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The Journal of Immunology, 2003, 170: 6209-6216.
Copyright © 2003 by The American Association of Immunologists

Fas (CD95) Induces Proinflammatory Cytokine Responses by Human Monocytes and Monocyte-Derived Macrophages1,2

David R. Park3,*, Anni R. Thomsen{dagger}, Charles W. Frevert*, Uyenvy Pham{dagger}, Shawn J. Skerrett*, Peter A. Kiener4,{ddagger} and W. Conrad Liles{dagger}

Divisions of * Pulmonary and Critical Care Medicine and {dagger} Allergy and Infectious Diseases, Department of Medicine, University of Washington School of Medicine, Seattle, WA 98104; and {ddagger} Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08540


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fas (CD95, APO-1) is regarded as the prototypical cell death receptor of the TNFR superfamily. Fas-induced apoptosis is generally considered to be a noninflammatory process, contributing to the silent resolution of immune and inflammatory responses. However, accumulating evidence indicates that Fas may also induce cellular activation signals. We hypothesized that Fas could activate proinflammatory cytokine responses by normal human monocytes and macrophages. Monocytes were isolated by negative immunoselection from the PBMC fraction of venous blood from healthy volunteers, and monocyte-derived macrophages were cultivated in vitro. Both monocytes and monocyte-derived macrophages released TNF-{alpha} and IL-8 following Fas ligation, and conditioned medium from Fas-activated monocytes and macrophages induced the directed migration of neutrophils in a chemotaxis assay. Fas-induced monocyte cytokine responses were associated with monocyte apoptosis, nuclear translocation of NF-{kappa}B, and cytokine gene expression and were blocked by caspase inhibition but not by inhibition of IL-1{beta} signaling. In contrast, Fas-induced macrophage cytokine responses occurred in the absence of apoptosis and were caspase independent, indicating maturation-dependent differences in the Fas signaling pathways that lead to proinflammatory cytokine induction. Rather than contributing to the resolution of inflammation, Fas ligation on circulating monocytes and tissue macrophages may induce proinflammatory cytokine responses that can initiate acute inflammatory responses and tissue injury.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Fas (CD95, APO-1) is generally regarded as the prototypical cell death receptor of the TNFR superfamily and initiates apoptosis following engagement by Fas ligand (FasL,5 CD95 ligand, TNFSF6) or by agonistic anti-Fas Abs (1, 2, 3). Fas triggers cell death pathways by aggregating cytosolic adapter proteins such as the Fas-associated death domain-containing protein (FADD), leading to the activation of caspase-8/FLICE and downstream cell death caspase cascades (4, 5). The prevailing notion has been that Fas-induced apoptosis functions as a noninflammatory pathway for the elimination of effete cells, especially during the regulation and resolution of immune and inflammatory responses (5, 6, 7, 8).

However, accumulating evidence indicates that Fas may also induce cellular activation signals (9). It is now well established that Fas signals parenchymal tissue cells to secrete proinflammatory cytokines and chemotactic factors. For instance, Fas ligation has been reported to stimulate chemokine production by a colonic epithelial cell line (10), early passage fetal human astrocytes (11), human synoviocytes (12), human bronchiolar epithelial cells (13), and human vascular smooth muscle cells (14). Many of these responses are associated with the activation of the transcription factor NF-{kappa}B (13, 15, 16, 17, 18, 19, 20).

Macrophages represent potentially important sources of Fas-induced proinflammatory mediators, but whether Fas ligation induces cytokine responses by normal macrophages is unclear. The evidence from animal and human studies is inconclusive. For instance, Fas ligation has been shown to induce IL-18 release by murine hepatic and splenic macrophages primed in vivo by IFN-{gamma} (21), IL-1{beta} release by murine bone marrow dendritic cells primed with LPS (22), TNF-{alpha} and IL-1{beta} release by a transformed murine dendritic cell line (22), and TNF-{alpha} release by a murine macrophage cell line (23). Only a single report has described Fas-induced proinflammatory cytokine release (IL-1{beta}) by normal unprimed macrophages (murine resident peritoneal macrophages) (24).

Human monocytes and monocyte-derived macrophages both express Fas but are differentially susceptible to Fas-induced apoptosis (25, 26, 27). There are no published reports of Fas-induced proinflammatory cytokine responses by human monocytes or macrophages. In fact, the sole published study of Fas-induced human monocyte cytokine responses examined only production of the anti-inflammatory cytokine IL-10 and concluded that Fas-induced monocyte cytokine production may represent an important anti-inflammatory mechanism (28).

