Differential effect of asparaginase on lymphocyte subpopulations and alleviation of the immunosuppression by lipopolysaccharide

Summary The effect of a range of E. coli L-asparaginase (EC2) concentrations on mouse splenocyte blastogenesis was examined at various concentrations of both T- and B-lymphocyte mitogens. At optimal mitogen concentration and with 0.1 unit EC2/ml inhibition of the Con A (T cell) response was 2.5 times as great as inhibition of the LPS (B cell) response. Another T-cell stimulant, periodate, was inhibited in a manner comparable to Con A while the PHA response was inhibited to an intermediate degree. In vivo the PFC response was suppressed to 19% of normal when 100 units EC2 was given with SRBC. However, when T-cell participation in this response was by-passed by simultaneously giving LPS, the residual activity rose from 19% to 52%, again suggesting that T-lymphocytes were the more severely depressed population. These observations demonstrate that a nonspecific stimulant of the immune system may alleviate the depression of immune responsiveness induced by a chemotherapeutic agent.     

Human recombinant interleukin-1 receptor antagonist inhibits lymphocyte blastogenesis induced by concanavalin A. Restorative effect of hrIL-1.

Interleukin-1 (IL-1), mainly produced by monocyte-macrophages, is a polypeptide cytokine with pleiotropic biological effects. IL-1 plays an important role in mediating immune response and inflammation. Recently a natural inhibitor to IL-1 has been discovered, interleukin-1 receptor antagonist (IL-1ra), produced by human monocytes cultured on adherent IgG which binds to the IL-1 receptors. In our study we found that the pretreatment of cells with serial dilutions of IL-1ra (250 ng/ml-2.5 pg/ml) inhibits, in a dose-dependent manner, lymphocyte DNA synthesis stimulated with Con A (10 micrograms/ml). IL-1ra did not have any effect on resting peripheral blood mononuclear cells (PBMC). Time course experiments show that IL-1ra at 250 ng/ml has its maximum inhibitory effect on lymphocyte blastogenesis when cells are pretreated 2 h before Con A. No effect was found when hrIL-1ra was added after Con A. Moreover, hrIL-1ra also inhibits the enhancing effects of exogenous hrIL-1 (400, 200, 100 and 50 ng/ml) on lymphocytes stimulated with Con A; while when hrIL-1ra was used on cells treated with only Con A, the inhibition was more pronounced. When PBMC were removed from monocytes, by adherence, the Con A-treated lymphocytes were not influenced by 2 h pretreatment of hrIL-1ra; while a strong inhibition was found when exogenous hrIL-1 was added at different concentrations. In addition, hrIL-1ra also inhibits the enhancing effect of hrIL-2 on lymphocyte DNA synthesis. In another set of experiments PBMC were pretreated with hrIL-1ra (250 ng/ml) for 2 h and then added LPs (10 ng/ml) and IL-1 alpha generation was determined using ELISA. In these experiments IL-1ra completely abolished the generation of IL-1 alpha. These data suggest that hrIL-1ra exhibits a dose-response inhibition of lymphocyte blastogenesis induced by Con A, probably through the down-regulation of IL-1 synthesis necessary as an early signal for T-cell activation and IL-2 production.     

Cellular cooperation in mitogen-induced human leukocyte inhibitory factor (LIF) synthesis.

Interactions between human T and B lymphocytes and between lymphocyte subpopulations and accessory cells in lymphokine synthesis were investigated. The cells were stimulated with leukoagglutinin (LA), concanavalin A (Con A), protein A (prot A) and anti-β₂-microglobulin (anti-β₂m). The presence of leukocyte inhibitory factor (LIF) in the culture supernatants was tested by the agarose-migration method. The results indicated that monocytes augmented LIF synthesis of T cells but suppressed that of B cells. Monocyte-helper effect was mediated by both cell-cell contact and soluble factors. In addition, T lymphocytes were found to augment B-cell LIF production. B lymphocytes enhanced Con A- but suppressed LA-induced LIF production by T cells. T-cell/B-cell collaboration was based on a direct cell-cell contact and no soluble factors were found.     

