Inflammatory neutrophils retain susceptibility to apoptosis mediated via the Fas death receptor Stephen A. Renshaw, Samantha J. Timmons, Vanessa Eaton, Lynne R. Usher, Mohammed Akil,* Colin D. Bingle, and Moira K. B. Whyte Respiratory Medicine and *Rheumatology, Division of Molecular and Genetic Medicine, University of Sheffield, Royal Hallamshire Hospital, United Kingdom
Abstract: Apoptosis and clearance of neutrophils is essential for successful resolution of inflammation. Altered signaling via the Fas receptor could explain the observed prolongation of neutrophil lifespan and associated tissue injury at inflammatory sites. We therefore compared inflammatory neutrophils extracted from joints of rheumatoid arthritis patients, with peripheral blood neutrophils. Inflammatory neutrophils underwent constitutive apoptosis in culture more rapidly than peripheral blood neutrophils; this was not explained by changes in surface expression of Fas or by induction of Fas ligand. Inflammatory neutrophils remained sensitive to Fas-induced death, at levels comparable to those seen in peripheral blood neutrophils. Similarly, granulocyte-macrophage colonystimulating factor reduced apoptosis but did not abolish signaling via Fas. These data provide evidence for the rate of apoptosis in inflammatory neutrophils being continually modulated by death and survival signals in the inflammatory milieu. This allows for rapid resolution of inflammation as levels of survival factors fall, and suggests new strategies for inducing resolution of inflammation. J. Leukoc. Biol. 67: 662–668; 2000. Key Words: inflammation · granulocyte-macrophage colonystimulating factor
INTRODUCTION Apoptosis, or programmed cell death, of the neutrophil granulocyte is recognized to be a critical event in the resolution of inflammation, leading to functional down-regulation [1] and to phagocytosis and clearance of intact apoptotic neutrophils by tissue macrophages [2, 3]. Neutrophils are exquisitely sensitive to apoptosis, which occurs constitutively during aging in vitro [2] and at inflamed sites in vivo [4, 5]. The mechanisms of constitutive neutrophil apoptosis, or of apoptosis of inflammatory neutrophils, are unknown but the control of neutrophil survival can be considered in terms of extrinsic or intrinsic signals. Death or survival signals in the inflammatory milieu that act on the neutrophil to modulate its survival constitute the extrinsic signals, whereas the intrinsic signals refer to unidentified components of the endogenous program of constitutive 662
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apoptosis. The interplay between these multiple signals will thus determine neutrophil longevity. A number of in vitro studies of peripheral blood neutrophils have shown inhibition of constitutive apoptosis by extrinsic signals, such as transmigration across an endothelial monolayer [6] or exposure to physiological doses of a range of cytokines and pro-inflammatory mediators, notably granulocyte-macrophage colonystimulating factor (GM-CSF) and lipopolysaccharide [7, 8]. More recently, neutrophil apoptosis was shown to be accelerated by ligation of so-called death receptors belonging to the tumor necrosis factor (TNF) receptor family [9]. Only two signaling members of this family have been described on the neutrophil cell surface, and ligation of the prototypic death receptor, Fas [10], or at early time points only, TNF-R1 [11], accelerates constitutive neutrophil apoptosis in vitro. Comparatively little is known about the potential for modulation of apoptosis in neutrophils that have migrated to inflamed sites because almost all studies of human neutrophil apoptosis have been confined to peripheral blood cells. There is some evidence that granulocyte lifespan at inflamed sites in vivo is susceptible to regulation by extrinsic signals. GM-CSF in the lavage from the lungs of patients with the adult respiratory distress syndrome (ARDS) has been shown to inhibit neutrophil apoptosis [12], and prolongation of neutrophil lifespan in vivo has been reported by circulating endogenous G-CSF [13] and GM-CSF [14]. Ligation of the Fas death receptor was also shown to induce eosinophil apoptosis in an animal model of allergic airway inflammation [15]. In addition, there are a number of studies that suggest there may be important phenotypical changes induced in neutrophils by migration into tissues. These inflammatory neutrophils may show alterations in lifespan, both in the lung [16] and the skin [17], and differences in priming [18] and receptor expression [17] compared with peripheral blood neutrophils. Re-programming of apoptosis in inflammatory neutrophils is particularly suggested by alterations in expression of caspases [19], cysteine proteases that are key executors of the endogenous cell death program [20]. We have sought to investigate whether inflammatory neutro-
Correspondence: Moira Whyte, Professor of Respiratory Medicine, Division of Molecular and Genetic Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, UK. E-mail:
[email protected] Received October 27, 1999; revised December 31, 1999; accepted January 3, 2000.
