Bruce H. Devens

The design and synthesis of a nonpeptide mimic of an immunosuppressing peptide

Abstract The synthesis and preliminary biological evaluation of a nonpeptide mimic of the immunosuppressant tripeptide (Lys-Pro-Arg) is described.

Investigations into the mechanism of immunosuppression caused by acute treatment with O,O,Sttrimethyl phosphorothioate. I. Characterization of the immune cell population affected

Acute administration of O,O,S-trimethyl phosphorothioate (OOS-TMP), an impurity in technical formulations of malathion, to female C57B1/6 mice was previously shown to suppress the generation of both cytotoxic T lymphocyte to alloantigen and antibody-secreting cells to sheep red blood cells. In this report, macrophages were shown to be the immune cell population most affected by acute OOS-TMP pretreatment by cell separation and reconstitution experiments. Macrophages from OOS-TMP-treated animals had increased levels of nonspecific esterases. In addition, the size distribution of macrophages from treated animals was slightly larger and more heterogeneous than macrophages from control animals. However, macrophages from OOS-TMP-treated animals did not exhibit tumoricidal activity. These data suggest that macrophages from OOS-TMP-treated animals were similar to those located in nonimmune inflammatory sites.

Organophosphorus pesticide immunotoxicity: effects of O,O,S-trimethyl phosphorothioate on cellular and humoral immune response systems

The time course of immunosuppression induced by acute treatment with O,O,S-trimethyl phosphorothioate (OOS-TMP), an impurity in technical formulations of malathion, was examined in female C57B1/6 mice. Both cell-mediated and humoral immune responses were examined and included allospecific cytotoxic T cells, proliferative response to mitogens, interleukin-2 production and antibody production to sheep red blood cells. OOS-TMP pretreatment led to a reversible suppression of the generation of cytotoxic T lymphocytes and antibody-secreting cells to sheep erythrocytes. However, the mitogenic response of splenocytes from animals treated with nontoxic doses of OOS-TMP (as measured by body weight loss, serum cholinesterase levels and splenic lymphocyte number) to concanavalin A was not significantly suppressed, but the response to the B cell mitogen lipopolysaccharide was slightly decreased on day 1 following treatment. In contrast, interleukin-2 production was elevated by 24 h following treatment, but had returned to control levels by day 7. These data suggest that OOS-TMP was able to block the generation of cytotoxic T lymphocytes and antibody responses at doses of OOS-TMP that did not affect body weight or splenic lymphocyte number and this suppression was reversible.

Investigations into the mechanism of immunosuppression caused by acute treatment with O,O,S-trimethyl phosphorothioate. II. Effect on the ability of murine macrophages to present antigen.

Acute administration of 10 mg/kg O,O,S-trimethyl phosphorothioate (OOS-TMP) for 24 h has been shown to suppress the in vitro generation of cytotoxic T lymphocyte responses and antibody-secreting cells to sheep red blood cells and to increase interleukin-2 production. Macrophages were shown to be the splenic cell population most affected by OOS-TMP pretreatment. In this report, the ability of macrophages from OOS-TMP-treated animals to function in antigen presentation was shown to be significantly decreased. In addition, macrophages from treated animals had increased phagocytic capability and interleukin-l production. However, the percentage of Ia-positive macrophages present in splenic populations was decreased following OOS-TMP treatment. A decrease in antigen presenting ability and the number of Ia-positive macrophages may explain the reversible suppression in cytotoxic T lymphocytes and antibody responses reported previously.

Effects of subchronic treatment with O,O,S-trimethyl phosphorothioate on cellular and humoral immune response systems.

The effect of a 14-day treatment with low doses of O,O,S-Trimethyl phosphorothioate (OOS-TMP), an impurity in technical malathion, on the generation of cell-mediated and humoral immune responses was examined in female C57B1/6 mice. At a dose of 0.5 mg/kg/day OOS-TMP, the generation of antibody-secreting cells to sheep red blood cells (SRBC), the production of interleukin 2 (IL-2), and proliferative responses to the mitogens concanavalin A (Con A) and lipopolysaccharide (LPS) were elevated. In contrast, the cytotoxic T-lymphocyte (CTL) response to alloantigen was unchanged. At 5.0 mg/kg/day OOS-TMP, both the CTL and specific antibody response were unchanged, but all other immune parameters examined were elevated. Data from cell separation and reconstitution experiments indicated that both macrophages and B cells were affected by this treatment regime. These data suggest that long-term exposure to low amounts of OOS-TMP may enhance the ability of an animal to generate an immune response.

