Regulation of immune functions by sperm-specific LDH and its differences with somatic isozyme in primary and secondary lymphocyte cultures

PROBLEM: Sperm-specific lactate dehydrogenase-C-4 (LDH-C-4) is an autoantig en that produces experimentally induced autoimmune orchitis in testes. In t he present study, immunological functions of B and T cells have been examin ed and compared after immunization with sperm-specific LDH and the LDH from somatic cells. METHODS: Three sets of experiments were performed. In the first set, effect s of Balb/C LDH isozymes at 10(-3) - 1 L mu g/well were investigated: (i) b y mixed lymphocyte cultures (MLC) using C-57 B1/6 female cells as responder s and AKR lymphocytes (irradiated) as stimulators, (ii) for regulatory T ce ll activity in MLC co-cultured along with Con-A-induced AKR lymphoblasts an d (iii) for modulation of lymphocyte activation by PHA in vitro. In the sec ond set of experiments, female mice (C-57 B1/6) were distributed in six gro ups for various treatments: i) saline las vehicle), ii) adjuvant, iii) LDH- B-4 (20 x 3 mu g), iv) LDH-B-4 (40 x 3 mu g), v) LDH-C-4 (20 x 3 mu g), and v) LDH-C-4 (40 x 3 mu g). Mice were hyperimmunized with -B-4 or -C-4 (Balb /c) with a primary dose of 20 or 40 mu g of protein per mouse, emulsified i n Freund's complete adjuvant (FCA) and two identical doses in Freund's inco mplete adjuvant (s.c.) within 22 days. Saline (group i) or adjuvant treated dams (group ii) served as controls. One week after the second booster, ser a were tested for IgG response and lymphocytes harvested for polyclonal act ivation in vitro using LPS and Con-A as mitogens. In the third set of exper iments, female Balb/c mice were divided into six groups as in the second ex periment and immunized with a single primary dose of isogenic LDH-B-4 or LD H-C-4 at 20 or 40 mu g of protein in FCA. On day 5, after sensitization wit h LDH, lymphocytes were evaluated for mitogenesis and for IgM production in vitro using LPS and Con-A as mitogens. RESULTS: i) Primary MLC(s) were non-specifically suppressed in the presence of 10(-3) - l mu g allogenic LDH-C-4 or -B-4, although LDH-C-4 tended to a bolish MLC completely. But MLC co-cultured with blast cells was suppressed by LDH-C-4 alone, indicating that sperm LDH suppresses induced formation of regulatory T cells. ii) FCA primed lymphocytes in situ were significantly inhibited for Con-A stimulation in vitro. Since LPS stimulation remained un affected, it appeared that FCA is immunosuppressive for T cell proliferatio n alone. iii) Cells primed with LDH increased mitogenic activity of LPS sev eral fold, although LDH-C-4 was less effective than LDH-B-4 in sensitizatio n of B lymphocytes. iv) However, effect of Con-A in mitogenesis was dose-de pendent, viz. cells primed at 70 x 3 mu g of each isozyme overcame the immu nosuppressive nature of FCA by bringing back the SI ( x 25) equivalent to s aline primed cells, while pre-treatment of cells with 40 x 3 mu g LDH-C-4 a bolished SI completely, indicating that -C-4 primed cells were immunologica lly suppressed for Con-A stimulation. Such a response was markedly visible when allogenic LDH-C-4 was used for hyperimmunization; lymphocytes challeng ed with somatic LDH under similar conditions did not react. Loss of T cell functions by LDH-C-4 was confirmed in the presence of PHA in primary cultur es, v) For antibody responses, although sperm LDH was highly reactive and d ose-dependent, somatic LDH was also immunogenic for Ige production in serum to a lesser degree. Besides, IgM antibody was also discernible by two isoz ymes in LPS-induced cultures. Significantly, -C-4 primed cells at the highe r dose, in comparison with the lower dose, were less responsive for IgM pro duction. CONCLUSIONS: It is concluded that LDH(s) from sperm and somatic cells share functionally related antigenic epitopes that can generate/modify immune re sponses in vivo and in vitro with qualitative differences. However, immunos uppressive determinant of LDH-C-4 is cell specific and dose selective.