent cells (171. Cyclic AMP, isoproterenol, epinephrine, histamine cholera toxin, or PGFnu did not activate the glass-adherent T-suppressor cell or only did so very poorly (181. Thus, it appeared that glass-adherent T cells were responsive to PGE in a rather unique way. Additional experiments have shown that PGE, or PGE, induce glass-adherent T cells to release a low molecular weight peptide which is highly suppressive of both T- and B-cell mitogenesis (19). Goodwin ef ul. examined the effects of prostaglandins on mitogen-stimulated [3H]thymidine incorporation in human peripheral blood mononuclear cells (PBMC) (20). They found that PGE, or PGE,, but not PGA, PGF,,, or PGF,,, inhibited 13H]thymidine incorporation when added in small concentrations (lo-” to lo-” M’l to concanavalin A (Con A)- or PHA-stimulated cultures of PBMC. PGE did not inhibit pokeweed mitogen (PWM) stimulation of PBMC, but did inhibit PWM stimulation of purified T cells. Since PHA and Con A stimulate T cells, while PWM is primarily a B-cell mitogen [though it stimulates T cells after B cells have been removed (21)], these data suggest that PGE inhibits T-cell, but not B-cell mitogenesis. PGE, is produced in PHA-stimulated cultures of PBMC in amounts that, when added exogenously, cause significant inhibition of mitogen stimulation. The amount of PGE, produced increases with increasing concentrations of PHA; levels of PHA that give optimal [:‘H]thymidine incorporation stimulate about 3 x lo-’ M PGE, production in 500,000 PBMC in 1 ml over 48 hr (20, 22). This confirmed the earlier report of Ferraris and DeRubertis (23). Since PGE, is produced in sufficient quantities in PHA cultures to cause an inhibition of [3H]thymidine incorporation in those cultures, one would expect that the addition of PG synthetase inhibitors to PHA cultures would result in increased

PROSTAGLANDINS

AND THE IMMUNE TABLE

PERCENTAGE

INCREASE

IN [3H]T~~~~~~~~ AS A FUNCTION

PHA concentration (wpiml) 0.2 0.4 1.0 2.0 4.0 8.0 20.0

Percentage increase 1,059 -t 233 ? 101 ? 702 71 k 40 k 4+3

134 86 33 10 10 6

1

INCORFQRATION OF

109

RESPONSE

DOSE OF

CAUSED

BY INDOMETHACIN

MITOGEN

cpm” Without indomethacin 411 4,823 18,164 27,768 33,079 43,428 52,111

5 k rt r f 2 2

213 1,556 3,410 5,569 5,876 8,923 16,428

With indomethacin 41764 16,069 36,533 47,205 56,630 60,777 54,196

2 t t t t t t

3,155 1,724 6,281 5,591 7,378 11,753 17,197

” The data are expressed as mean + SEM of cpm, and of percentage increase in [“Hlthymidine incorporation, in three experiments on different subjects. (Reprinted with permission from the Journal of Clinical Investigation.)