Suppression of lymphocyte reactivity by culture supernatant from horse embryos and endometrium

The mechanisms that permit maternal tolerance of the conceptus allograft during early pregnancy in the mare have not been investigated. Embryos and endometria were collected from mares 14 days after ovulation and cultured for 20.5 h. The effect of addition of culture supernatant on incorporation of [3H]thymidine by equine peripheral blood lymphocytes was studied. Culture supernatant from endometrium of nonpregnant mares did not affect lymphocyte blastogenesis, but supernatant from both embryos and endometrium of pregnant mares reduced concanavalin A (Con A)- and phytohemagglutinin-induced blastogenesis. Five of six cultures performed in the present of indomethacin did not contain immunosuppressive factors. The suppressive effect on Con A-induced blastogenesis was eliminated by charcoal treatment of the supernatants and reduced by treatment with trypsin or heat. Blastogenesis of bovine lymphocytes was inhibited by culture supernatant of endometrium from pregnant mares, but not by embryo supernatant. Preincubation of blood lymphocytes with supernatants from endometrium of pregnant mares enhanced subsequent incorporation of [3H]thymidine by lymphocytes. A 24-h delay in addition of embryo culture supernatants significantly reduced the degree of immunosuppression. These results suggest that probably more than one substance interacts with the lymphocyte cultures and the observed blastogenesis reflects the end result of the interaction between suppressive and stimulating factors. The lymphocyte inhibitory effect evident in supernatants from embryos and endometrium from pregnant mares may be important in local immunosuppression and maternal acceptance of pregnancy.     

Selective suppression of blastogenesis induced by different mitogens: effect of noncyclic adenosine-containing compounds

Lymphocyte responses to the galactosyl binding lectins, SBA and PNA, and responses to Con A are suppressed by cAMP analogs and agents that increase intracellular cAMP to a greater extent than are responses to PHA. Adenosine shares with cAMP this selective inhibitory effect. ATP inhibits lymphocyte proliferation via generation of adenosine. This conclusion is based on the findings that (i) β,γ-methylene ATP (β,γ-met ATP) is similar to ATP in its inhibitory effects, whereas α,β-met ATP has little effect on lymphocyte proliferation, (ii) α,β-met ATP reverses both ATP and β,γ-met ATP induced suppression, but does not reverse adenosine-mediated suppression, and (iii) inhibition of adenosine deaminase potentiates ATP and β,γ met ATP-mediated suppression of blastogenesis. The relative potency of various noncyclic adenosine compounds in suppressing mitogen-induced blastogenesis is: 2-chloradenosine > ATP ≥ ADP > AMP > adenosine.     

Suppression of mitogen- and tumor-cell-induced lymphocyte stimulation by tumor-associated fetal antigens

The ability of tumor-associated fetal antigens (TAFA) to suppress mitogen and tumor-cell-induced blastogenesis was investigated. Three different TAFA (I, III, and IV) were tested in PHA and Con A lymphocyte proliferation assays. Spleen cells from New Zealand black rats (NBR) were fractionated over nylon wool; and nonadherent (NA) and adherent (AD) cells were compared with unfractionated (UF) cell responses. Preincubation of NA cells with TAFA-I was followed by addition of phytohemagglutinin (PHA) elicited suppression in a 3-to 4-day assay. UF cells were unresponsive to TAFA and/or PHA at all concentrations tested. TAFA dose—response titration curves in Con A proliferation assays revealed that all TAFA tested (TAFA I, -III, and -IV from fibrosarcomas; TAFA-I and -III from osteosarcomas) were suppressive. For some TAFA, nanogram quantities produced significant suppression. In mixed leukocyte tumor cell assays (MLTC) both UF and NA normal rat spleen cells were tested for proliferative responses to syngeneic tumor cells. Four distinct TAFA, purified by high-pressure liquid chromatography, suppressed lymphocyte proliferation when added to MLTC cultures in 5-day assays. Specificity experiments demonstrated that trinitrophenol-bovine serum albumin did not produce similar immunosuppression. TAFA did not block recognition of tumor antigen nor produce nonspecific cytotoxicity of the spleen cells. Significant suppression of DNA synthesis was produced by TAFA-1 following cocultivation with spleen and tumor cells for 1, 2, and 3 days, compared to no suppression when spleen and tumor cells were washed free of TAFA-I prior to tumor cell addition at Day 0. Similar experiments using rat embryo fibroblasts (REF) as stimulators demonstrated that pre-REF cocultivation treatment of lymphocytes with TAFA-I significantly reduced subsequent blastogenic responses. This effect was not reversible; however, if TAFA-I was added to responders previously stimulated by REF, a suppressive response was not seen. Experiments were also carried out to determine the reversibility of TAFA-I effects. Cells were treated with TAFA-I from 1 to 5 days, followed by determination of lymphocyte blastogenesis. TAFA-I effects are reversible and antigen presence is required to completely suppress (or inhibit) stimulation by tumor cells.     