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phils do or do not show altered responses to extrinsic signals, which might imply a resetting of the apoptosis program. We were particularly interested to examine the effects of the death receptor, Fas, because previous studies have suggested that neutrophils express both Fas and its endogenous ligand, FasL, and that Fas/FasL interactions may represent a mechanism of autocrine/paracrine neutrophil death that underpins the short lifespan of the neutrophil [10]. We also compared the ability of a prototypic suppressor of neutrophil apoptosis, GM-CSF, to inhibit both constitutive and Fas-mediated apoptosis in peripheral blood and inflammatory neutrophils.
METHODS Materials All chemicals were of analytical reagent grade and were purchased from Sigma (Poole, UK) unless stated otherwise, and were of the highest grade purity available. Percoll was obtained from Pharmacia Biotech (St. Albans, UK); culture media [Hanks’ balanced salt solution (HBSS), RPMI 1640] and supplements [fetal calf serum (FCS), penicillin, streptomycin, glutamine] were from Life Technologies (Paisley, UK). CH-11 and ZB4 were obtained from Upstate Biotechnology (Lake Placid, NY); UB2-fluorescein isothiocyanate (FITC) anti-Fas mAb, and goat anti-mouse anti-IgG-phycoerythrin were from Immunotech (Marseilles, France); KB8301 was from PharMingen (San Diego, CA); anti-mouse horseradish peroxidase (HRP)-conjugated antibodies was from Santa Cruz (Santa Cruz, CA); and recombinant human (rh) GM-CSF was from Roche Molecular Biochemicals (Lewes, UK). Alf 2.1 (anti-FasL mAb), and the KFL-9 cell line were a kind gift of Prof. D. Kaplan (Dept. Pathology, Case Western Reserve University, Cleveland, OH). FasL enzyme-linked immunosorbent assay (ELISA) was from Stratatech Scientific (Luton, UK).
Neutrophil isolation and culture Human peripheral blood neutrophils were isolated from venous blood of healthy volunteers by dextran sedimentation and centrifugation through a discontinuous Plasma-Percoll gradient as previously described [2, 21]. Inflammatory neutrophils were purified from synovial fluid drawn therapeutically from the joints of rheumatoid arthritis patients. Ethical approval for the study was obtained from the South Sheffield Regional Ethics Committee, and all patients gave fully informed consent. Synovial fluid was spun for 10 min, and the cell pellet was washed three times with room temperature sterile phosphatebuffered saline (PBS). Thereafter the cells were centrifuged through a Plasma-Percoll gradient and washed identically to those obtained from peripheral blood [2]. Purity was assessed by counting .500 cells on duplicate cytospin preparations, and was always .95%. The contaminating cells were almost exclusively eosinophils in the case of peripheral blood. Neutrophils prepared from synovial fluid usually exceeded 98.5% purity, and the small numbers of contaminating cells were usually mononuclear cells. Neutrophils were incubated at 37°C in a 5% CO2 atmosphere, in RPMI, containing penicillin and streptomycin (100 U/L) and supplemented with 10% FCS, in 96-well non-tissue culture-treated Falcon Flexiwell plates (Becton Dickinson, UK). Where cells were cultured in the presence of additives, control cells were cultured in the presence of the appropriate vehicle. KFL-9 and K562 cells were cultured in the same medium in tissue culture-treated plasticware.
Assessment of cell viability and morphology At the time points indicated, apoptosis was quantified by morphology on Giemsa-stained cytospins, counting .300 cells per slide on duplicate cytospins. This method has been shown to correlate closely with other measurements of neutrophil apoptosis, including annexin V binding [22] and shedding of CD16 [23]. In addition, necrosis was assessed at all time points by exclusion of the vital dye trypan blue and was ,2% in all cases.