[3H]thymidine incorporation. This phenomenon is demonstrated in Table 1 at seven different concentrations of PHA (22). It is clear that addition of indomethatin enhances [3H]thymidine incorporation in PHA cultures, and that indomethacin causes a greater percentage increase in [3H]thymidine incorporation as the dose of PHA is decreased. The percentage increase in [3H]thymidine incorporation ranges from 1059 + 134% at the lowest dose of PHA (0.2 Fg/ml) to 4 + 3% at the highest dose (20 &ml). Similar results were obtained with RO-20-5720, an experimental drug developed at Roche which is chemically unrelated to indomethacin (24). The only known action of RO-20-5720 is reversible inhibition of prostaglandin synthetase. One possible explanation for this varying response to prostaglandin synthetase inhibitors might be that the PBMC are more sensitive to the inhibiting effects of endogenous PGEz at lower concentrations of mitogen. This indeed appears to be the case. Figure 1 graphs the percentage inhibition caused by four concentrations of PGE, of r3H]thymidine incorporation induced by different concentrations of mitogen. A family of curves is generated, cultures with the lowest concentrations of PHA showing the greatest sensitivity to exogenously added PGE,. More recent evidence would suggest that this is a general rule for all inhibitors: The lower the mitogen dose, the more sensitive the culture is to inhibition (25). Removal of glass-adherent cells by passage of PBMC over glass wool eliminated the enhancing effect seen with PG synthetase inhibitors and reduced PGE, production in those cultures to less than 20% of normal. In human peripheral blood it would appear that the predominant prostaglandin-producing cell is the macrophage (26)) though there is evidence from experimental animals that B cells (27)) T cells (17, 28), and macrophages (29-31) all can produce prostaglandins. Several other laboratories have been engaged in the study of prostaglandins and the immune response. Mendelsohn et al. (32) and Berenbaum et al. (33) have shown that PGE, and cortisol are synergistic in their inhibitory effects on the mitogen response. Muscoplat et al. have described a prostaglandin-mediated suppression system in peripheral blood, spleen, and lymph node cells from swine (34). Addition of indomethacin to Con A-stimulated cultures of PBMC or lymph node

110

GOODWIN

PGE;!

AND

WEBB

CONCENTRATION

I. Percentage inhibition of [“Hlthymidine incorporation caused by PGEz at six concentrations of PHA. Data is from one experiment, and shows that, at any given level of PGE,, more inhibition is caused in PHA cultures as the concentration of the mitogen is decreased. In this experiemnt, the PHA concentration of 4.0 &ml gave optimal stimulation. (Reprinted with permission from the Journal of Clinical Investigation.) FIG.

cells resulted in an increase in [“Hlthymidine incorporation. In contrast to the findings with human PBMC, these workers reported that removal of glassadherent cells did not decrease the enhancement in [“Hlthymidine incorporation by indomethacin. Thus their prostaglandin-producing cell would appear to be nonadherent. Novogrodsky and co-workers have shown that prostaglandins produced by adherent cells suppress the response of human PBMC to peanut agglutinin, hepatic binding protein, and soybean agglutinin (35). Addition of prostaglandin synthetase inhibitors or removal of the glass-adherent cells caused a twofold or greater increase in [“Hlthymidine incorporation. Interestingly, these authors found no enhancement of PHA- or Con A-induced blastogenesis by PG synthetase inhibitors. They attributed this divergence from the results of Goodwin ef ul. (20) to differences in methodology. Page et ul. (36) and Rice et al. (37) have confirmed the finding of enhancement by indomethacin of PHA-induced proliferation in human PBMC. Rice and co-workers (37) have recently compared the prostaglandin-producing suppressor cell system in human PBMC to the adherent suppressor cell system described earlier by the same investigators (38). They confirmed the finding of a prostaglandin-mediated suppression by adherent cells in mitogen-stimulated cultures of human PBMC, and also described an adherent suppressor cell system that was independent of prostaglandin synthesis. Thus adherent PBMC suppress the mitogen response of the nonadherent cells by at least two mechanisms, only one of which is dependent on prostaglandin synthesis. There are a few reports implicating prostaglandins in the control of T-cell functions other than the proliferative response to mitogens. Morley and co-workers have shown that PGE inhibits and indomethacin augments lymphokine production as well as proliferation in cultures of guinea pig lymphocytes (39,40). Droller et al. showed that inhibition of prostaglandin synthesis by a variety of agents resulted in increased natural and antibody-dependent cytotoxicity of two tumor cell lines caused by human PBMC (41).

