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a Department of Animal Science, Cornell University, Ithaca, New York 14853
ABSTRACT
Regression of the corpus luteum (CL) occurs by apoptosis. The Fas antigen (Fas) is a cell surface receptor that induces apoptosis in sensitive cells when bound to Fas ligand or agonistic anti-Fas monoclonal antibodies (Fas mAb). A potential role for Fas to induce apoptosis in dispersed CL cell preparations was tested in cells isolated from mice on Days 2–4 of pseudopregnancy. Total CL dispersates, containing steroidogenic luteal cells, fibroblasts, and endothelial cells, were cultured. The effect of pretreatment of cultures with cytokines interferon
(IFN) and tumor necrosis factor 
(TNF) was examined because these cytokines demonstrated effects on Fas-mediated apoptosis in other cell types. Fas mAb had no effect on viability of CL cells cultured in 5% fetal bovine serum (FBS) and pretreated with or without IFN or TNF, but Fas mAb did kill 23% of the cells in cultures pretreated with IFN + TNF. Fas mRNA was detectable in cultured CL cells and was increased 2.1-, 2.0-, and 11.8-fold by treatment with TNF, IFN, or IFN + TNF, respectively. CL cells treated with the protein synthesis inhibitor cycloheximide (CX) were killed by Fas mAb in the absence of cytokine pretreatment (34%); pretreatment with IFN or IFN + TNF further potentiated killing (62% and 96%, respectively), whereas pretreatment with TNF had no effect (42%). Cells cultured in medium supplemented with insulin, transferrin, and selenium instead of FBS were killed by Fas mAb in the presence of IFN (23%) or IFN + TNF (29%) but not in the presence of TNF. Cells derived from the mouse CL have a functional Fas pathway that is inhibited by FBS and activated by treatment with CX, IFN, and IFN + TNF.
apoptosis, corpus luteum, corpus luteum function, cytokines, ovary
INTRODUCTION
Involution of the corpus luteum (CL) occurs by the process of apoptosis in mice and other species [1–6]. The molecular signals initiating luteolysis have not been delineated, but products of genes that regulate apoptosis have been associated with luteolysis [4, 7, 8]. The Fas antigen (Fas) is a cell surface receptor that triggers apoptosis in sensitive cells when bound to Fas ligand (FasL) or agonistic anti-Fas monoclonal antibodies (Fas mAb). The Fas signaling pathway involves a series of intracellular protein-protein interactions that results in a cascade of protease activation, cleavage of cellular substrates, and cell death [9]. Fas expression has been detected in mouse, rat, and human CL [2, 10, 11], and FasL expression has been demonstrated in rat and mouse CL [2, 10]. Studies to assess the responsiveness of luteal cells to Fas-mediated killing have not been done.
We previously showed that mouse ovarian surface epithelial cells (OSE) present as purified cultures and in mixed culture with dispersed CL cells were sensitive to Fas-mediated apoptosis when pretreated with interferon (IFN). However, CL cells in the mixed CL/OSE cultures were unaffected [12]. In subsequent experiments, we found that mouse granulosa cells were resistant to Fas mAb-induced killing in the presence and absence of IFN but became sensitive when treated with a combination of IFN + tumor necrosis factor (TNF) or with the protein synthesis inhibitor cycloheximide (CX) [13]. The current studies were undertaken to determine conditions under which dispersed cells from mouse CL may be responsive to Fas-mediated apoptosis in vitro.
MATERIALS AND METHODS
Materials
Mice were obtained from Charles River (Wilmington, MA). All culture media, additives, enzymes, and cytokines were obtained from Gibco BRL (Grand Island, NY), except that L-glutamine was from Sigma Chemical Co. (St. Louis, MO). Tissue culture plates were obtained from Corning-Costar (Cambridge, MA), and slide-well chambers were from Nunc-Intermed (Naperville, IL). Monoclonal hamster anti-mouse Fas antibody (clone Jo-2) was from PharMingen (San Diego, CA), and hamster IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA). Neutravidin-Oregon Green 488 was obtained from Molecular Probes (Eugene, OR). Terminal deoxynucleotidyl transferase (TdT) and avian myeloblastosis virus reverse transcriptase were obtained from Promega (Madison, WI), random hexamer was from Pharmacia (Piscataway, NJ), Taq polymerase was from Fisher (Pittsburgh, PA), biotin-dUTP was from Boehringer Mannheim (Indianapolis, IN), and 32P-dCTP was from NEN Life Sciences (Boston, MA).
