Regulation of the in vitro antibody response by neuroendocrine hormones ( corticotropin / endorphins / enkephalins / pro - opiomelanocortin / immunoendocrinology ) HOWARD

Treatment of lymphocytes with inducers of interferon a (IFN-a) results in the production of corticotropin (ACTH) and endorphin-like activities. The pro-opiomelanocortinderived hormones ACTH and a-, (-, and y-endorphin and the structurally related hormones [Leu]and [Metlenkephalin were therefore tested for their effects on the in vitro antibody response ofmouse spleen cells. ACTH and a-endorphin were potent inhibitors (.80% suppression) of the antibody response to the T-celldependent antigen sheep erythrocytes at a concentration of 0.5 p.M. [Met]and [Leu]enkephalin were moderate inhibitors (approximately 60% suppression) at 0.2-2 FM, and (and y-endorphin were minimal inhibitors (approximately 20% suppression) at 5-6 pAM. At higher concentrations ACTH also inhibited the antibody response to the T-cell-independent antigen dinitrophenylFicoll, suggesting that T-cell function was more sensitive to blockage by these hormones than was B-cell function. ACTH and IFN had similar suppression properties; thus, the hormone-like activities associated with IFN-a may play a role in IFN-induced immunosuppression. a-Endorphin immunosuppression was blocked by naloxone, which suggested that a-endorphin exerted its effects through binding to opiate-like receptors on the spleen cells. The failure of (3-endorphin to suppress the immune response significandy was not due to its failure to bind to the opiate-like receptors because it blocked a-endorphin-induced suppression. Direct evidence for both opiate and ACTH receptors on the spleen cells was obtained in binding studies with labeled enkephalin and ACTH. Such studies revealed the presence of both highand low-affminty receptors. The data show that neuroendocrine polypeptide hormones can regulate the immune response. It has been shown recently that interferons (IFNs) have hormonal or hormone-like activities and share common pathways of cell activation with polypeptide hormones (1-3). Additionally, based on biological, biochemical, and antigenic characterization, we have shown that human lymphocytes stimulated with IFN-a inducers produce corticotropin (ACTH) and endorphin-like substances (4-6). The findings support a regulatory circuit between the immune and neuroendocrine systems which operates by known hormones that are common to both systems. To test if the putative regulatory circuit could operate in the opposite direction, we determined if the pro-opiomelanocortin-derived neuroendocrine polypeptide hormonesACTH and endorphins (for review, see ref. 7) and the structurally related enkephalins were capable of modulating cells of the immune system (in vitro antibody production). The data presented show that ACTH, a-endorphin, and enkephalins are capable of directly suppressing lymphocyte function, thus establishing a complete regulatory circuit between the immune and neuroendocrine systems. MATERIALS AND METHODS Mice. C57BL/6 female mice, 8-12 weeks old, were obtained from The Jackson Laboratory. Antigens. Sheep erythrocytes (SRBC) were obtained from the Colorado Serum Company (Denver, CO). Dinitrophenylated Ficoll (DNP-Ficoll) was obtained from D. Mosier (8). Reagents. Highly purified ACTH (40 units/ml) was obtained from Armour (Phoenix, AZ). Another ACTH preparation (69 units/mg of protein) was obtained from Sigma. Synthetic endorphins and enkephalins were obtained from Boehringer Mannheim. Reagent grade 2-mercaptoethanol was obtained from Eastman. Cultures. Dissociated mouse spleen cells were cultured for in vitro plaque-forming cell (PFC) responses to SRBC and DNP-Ficoll as described (9). Cultures consisted of 1.5 x 107 cells in 1 ml. All PFC responses were determined on day 5 except when kinetic studies were performed. All results are expressed as the mean of duplicate or triplicate cultures. RESULTS AND DISCUSSION ACTH suppressed the C57BL/6 mouse spleen cell PFC response to both SRBC (T-cell-dependent antigen) and DNP-Ficoll (T-cell-independent antigen) as shown in the dose-response curves (Fig. 1). The slopes for ACTH suppression of the antibody response to the two antigens were essentially the same, but the anti-SRBC response was more sensitive to suppression. Approximately 80% of the anti-SRBC PFC response was suppressed with 0.5 AM ACTH, whereas 2 A.M was required for comparable suppression of the anti-DNP-Ficoll response. Treatment of spleen cells with anti-Thy 1.2 antibody in order to remove T cells did not alter ACTH suppression of the antiDNP-Ficoll PFC response (data not shown). Thus, ACTH suppressed the antibody response to T-cell-dependent and T-cellindependent antigens and this suppression did not require the presence ofT cells. Further, the data suggest that T-cell function was more sensitive to blockage by ACTH than was B-cell function. The close association of an ACTH-like substance with IFNa (4, 6) prompted us to determine if the immunosuppressive properties of ACTH were similar to those of IFN. The immunosuppressive effects of IFN are blocked by thiol-reducing agents such as 2-mercaptoethanol (10), so we tested the effect of 2-mercaptoethanol on ACTH suppression of the PFC response. 2-mercaptoethanol completely reversed the immunosuppressive effects ofACTH in both the SRBC and DNP-Ficoll PFC responses (Table 1). 2-mercaptoethanol significantly blocked ACTH suppression in the SRBC PFC system when Abbreviations: IFN, interferon; ACTH, corticotropin; SRBC, sheep erythrocytes; DNP, dinitrophenyl; PFC, plaque-forming cell(s). The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.