PROSTAGLANDINS

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THE

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111

RESPONSE

Parker and his associates have recently published a series of papers showing that products of arachidonic acid metabolism other than PGs (e.g., thromboxanes) stimulate mitogenesis in human lymphocytes (42-44). Addition of compounds that selectively inhibit thromboxane synthesis without affecting prostaglandin synthesis resulted in a depressed PHA response (43). The overall effect of the addition of arachidonic acid to PHA cultures was stimulatory (42). PROSTAGLANDIN

REGULATION OF HUMORAL RESPONSES IN VITRO

IMMUNE

Webb and co-workers have demonstrated a role for prostaglandin in B-cell regulation. Their demonstration of increased plaque-forming cells after sRBC immunization in mice pretreated with indomethacin was described earlier in this review (13). Zimecki and Webb also studied the in vitro response of mouse splenocytes to the T-independent antigens polyvinyl pyrolidone and DNP-Ficoll (27). They found that addition of prostaglandin synthetase inhibitors caused a 50-300% increase in the number of plaque-forming cells produced. This enhancing effect of the PG synthetase inhibitors persisted in highly purified B-cell preparations with no T cells and less than 5% macrophages. They concluded from these findings that B cells may regulate their response to certain antigens by producing prostaglandins. Another interesting aspect of this study was the finding that the PG synthetase inhibitors were more stimulatory when the initial response to the T-independent antigen was less than optimal. This parallels the finding of Goodwin et al. of greater increases in PHA-induced [3H]thymidine incorporation at concentrations of the mitogen that elicit a suboptimal response in PBMC (22). Other investigators have shown that PGE, and PGE, can stimulate the anamnestic antibody response to KLH in vitro, but the high concentrations of PGE employed (> lo-” M) make it difficult to rule out a nonspecific toxicity on a regulatory cell population (45). If suppressor T-cell populations are defined by their increased sensitivity to radiation (46), cyclophosphamide (47), or preincubation (48), it is possible to postulate that these same cells are more sensitive to toxicity by high concentrations of prostaglandins and other agents. It is clear that the strongest arguments for a role for prostaglandins in a particular immunologic phenomenon are made when opposite effects are demonstrated by the addition of exogenous prostaglandin versus the removal of endogenous prostaglandin, i.e., addition of PG synthetase inhibitors. ROLE

OF PROSTAGLANDINS ASSOCIATED

IN DISORDERED WITH DISEASE

IMMUNOREGULATION STATES

Immunosuppression Associated with Malignancy Several groups of investigators have studied the possible contributions of prostaglandin-mediated suppressor systems to the depressed cellular immunity associated with tumors. The initial experiments of Plescia et al. showing reversal of tumor-mediated immunosuppression in vitro with PG synthetase inhibitors were described earlier (14). In a more recent report, these same investigators have shown that MC 16 tumor cells (a PGE,-secreting tumor cell line) inhibit the antibody response to sRBC of mouse splenocytes in vitro and in vivo (49). Mice injected with these tumors have a greatly diminished antibody response after

112

GOODWIN

AND WEBB

intraperitoneal injections of sRBC. lndomethacin and other inhibitors of PG synthetase completely reversed the tumor-induced immunosuppression in vitro and in viva. Indomethacin in vitro and in Lhw also enhanced the depressed mitogen responses of splenocytes from mice bearing a variety of tumors (50). Lynch and Salomon (51) have recently confirmed the tinding of Plescia ct ul. (14) that indomethacin administration would result in a slowing of tumor growth in mice. Indomethacin also enhanced the immunotherapeutic effect of corynebacterium parvum or BCG injections in tumorous mice (51). While the experiments cited above would suggest that PGE aids tumor growth by decreasing immune surveillance. there is also considerable evidence that PGE can directly inhibit the growth of some tumor cells in \*itro and that indomethacin and other PG synthetase inhibitors can enhance tumor growth (52, 53). Further in vilv experiments on the effects of PG synthetase inhibitors on the growth of many different tumors are necessary before we can conclude whether endogenous PGE predominantly enhances or inhibits tumor growth. It is likely that PGE does have a function in the growth of most human tumors, since high concentrations of PGE are found in human malignancies (54). Goodwin et al. have investigated the role of the prostaglandin-producing suppressor cell in the hyporesponsiveness to PHA of PBMC from patients with Hodgkin’s disease (55). Previous investigations by Twomey ct ~1. (561, and Sibbitt el al. (57) had suggested that the defect in cell-mediated immunity in Hodgkin’s disease was caused by circulating suppressor cells. Six patients with untreated Hodgkin’s disease, nodular sclerosing type, were studied. Figure 2 shows the