In the present study, we sought to determine whether Fas induces proinflammatory cytokine responses by normal human monocytes, to determine the effect of macrophage maturation on these responses, to characterize the net biological proinflammatory activity of the mediators released, and to begin to examine the mechanisms of signaling of these responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Recombinant human soluble Fas ligand (sFasL) was prepared as previously described (25, 29). Fas-specific mAbs CH-11 and ZB4 were obtained from Coulter Immunotech (Miami, FL). Recombinant human IL-1R antagonist protein and human IL-1{beta}-specific polyclonal goat IgG were obtained from R&D Systems (Minneapolis, MN). Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) and benzyloxycarbonyl-Phe-Ala-fluoromethylketone (z-FA-fmk) were obtained from Enzyme Systems Products (Livermore, CA). All of these reagents were LPS free (<0.01 endotoxin units/ml) by the Limulus amebocyte assay (ECL-1000; BioWhittaker, Walkersville, MD). LPS (derived from Escherichia coli 055:B5) and other standard chemical reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated.

Cell preparations

Normal human PBMC and neutrophils were isolated from the EDTA-anticoagulated venous blood of healthy volunteers using density gradient centrifugation over Histopaque-1077 (Sigma-Aldrich) as previously described (25). Monocytes were isolated from the PBMC fraction by negative immunoselection using the Monocyte Isolation kit and Depletion Column Type BS (Miltenyi Biotec, Auburn, CA) according to the supplier’s instructions. The monocyte preparations were >95% viable and contained >95% mononuclear cells on cytocentrifuge specimens (Shandon Southern Cytospin; Shandon, Pittsburgh, PA) stained with a modified Wright-Giemsa stain (Diff-Quick; American Scientific Products, McGaw Park, IL). More than 95% expressed CD14 as determined by immunofluorescence flow cytometry. The neutrophil preparations were >95% viable and contained >98% polymorphonuclear cells.

Monocyte-derived macrophages were prepared as previously described (25) by culturing freshly isolated monocytes in RPMI 1640 supplemented with both 10% FCS and 10% human AB serum, 2 mM L-glutamine, 25 U/ml penicillin, and 25 µg/ml streptomycin (all from BioWhittaker) in Teflon PFA Vials (Cole-Parmer, Vernon Hills, IL) at 37°C in a humidified 5% CO2 incubator. After 7 days, dead cells were removed by centrifugation over Histopaque-1077. The remaining cells all had macrophage morphology and were >95% viable.

For experimental use, purified monocytes and monocyte-derived macrophages were suspended at a concentration of 1 x 106 cells/ml in RPMI 1640 supplemented with 2 mM L-glutamine, 25 U/ml penicillin, and 25 µg/ml streptomycin. Unless otherwise indicated, the monocyte culture medium was routinely supplemented with 10% human AB serum to lessen Fas-induced monocyte apoptosis (25). The cells were cultured with test reagents at 37°C in a humidified 5% CO2 incubator in either polypropylene tubes or cell culture plates (Costar, Cambridge, MA). At selected time points, the conditioned culture medium was aspirated, filtered, and frozen at -80°C for later analysis of cytokine levels and chemotactic activity. Cell pellets were harvested for apoptosis determination and preparation of RNA and nuclear proteins as described below.

Apoptosis

Monocyte and monocyte-derived macrophage apoptosis was assessed using the binding of FITC-annexin V (Annexin VFITC Apoptosis Detection kit; R&D Systems). Treated monocytes or macrophages (5 x 105/0.5 ml) were washed and incubated with saturating concentrations of FITC-annexin V for 30 min at room temperature according to the manufacturer’s instructions. The labeled cells were analyzed immediately using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ). Apoptotic cells were defined as those with green fluorescence >98% of unlabeled cells, and this was confirmed by morphological analysis.

Cytokine ELISA

TNF-{alpha}, IL-1{beta}, IL-8, IL-10, and TGF-{beta}1 immunoreactivity were measured in conditioned tissue culture supernatants using specific human ELISAs according to the supplier’s protocols (R&D Systems). These monocyte/macrophage cytokine products were chosen to represent acute phase proinflammatory cytokines (TNF-{alpha} and IL-1{beta}), neutrophil chemoattractants (IL-8), and anti-inflammatory cytokines (IL-10 and TGF-{beta}1). The lower limits of detection of these assays were 8 pg/ml for TNF-{alpha}, IL-1{beta}, and TGF-{beta}1; 15 pg/ml for IL-8; and 31 pg/ml for IL-10. The TGF-{beta}1 assay recognized only the active form of TGF-{beta}1.

Neutrophil chemotaxis

Neutrophil migration toward monocyte- and macrophage-conditioned tissue culture medium was assessed with a fluorescence-based assay using 96-well chemotaxis chambers containing polycarbonate filters with 8-µm pores (ChemoTx; Neuro Probe, Gaithersburg, MD) (30). Normal human neutrophils were incubated in RPMI 1640 containing 10% FCS and 5 µg/ml calcein AM (Molecular Probes, Eugene, OR), then washed and suspended at 4 x 106 cells/ml in phenol red-free RPMI 1640 (Sigma-Aldrich). Quadruplicate chamber wells were filled with varying dilutions of conditioned medium, fresh medium containing mAb CH-11 or LPS, or fresh medium alone. Wells containing PBS were used as the negative control. Zymosan-activated serum (prepared by incubating human AB serum with 100 mg/ml zymosan (ICN Pharmaceuticals, Cleveland, OH) for 30 min at 37°C) served as a positive control for chemotaxis. The chemotaxis membrane was applied and labeled neutrophils (1 x 105 cells/25 µl) were placed directly onto the membrane. The chambers were incubated for 60 min at 37°C, nonmigrating cells on the top side of the membrane were removed, and fluorescence was determined in a fluorescence microtiter plate reader (CytoFluor II; PerSeptive Biosystems, Framingham, MA) in the bottom-read position. The data are reported as a chemotaxis index, normalized to neutrophil migration toward PBS.