Cell Culture and Animals
Ovaries were obtained from pseudopregnant Crl:CD-1(ICR)BR mice 2 to 4 days after breeding with a vasectomized male. Procedures were approved by the Cornell University Institutional Animal Care and Use Committee and are in accord with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Ovaries were trimmed, incubated for 30 min at 37°C in 0.25% trypsin/1 mM EDTA, vortexed three times for 5 sec each, and rinsed in Dulbecco's minimum essential medium (DMEM)-F12. This step was performed to remove OSE from the ovaries because our previous work showed that OSE are present in CL cultures prepared without this step. CL cultures prepared from ovaries with OSE removed rarely contained patches of OSE (<1 patch/culture well), which are highly distinctive, whereas in our previous studies we routinely identified >5 patches/culture well. Individual CL were isolated and enzymatically dispersed as previously described [12]. Cells were resuspended in basal medium (DMEM-F12 containing 88 µg/ml pyruvate, 292 µg/ml L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone) plus 10% fetal bovine serum (FBS) and plated on Day 0 at a density of 6 x 104 cells per well in 24-well (12 mm) culture plates or 2 x 105 cells/well in 6-well (35 mm) culture plates. For cytochemical analyses, cells were plated at 6 x 104 cells/well in 8 x 20-mm slide-well chambers. Serum concentration in medium was reduced to 5% on Day 1, and thereafter medium was changed daily. Use of 10% medium on Day 0 was intended to promote initial plating of the cells.
Cell Cultures: Experimental Design
In the first experiment, the ability of Fas mAb to induce cell death was assessed in the presence or absence of IFN and TNF. CL cultures were preincubated on Day 2 in basal medium plus FBS with no cytokines, with 200 U/ml IFN, with 10 ng/ml TNF, or with 200 U/ml IFN + 10 ng/ml TNF. On Day 3, cultures from each cytokine pretreatment group were treated with the same cytokines and either 2 µg/ml Fas mAb or 2 µg/ml hamster IgG (control). Pretreatment and treatment were performed with fresh medium changes. On Day 4, cells were rinsed, trypsin was added, and live cells were counted in a hemacytometer by trypan-blue exclusion. Percentage of killed cells for cultures treated with Fas mAb was calculated by comparison with IgG-treated cultures given the same pretreatment. In additional cultures (n = 3), cells were pretreated exactly as described above on Day 2 and were frozen at -80°C on Day 3 for subsequent analysis of Fas mRNA.
A second experiment was performed exactly as above except that 0.5 µg/ml CX was added to cells 2 h prior to treatment with Fas mAb or IgG on Day 3. The medium was changed with CX treatment, and Fas mAb or IgG were added in small volumes to the existing medium. Cells were counted on Day 4 as described above.
The effects of medium containing FBS versus defined medium on Fas-mediated killing were tested in cultures prepared as described above. On Day 2, cultures were pretreated with the same cytokine treatments (no cytokines, TNF, IFN, IFN + TNF) in either basal medium containing FBS or in basal medium containing 100 ng/ml insulin, 5 µg/ml transferrin, 20 nM selenium, and 0.1% BSA (ITS medium). On Day 3, medium was replaced with medium containing the same pretreatment and either 2 µg/ml Fas mAb or 2 µg/ml IgG. On Day 4, cells were counted as described above.
In each experiment, treatments were performed in three wells and the experiment was repeated with three separate CL preparations. Percentage of cells killed was determined by subtracting the mean cell counts of Fas mAb-treated wells from the mean counts in IgG-treated wells given the same pretreatment and then dividing by the mean counts in the IgG-treated wells. Percentage of cells killed was analyzed by one-way randomized complete block ANOVA, and Duncan's procedure was used to compare individual means [14]. A supplemental analysis of cell counts in IgG-treated wells given various pretreatments was performed by randomized complete block ANOVA. All data are presented as mean ± SEM.