140

PERCENT INCREASE

120

.~ 1

100 80

I. . 8

60 r 40

-L 5

20

0-

NORMALS

tic

FIG. 2. Percentage increase in phytohemagglutinin-stimulated [“Hlthymidine incorporation caused by indomethacin in mononuclear cells from 29 controls and six patients with Hodgkin’s disease. (Reprinted with permission from the New England Journal of Medicine.)

PROSTAGLANDINS

AND THE IMMUNE

RESPONSE

113

percentage increase in PHA-induced [SH]thymidine incorporation caused by indomethacin in the 6 patients with Hodgkin’s disease and 29 controls. The average increase in cpm with indomethacin was 182 ? 60% for the patients and 44 t 18% for the controls (mean f. SD, P < 0.001). Using RO-20-5720 there were similar results, with a 95 + 29% increase in PHA stimulation of Hodgkin’s disease lymphocytes versus a 28 c 10% increase in controls. Figure 3 depicts these data in raw counts per minute. The mean response of the Hodgkin’s disease lymphocytes was 48% of the mean control response. This increased to 94% of normal with addition of indomethacin. Thus indomethacin almost totally reversed the depressed PHA response in Hodgkin’s disease lymphocytes. The suppression of the PHA response in the patients with Hodgkin’s disease was aIso reversed by removal of glass-adherent cells prior to culture. This was expected, since the PG-producing suppressor cells are glass adherent (20). Two possible explanations for the increased response of Hodgkin’s disease lymphocytes to indomethacin are apparent. First, the Hodgkin’s disease lymphocytes could be more sensitive to the PGs produced by the suppressor cells. Second, the suppressor cells could be producing more PGs. To investigate the first possibility, PGE, was added to cultures of Hodgkin’s disease versus controls. No difference in sensitivity to inhibition by exogenous PGEz between Hodgkin’s disease and normal lymphocytes was found. PGE, production was then measured in mitogen cultures of PBMC (5OO,OOO/ml) from three patients with Hodgkin’s disease and three age- and sex-matched controls. The mean PGE, production of the Hodgkin’s disease PBMC was 20,600 t 7620 pg/ml (6 x 10e8 M) in 48 hr versus 5368 t 2944 pg/ml (1.6 x lO-8M) for the controls (mean F SD, P < 0.02). The amount of PGE, produced in the cultures of Hodgkin’s disease lymphocytes 2o,om 15,000

F

CPM

1

I PHA

I Pn4 -+ HKMTHAClN

FIG. 3. Phytohemagglutinin (PHA) response (mean ? SEM) in counts per minute (cpm) of six patients with Hodgkin’s disease and 20 normal controls with or without the addition of indomethacin. Without indomethacin, the patient and control groups are significantly different (r = 3.01, P < 0.002). After indomethacin there is no significant difference (t = 0.33, P z 0.6 by one-tailed t test). Counts per minute are graphed on a log scale. (Reprinted with permission from the New England Journal of Medicine.)