Flow cytometry Cells were assessed for Fas and FasL expression by immunofluorescent labeling and flow cytometric analysis. For studies of Fas expression, cells were
resuspended in 100 µL of PBS, co-incubated with 10 µL of an FITC-conjugated anti-Fas mAb, UB2, or a control isotype-matched, FITC-conjugated antibody for 30 min at 4°C. Cells were then washed and resuspended in 200 µL of PBS. For detection of FasL expression, cells were suspended in 100 µL of PBS with 10% goat serum, co-incubated with 400 ng of an anti-FasL mAb, Alf2.1, then washed and incubated on ice for 30 min with a phycoerythrin-labeled secondary antibody in PBS with 10% goat serum. Cells were again washed, then resuspended in 200 µL of PBS, and analyzed by a Becton Dickinson FACScan flow cytometer. Use of an isotype control antibody for both neutrophils (n 5 4) and KFL-9 cells (n 5 3) confirmed lack of nonspecific binding as previously reported [24 and data not shown]. All experiments were repeated a minimum of four times.
Ribonuclease protection assay Neutrophils were cultured in Teflon-lined flasks at 5 3 106/mL as described above. At the time points indicated neutrophils were gently resuspended and washed once in PBS before lysis. Cells were lysed, and subsequent RNA extraction performed by RNeasy (Qiagen), according to the manufacturer’s protocol. The probes for ribonuclease protection assay were the riboquant APO-3c probe set (PharMingen, San Diego, CA). Probe synthesis, hybridization, proteinase K, and RNase digestion were carried out according to the protocol supplied by PharMingen. Samples were run on a 5% acrylamide gel, which was then dried. The gel was placed on film in a cassette with an intensifying screen at 280°C for 1–7 days.
Fas ligand ELISA After culture for the times indicated, cell suspensions of neutrophils, or control cell lines K562 and KFL-9, were gently centrifuged at 1000 rpm for 5 min. The supernatants were removed and subjected to analysis by ELISA for the soluble form of FasL according to the manufacturer’s instructions.
Statistical analysis The results are expressed as mean 6 standard error of the mean of the number (n) of independent experiments, each using cells from separate donors and with each experiment performed in duplicate. Statistical comparisons within the same populations were performed by the paired Student’s t test, and comparisons between peripheral blood and inflammatory neutrophils were made using the unpaired Student’s t test. Where ratios of rates of apoptosis are shown, the figures given are the median 6 interquartile range. Statistical comparisons between ratios are made by the Mann-Whitney test. Significance was assumed at levels of P , 0.05.
RESULTS Constitutive apoptosis is accelerated in inflammatory compared with peripheral blood neutrophils After neutrophil preparation, apoptosis was readily quantified by morphological criteria, with nonapoptotic neutrophils (Fig. 1a) being readily distinguishable from cells of apoptotic morphology (Fig. 1b, arrows). Cytocentrifuge preparations of synovial aspirate showed frequent apoptotic neutrophils (Fig. 1c, arrow), and occasional apoptotic bodies, presumably neutrophils, within macrophages, so-called Reiters’ cells (Fig. 1b). The proportion of neutrophils demonstrating light microscopic features of apoptosis in synovial fluid was consistently greater than in peripheral blood samples. In a series of eight samples, the percentage of apoptotic neutrophils in synovial fluid was 2.05 6 0.39%, and 0.43 6 0.11% in peripheral blood (P 5 0.004). When cultured in vitro, inflammatory neutrophils underwent constitutive apoptosis more rapidly than peripheral blood neutrophils (Fig. 1). In this series of experiments, the percentage of inflammatory neutrophils exhibiting the charac-
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Fig. 1. Accelerated constitutive apoptosis in inflammatory compared with peripheral blood neutrophils. Time course of culture in vitro of neutrophils extracted from peripheral blood (filled circles) or synovial fluid (filled triangles). Results are mean 6 SEM for five experiments. P , 0.04 for t 5 8; P , 0.01 for t 5 20. Photomicrographs of cytocentrifuge preparations show: (a) unprocessed synovial fluid cells; (b) a synovial macrophage seen engulfing an apoptotic neutrophil (arrow); (c) purified inflammatory neutrophils of nonapoptotic morphology; (d) purified inflammatory neutrophils aged in vitro, of predominantly apoptotic morphology.