Analysis of Fas Antigen mRNA
Fas mRNA was quantified by a competitive reverse-transcriptase polymerase chain reaction (RT-PCR) assay as previously described [12]. RNA was reverse transcribed in the presence of various concentrations of an internal Fas standard RNA. The standard RNA was prepared by in vitro transcription of a mutated Fas cDNA containing a 50-base pair (bp) deletion internal to the PCR primer binding sites. cDNA in the RT reaction was amplified by PCR in the presence of 32P-dCTP using primers designed to span two introns and to generate a 264-bp fragment for the test RNA and a 214-bp fragment for the internal RNA standard. RT-PCR products were fractionated on a 2% agarose gel, and radioactive signal was quantified on a Fuji BAS1000 PhosphoImager. The concentration of Fas mRNA in each sample was calculated by regression of the log of the ratio of sample signal intensity to standard signal intensity versus standard concentration. All samples from a single experiment were analyzed together. Data were analyzed by a one-way randomized complete block ANOVA with supplemental analyses by Duncan's procedure [14].
Detection of Membrane-Associated Phosphatidylserine
Detection of phosphatidylserine on the outside of the cell membrane, a unique and early marker for apoptosis [15], was performed using a commercial kit (TACS annexin V-Oregon Green; Trevigen, Gaithersburg, MD) as previously described [13]. Cells were cultured as described above, pretreated with IFN + TNF, and tested 5 h after Fas mAb or IgG treatment in the presence of CX or 9 h after Fas mAb or IgG treatment in the absence of CX for staining with annexin V or propridium iodide (PI, a vital stain). Test times were defined as the earliest time when an increase in dying cells was observed. Healthy cells were unstained by either dye, apoptotic cells were stained only by annexin V, and dead cells were stained by annexin V and PI. The assay was repeated on three separate CL preparations. Previous studies have utilized flow cytometry to quantify annexin V staining of nonadherent cells. Flow cytometry was not attempted in the present study because of concerns that removal of cells from the culture dish by trypsinization or other methods would affect membrane staining.
In Situ End-Labeling of DNA
Fragmentation of DNA typical of apoptosis was detected by the TUNEL technique [16]. Cells were fixed in Carnoy's fixative for 15 min at -20°C and hydrated in PBS. Cells were incubated with 10 µM biotin-dUTP and 200 U/ml TdT enzyme for 30 min at room temperature. Cells were rinsed, blocked with PBS-1% NGS for 5 min, incubated with neutravidin-Oregon Green 488 in PBS-1% normal goat serum (NGS), and observed under phase contrast and epifluorescent illumination using a 495-nm excitation filter and a 520-nm absorption filter. Cells with distinct bright nuclear staining, indicative of numerous strand ends labeled by TdT, were considered positive, and cells with diffuse or no nuclear staining were considered negative. The percentage of positive cells was calculated from randomly chosen fields (two fields/well and two wells/treatment for each of three replicates) photographed under fluorescent light using a Spot II CCD camera (Diagnostic Instruments, Sterling Heights, MI). In one experiment, cells were pretreated with IFN + TNF on Day 2 as described above and treated with IFN + TNF with or without CX and with either Fas mAb or IgG on Day 3. End-labeling was performed 12 h after treatment, when cell death was actively occurring. A second experiment was performed to determine whether any apoptosis was occurring due to the IFN + TNF pretreatment, because results of a previous experiment suggested that the combination of these two cytokines may cause apoptosis in mouse CL cells [17]. Cells were treated with IFN + TNF on Day 2 as described, and end-labeling was performed 12 and 36 h later. Data from both experiments were log transformed to equalize variance and were analyzed by randomized complete block ANOVA and Duncan's test.
RESULTS
Fas mAb-Mediated Killing of Mouse CL Cells
To determine if cells from dispersed CL have a functional Fas pathway, cultures were pretreated with or without the cytokines IFN and TNF for 24 h and then treated with Fas mAb for 24 h in the presence or absence of cytokines. CL cells pretreated without cytokines or with IFN or TNF were resistant to Fas mAb-induced killing. However, 23% killing occurred in response to Fas mAb in cells pretreated with IFN + TNF (Fig. 1). Analysis of cell numbers in IgG-treated cultures on Day 4 of culture indicated no difference in cells pretreated without cytokines and those pretreated with IFN or TNF. IgG-treated cultures pretreated without cytokines contained 10.7 ± 0.8 x 104 cells/well, 78% more cells than were plated on Day 0. Cultures treated with IFN + TNF had 9.1 ± 1.1 x 104 cells/well, 53% more than the number of cells plated but only 86% of the number of cells in the IgG-treated cultures not pretreated with cytokines (P < 0.05). On observation by phase contrast microscopy (Fig. 2), these control cultures appeared healthy and did not have increased numbers of apoptotic cells.