Cytokine mRNA expression

Expression of monocyte cytokine mRNA was analyzed using a ribonuclease protection assay. Total cellular RNA was isolated from 5 x 106 monocytes using a column-based system (RNeasy Minikit;, Qiagen, Valencia, CA) and treated with RNase-free DNase (DNAfree; Ambion, Austin, TX) to remove any contaminating DNA. A biotinylated RNA probe mixture was prepared by in vitro transcription using a custom template set containing probes for proinflammatory (TNF-{alpha} and IL-1{beta}), chemotactic (IL-8 and monocyte chemoattractant protein-1 (MCP-1), and anti-inflammatory (IL-10 and TGF-{beta}) cytokines and standard housekeeping gene sequences (RiboQuant; BD PharMingen, San Diego, CA). The supplier’s protocol was modified to include biotinylated nucleotide RNA labeling mixture (Roche Molecular Systems, Pleasanton, CA) instead of 32P-labeled nucleotides. Hybridization with sample RNA and ribonuclease digestion were performed according to the RiboQuant protocol, and the protected probe fragments were purified using RNase inactivation/precipitation solution (Ambion). The samples were resolved using 5% denaturing polyacrylamide gel electrophoresis (Protean IIxi; Bio-Rad, Hercules, CA), transferred to a nylon membrane (Roche) using semidry transfer apparatus (Bio-Rad), and developed using a streptavidin-HRP conjugate and chemiluminescent detection system (North2South ECL kit, Pierce, Rockford, IL).

NF-{kappa}B activation

Monocyte NF-{kappa}B activation and nuclear translocation were assessed using an EMSA. Nuclear proteins were extracted from 5 x 106 human monocytes according to the protocol of Dignam et al. (31) as modified by Brophy and Sibley (32). The Bradford assay (Bio-Rad) was used to determine the protein concentrations of the extracts. The double-stranded NF-{kappa}B consensus oligonucleotide, 5'-AGT TGA GGG GAC TTT CCC AGG C-3' (Promega, Madison, WI), was end-labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase using the Promega Gel Shift Assay kit. For supershifts, 1 µl of anti-NF-{kappa}B p65 rabbit antiserum (provided by Dr. K. Bomsztyk, University of Washington, Seattle, WA) or 4 µg of anti-NF-{kappa}B p50 rabbit polyclonal IgG Ab (Santa Cruz Biotechnology, Santa Cruz, CA) were preincubated with the nuclear extracts in binding buffer for 30 min before addition of the labeled probe. For cold competition, a 50-fold excess of unlabeled probe was added before the 32P-labeled probe. Electrophoresis of the DNA protein complexes was performed in 6% nondenaturing polyacrylamide gels as described in the Promega Gel Shift Assay System protocol and the resulting gels were dried and exposed to film.

Statistical analysis

Most experiments were performed four times using cells from different donors, and results are reported as the mean ± SEM or as designated. Statistical comparisons were made using Student’s t test or ANOVA with the Tukey-Kramer post hoc test for multiple comparisons using GraphPad InStat software (GraphPad Software, San Diego, CA). Differences were regarded as significant if p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fas ligation induces human monocyte and monocyte-derived macrophage proinflammatory cytokine production

Fas ligation has previously been reported to induce production of the anti-inflammatory cytokine IL-10 by human monocytes (28). The main purpose of our studies was to determine whether Fas induces human monocyte and macrophage proinflammatory cytokine responses. The agonistic Fas mAb CH-11 induced a dose-dependent accumulation of TNF-{alpha} and IL-8 that became evident after 4 h and further increased after 18 h in both monocyte and monocyte-derived macrophage cultures (Fig. 1). CH-11-induced TNF-{alpha} and IL-8 levels approached the levels induced in response to LPS used as a positive control stimulus. The same dose-response patterns of TNF-{alpha} and IL-8 production were observed consistently in four independent experiments. The IL-8 responses to mAb CH-11 stimulation did not reach statistical significance in this series of experiments due to the higher background IL-8 release by unstimulated cells and the greater variability between individual experiments. Statistically significant IL-8 responses were observed after 18 h in other experiments (see Figs. 2 and 7).



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  		FIGURE 1. Fas induces human monocytes and monocyte-derived macrophages to produce predominantly proinflammatory cytokines. Monocytes (left panels) and monocyte-derived macrophages (right panels) were stimulated with a range of concentrations of agonistic Fas mAb (CH-11, 5–500 ng/ml) or LPS (1 µg/ml) as indicated. Conditioned medium was harvested from parallel wells after 1, 4, and 18 h and analyzed for levels of TNF-{alpha} (top panels), IL-8 (middle panels), and IL-10 (bottom panels) using cytokine-specific ELISAs. The bars show the mean values ± SEM of four independent experiments using different donors. The asterisks indicate results significantly different (p <.05) from the unstimulated condition at each time point.