teristic morphological appearance of apoptosis at 8 h was 11.6 6 2.3 (n 5 5), compared to 6.2 6 1.1 in peripheral blood neutrophils (n 5 6, P , 0.05). At 20 h 65.2 6 4.7% of inflammatory neutrophils were apoptotic (n 5 8) compared to 38.0 6 7.3 of those from peripheral blood (n 5 6, P , 0.01).
Human peripheral blood and inflammatory neutrophils express Fas but not FasL To investigate whether the accelerated apoptosis of inflammatory neutrophils was mediated via the Fas death receptor pathway, we first examined expression of Fas on peripheral blood and inflammatory neutrophils. Fas was expressed at equivalent levels in the two populations, as assessed by flow cytometry, and was not altered in either population by aging in vitro (Fig. 2A). We next sought evidence for expression of the endogenous ligand for Fas, FasL, in peripheral blood and inflammatory neutrophils. Using an antibody previously shown to detect FasL specifically [24], FasL could not be identified in either freshly isolated or aged peripheral blood or inflammatory neutrophils by flow cytometry (Fig. 2C). We were able, however, to demonstrate the presence of FasL on a Fas ligand transfected control cell line, KFL-9, but not on the untransfected control line, K562 (Fig. 2B). To exclude the possibility that FasL was being cleaved from the cell membrane by a matrix metalloproteinase (MMP), we pretreated neutrophils with an MMP inhibitor, KB-8301, shown to inhibit shedding of FasL [25]. FasL was not detectable on KB-8301-treated neutrophils by flow cytometry (Fig. 2D). Moreover, soluble FasL (sFasL) was not detected in neutrophil culture supernatants by ELISA (Table 1). Finally, we were unable to detect FasL mRNA by RNase protection assay, using appropriate positive controls (Fig. 3).
Inflammatory neutrophils retain susceptibility to Fas-mediated apoptosis Neutrophil apoptosis is induced by the cross-linking anti-Fas antibody, CH-11, and this CH-11-mediated death is inhibited by a range of pro-inflammatory cytokines [10]. We confirmed a time-dependent acceleration of peripheral blood neutrophil apoptosis by CH-11, which was abolished by treatment with 664
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ZB4, a blocking anti-Fas antibody (Fig. 4). Inflammatory neutrophils appeared to show Fas-sensitivity that was equivalent to peripheral blood neutrophils (Fig. 4), with a median fold increase in apoptosis in response to CH-11 of 5.3 (interquartile range 2.8–8.9) at 8 h, and 1.4 (1.3–1.5) at 20 h compared with 3.2 (1.9–6.1) and 1.8 (1.7–1.9), respectively, in peripheral blood neutrophils (P . 0.15 for each comparison). As would be predicted from the lack of FasL expression on neutrophils, ZB4 had no effect on the rates of constitutive apoptosis in culture of peripheral blood or inflammatory neutrophils (Fig. 4).
GM-CSF reduces absolute levels of apoptosis, but does not prevent apoptosis induction by ligation of Fas To investigate whether apoptosis of inflammatory neutrophils was suppressed by a prototypic survival factor, GM-CSF, we examined constitutive and Fas-mediated apoptosis, in the presence or absence of GM-CSF, in peripheral blood and inflammatory neutrophils. In both neutrophil populations the presence of GM-CSF reduced rates of constitutive and Fasmediated apoptosis (Fig. 5). In the in vivo setting, where neutrophils have transmigrated into a site of inflammation, they are exposed to levels of GM-CSF equal to or greater than those used in our study [12], hence their response to Fas ligation in these circumstances is of interest. When Fas is ligated in the presence of GM-CSF, the increase in apoptosis seen with inflammatory neutrophils is broadly comparable to the increases seen in peripheral blood neutrophils (Table 2). This strongly suggests that inflammatory neutrophils retain the ability to respond to death signals, even in the presence of physiological concentrations of survival factors.