114

GOODWIN

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caused 65% inhibition when added exogenously to Hodgkin’s disease glassnonadherent cells, while the amount of PGE, produced by the controls caused 35% inhibition when added to normal glass-nonadherent cells. Bockman has recently confirmed that mononuclear cells from patients with Hodgkin’s disease produce fourfold more PGE, than normal. He also showed that addition of indomethacin increases the number of T-lymphocyte colonies formed on soft agar after PHA-stimulation of PBMC from patients with Hodgkin’s disease (58). Thus, it would appear that a prostaglandin-producing suppressor cell is partly responsible for the hyporesponsiveness to PHA seen in Hodgkin’s disease, and this suppression can largely be reversed in r,irro by indomethacin, a PG synthetase inhibitor. PHA cultures of Hodgkin’s disease lymphocytes produce - fourfold more PGEp than do cultures of normal lymphocytes. When this PGE production is eliminated by the addition of PG synthetase inhibitors or the removal of glassadherent cells, the response of the Hodgkin’s disease lymphocytes to PHA increases into the normal range. PG-Producing suppressor cells were also studied in the depressed immune response associated with disseminated malignancy (59). In 10 anergic patients with a variety of metastatic solid tumors, indomethacin did not reverse the depressed PHA response in vitro. Prostaglandin-mediated immunosuppression would not appear to be an important factor in the anergy of these patients. Immunosl4ppressior? Inflammation

Associated

n*ith Chronic

lnjkction

or Chronic

Suppressor cell mechanisms have been invoked as contributing to the maintenance of some chronic infections, including chronic fungal disease (60) and tuberculosis (61). Little work has been reported on the role of prostaglandins in chronic infections. Muscoplat’s group has used in \‘itro prostaglandin blockade as a tool to distinguish cattle previously exposed to Bracelia abortas but unresponsive to the antigen from nonexposed cattle (62). PBMC from previously exposed but anergic cattle, as well as from nonexposed animals will not respond to brucella antigen ilz vitro. After addition of indomethacin to the cutlures PBMC from the previously exposed anergic animals give a positive response to brucella while PBMC from the unexposed animals remain unresponsive. This would suggest that prostaglandins are involved in specific immunosuppression against brucella in the anergic animals. A recent abstract suggests that the depressed PHA response of PBMC from patients with disseminated coccidiomycosis can be reversed with indomethacin (63). However, while the depressed mitogen responses of individuals with disseminated fungal disease can be reversed in vitro with PG synthetase inhibitors it is not possible to reverse the specific anergy to the infecting agent (64). Thus PBMC from subjects with disseminated coccidiomycosis did not respond to coccidioidin in k’itro even with indomethacin in the cultures. This is clearly different from the case with brucellosis in cattle described above. Prostaglandin-mediated suppression has also been studied in the depressed cellular immunity associated with sarcoidosis (65). The depressed PHA response of patients with sarcoidosis could be largely reversed by removing the glassadherent cells but not by inhibition of prostaglandin synthesis. This lends further

PROSTAGLANDINS

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support to the contention of Rice et al. that there are at least two glass-adherent suppressor cell mechanisms in human PBMC, only one of which is dependent on prostaglandin synthesis (37). Zurier and his colleagues have been studying the pathogenesis of disease in NZB/NZW mice which spontaneously develop a complex series of autoimmune diseases (66-69). Perhaps of greatest interest are data suggesting that treatment of NZB/NZW male or female mice with subcutaneous injections of PGE, can delay the appearance of disease and enhance survival (66). Of related interest, Webb and co-workers investigated the ability of NZB mice to develop increases in splenic PG and CAMP following the iv injection of sRBC (70). These data revealed that young NZB mice could show normal or supernormal increases in PG levels in response to sRBC. However, in old NZB mice (>4 months) showing signs of disease (splenomegaly) the injection of sRBC had no effect on PG or CAMP levels in the spleen. This was in part due to the fact that baseline levels of PGF,, were elevated in mice showing splenomegaly compared to age-matched controls. That this high background level of PGF,, was not simply a function of age was shown by data obtained using old C57B1/6 mice, in which sRBC-induced increases still occurred (albeit with a lower maximum). Both old and young NZB mice fail to respond well to sRBC by forming plaque-forming cells in a Mishell-Dutton system; also, the addition of PG synthetase inhibitors has no effect on the appearance of plaque-forming cells. Skelly et al. (71) have confirmed these results using NZB/NZW hybrids and measuring changes in splenic CAMP levels following sRBC injection. Furthermore, they were able to transfer the unresponsiveness of old mice to young mice using adoptive transfer of spleen cells. CHANGES