 


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  		FIGURE 2. Fas-induced human monocyte cytokine responses are equivalent after stimulation with either sFasL or mAb CH-11. Monocytes were incubated 18 h with agonistic Fas mAb (CH-11, 500 ng/ml), sFasL (500 ng/ml), CH-11 plus inhibitory Fas mAb (ZB4, 10 µg/ml, added 1 h before CH-11), or LPS (1 µg/ml) as indicated. Conditioned medium was harvested and analyzed for levels of TNF-{alpha} (top) and IL-8 (bottom) using cytokine-specific ELISAs. The bars show the mean values ± SEM of four independent experiments using different donors. The asterisks indicate results significantly different (p < 0.05) from the unstimulated condition. ZB4 had no effect on spontaneous or LPS-induced cytokine responses (data not shown).

 


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  		FIGURE 7. Fas-induced proinflammatory cytokine responses of monocytes and monocyte-derived macrophages are differentially susceptible to inhibition of caspases. Monocytes (top panels) and monocyte-derived macrophages (bottom panels) were pretreated for 1 h with the pan-caspase inhibitor z-VAD-fmk or the noncaspase cysteine protease inhibitor z-FA-fmk (both 20 µM) and then stimulated with agonistic Fas mAb (CH-11, 500 ng/ml, gray bars) or LPS (1 µg/ml, black bars) as indicated. Conditioned medium was harvested after 18 h and analyzed for levels of TNF-{alpha} (left panels) and IL-8 (right panels) using cytokine-specific ELISAs. The bars show the mean values ± SEM of four independent experiments using different donors. The symbols indicate results significantly different (p < 0.05) from the unstimulated condition (*), the CH-11-stimulated condition ({dagger}), and the LPS-stimulated condition ({ddagger}).

 
In contrast to the TNF-{alpha} and IL-8 responses, Fas-induced IL-10 responses were comparatively minor (Fig. 1). Cells from only two of four donors produced detectable levels of IL-10. Furthermore, this IL-10 response was evident only at the highest dose of mAb CH-11 and, in monocytes, only at the later time point. CH-11 failed to induce detectable increases in monocyte or macrophage TGF-{beta}1 release (data not shown).

In the next series of experiments, we compared the responses of monocytes to stimulation with either mAb CH-11 or recombinant sFasL and tested the specificity of the responses using the blocking anti-Fas mAb ZB4. sFasL and mAb CH-11 were equally potent stimuli for monocyte TNF-{alpha} and IL-8 production (Fig. 2). Also, Fas blockade by mAb ZB4 completely inhibited CH-11-induced TNF-{alpha} and IL-8 responses, indicating that the effect of mAb CH-11 was mediated through the Fas receptor and not through a nonspecific effect of the IgM Ab or a contaminant. ZB4 had no effect on unstimulated monocyte cytokine production and did not alter monocyte cytokine responses to LPS (data not shown).

Fas-induced monocyte/macrophage production of neutrophil chemoattractant activity

Our Fas-induced proinflammatory cytokine data were generated using immunoassays that may potentially detect proteolytically cleaved and biologically inactive cytokine fragments released from apoptotic cells. To determine whether Fas induced the release of biologically active mediators with physiologically significant effects, the neutrophil chemoattractant activity of conditioned medium from mAb CH-11-stimulated monocytes and macrophages was determined using a quantitative fluorescence-based chemotaxis assay (Fig. 3). Conditioned medium from monocytes stimulated overnight with mAb CH-11 or LPS contained high levels of neutrophil chemotactic activity, exceeding that of 10% zymosan-activated serum (chemotactic index, 4.5 ± 0.1). This activity was detectable even after 10-fold dilution. Monocytes incubated with medium alone elaborated minimal chemotactic activity that was evident only in undiluted samples. Conditioned medium from parallel cultures of mAb CH-11-stimulated monocyte-derived macrophages produced nearly identical neutrophil chemotactic responses (data not shown). mAb CH-11 alone, at the 500 ng/ml concentration used in these studies, had no direct neutrophil chemotactic activity (chemotactic index, 1.2 ± 0.2).



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  		FIGURE 3. Fas induces monocyte release of neutrophil chemotactic activity. Monocytes were incubated for 18 h with agonistic Fas mAb (CH-11, 500 ng/ml) or LPS (1 µg/ml). Conditioned medium was harvested, diluted in medium, and analyzed for chemotactic activity using calcein AM-labeled normal human neutrophils and a fluorescence-based microtiter plate assay. A chemotactic index was calculated by dividing the mean fluorescence in wells containing conditioned medium by that in wells containing PBS. The points show the mean values from three independent experiments using different monocyte supernatant samples and different neutrophil donors. No error bars are shown because they are obscured by the data markers. The monocyte-activating reagents (mAb CH-11 and LPS) had no intrinsic neutrophil chemotactic activity.