DISCUSSION Inflammatory neutrophils from synovial fluid show enhanced rates of apoptosis in vitro These studies demonstrate that human inflammatory neutrophils, harvested from rheumatoid joint synovial fluid, undergo http://www.jleukbio.org
TABLE 1. Neutrophils, K562 Cells (Negative Control) and KFL-9 Cells (Positive Control) were Cultured for 24 h in the Presence or Absence of the Metalloproteinase Inhibitor, KB-8301 and the Presence of Soluble Fas-L Detected by ELISA FasL concentration, pg/µL
Neutrophils Neutrophil and KB-8301 K562 K562 and KB-8301 KFL-9 KFL-9 and KB-8301
0.003 6 0.0 0.003 6 0.0 0.003 6 0.0 0.040 6 0.0 17.0 6 1.0 14.5 6 1.0*
The data shown represent the mean 6 SE of three separate experiments. * P 5 0.02 compared with KFL-9 alone.
constitutive apoptosis in vitro more rapidly than peripheral blood neutrophils. There was an increase in the proportion of apoptotic cells in freshly isolated inflammatory neutrophils compared with peripheral blood, at levels comparable to those described in other inflammatory exudates [12]. Although absolute numbers are small, this is likely to indicate a significant excess of apoptotic deaths in the inflammatory population because apoptotic neutrophils are thought to be identifiable for only short periods of time in inflammatory exudates, before ingestion and degradation by macrophages [4, 26]. Furthermore there was an accelerated rate of apoptosis after culture in vitro over 20 h. This was comparable to the accelerated in vitro apoptosis of synovial fluid neutrophils previously described by Bell et al. [27] and Savill et al. [2].
Signaling via the Fas death receptor pathway does not regulate the altered survival of inflammatory neutrophils To investigate whether the accelerated apoptosis of inflammatory neutrophils was mediated via death-receptor pathways, we
Fig. 2. Expression of Fas and FasL on neutrophils by flow cytometry. All results are representative of four separate experiments performed with a different donor each time. (A) Peripheral blood and inflammatory neutrophils, either freshly isolated or cultured for 18 h were labeled with anti-Fas antibody (UB2-FITC). Isotype control antibody is shown as a shaded curve. Specific binding of Fas is shown as an outline curve. (a, b) Peripheral blood neutrophils; (c, d) inflammatory neutrophils. (B) KFL-9 cells (K562 cells transfected with a FasL-expressing vector) are shown as a positive control for Fas ligand expression and K562 cells as a negative control for FasL expression, as previously described [24]. (C) Peripheral blood and inflammatory neutrophils, either freshly isolated or cultured for 18 h were labeled with anti-Fas-L antibody (Alf2.1) and detected with a phycoerythrin-conjugated goat anti-mouse polyclonal Ab, as described in Methods. Secondary antibody alone is shown as a shaded curve. Specific binding of Fas-L is shown as an outline curve. The results are representative of at least four separate experiments from independent donors. (a, b) Peripheral blood neutrophils; (c, d) inflammatory neutrophils. (D) To exclude metalloproteinase cleavage of FasL from the cell surface masking the presence of FasL, we cultured the neutrophils in the presence of the metalloproteinase inhibitor KB-8301 at a concentration of 10 mM.
Fig. 3. Ribonuclease protection assay showing expression of Fas ligand in control KFL-9 cells only, and expression of Fas and caspase-8 in human neutrophils. Loading is quantified by the housekeeping genes, L32 and GAPDH. The result shown is representative of two independent experiments.
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TABLE 2.
Fold Increases in Percentage of Apoptotic Neutrophils After Fas Ligation in Presence of GM-CSF
Peripheral blood neutrophils Inflammatory neutrophils
8h
20 h
9.5 (2.7–16.4) 10.5 (2.2–13.7)
3.4 (2.6–4.6) 3.8 (2.4–4.1)
Median (interquartile ranges) are shown. P . 0.8 for comparisons between peripheral blood and inflammatory neutrophils.