Short-term stimulation of lymphocyte proliferation by indomethacin in vitro and in vivo

The effect of short-term (up to 24 h) in vitro and in vivo treatment with indomethacin was studied on the blastogenesis of mouse spleen cells. Indomethacin in itself induced a strong proliferation of the lymphocytes starting after 6 h treatment both in vitro and in vivo. Besides, significantly enhanced the blastogenesis of splenocytes in response to various doses of PHA and Con A. The stimulation of lectin-induced lymphocyte proliferation occurred after indomethacin treatment both in vitro and in vivo. Indomethacin had no major effect on the distribution of Lyt-1+ and Lyt-2+ subsets within the spleen cell population. An important role of the prostaglandins in the early phase of lymphocyte activation is suggested.     

Ontogeny of T-cell mitogen response in Lewis rats. III. Juvenile adherent suppressor cells block adult mitogen responses

Spleen cells from suckling female Lewis rats (4 to 20 days old) were able to suppress mitogenic responses to concanavalin A (Con A) and phytohemagglutinin (PHA) of spleen or thymus cells from adult female Lewis rats and thymus cells from suckling Lewis rats. Thymus cells from suckling rats were unable to suppress adult spleen cell mitogenic responses to Con A. Removal of carbonyl iron (cFe)-, plastic-, or nylon-wool-adherent cells removed the suppressive action of juvenile spleen cells, but irradiation did not. Separated plastic-adherent spleen cells from suckling animals suppressed adult mitogenic responses to Con A. at optimal Con A doses 2-mercaptoethanol (2-ME, 2 X 10(-5) M) abolished the suppressive effect of juvenile cells, however, at the hyperoptimal dose of Con A (125 micrograms/ml) even higher doses of 2-ME did not relieve suppression by juvenile cells. These suppressor cells in suckling pups were affected by early weaning which decreased suppression, resulting in enhanced mitogenic responses of juvenile cells and removal of the ability to suppress adult mitogenic response.     

The effect of a high external temperature on cellular immunity]

The study was made of spleen cells proliferative response to mitogens PHA, Con A or alloantigens in relation to hyperthermia effects. Acute hyperthermia (rectal temperature 42 degrees) enhanced lymphocyte function, proliferative responses to allo-antigens, PHA and Con A increased. Thermal shock was associated with suppression of the spleen cell response. Mice suffering from hyperthermia for 20 min (43-44 degrees) daily during 10, 20 and 30 days showed suppressed T-cell immune response. Normal splenocyte proliferation recovered 40 days after hyperthermia induction.     

Peritoneal macrophages exposed to purified macrophage colony-stimulating factor (M-CSF) suppress mitogen- and antigen-stimulated lymphocyte proliferation

The effect of M-CSF-exposed macrophages on murine splenic lymphocyte responses was determined. Resident peritoneal macrophages incubated with purified M-CSF for 48 hr inhibited lymphocyte proliferation to Con A, PHA, and listerial antigen as determined by [3H]TdR uptake, and inhibited Con A-stimulated lymphocyte IL 2 production. The inhibition was similar to that observed with macrophages from BCG-infected mice. Maximal suppression occurred at M-CSF concentrations of 500 U/ml or greater and when the incubation time with M-CSF was 48 hr or more. M-CSF effect was specific because rabbit anti-M-CSF IgG blocked the suppression whereas control rabbit IgG did not. Secretory products of macrophages could not be implicated in this interaction. Catalase and indomethacin, alone or together, did not reverse the inhibition. In addition, putative suppressive factors were not detected in supernatants of M-CSF-stimulated macrophages. Lymphocytes that were removed from macrophage monolayers and were recultured in medium plus Con A were able to proliferate. Macrophages stimulated by M-CSF therefore appear to have inhibitory activity for proliferating lymphocytes, and may play a role in immunoregulatory mechanisms.     