Investigations into the mechanism of immunosuppression caused by acute treatment with O,O,S-trimethyl phosphorothioate: generation of suppressive macrophages from treated animals.

At concentrations normally found in the spleen, macrophages from animals treated with O,O,S-trimethyl phosphorothioate (OOS-TMP) for 24 hr were previously shown to be immunosuppressive (Rodgers et al., 1985b). In addition, it was shown that macrophages from OOS-TMP-treated animals had a diminished capacity to present antigen (Rodgers et al., 1985c). In this report, it was shown that lowering the number of splenic adherent cells (95% macrophages by morphology) utilized in cell-mixing experiments to reconstitute the nonadherent splenic populations returned the humoral immune response to control levels. One day following acute administration of OOS-TMP, resident peritoneal cells were able to suppress the proliferation of P815 tumor cells. In addition, proliferative responses to concanavalin A and lipopolysaccharide were decreased at suboptimal concentrations of mitogen. Fresh supernatants from splenocytes cultured for 24 hr from OOS-TMP-treated animals blocked the generation of a humoral immune response. However, supernatants from splenocytes of control animals generated in the same manner did not block the generation of a humoral immune response. These data suggest that OOS-TMP induced the generation of suppressive macrophages which may potentially act through the release of labile factors which block proliferation or antigen- or mitogen-induced lymphocyte stimulation.

Transduction of the IFN-γ signal for HLA-DR expression in the promonocytic line THP-1 involves a late-acting PKC activity

Interferon gamma (IFN-gamma) is the most potent known lymphokine for activating macrophages and has been shown to induce expression of HLA-DR in THP-1 cells, a monocytic tumor cell line which expresses many of the properties of monocytes, in a dose- and time-dependent manner. Experiments were designed to examine, by FACS analysis and by measurement of messenger RNA levels, the molecular mechanism regulating the expression of HLA-DR molecules. The expression of HLA-DR molecules induced by IFN-gamma was blocked by the protein kinase C (PKC) inhibitors sphingosine, staurosporine, and H7. H7 when added up to 20 hr after the initial stimulation with IFN-gamma prevented the further expression of HLA-DR. The general kinase inhibitors H8, H9, and HA1004, all less potent PKC inhibitors than H7, did not block the IFN-gamma-induced expression of HLA-DR at the concentrations employed. W7, a calmodulin antagonist, but not a PKC inhibitor, was also unable to prevent the IFN-gamma-induced expression of HLA-DR. Treatment of THP-1 with phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC, alone or with Ca2+ ionophore A23187, was unable to induce HLA-DR expression. However, pretreatment with PMA for 24 hr prior to IFN-gamma stimulation decreased the IFN-gamma-induced expression of HLA-DR without decreasing IFN-gamma receptor levels. These results suggest that PKC plays a significant role in the IFN-gamma-induced signal transduction pathway leading to the expression of HLA-DR in cells of the mononuclear phagocytic lineage, and that PKC activity is required throughout the course of events leading to the actual expression of HLA-DR.

lnterleukin-2 Stimulates the Development of Anergy via the Activation of Nonspecific Suppressor T Cells

We have previously demonstrated that incubation of splenic lymphocytes from an unimmunized mouse with IL-2 IFN-α or γ resulted in the development of a population of nonspecific regulatory cells (Ts). These cells were shown to block the ability of lymphocytes to generate mixed-lymphocyte responses in vitro. In the studies reported here, we have investigated the cell populations involved in this phenomenon. The Ts cells develop over a period of 2 or more days in culture with IL-2. Antibody to the 55-kD chain of the IL-2 receptor blocks Ts generation while stimulating T-cell proliferation. Although NK activity develops in these cultures, the mechanism of suppression is not via a lytic mechanism. Generation of the Ts in culture in the presence of IL-2 requires adherent cells as well as CD8+ cells. In studies using Con-A as the stimulus, the generation of Ts clearly requires both CD4+ T cells as well as CD8+ T cells and adherent cells. The evidence suggests that the CD4+ T cells serve as a source of IL-2 that is necessary in the activation of the IL-2-responsive CD8+, nonspecific Ts.