Fig. 4. CH-11 (cross-linking anti-Fas antibody) accelerates apoptosis of peripheral blood and inflammatory neutrophils. Both peripheral blood and inflammatory neutrophils undergo time-dependent constitutive apoptosis in culture, as assessed morphologically. This was unaffected by a Fas antagonistic antibody, ZB4. The Fas agonistic antibody, CH-11, at 500 ng/mL significantly accelerated apoptosis in both populations (e.g., at 20 h, P 5 0.001 for peripheral blood neutrophils (n 5 6), and P 5 0.002 for inflammatory neutrophils, n 5 8). This effect was completely blocked by ZB4 (500 ng/mL).
chose to examine the prototypic death receptor, Fas [9]. Fas ligation is known to significantly accelerate neutrophil apoptosis [10, 28]. The only other member of the death receptor family known to be expressed on neutrophils, TNF-R1, produces a more modest acceleration of apoptosis at early time points, which is potentially confounded by inhibition of apoptosis at later time points [11] mediated via activation of NF-kB [29]. Moreover, a recent study has suggested that TNF-R1 expression on neutrophils is markedly down-regulated after neutrophil extravasation, whereas expression of Fas is maintained [17]. Having observed no differences in Fas expression, we addressed the possibility that inflammatory neutrophils express its ligand, FasL, which might permit an autocrine/paracrine induction of apoptosis. There have been reports of FasL expression on human peripheral blood neutrophils, using a
Fig. 5. Effect of GM-CSF on Fas-mediated apoptosis of peripheral blood and inflammatory neutrophils. In peripheral blood neutrophils, CH-11 accelerated neutrophil apoptosis at all time points. e.g., at t 5 20 h P 5 0.001 for CH-11 vs. control (peripheral blood n 5 6), and P 5 0.0005 for inflammatory neutrophils (n 5 8). This acceleration was inhibited by GM-CSF (50 units/mL). At 20 h P 5 0.003 for peripheral blood neutrophils (n 5 8), and P 5 0.009 for inflammatory neutrophils (n 5 6), for GM-CSF 1 CH-11 vs. CH-11 alone.
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Fas-Fc fusion protein [10] or an anti-FasL antibody [30] subsequently shown to be unreliable for flow cytometry [24]. More recently, however, Brown et al. [28], using highly purified neutrophil populations, failed to detect FasL expression by reverse transcriptase-polymerase chain reaction (RT-PCR) or Western blot. In our experiments, neither peripheral blood nor inflammatory neutrophils expressed FasL, either when freshly isolated or after aging in vitro for 18 h, as assessed by flow cytometry. Because cleavage of both FasL and TNF-a from the cell surface by MMP has been described [25, 31], we excluded shedding of FasL both by flow cytometry of MMPI-treated neutrophils and by ELISA. We therefore concur with Brown et al. [28] that human peripheral blood neutrophils, at least under the culture conditions employed in this study, do not express FasL. More importantly, we could find no evidence for up-regulation of FasL expression in inflammatory neutrophils, arguing against a role for paracrine or autocrine FasL/Fas interactions as a mechanism of inflammatory neutrophil apoptosis. Fas may, however, be a physiologically relevant death signal for inflammatory neutrophils because monocyte-macrophages express high levels of FasL [32] and can induce Fas-mediated neutrophil death [28]. Moreover, sFasL is detectable in synovial fluid [33] and addition of synovial fluid to peripheral blood neutrophils has been shown to accelerate their death by apoptosis [27]. Cells expressing equivalent levels of cell-surface Fas may differ in susceptibility to Fas-mediated death [10], and this susceptibility may be altered within a cell type, e.g., by stimulation of cytokine receptors [34–36]. We therefore examined the induction of apoptosis by Fas ligation in inflammatory compared with peripheral blood neutrophils, and the suppression of this death by co-incubation with a neutrophil survival factor. We chose GM-CSF because it inhibits both constitutive [7, 8] and Fas-mediated [37] apoptosis of peripheral blood neutrophils and has been identified in vivo, both in synovial fluid samples from inflamed rheumatoid joints [27, 38] and in lung lavage fluid from ARDS patients [12]. Induction of neutrophil apoptosis by Fas ligation is an attractive therapeutic strategy, by analogy with ligation of Fas on airway eosinophils, which has been shown to accelerate eosinophil apoptosis and to abrogate airway inflammation in an animal model of allergic inflammation [15]. However, as we have shown, the amount of apoptosis induced by Fas ligation in inflammatory neutrophils is reduced by GM-CSF. This ability of a survival signal to suppress apoptosis in cells that are already Fas-sensitive is not seen in other cell types, such as mesangial cells [39]. Therapeutic induction of neutrophil apoptosis may thus require inhibition of survival factor signaling to permit death receptormediated apoptosis. http://www.jleukbio.org