 
Fas-induced monocyte cytokine gene expression

To determine whether Fas-induced monocyte cytokine production involved increased cytokine gene expression, steady-state cytokine mRNA levels were measured using a multiprobe ribonuclease protection assay containing probes for proinflammatory, chemotactic, and anti-inflammatory cytokines. Freshly isolated monocytes demonstrated little constitutive cytokine gene expression (Fig. 4). Unstimulated monocytes incubated for 1 h in medium alone had no detectable levels of message for TNF-{alpha} and only modest levels of that for IL-1{beta}, MCP-1, and IL-8. The IL-1{beta} and MCP-1 message levels declined after 4 and 8 h, whereas the IL-8 signal increased over this time period. CH-11 stimulation led to a marked but transient increase in TNF-{alpha} message expression, a sustained increase in IL-1{beta} and MCP-1 message expression, and an earlier and greater increase in IL-8 message levels compared with the unstimulated cells. We were unable to confirm Fas-induced monocyte IL-10 gene expression as reported by Daigle et al. (28) using a PCR technique. We suspect that the IL-10 mRNA signal was below the level of detection of our ribonuclease protection assay and therefore quantitatively much smaller than the TNF-{alpha}, IL-1{beta}, MCP-1, and IL-8 mRNA responses. No TGF-{beta}1, IL-12, or IFN-{gamma} signals were detected at any of the time points.



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  		FIGURE 4. Fas ligation induces monocyte proinflammatory cytokine gene expression. Monocytes were incubated for 2.5 h with either agonistic Fas mAb (CH-11, 500 ng/ml) or LPS (1 µg/ml), total cellular RNA was isolated, and cytokine gene expression was analyzed using a multiprobe cytokine ribonuclease detection assay. Lane 1 contains the undigested probe mixture. The remaining lanes contain ribonuclease-digested probe from reactions containing 5 µg of positive control RNA supplied by the probe manufacturer (lane 2), yeast tRNA-negative control (lane 3), RNA from freshly isolated monocytes (lane 4), from unstimulated monocytes (lanes 5–7), and from mAb CH-11-stimulated monocytes (lanes 8–10). Bands indicating the positions of the undigested probes are identified on the left. Protected probe fragments (sample mRNA signal) are identified by arrows on the right. Not all of the mRNA species are represented in the positive control RNA sample. Nonspecific bands can be identified in the yeast RNA negative control lane (lane 3). This figure is representative of three independent experiments using different monocyte donors.

 
NF-{kappa}B activation

To determine whether Fas-induced monocyte proinflammatory cytokine gene expression was associated with activation and nuclear translocation of NF-{kappa}B, EMSAs were performed using monocyte nuclear extracts and a NF-{kappa}B-specific oligonucleotide probe (Fig. 5). Constitutive NF-{kappa}B DNA-binding activity was detected in unstimulated monocytes. Stimulation of the monocytes with mAb CH-11 caused a consistent but modest increase in NF-{kappa}B nuclear translocation in each of six independent experiments, although always to a lesser extent than after stimulation with LPS. Specificity of the signal was confirmed by delay of the probe migration by Abs recognizing two NF-{kappa}B subunits (p50 and p65) and by effective competition for the DNA-binding activity by excess cold oligonucleotide probe.



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  		FIGURE 5. Fas induces NF-{kappa}B nuclear translocation in normal human monocytes. Monocytes were incubated for 1 h with either agonistic Fas mAb (CH-11, 500 ng/ml) or LPS (1 µg/ml). Nuclear extracts were prepared and incubated with a 32P-labeled DNA probe having the consensus NF-{kappa}B binding sequence, then resolved by PAGE and imaged using autoradiography. Each lane contains labeled probe. The probe was incubated without nuclear extract (lane 1), with extract from unstimulated monocytes (lane 2), with extract from LPS-stimulated monocytes (lane 3), and with extract from mAb CH-11-stimulated cells (lanes 4–7). The samples in lanes 5 and 6 were incubated with Abs recognizing the p65 and p50 NF-{kappa}B subunits, respectively. The sample in lane 7 was incubated with an excess of cold probe. Bands representing free probe, NF-{kappa}B-bound probe, and the supershifted probe are indicated. This figure is representative of six independent experiments using different donors.

 
Differential sensitivity of normal human monocytes and monocyte-derived macrophages to Fas-induced apoptosis

In most experimental models of Fas-induced proinflammatory responses, simultaneous apoptosis of the target cells has been a prominent feature (10, 13, 14, 21, 23, 24). We have previously shown, using a propidium iodide-staining assay, that monocytes but not monocyte-derived macrophages are susceptible to Fas-induced apoptosis (25). For this study, we sought to determine the extent of Fas-induced monocyte and monocyte-derived macrophage apoptosis using the more sensitive annexin V-binding assay (Fig. 6). Monocytes underwent considerable spontaneous apoptosis during overnight incubation without serum, and monocyte apoptosis was significantly augmented by mAb CH-11 as previously reported (25, 33). Serum inhibited, but did not completely block, Fas-induced monocyte apoptosis. In contrast, monocyte-derived macrophages were highly resistant to both spontaneous and Fas-induced apoptosis, regardless of the presence of serum.