IN SENSITIVITY TO INHIBITION BY PGE, IN LYMPHOCYTES FROM OLD PEOPLE AND OTHERS

Aging is associated with depressed humoral and cellular immunity in humans (72-76) and experimental animals (77). There seems to be a subpopulation of apparently healthy old people who are anergic to skin testing (72, 76), and this anergy has been associated with decreased survival (72). The cause of this T-cell dysfunction is unknown. Recent work by Inkeles et al. has demonstrated that the hyporesponsiveness of lymphocytes from aging humans to PHA is actually a sum of two deticiencies (73). First the number of mitogen-responsive cells is reduced in lymphocyte preparations from old persons. Second, the mitogen-responsive cells from old persons fail to divide as rapidly as cells from younger subjects. Goodwin and Messner studied the activity of the PG-producing suppressor cell in subjects of different ages and found that PBMC from healthy subjects over 70 are much more sensitive to inhibition by exogenous PGEz than are PBMC from young adults (78). The amount of PGE, required to cause 50% inhibition of the PHA (4.0 pg/ml) response of 12 healthy subjects over age 70 was -lo-* M. This was more than two orders of magnitude lower than the amount required for 50% inhibition in 17 young adults (>3 x lo+ M). This increased sensitivity to PGE would appear to account for much of the depression in cellular immune function found in healthy old people. Addition of indomethacin to the cultures caused a 140 c 16% increase in the cultures from the subjects over 70 versus a 36 2 3%

116

GOODWIN

AND

WEBB

increase for the young controls. The mean PHA response of the old people was 40% of the control response without indomethacin. After addition of indomethacin the mean response of the older subjects rose to 72% of the mean response of the young controls. Several other conditions have been described where there are changes in sensitivity of leukocytes to PGE,. Kirbey et al. found that PGE, did not reverse PHA-induced inhibition of leukocyte migration in buffy coat preparations from patients with multiple sclerosis (79). They examined the migration of leukocytes out of capillary tubes into soft agar. PHA inhibits this migration, presumably by stimulating the production of migration inhibition factors in lymphocytes. PGE reverses the PHA-induced inhibition, once again presumably by its action on the lymphocyte. Kirby et cd. found that PGE would not reverse the PHA-induced inhibition in leukocytes from multiple sclerosis patients. They suggested that lymphocytes from patients with multiple sclerosis are insensitive to inhibition by PGE and this lack of sensitivity to a feedback inhibitor might explain the chronic inflammatory nature of the disease. Goodwin and Messner examined sensitivity to PGE, of PHA-stimulated PBMC from multiple sclerosis patients and normals and found no difference between the two groups (80). These conflicting results in two very different assay systems would suggest that the role of PGE in multiple sclerosis is not straightforward. Further evidence for an actual role for PGE in this disease is provided by the recent study from Zurier’s laboratory that showed that the increased binding of lymphocytes from patients with multiple sclerosis to measles-infected cells could be reversed in vitro or in V~VOby the administration of prostaglandin synthetase inhibitors (81). Page and co-workers have reported that lymphocytes from patients with juvenile periodontitis are less sensitive to inhibition by PGE, (IO-,; M) than are cells from patients with adult periodontitis or normal controls (36). They postulate that a defect in the response of lymphocytes to PGE, allows for the exuberant immune reactivity seen in subjects with juvenile periodontitis. MECHANISM