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Expression of Fas mRNA in Mouse CL Cells
Levels of Fas mRNA were measured using a quantitative competitive RT-PCR assay (Fig. 3). Fas mRNA was detectable in CL cells cultured for 48 h and was increased 2.1-, 2.0-, and 11.8-fold in cells treated for 24 h with TNF, IFN, or IFN + TNF, respectively (Fig. 4).
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Effect of CX on Responsiveness of Mouse CL Cells to Fas mAb
To determine if labile protein inhibitors of the Fas pathway prevent responsiveness to Fas mAb, cells were treated with the protein synthesis inhibitor CX. Cycloheximide induced responsiveness to Fas mAb-induced killing in CL cells cultured in the absence of cytokines (34.0% killing; Fig. 5). Pretreatment of CL cells with TNF alone did not alter Fas mAb-induced killing in the presence of CX (42.3% killing), whereas pretreatment with IFN or IFN + TNF potentiated killing (62.4% and 96.0% killing, respectively). On Day 4 of culture, IgG-treated cultures not pretreated with cytokines contained 6.6 ± 0.9 x 104 cells/well, 10% more cells than the number of cells originally plated. Cell number in cultures pretreated with IFN + TNF and treated with IgG was slightly lower (91%, p < 0.05) than cell number in IgG-treated cultures not pretreated with cytokines. These cultures appeared healthy (Fig. 2) and did not have increased numbers of apoptotic cells. Cell number in cultures pretreated with either IFN alone or TNF alone was not different from cell number in cultures receiving no pretreatment.
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Fas mAb-Induced Killing of CL Cells Occurs by Apoptosis
A specific and early marker of cells undergoing apoptosis is the translocation of phosphatidylserine from the inside to the outside of the cell membrane. This translocation can be detected by the binding of fluorescent-labeled annexin V to the external membrane of intact cells that exclude the nuclear dye PI [15]. Positive annexin V staining was observed after Fas mAb treatment in CL cells pretreated with or without CX in the presence of IFN + TNF (Fig. 6). IgG-treated cultures pretreated with IFN + TNF or CX + IFN + TNF were not stained with annexin V (Fig. 6). Apoptotic cells that appear highly condensed under phase contrast microscopy stain uniformly bright with annexin V, whereas cells that are not as condensed have a more punctate staining pattern. Staining with PI was absent (not shown) because dead cells with disrupted membranes do not adhere to culture dishes.
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A second characteristic of apoptotic cells is the degradation of cellular DNA into oligonucleosomal units. This process can be detected by the TUNEL technique, which labels the ends of DNA strands with fluorescent dUTP. Apoptotic cells labeled by the TUNEL technique have brightly fluorescent, condensed nuclei. In cells pretreated with IFN + TNF and treated with Fas mAb for 12 h, nuclei of 10% of cells had positive end-labeling by TUNEL versus 3% of nuclei in cells pretreated with IFN + TNF and treated with IgG (P < 0.05, Fig. 7). When cells were treated as above in the presence of CX, 33% were positive in Fas mAb-treated cultures versus 5% in IgG-treated controls (P < 0.05).
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We also determined the number of cells with positively labeled nuclei by TUNEL after 12 and 36 h of IFN + TNF treatment in the absence of Fas mAb. Twelve hours after treatment with IFN + TNF, 3.1% ± 1.0% of cells were positive versus 3.0% ± 0.8% in controls, and at 36 h, 2.5% ± 0.5% were positive versus 3.4% ± 0.3% in controls.
Fas mAb-Induced Killing of CL Cells in Serum-Free Medium
Resistance of CL cells to Fas mAb-induced killing in the absence of IFN + TNF or CX suggested that the Fas pathway is present in cells from the CL but may be inhibited under the culture conditions employed. The effect of culturing CL cells in basal medium supplemented with either FBS or ITS was tested. Whereas cells cultured in FBS were resistant to Fas mAb unless pretreated with IFN + TNF (29.9% killing), cells cultured in ITS became responsive in the presence of IFN alone or IFN + TNF (23.4% and 28.9% killing, respectively; Fig. 8).