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  		FIGURE 6. Fas-induced monocyte apoptosis is inhibited by serum and by maturation into monocyte-derived macrophages. Monocytes (left) and monocyte-derived macrophages (right) were incubated for 18 h in the presence or absence of 10% human AB serum and agonistic Fas Ab (mAb CH-11, 500 ng/ml). Apoptosis was measured using FITC-annexin V binding and flow cytometry. The bars show the percentages of annexin V-positive cells (mean ± SEM from three different experiments using different donors). The asterisks indicate results significantly different (p < 0.05) from the rate of spontaneous apoptosis in the control unstimulated condition.

 
Effect of caspase inhibition on Fas-induced cytokine responses

Fas-induced apoptosis is triggered by the activation of cell death caspase cascades (4, 5). These caspase cascades can indirectly induce proinflammatory cytokine responses via caspase-mediated proteolytic processing and activation of IL-1. Mature IL-1 can then stimulate cytokine gene expression via autocrine feedback through the IL-1R (14, 21, 24, 34). Based on our finding that Fas-induced monocyte cytokine responses were associated with substantial rates of apoptosis and reports that monocyte apoptosis is associated with the activation of caspases 8 and 3 (35, 36), we suspected that monocyte cytokine responses would be at least partially dependent on caspase and IL-1{beta} activity. Based on their differential sensitivity to Fas-induced apoptosis, we hypothesized that Fas-induced monocyte cytokine responses would be more sensitive to caspase inhibition than that of monocyte-derived macrophages. Therefore, we tested the effects of pretreatment with the tetrapeptide pan-caspase inhibitor z-VAD-fmk on mAb CH-11-induced cytokine production. Monocyte TNF-{alpha} responses were markedly inhibited and IL-8 responses were partially inhibited by z-VAD-fmk (Fig. 7). In contrast, macrophage TNF-{alpha} and IL-8 responses to mAb CH-11 stimulation were unaffected by caspase inhibition. Interestingly, z-VAD-fmk also selectively inhibited monocyte cytokine responses to LPS stimulation but did not influence the responses of macrophages to LPS (Fig. 7). As a control for the specificity of the z-VAD-fmk reagent, we pretreated parallel cultures with a peptide inhibitor of noncaspase cysteine proteases (z-FA-fmk). This inhibitor had no significant effect on either Fas-induced or LPS-induced cytokine responses (Fig. 7).

Fas-induced IL-1{beta} production and the effects of IL-1{beta} inhibition on Fas-induced cytokine responses

The dependence of monocyte cytokine responses on caspase activity suggested that caspase-mediated IL-1{beta} activation might play a role in initiating or amplifying other cytokine responses via an autocrine/paracrine feedback pathway (14, 21, 34). To determine the role of IL-1{beta} in mediating Fas-induced monocyte and macrophage cytokine responses, we measured Fas-induced IL-1{beta} release and tested the effects of two different strategies to block IL-1 signaling. Monocytes produced a substantial IL-1{beta} response after mAb CH-11 stimulation, whereas macrophages produced comparatively little IL-1{beta}, regardless of the stimulus (Fig. 8A). Surprisingly, inhibition of monocyte IL-1 signaling pathways by pretreatment with either a polyclonal neutralizing anti-IL-1{beta} Ab or with recombinant human IL-1R antagonist protein had little effect on Fas-induced monocyte TNF-{alpha} or IL-8 responses (Fig. 8B).



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  		FIGURE 8. Fas-induced monocyte and monocyte-derived macrophage IL-1{beta} responses. A, Monocytes (left) and monocyte-derived macrophages (right) were stimulated with a range of concentrations of agonistic Fas mAb (CH-11, 5–500 ng/ml) or LPS (1 µg/ml) as indicated. Conditioned medium was harvested from parallel wells after 1, 4, and 18 h and analyzed for levels of IL-1{beta} using a cytokine-specific ELISA. The bars show the mean values ± SEM of four different experiments using different donors. The vertical axis scale is the same in both panels to emphasize the difference in the responses. The asterisks indicate results significantly different (p < 0.05) from the unstimulated condition at each time point. B, Monocytes were pretreated for 1 h with a neutralizing polyclonal anti-IL-{beta} Ab (IL-1Ab, 1 µg/ml) or IL-1R antagonist protein (IL-1RA, 100 ng/ml) and then stimulated with agonistic Fas mAb (CH-11, 500 ng/ml) or LPS (1 µg/ml) as indicated. Conditioned medium was harvested after 18 h and analyzed for levels of TNF-{alpha} (B, top panel) and IL-8 (B, bottom panel) using cytokine-specific ELISAs. The bars show the mean values of two independent experiments using different donors and yielding similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we investigated Fas-induced cytokine responses of normal human blood monocytes and monocyte-derived macrophages. Our principal finding was that Fas ligation on these cells induces predominantly proinflammatory cytokine production, resulting in potent neutrophil chemoattractant bioactivity. This represents the first evidence that Fas ligation activates human monocyte and macrophage proinflammatory cytokine responses. These observations are especially significant because they run counter to the prevailing notion that Fas ligation on phagocytes and Fas-induced phagocyte apoptosis result in predominantly anti-inflammatory effects that contribute mainly to the resolution of inflammation (5, 6, 7, 8, 28).

