Identification of IL-6 as one of the important cytokines responsible for the ability of mononuclear cells to stimulate Sertoli cell functions.

There is increasing evidence that locally produced cytokines may play an important role in the control of testicular function. In a previous report we demonstrated that medium conditioned by activated human peripheral blood mononuclear cells (PBMC-CM), which is a rich source of cytokines, has extremely potent effects on Sertoli cell transferrin and cGMP secretion. Part of this activity could be explained by interleukin-1beta (IL-1beta) but additional cytokines were evidently involved. In the present study we tried to characterize and purify additional components active on Sertoli cells from PBMC-CM. To this end PBMC-CM was subjected to a purification procedure involving successively: adsorption to silicic acid, affinity chromatography with an antiserum recognizing a mixture of cytokines except IL-1beta, gel-filtration, reversed-phase HPLC and cation-exchange FPLC. Throughout this protocol a Sertoli cell bioassay was used to monitor the effects on transferrin and cGMP production. After cation-exchange FPLC, SDS-PAGE using silver staining showed a single protein band in the bioactive fractions. NH2-terminal amino-acid sequencing revealed that the active principle(s) in this band corresponded to four truncated forms of IL-6 missing the first 13, 14, 17 and 18 N-terminal amino-acids, respectively. The truncated IL-6 molecules were as active as intact IL-6 in the Sertoli cell bioassay. Since neither IL-1beta nor IL-6 alone or in combination could account for the extremely potent effect of PBMC-CM, we tested a series of additional cytokines (IL-1alpha, INF-alpha, IL-4, TGF-beta, IFN-gamma) alone and in combination with IL-1beta and IL-6. These data suggest that IL-1beta, IL-6 and TNF-alpha display more than additive effects on Sertoli cell transferrin and cGMP secretion and that the combination of these cytokines may explain the major part of the effects observed with crude PBMC-CM. The observation that the latter effects could be observed with murine as well as human IL-1beta, IL-6 and TNF-alpha further supports the potential physiological relevance of these findings.