Suppressor macrophages in lymphocyte proliferation of cynomolgus monkeys

The present study demonstrated the presence of cells belonging to monocyte/macrophage lineage which suppressed mitogen-induced blastogenesis of peripheral blood lymphocytes in cynomolgus monkeys. Depletion of adherent or phagocytic cells from peripheral mononuclear cells caused a substantial increase in the blastogenic response of cynomolgus monkey lymphocytes whereas the same treatment led to marked reduction rather than enhancement in human lymphocyte blastogenesis. Addition of thioglycollate-elicited peritoneal exudate adherent cells as macrophages suppressed the blastogenic response of nonadherent lymphocytes in a dose-dependent manner. The suppressive effect was observed not only in autologous but also in allogeneic macrophages to the responder lymphocytes. Treatment of macrophages with silica, carrageenan or freezing-thawing reduced their suppressive effect but there was no reduction with mitomycin C or indomethacin. No suppressive activity was detected in the cell-free supernatant of macrophages cultured in the presence or absence of mitogens for up to 4 days. From these findings, it appeared that monocyte/macrophage lineage might be responsible for the observed suppressive effect on mitogen-induced blastogenesis of cynomolgus monkey lymphocytes.     

Inhibition of murine lymphokine-activated killer (LAK) cell activity by adherent cells

The effects of adherent cell depletion, indomethacin, and prostaglandin E2 (PGE2) on murine LAK cell activity were investigated. Removal of plastic adherent cells from splenocyte suspensions either prior to 5-day culture with 1000 U/ml of recombinant human IL-2 (rIL-2) or prior to assay resulted in an enhanced LAK cell cytotoxicity compared to that of whole spleen cell suspensions. Indomethacin enhanced LAK cell cytotoxicity of whole splenocyte suspensions if present during the culture period, but had no effect on whole splenocyte or adherent cell-depleted cell suspensions if added just prior to assay. PGE2 suppressed LAK cell activity of nonadherent splenocyte but not whole splenocyte suspensions when present during the culture period. In vivo treatment of mice with indomethacin enhanced cytotoxicity directed toward both LAK sensitive, natural killer (NK) resistant (P-815) and LAK, NK sensitive (YAC-1) tumor cell targets. Splenocytes from indomethacin-treated mice cultured with additional indomethacin and rIL-2 exhibited highest LAK cell activity. The results from this study indicate that LAK cells are regulated by adherent cells which suppress LAK cell activity. This suppression can be reversed both in vitro and in vivo by indomethacin. This study has important implications for the possible clinical use of indomethacin in the potentiation of in vivo and in vitro LAK cell activity for immunotherapeutic protocols.     

Immunoregulation in experimental filariasis. I. In vitro suppression of mitogen-induced blastogenesis by adherent cells from Jirds chronically infected with Brugia pahangi

Nonspecific immunoregulatory events were examined in inbred jirds chronically infected with Brugia pahangi. The responsiveness of spleen cells from infected animals to the T cell mitogens PHA and Con A and to the B cell mitogens, LPS and PWM, was found to be suppressed by as much as 90% when compared with the reactivity of lymphocytes from normal animals. Furthermore, spleen cells from infected jirds were capable of suppressing the mitogen reactivity of normal spleen cells. Depletion of cells adherent to nylon wool, glass wool, or plastic alleviated the regulatory activity exerted by spleen cells from infected jirds. Addition of indomethacin, an inhibitor of prostaglandin synthetase, to cultures of spleen cells from infected animals did not alter the suppression observed. In contrast, lymphocytes from the peripheral lymph nodes of infected jirds did not exhibit depressed T cell mitogen reactivity and were incapable of suppressing the PHA or Con A responsiveness of normal lymph node cells. However, the reactivity of lymph node cells from infected jirds to B cell mitogens, LPS and PWM, was suppressed. These results imply the existence of multiple regulatory mechanisms, at least one of which is restricted to the spleen. The relevance of nonspecific regulation to development of parasite-specific immunologic reactivity and to the infection is discussed.     

The suppressive effect of a supernate from concanavalin A-activated human lymphocytes: effects of concanavalin A-activated lymphocytes and their supernates on cytotoxic and mixed lymphocyte reactions.

The effects of Concanavalin A-treated human peripheral blood lymphocytes and their supernatants were evaluated on the MLC reaction and on the generation of cytotoxic lymphocytes assessed by cell-mediated lympholysis (CML). Experiments were performed with both allogeneic and xenogenic sensitization. It was found that Con A-activated cells suppressed the MLC and CML reactions in allogeneic and xenogeneic systems. On the other hand, the SIRS-like supernate was able to suppress the MLC reaction and blastogenesis, but had no suppressive effect on the generation of cytotoxic lymphocytes. We found no difference in the magnitude of suppression, whether or not Con A-activated lymphocytes were syngeneic to the responder cells. This finding suggests that there is no requirement for allogeneic restriction in the interaction between suppressor and suppressed cells, and demonstrates a soluble human suppressor substance capable of suppressing some cell-mediated reactions.     