What determines the lifespan of the inflammatory neutrophil? Previous studies of neutrophil apoptosis have been almost exclusively confined to peripheral blood cells and there is little information on the phenotype of the inflammatory neutrophil and its susceptibility to apoptosis. The lifespan of the peripheral blood neutrophil is short, with a half-life of 6–8 h in vivo [40]. There is some in vivo evidence that the ‘‘time-clock’’ of neutrophil apoptosis can be modulated at inflamed sites, with extended survival of neutrophils that have migrated into skin windows [17] or into the lung, as assessed by positron emission tomography [16] or using cells harvested by bronchoalveolar lavage [19]. It is not clear, however, whether this altered survival at inflamed sites is due to resetting of the endogenous time-clock of apoptosis or to survival signals from the inflammatory milieu that are mediated via cell-surface receptors. External stimuli including soluble mediators [7, 8], engagement of adhesion receptors [6], and ligation of death receptors [10, 28] modulate apoptosis of peripheral blood neutrophils. Moreover, Matute-Bello et al. have shown that GM-CSF and G-CSF, present in bronchoalveolar lavage fluid of ARDS patients, inhibit neutrophil apoptosis [12]. We have now shown that human inflammatory neutrophils, when cultured in vitro, respond to Fas-mediated induction of apoptosis, and GM-CSFmediated inhibition, in a manner broadly equivalent to peripheral blood neutrophils. This argues against a resetting of the intrinsic program of neutrophil apoptosis. The observed increase in rates of in vitro apoptosis of inflammatory neutrophils, however, is intriguing and suggests that, as a population, they are at a more advanced stage of commitment to the apoptotic program. This may be due in part to the selection of a population of increased average age in the inflammatory samples, but the contribution of this differential sampling is hard to quantify. A more attractive hypothesis (which would not be any less valuable even if the differential sampling hypothesis is true) is that, during transmigration, neutrophils have their internal program of apoptosis accelerated, but that this is continually suppressed by survival factors in the inflammatory environment. Thus, when removed from that environment, they display an acceleration of apoptosis compared to their peripheral blood contemporaries. In vivo, however, they are exposed to high concentrations of survival factors such as GM-CSF, which allows extension of their lifespan while inflammation persists, but permits a rapid exit into apoptosis as survival factors decrease. This is thus permissive for rapid resolution of inflammation, in which maintenance of Fas sensitivity may well be important.
Summary To conclude, these studies show that inflammatory neutrophils express the death receptor, Fas, at equivalent levels to peripheral blood neutrophils and remain equally sensitive to apoptosis mediated via Fas ligation. Apoptosis suppression by the survival factor, GM-CSF, was similarly retained in inflammatory neutrophils. We could find no evidence for expression of FasL on inflammatory neutrophils. Regulation of inflammatory neutrophil apoptosis by extrinsic signals, pro- and anti-apoptotic factors present in the inflammatory milieu, on the background
of an accelerated intrinsic program of apoptosis, perhaps permits rapid apoptosis and clearance of inflammatory neutrophils and thus facilitates resolution of inflammation.
ACKNOWLEDGMENTS This work was supported by the Wellcome Trust (Medical Graduate Fellowship to S. A. R.) and by project grants from Trent Region NHS Research and Development. The authors would like to thank Prof. D. Kaplan for his kind gift of anti-Fas ligand antibodies and control cell lines, and Olivia Smith and John Lowry of the Institute of Cancer Studies, University of Sheffield, for assistance with flow cytometry.
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