POTENTIATION OF FAS-MEDIATED APOPTOSIS OF MURINE GRANULOSA-CELLS BY INTERFERON-GAMMA, TUMOR-NECROSIS-FACTOR-ALPHA, AND CYCLOHEXIMIDE

The Fas antigen is a transmembrane receptor belonging to the tumor nec rosis factor-alpha (TNF) receptor family that, when activated by Fas l igand or agonistic antibodies, induces death by apoptosis. Although th e presence of Fas antigen in ovarian tissues has been demonstrated, li ttle is known about whether Fas antigen is functional in the ovary. Th is report shows that murine granulosa cells are initially resistant to antibody-induced Fas-mediated apoptosis, but will undergo apoptosis w hen cotreated with TNF and interferon-gamma (IFN) or cycloheximide (CX ). Granulosa cells were obtained from follicles of 23-day-old mice 2 d ays after injection of PMSG. Twenty-four hours after plating, cells we re pretreated with either 0 or 200 U/ml IFN, which has been shown to i nduce Fas antigen expression and is required for Fas-mediated killing in many cell types. At 48 h, cells were treated with 2 mu g/ml control IgG, 2 mu g/ml anti-Fas antigen antibody (Fas mAb), 10 ng/ml TNF, or Fas mAb and TNF. Cytotoxicity (percent killing) relative to control Ig G was determined at 72 h by counting granulosa cells after trypsinizat ion. In the absence of IFN, no cytotoxicity was observed. In the prese nce of IFN, neither TNF or Fas mAb alone was cytotoxic, but the combin ation of Fas mAb and TNF resulted in 25% killing (P < 0.05). Fas antig en messenger RNA (mRNA) was detectable in cultures not treated with cy tokines and was increased 5-fold by TNF, 2-fold by IFN, and 17-fold by the combination of IFN and TNF. To test whether the presence of a lab ile inhibitor(s) of Fas-mediated killing in granulosa cells is the cau se of resistance to Fas mAb, the protein synthesis inhibitor CX was us ed. Experiments were performed as described above, except that cells w ere treated with 0.5 mu g/ml CX in conjunction with other treatments a t 48 h. Fas mAb treatment in the presence of CX induced 25% cell death without IFN pretreatment and 38% with IFN (P < 0.05). TNF treatment i n the presence of CX had no effect alone, but potentiated the effects of Fas mAb, resulting in 56% killing in the absence of IFN and 86% kil ling in the presence of IFN (P < 0.05). Cells stained positively for D NA fragmentation and annexin V binding, features characteristic of apo ptosis. Because initial experiments showed that treatment with TNF alo ne increased Fas mRNA levels, the effect of pretreating cells for 24 h with TNF before treatment with Fas mAb was tested. Pretreatment with TNF or IFN alone did not promote Fas mAb-mediated killing, but combine d pretreatment with TNF and IFN resulted in 25% killing in response to Fas mAb. Treatment of cells with the combination of IFN and TNF induc ed a 19-fold increase in Fas antigen mRNA levels. Corresponding increa ses in Fas antigen protein expression on the surface of cells in respo nse to cytokine treatments were detected by immunocytochemistry. Human TNF did not duplicate the effects of mouse TNF in inducing Fas antige n mRNA expression and Fas mAb-induced killing. As human TNF interacts exclusively with the type I, but not the type II, TNF receptor in the mouse, potentiating effects of mouse TNF on the Fas pathway are probab ly mediated via the type II TNF receptor. The effects of cytokine trea tments on levels of mRNA for FAP-1, an inhibitor of Fas-mediated apopt osis, were determined. FAP-1 mRNA was detectable in untreated granulos a cells, and levels were not altered by treatment with TNF and/or IFN. In summary, the Fas-mediated pathway of apoptosis is functional in mo use granulosa cells that are stimulated with IFN and. TNF. These cytok ines may function at least partially by increasing Fas antigen express ion. Granulosa cells appear to have inhibitors of the Fas antigen path way, as treatment with CX potentiates Fas-mediated death. TNF promotes Fas-mediated killing in the presence and absence of CX. Therefore, TN F is not likely to act simply by increasing Fas antigen expression or decreasing protein inhibitors of the Fas pathway, because TNF remains effective when these processes are blocked by CX.