In fact, relatively little is known about Fas-induced human monocyte/macrophage cytokine responses. In the single previous report on this topic, Daigle et al. (28) reported that Fas ligation on human monocytes induced expression of the anti-inflammatory cytokine IL-10, which inhibited CD3-induced lymphocyte proliferation in mixed PBMC cultures. The authors concluded that Fas-induced monocyte responses may represent an important anti-inflammatory mechanism, at least in the context of lymphocyte proliferation during secondary immune responses (28). Our data indicate that the monocyte/macrophage cytokine response to Fas ligation is more complex.

We investigated a broader array of Fas-induced monocyte/macrophage cytokine responses that are relevant to acute inflammatory responses and innate immunity. We found low levels of Fas-induced IL-10 production similar to those reported by Daigle and colleagues (28). However, we also observed earlier and quantitatively much greater release of TNF-{alpha}, IL-8, and IL-1{beta} (Fig. 1). The net proinflammatory biological activity of this response was confirmed by the demonstration of potent neutrophil chemoattractant activity (Fig. 3). Thus, Fas ligation activates human monocytes and monocyte-derived macrophages to express and secrete predominantly proinflammatory cytokines. In vivo, Fas agonists would be expected to initiate macrophage activation and cytokine production, resulting in leukocyte recruitment and acute inflammation.

We view our findings and those of Daigle et al. (28) as complementary, not contradictory. The combined results suggest that the consequences of Fas-induced monocyte/macrophage activation may vary depending on the biological context. The same complex mixture of cytokine mediators released by Fas-stimulated monocytes may inhibit lymphocyte proliferation, yet activate neutrophilic inflammation.

The signaling pathways that connect Fas to monocyte/macrophage cytokine production remain unclear. We found that Fas-induced monocyte cytokine responses were associated with rapid expression of proinflammatory cytokine genes, suggesting at least partial regulation at the transcriptional level (Fig. 4). However, we found interesting differences in the Fas-induced cytokine responses of blood monocytes compared with monocyte-derived macrophages, suggesting important maturation-dependent differences in these signaling pathways.

Fas-induced cytokine responses of monocytes were blocked by the pan-caspase inhibitor z-VAD-fmk, indicating that Fas-induced monocyte cytokine production is dependent on caspase activity (Fig. 7). Surprisingly, despite that fact that monocytes produced large amounts of IL-1{beta}, Fas-induced monocyte cytokine production was unaffected by two different IL-1{beta} neutralization strategies (Fig. 8). Thus, Fas-induced monocyte proinflammatory cytokine responses appear to be caspase dependent, but independent of extracellular IL-1{beta} feedback. The precise mechanism remains unknown, but caspases are increasingly recognized for functional regulation of processes other than the execution of programmed cell death. For instance, caspase activity appears to be necessary for monocyte-macrophage differentiation (37). In terms of cytokine responses, caspase-3 has been reported to induce hepatocyte chemokine production via the activation of the transcription factor AP-1 (38), and caspase-3 can proteolytically activate kinase signaling cascades (39, 40) that contribute to the regulation of cytokine expression.

The potent inhibitory effect of z-VAD-fmk on LPS-induced monocyte TNF-{alpha} responses was an unexpected finding (Fig. 7). Caspase inhibition has been reported to block LPS-induced NF-{kappa}B activation, inducible NO synthase expression, and NO production in murine macrophage RAW264.7 cells (41). Using the same experimental model, another group found that z-VAD-fmk inhibited LPS-induced mitogen-activated protein kinase phosphorylation but not NF-{kappa}B translocation (42). In another model, z-VAD-fmk had no effect on LPS-induced NF-{kappa}B-dependent cytokine gene expression in murine Mf4/4 and pU5.18 macrophage cell lines (43). At this point, the precise role of caspases in signaling cytokine responses to LPS stimulation remains unresolved. Our results suggest that caspases, or other pathways inhibited by z-VAD-fmk, are important in normal human monocyte responses to both Fas ligation and LPS stimulation and suggest that caspases may have a generalized role in signaling monocyte cytokine responses.

Others have reported Fas-induced proinflammatory cytokine responses by a variety of transformed cell lines and parenchymal tissue cells (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 38) and by murine macrophages (21, 22, 23, 24). However, in most of the experimental models in which it has been measured, simultaneous apoptosis has been a prominent feature, affecting up to 85% of the target cell population unless the cells were first primed with an antiapoptotic stimulus (10, 13, 14, 21, 22, 23, 24, 38).