Stimulation of suppressor cells in the bone marrow and spleens of high dose cyclophosphamide-treated C57Bl/6 mice

Systemic administration of a single dose (300 mg/kg) of cyclophosphamide (Cy) induced the appearance of a population of suppressor cells in the bone marrow and spleens of mice. Suppressor cells were assayed by their capacity to inhibit the concanavalin A (Con A) blastogenesis or the mixed-lymphocyte response of normal C57Bl/6 spleen cells. Cy-induced bone marrow (Cy-BM) suppressor cells were present as early as 4 days following Cy therapy and their activity gradually decreased over the next 2 weeks. Cy-induced splenic (Cy-Sp) suppressor cells were maximally present on Days 6 through 10 following Cy therapy. Studies were performed to characterize the suppressor cells of bone marrow obtained 4 days after Cy treatment and of normal bone marrow (N-BM). Some suppressor activity was present in normal bone marrow. N-BM suppressor cells resembled cells of the monocyte/macrophage lineage in that they were slightly adherent to Sephadex G-10, sensitive to L-leucine methyl ester (LME), and insensitive to treatment either with anti-T-cell antibody and complement or with anti-immunoglobulin antibody and complement. Their suppressive activity was abrogated by incubation with either indomethacin or catalase. Cy-BM suppressor cells were also resistant to treatment with anti-T-cell and anti-immunoglobulin antibody and complement but were not adherent to Sephadex G-10 and not sensitive to LME. Their suppressive activity was partially eliminated by indomethacin alone or in combination with catalase. We conclude that Cy chemotherapy induces the appearance of a population of immune suppressive cells and that these cells appear first in the bone marrow and subsequently in the spleen.     

Suppressor and protector factors derived from Trypanosoma lewisi.

Trypanosoma lewisi is a specific protozoan blood parasite of rats. Normal rats infected 2 h after treatment with plasma from day 8 irradiated (8.5 Gy) infected rats had significantly higher parasitaemia; in contrast, animals infected 7 days post-plasma treatment were significantly protected. Trypanolytic and ablastic antibodies could be demonstrated in the serum of normal rats treated with the plasma; the trypanolytic antibodies were stage-specific. Suppression of normal rat splenocyte responses to Con A and lipopolysaccharide (LPS) stimulation were also observed in the presence of different protein concentrations of whole lysate from epimastigote forms. The suppression by mitogen Con A was ablated by the addition of exogenous IL 2, or by washing cells incubated with the lysate prior to mitogen stimulation. These results indicate that immunoregulatory factors are present in the plasma of rats infected with T. lewisi, and the effect of these factors can be demonstrated in vitro with whole parasite lysate. The restoration of normal splenocyte responses to Con A by addition of exogenous IL 2 or by washing cells suggests that the suppressor factor(s) act(s) on the T cells by inhibiting their proliferation and IL 2 production, and the continued presence of these products is essential in the maintenance of suppression.     

Glucocorticosteroid modification of lymphocyte blastogenesis in tumor-bearing hosts

The present study was done to evaluate glucocorticosteroid modulation of the individual cells contributing to tumor-induced dualistic (macrophage and suppressor T-cell) suppression in mitogen-induced lymphocyte proliferation. Steroid-sensitive T cells from normal mice showed a characteristic decrease in phytohemagglutinin responsiveness in the in vitro presence of 0.1 μg hydrocortisone sodium-21 succinate (HC), while tumor-bearing host (TBH) T cells demonstrated little or no decrease. This correlates well with kinetic study results demonstrating decreased HC-mediated suppression of mitogen-induced TBH T-cell DNA synthesis at 8 to 10 days posttumor cell inoculation. Hydrocortisone-treated cells from mice with advanced tumors had significantly higher degrees of blastogenesis than untreated TBH cells. In the optimal presence of both macrophages (Mφ) and HC, TBH cells had significantly greater blastogenesis than their normal counterparts. Alone, Mφ or HC depressed normal host PHA responsiveness. Experiments were carried out to determine if the increased blastogenesis was due to the additional Mφ presence in TBH. In vivo administration of methylprednisolone acetate demonstrated that steroids can inhibit tumor growth. A significant delay in tumor appearance occurred when the steroid was administered 4 to 7 days after tumor transplant perhaps due to the measurable lack of suppressor T cells. These studies indicated that steroid suppression was directed against a sensitive suppressor T cell and possibly acts by preventing its differentiation. The role of the Mφ in the afferent limb of the cell-mediated immune response seems less clear, but the results suggest nonspecific inhibition of TBH T-cell response to blastogenic stimulus.     