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DISCUSSION
These data demonstrate that engagement of Fas with Fas mAb induces apoptosis of mouse CL cells cultured under certain conditions. Results of TUNEL and annexin V staining indicate that the cells were dying by apoptosis in response to Fas mAb, and cell counts used to calculate percentage of cells killed demonstrate the magnitude of cell death in these cultures. The fact that the percentage of cells positive by TUNEL in Fas mAb-treated cultures was less than half of the percentage killed as determined by cell counts is to be expected because the TUNEL technique detects only cells in the advanced stages of apoptosis after degradation of DNA has occurred. Cells that have not progressed to this stage or that have died and sloughed off the plate will not be detected. Thus, actual cell counts are very important in quantifying cell death.
The CL cultures used were total dispersates of mouse CL, which would be expected to contain steroidogenic luteal cells, fibroblasts, and endothelial cells. In a previous study, we used Nile red staining for lipid droplets to determine the presence of steroidogenic luteal cells in cultures that were prepared in a similar fashion [12]. After 4 days, the majority of cells in the cultures stained positively for lipid droplets. CL cultures were also incubated with octadyl indocarbocyanine-acetylated low-density lipoprotein, which is taken up specifically by endothelial cells and monocytes. After 4 days, less than 10% of the cells in cultures were endothelial cells. Therefore, the predominant cell type present in culture was the steroidogenic luteal cell. No attempt was made to determine the number of large versus small luteal cells present. However, large rat luteal cells are reported to be more fragile than small luteal cells and more susceptible to degradation during enzymatic dispersion [18].
Responsiveness of CL cells to Fas mAb-induced killing was affected by the presence of FBS, the cytokines IFN and TNF, and the protein synthesis inhibitor CX. In the presence of FBS, pretreatment with IFN + TNF was required for Fas mAb-induced killing. Fas mRNA levels were increased two-fold by IFN or TNF, but the combination of IFN + TNF induced a 12-fold increase. This finding suggests that combined treatment with IFN + TNF may have been necessary to induce an effective level of Fas expression, whereas doubling of Fas expression by IFN or TNF independently was insufficient. However, CL cells became responsive to Fas mAb in the absence of cytokines when treated with CX, suggesting that labile protein inhibitors of the Fas pathway may be present in CL cells and may be more important than the level of Fas expression in limiting responsiveness to Fas-mediated killing. Because treatment with CX has also been shown to promote Fas-mediated killing in a number of nonovarian cell types [19], this effect of labile protein inhibitors may be a general characteristic of the pathway. Protein inhibitors of Fas-mediated killing, such as FLIP and FAP-1, have been identified. FLIP is expressed in ovarian tissue [20], and FAP-1 is expressed in granulosa cells [13]. Another class of inhibitors, the inhibitor of apoptosis proteins (IAPs), has been localized to ovarian follicular cells [21]. The presence of inhibitory proteins in ovarian cells may be of primary importance in limiting Fas-mediated apoptosis.
Fas mAb-induced killing in the presence of CX was higher in CL cells pretreated with IFN than in cells that were not pretreated with cytokines or that were pretreated with TNF. The fact that Fas mRNA levels were increased similarly in cells treated with IFN or TNF again suggests that levels of expression of Fas may not be the primary factor determining cellular responsiveness. IFN probably has other effects on the Fas pathway in addition to increasing expression. IFN has been shown to increase expression of caspases, which become activated during apoptosis, and of proapoptotic Bcl-2 family members [22–24].
In a previous study, we reported that OSE present in cultures prepared from mouse CL, as well as purified preparations of OSE, were susceptible to Fas mAb-induced killing when pretreated with IFN in medium containing FBS; however, the CL cells surrounding the patches of OSE were resistant under these conditions [12]. The results reported here confirm that mouse CL cells cultured in serum-supplemented medium are resistant to Fas mAb-induced killing in the presence and absence of IFN and require pretreatment with IFN + TNF to promote responsiveness. CL cells cultured in serum-free medium supplemented with ITS only required pretreatment with IFN to become responsive to Fas mAb. FBS may contain factors that inhibit Fas-mediated killing. Growth factors such as insulin-like growth factor (IGF) that are present in serum have been shown to attenuate the spontaneous apoptosis of granulosa cells cultured in serum-free medium [25]. The concentration of insulin used in the ITS medium in the present study (100 ng/ml) is 10-fold less than what is used in commercial ITS preparations and would have minimal IGF activity. Our recent results showed that bovine granulosa cells are killed by recombinant human FasL in serum-free medium (supplemented with ITS) in the absence of cytokines [26]. However, in medium containing FBS, pretreatment with IFN alone was required to promote FasL-induced apoptosis; TNF alone had no effect, and cotreatment with IFN + TNF did not further enhance killing [27]. Therefore, bovine granulosa cells may be somewhat more responsive to Fas-mediated killing than are mouse CL cells. However, in cells from both species, inhibitory effects of FBS on Fas-mediated killing were observed.