OF ACTION OF PROSTAGLANDINS

It is widely accepted that PGE, exerts its actions on various tissues through activation of membrane-bound adenyl cyclase. The evidence for CAMP as a second messenger for PGE, in lymphocytes has been extensively reviewed (82, 83). Bromberg et al. recently reexamined this question and found the following (84): (i) PGE, in concentrations of lo-” to lOUs M caused significant increases in CAMP in human PBMC. (ii, The effect of PGE, on CAMP paralleled its inhibitory effect on inhibition of mitogenesis. (iii) PGF,,, which does not inhibit mitogenesis, does not stimulate CAMP increases. (iv) PBMC that have been preincubated overnight before addition of mitogen are no longer inhibited by PGE, (22). These preincubated PBMC also do not have CAMP increases after exposure to PGE,. Cell surface receptors for PGE have been described in various tissues from experimental animals. Schaumberg, and later Grunnet and Bojesen have studied

PROSTAGLANDINS

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117

binding of 3H-PGE, to rat thymocytes (85,86). They found a Kd of 2 x lo+’ M with an average of 300 binding sites per cell. Goodwin et al. studied the binding of 3H-PGA,, El, Ez, F1,, and F,, to human PBMC (87). They found specific reversible binding for 3H-PGE, and Ee with a kd of -2 x lops M and a maximum number of binding sites of 200 per cell, assuming uniform distribution. No specific binding of 3H-PGA, F1,, or Fza to lymphocytes was detected. Also, the addition of lo- to lOOO-fold greater amounts of unlabeled PGA, F,,, or F,, did not inhibit the binding of 3H-PGE. Glass-adherent PBMC had fewer binding sites than nonadherent cells. Preincubation of the cells overnight resulted in a loss of binding sites. Thus there are distinct parallels between the functional effect of PGE (inhibition of mitogenesis), the CAMP response to PGE, and the binding of 3H-PGE to lymphocytes. It should be noted that the link between PGE and CAMP is not entirely established, however. Sinha and Colman have recently presented compelling evidence that PGE, inhibits platelet aggregation by a mechanism independent of CAMP (88). In addition to cell surface receptors for PGE that would appear to be coupled to adenylate cyclase, there exist uptake mechanisms for PGE. Bito has demonstrated saturable active uptake of 3H-PGs into many animal tissues, both in viva and in vitro (89, 90). This uptake can be inhibited by metabolic poisons, inhibitors of biotransport such as probenecid, and anti-inflammatory drugs (91). The possibility still exists then, that many of the effects of PGs on immune function are mediated in ways independent of adenylate cyclase stimulation. MANIPULATION

OF PROSTAGLANDIN-MEDIATED IN V/V0

IMMUNOREGULATION

Indomethacin, which blocks prostaglandin synthesis in vitro at low concentrations (0.1 - 1.O p&ml) (92), can be given in vivo with relative safety (93) and can achieve serum concentrations of 0.5-3.0 &ml (94). Robinson and his co-workers reported in 1968 that pretreatment of mice with indomethacin enhanced their ability to resist some infections (95), but this result aroused little attention, presumably because the mechanism of action of indomethacin was not known at that time. It is logical to assume that some of the effects of indomethacin and other PG synthetase inhibitors noted in vitro could be reproduced in vivo. Some investigations of in vivo effects of indomethacin have been described earlier in this review. Webb showed that administration of indomethacin or RO-20-5720 to mice resulted in a greater number of direct splenic plaque-forming cells after injection of sRBC (13). Kazimiera repeated these experiments and showed that indomethacintreated mice had an increased antibody titer to sRBC (96). Pelus and Strausser reported that indomethacin in vitro and in vivo would enhance the mitogen response of splenocytes from tumorous mice (50). Muscoplat et al. studied the effect of indomethacin administration on delayed hypersensitivity skin responses in guinea pigs previously sensitized to mycobacterium bovis (97). Oral administration of 1 mg indomethacin simultaneous with indradermal injection of Mycobacterium bovis antigen resulted in a more than twofold increase in skin thickness compared to the guinea pigs not given indomethacin. This shows that it is possible to enhance delayed hypersensitivity in vivo. In addition, it suggests that the major