Interaction of reovirus with cell surface receptors. II. Generation of suppressor T cells by the hemagglutinin of reovirus type 3

In studying reovirus interactions with lymphocytes, we have found that reovirus type 3, but not type 1, inhibits the in vitro proliferative response of murine splenic lymphocytes to concanavalin A (Con A). By analyzing recombinant clones containing genes from both reovirus types 1 and 3, we found that the S1 gene, the gene that encodes the viral hemagglutinin, is responsible for the inhibitory effect. In addition we found that type 3, but not type 1, generates suppressor T cells in vitro capable of suppressing Con A proliferation. By analyzing recombinant clones, we also found that the viral hemagglutinin is responsible for the generation of suppressor T cells by reovirus type 3. These effects were observed whether UV-inactivated or live virus was used. Reovirus type 3 inhibition of the proliferative response of murine splenic lymphocytes to Con A was blocked by anti-reovirus type 3 antibody but not by anti-reovirus type 1 antibody. Antiviral antibody had no effect on the ability of reovirus type 3 induced suppressor cells to inhibit Con A proliferation. We have previously demonstrated a receptor on murine lymphocytes for the hemagglutinin of reovirus type 3, and our results suggest that the in vitro suppression of Con A proliferation of murine lymphocytes by reovirus type 3 is secondary to the interaction of the viral hemagglutinin with a receptor on the surface of murine lymphocytes, which results in the generation of functionally active suppressor T cells.     

Activation of human B lymphocytes. V. Kinetics and mechanisms of suppression of plaque-forming cell responses by concanavalin A-generated suppressor cells.

The kinetics and mechanisms of suppression of the PWM-induced PFC response of human PB lymphocytes by Con A-activated suppressor cells were investigated. It was necessary that Con A suppressor cells be present early in the process of activation of human B cells toward antibody syntheses, but maximal suppression of the PFC response occurred later in the culture period. In addition, Con A-activated cells, although suppressing the PFC response to PWM greater that 90% of control, did not significantly suppress the blastogenic response to PWM after 3 or 5 days in culture. On the contrary, after 3 days in culture, background tritiated thymidine incorporation as well as tritiated thymidine incorporation to PWM stimulation was increased when Con A suppressor cells are added to fresh autologous peripheral blood lymphocytes. This increased blastogenic response after three days most likely represented an autologous mixed lymphocyte reaction (MLR) or Con A suppressor cells against fresh autologous non-T cells. The induction of autoreactive cells may be one of several modes of suppression of PFC responses by Con A activated suppressor cells.     

Suppression of T lymphocyte chemotactic factor production by the opioid peptides beta-endorphin and met-enkephalin

The opioid peptides beta-endorphin and met-enkephalin have been shown to modulate human lymphocyte proliferation, mononuclear cell locomotion, natural killer cell activity, and neutrophil locomotion. This study demonstrates that beta-endorphin and met-enkephalin inhibit the production of a T lymphocyte chemotactic factor (LCF) by concanavalin A (Con A)-stimulated peripheral blood mononuclear cells. Inhibition of LCF production was observed by using concentrations of 10(-11) to 10(-6) M beta-endorphin or met-enkephalin but not alpha-endorphin. A bimodal pattern of suppression of LCF production was observed with both met-enkephalin and beta-endorphin when titrated from 10(-12) to 10(-6) M concentrations, with the peaks of suppressive activity occurring at concentrations of 10(-11) M and 10(-6) M. Timed studies of the production of LCF over a 54-hr period showed that there was an appreciable lag in the onset of measurable LCF activity in mononuclear supernatants produced in the presence of beta-endorphin and met-enkephalin. The suppression of LCF production mediated by opioid peptides in mononuclear supernatants was abrogated by depletion of glass-adherent mononuclear cells before culturing with opioids and Con A. The inhibitory effect of opioid peptides on LCF production was prevented by the addition of indomethacin to cell cultures. Additional experiments showed that exogenous prostaglandin E2 (PGE2) suppressed Con A-stimulated LCF production when added at concentrations ranging from 10(-6) to 10(-8) M. Other studies suggested that the mechanism of opioid peptide-mediated suppression of LCF production was due to an enhanced sensitivity of mononuclear cells to the inhibitory action of PGE2. These data provide further evidence for modulation of the immune response in humans by the neuroendocrine hormones beta-endorphin and met-enkephalin and further suggest a link between this modulation and arachidonic acid metabolism.