The Fas antigen is expressed in rat, mouse, and human CL [2, 10, 11], and FasL has been detected in rat and mouse CL [2, 10]. Expression of Fas protein was higher in regressing CL than in newly developed CL of mice and humans [10, 11], and expression of FasL protein was elevated in the regressing postpartum rat CL [2]. These studies demonstrate the presence of the Fas pathway in CL cells and modulation of expression during different physiological states. The present experiments are the first to directly study responsiveness of CL cells to Fas-mediated apoptosis and to assess the effect of culture conditions and cytokines. Because the presence of serum in culture medium inhibited Fas-mediated killing, it is possible that factors that support viability of CL cells in vivo also prevent Fas-mediated killing. At the time of luteal regression, removal of trophic factors may allow activation of the Fas pathway. The Fas pathway may also become activated by cytokines within the CL. IFN is expressed within bovine CL [28], and TNF has been shown to be expressed by luteal and endothelial cells within the CL of the mouse and other species [29]. In addition, increased infiltration of macrophages and lymphocytes capable of secreting TNF and IFN occurs during natural regression of the CL [30] and could potentially contribute to activation of the Fas pathway.
Exposure of IgG-treated cultures to IFN (200 U/ml) and TNF (10 ng/ml, equivalent to 1000 U/ml) for 48 h caused a small reduction in cell number (86% of controls in the absence of CX and 91% in the presence of CX). The decrease in cell number was not attributable to apoptosis. Cells incubated with IFN + TNF for 12 or 36 h had the same low percentage of cells positive for TUNEL as did control cultures. Positive staining with annexin V was not detected in cells exposed to IFN + TNF for 29 h. On visual observation, cultures treated with IFN + TNF appeared healthy and did not have increased numbers of dead cells. A possible explanation is that growth of cells in culture may be inhibited by IFN + TNF. This possibility is supported by the fact that cell number at the end of culture on Day 4 was 78% greater than the number originally plated on Day 0 in IgG-treated cultures but was only 53% greater in cultures treated with IFN + TNF. The identity of the cell type(s) that increased in number during culture was not determined. Previous studies showed that fibroblast growth factor and serum increased proliferation in bovine CL cultures [31]. Evidence for the presence of steroidogenic cells in the cultures was presented, but subsequent studies by others questioned whether proliferation might be attributable to contaminating nonsteroidogenic cell types such as endothelial cells [18]. In a previous study, the effects of higher concentrations of cytokines for longer periods of time on viability of CL cells from pregnant mice were tested; 5000 U/ml of TNF was required to reduce cell number by Day 3 of culture in the presence of 1000 U/ml IFN. By Day 6 of culture, 1000–5000 U/ml of TNF in the presence of IFN decreased cell number. The number of apoptotic cells, assessed by in situ analysis of DNA fragmentation, increased in response to IFN + TNF on Day 6 but not on Day 3 of culture [17]. In bovine CL cells, TNF (at 1000 ng/ml but not 10 ng/ml) in the presence of IFN (100 U/ml) reduced cell number on Day 7 of culture [32]. The ability of IFN + TNF to induce apoptosis of CL cells may require extended exposure and/or relatively high doses.
In summary, the Fas pathway is present in cells within mouse CL and can be activated under certain conditions. Serum reduces responsiveness of cultured CL cells to Fas-mediated apoptosis, whereas CX and the cytokines IFN and TNF increase responsiveness. A future challenge is to elucidate the physiological factors that result in suppression and activation of the Fas pathway in vivo.
FOOTNOTES
First decision: 29 December 1999.
1 This work was supported by NIH grant HD 32535. 
2 Correspondence: Susan M. Quirk, 258 Morrison Hall, Cornell University, Ithaca, NY 14853. FAX: 607 255 9829; smq1{at}cornell.edu
Accepted: February 9, 2000.
Received: November 29, 1999.
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