PRODUCTION OF INTERLEUKIN-6 BY RAT OVARIAN GRANULOSA-CELLS FOLLOWING THE REMOVAL OF RESIDENT MACROPHAGES IN-VITRO

Recently, we demonstrated that significant levels of interleukin-6 (IL -6) are detectable in the conditioned media obtained from cultured gra nulosa cells following treatment with FSH, IL-1 or lipopolysaccharide (LPS). On the basis of these findings, we suggested that the granulosa cell is capable of secreting IL-6. However, because of the invasive n ature of the procedure used to isolate the cells, resulting population s of granulosa cells though considered relatively homogeneous may cont ain a number of immune cell types capable of IL-6 production. Thus, a primary concern has been that the cellular source of IL-6 may not be t he granulosa cell but rather one of the concomitantly isolated immune cell types, in particular, the macrophage. Consequently, we assessed t he ability of the granulosa cell to produce IL-6, progesterone and est rogen under conditions in which ovarian macrophages have been depleted from the isolated population. Briefly, populations of freshly isolate d granulosa cells were placed either directly in culture or further pr ocessed for removal of macrophages by adherence methods. The degree to which macrophages were removed was assessed by flow cytometric analys is using highly specific, fluorescently-tagged monoclonal antibodies a s markers for macrophages. The results indicated that following our se paration procedures less than 1% of the cells within the recovered gra nulosa cell population were labelled as macrophages. Treatment of both the unseparated and macrophage-depleted granulosa cell populations wi th increasing amounts of FSH caused significant dose-dependent increas es in IL-6, progesterone and estrogen. Most interestingly, both proges terone and estrogen steroidogenesis as well as IL-6 release were more responsive to FSH in the presence of macrophages than following their removal. Taken together, the present results demonstrate that (1) a hi ghly purified population of granulosa cells can indeed produce interle ukin-6 and (2) a functional link exists between the immune system and granulosa cell function.