In this context, our demonstration that Fas-induced monocyte-derived macrophage cytokine responses occurred in the absence of detectable apoptosis is a novel and important finding (Figs. 1 and 6). Our results imply that Fas-induced proinflammatory signaling can occur independently of Fas-induced cell death pathways once maturation-dependent changes in the monocyte signaling repertoire have occurred. The biological relevance of our findings using monocyte-derived macrophages is supported by similar responses to Fas agonists that we have observed in normal human alveolar macrophages (44).

Fas-induced macrophage proinflammatory cytokine production occurred in the absence of apoptosis and was caspase independent (Fig. 7). Among potential explanations, there may be a direct mechanistic link between resistance to apoptosis and transduction of an activating signal. Caspase-8/FLICE inhibitory protein (FLIP) is one candidate for this signaling link that we are actively investigating. FLIP associates with FADD in the death-inducing signaling complex and blocks the activation of caspase-8/FLICE (45). Monocyte maturation and activation are associated with the up-regulation of FLIP and increased FLIP expression accounts for the resistance of mature macrophages and activated monocytes to Fas-induced apoptosis (22, 27, 36, 46). Importantly, FLIP not only blocks cell death pathways, but may contribute directly to proinflammatory cytokine responses by linking Fas with the activation of NF-{kappa}B and the extracellular signal-regulated kinases (20, 47, 48, 49).

In addition to FLIP, other adapter proteins capable of proinflammatory signaling may interact with the Fas-FADD signaling complex. Components of the TNF signaling pathway have been reported to interact with FADD in addition to the TNFR (3, 40, 49). FADD has also been reported to associate with the Toll-like receptor (TLR) adapter protein myeloid differentiation factor 88, linking TLR and IL-1R signaling with cell death pathways (50, 51). Conceivably, myeloid differentiation factor 88 could interact with Fas-associated FADD, leading to signaling in the reverse direction from Fas through the TLR/IL-1R signaling cascade.

Fas can also activate NF-{kappa}B independently of FADD recruitment (16), probably via interactions with accessory proteins having kinase activities (40). The phosphatidylinositol 3-kinase/Akt pathway may be particularly relevant (52), because it can augment NF-{kappa}B transcriptional activity without increasing NF-{kappa}B nuclear translocation (53). This could account for the robust expression of NF-{kappa}B-dependent cytokine genes despite the modest increases in NF-{kappa}B nuclear translocation signal that we observed.

For our studies, we chose to use mAb CH-11 and sFasL rather than membrane-bound or immobilized forms of Fas agonists. In some models, sFasL has less potent apoptosis-inducing and proinflammatory signaling activity than membrane-associated FasL (54, 55, 56, 57). mAb CH-11 may mimic the receptor-aggregating functions of membrane FasL due to its pentavalent IgM structure, but we found similar results using either mAb CH-11 or sFasL. The effects of membrane FasL on human monocyte/macrophage cytokine responses are unknown and are worthy of investigation. However, our findings using soluble agonists are important because sFasL is known to be released from activated monocytes (58) and other cells, and is present in large quantities in important inflammatory diseases such as the acute respiratory distress syndrome (59).

In conclusion, Fas induces proinflammatory cytokine responses by human monocytes and monocyte-derived macrophages. Fas-induced cytokine responses are associated with increased cytokine gene expression and appear to involve NF-{kappa}B activation, but there are important maturation-dependent differences in the Fas-induced signaling pathways. Rather than inducing apoptosis and the silent elimination of inflammatory cells, Fas may serve to activate circulating monocytes and resident and recruited macrophages to produce proinflammatory mediators that can initiate and perpetuate acute inflammation and tissue injury. As a result, Fas-induced monocyte/macrophage responses may play an important role in the regulation of innate immune responses and may contribute to the pathogenesis of a variety of clinically important inflammatory diseases.


   Acknowledgments
 
We thank Dr. Jeanette Crisostomo, Michele Timko, and Venus Wong for their expert technical assistance.


   Footnotes
 
1 This work was supported in part by the National Institutes of Health (National Heart Lung and Blood Institute Grants K08 HL03577 and R01 HL62995), the Northwest Affiliate of the American Heart Association, and the American Lung Association of Washington. Back

2 Portions of this work have been presented in abstract form at the International Scientific Conference of the American Thoracic Society, May 2000, in Toronto, Ontario, Canada (abstract A652). Back

3 Address correspondence and reprint requests to Dr. David R. Park, Harborview Medical Center, Box 359640, 325 9th Avenue, Seattle, WA 98104. E-mail address: drp{at}u.washington.edu Back

4 Current address: MedImmune Inc., 35 West Watkins Mill Road, Gaithersburg, MD 20878. Back

5 Abbreviations used in this paper: FasL, Fas ligand; sFasL, soluble FasL; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; z-FA-fmk, benzyloxycarbonyl-Phe-Ala-fluoromethylketone; FADD, Fas-associated death domain-containing protein; TLR, Toll-like receptor; MCP, monocyte chemoattractant protein. Back

Received for publication February 25, 2003. Accepted for publication April 4, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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