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, Tumor Necrosis Factor-
, and Cycloheximide1
Department of Animal Science, Cornell University, Ithaca, New York 14853
Address all correspondence and requests for reprints to: Dr. Susan M. Quirk, 258 Morrison Hall, Cornell University, Ithaca, New York 14853. E-mail: smq1{at}cornell.edu
The Fas antigen is a transmembrane receptor belonging to the tumor
necrosis factor-
(TNF) receptor family that, when activated by Fas
ligand or agonistic antibodies, induces death by apoptosis. Although
the presence of Fas antigen in ovarian tissues has been demonstrated,
little is known about whether Fas antigen is functional in the ovary.
This report shows that murine granulosa cells are initially resistant
to antibody-induced Fas-mediated apoptosis, but will undergo apoptosis
when cotreated with TNF and interferon-
(IFN) or cycloheximide (CX).
Granulosa cells were obtained from follicles of 23-day-old mice 2 days
after injection of PMSG. Twenty-four hours after plating, cells were
pretreated with either 0 or 200 U/ml IFN, which has been shown to
induce Fas antigen expression and is required for Fas-mediated killing
in many cell types. At 48 h, cells were treated with 2 µg/ml
control IgG, 2 µg/ml anti-Fas antigen antibody (Fas mAb), 10 ng/ml
TNF, or Fas mAb and TNF. Cytotoxicity (percent killing) relative to
control IgG was determined at 72 h by counting granulosa cells
after trypsinization. In the absence of IFN, no cytotoxicity was
observed. In the presence of IFN, neither TNF or Fas mAb alone was
cytotoxic, but the combination of Fas mAb and TNF resulted in 25%
killing (P < 0.05). Fas antigen messenger RNA
(mRNA) was detectable in cultures not treated with cytokines 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 labile inhibitor(s) of Fas-mediated killing in granulosa cells is the cause of resistance to Fas mAb, the protein synthesis inhibitor CX was used. Experiments were performed as described above, except that cells were treated with 0.5 µg/ml CX in conjunction with other treatments at 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 in 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% killing in the presence of IFN (P < 0.05). Cells stained positively for DNA fragmentation and annexin V binding, features characteristic of apoptosis.
Because initial experiments showed that treatment with TNF alone 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 combined pretreatment with TNF and IFN resulted in 25% killing in response to Fas mAb. Treatment of cells with the combination of IFN and TNF induced a 19-fold increase in Fas antigen mRNA levels. Corresponding increases in Fas antigen protein expression on the surface of cells in response to cytokine treatments were detected by immunocytochemistry. Human TNF did not duplicate the effects of mouse TNF in inducing Fas antigen 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 probably mediated via the type II TNF receptor.
The effects of cytokine treatments on levels of mRNA for FAP-1, an inhibitor of Fas-mediated apoptosis, were determined. FAP-1 mRNA was detectable in untreated granulosa cells, and levels were not altered by treatment with TNF and/or IFN.
In summary, the Fas-mediated pathway of apoptosis is functional in mouse granulosa cells that are stimulated with IFN and TNF. These cytokines may function at least partially by increasing Fas antigen expression. Granulosa cells appear to have inhibitors of the Fas antigen pathway, as treatment with CX potentiates Fas-mediated death. TNF promotes Fas-mediated killing in the presence and absence of CX. Therefore, TNF 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.
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