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role for prostaglandins in delayed hypersensitivity skin testing is antiinflammatory, not proinflammatory. Goodwin et al. examined the effect of indomethacin administration on humoral immunity in man by administering indomethacin (100 mg/day) to 15 normal subjects for 2 days before and 10 days after they received a bivalent influenza vaccine (98). Compared to 15 age- and sex-matched controls, the indomethacin-treated group had a significantly increased antibody titer to A-Victoria (P < 0.025). There was no difference in titer to A-New Jersey. Since 90% of the subjects had antibody titers to A-Victoria prior to vaccination, while none had prior titers to A-New Jersey, the results suggested that indomethacin administration enhanced the secondary but not the primary humoral immune response. Effect of indomethacin administration in man on delayed hypersensitivity skin testing has also been studied (98, 99). Indomethacin administration (100 mgday) for 5 days did not influence the amount of induration caused by a battery of three common antigens in 10 normal subjects (98). When indomethacin was administered to subjects with depressed cellullar immunity there was an enhancement of delayed hypersensitivity (99). Two previously anergic patients with common variable immunodeficiency became reactive to skin tests while on indomethacin (100 mg/day) and became anergic again after the medication was stopped. The in llitro response of the patients’ lymphocytes to PHA also rose while they were taking indomethacin (Figure 4). SOME

PITFALLS

OF RESEARCH IMMUNE

ON PROSTAGLANDINS RESPONSE

AND THE

It is important to realize that not all effects attributed to PGs have been proven conclusively. Work with prostaglandins is amenable to certain artifacts. First, most early reports, and even many reports in the past 2 years have used very high concentrations of PGs in vitro to demonstrate an effect on the immune response. When lo-” to 1O-6 M concentrations are used, it is not possible to conclude that the effect noted has any relevance to in rGvo regulation. It is at least equally likely that at these concentrations PGs are altering immune function by a nonspecific toxicity on the cells involved. Second, the “specific” PG synthetase inhibitors employed have actions other than blockade of PG synthesis. Some studies (43,100) employ high indomethacin concentrations that are toxic to lymphocytes (101), and attribute the effects to a specific blockade of PG synthesis. Even in submicromolar concentrations, indomethacin is not a specific PG synthetase inhibitor. Kantor and Hampton have recently reported that indomethacin at 10-R M inhibits CAMP-dependent protein kinase (102). At higher concentrations indomethacin inhibits phosphodiesterase (103). In addition, many of the inhibitors employed also block thromboxane and endoperoxide synthesis (42). Thus it is not sufficient to add a PG synthetase inhibitor to a system and then ascribe any effects noted to inhibition of prostaglandin synthesis. As a minimum, one should use two structurally unrelated PG synthetase inhibitors. Further evidence is provided if the readdition of specific PGs into the system abrogates the effect of the PG synthetase inhibitors.

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RESPONSE

119

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POSITIVE SKIN TESTS FIG. 4. Effect of indomethacin administration in rive on the response to PHA in rirro and on the response to skin testing in a patient with adult-acquired immunodeficiency. The two shaded areas represent the period of indomethacin administration (25 mg, q.i.d.). The in vitro response of the patients’ peripheral blood mononuclear cells to an optima) concentration of PHA and to PHA plus indomethacin (1 ~p/ml) is graphed. (Reprinted with permission from the Journal of Clinical and Laboratory Immunology).

A final pitfall of work on prostaglandin immunoregulation is the determination of the cell responsible for the PG production. This is an especially difficult task in human PBMC, where techniques for separating subfractions of cells are far from perfect. It is clear that platelets produce large amounts of PGs, so care must be taken during separation of PBMC to remove the platelets. This must be done without losing the low density monocytes, which are high PG producers (104). In mouse spleens a glass-adherent T lymphocyte produces large amounts of PGE (17). In human PBMC the prostaglandin-producing cells are also glass adherent, but most investigators have concluded that they are monocytes (26, 37). Since the techniques of monocyte separation employed all use adherence, it has been impossible thus far to rule out a prostaglandin-producing, glass-adherent T cell in human PBMC. REFERENCES 1. 2. 3. 4.

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