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. 2000 Sep 4;192(5):647-58.
doi: 10.1084/jem.192.5.647.

Fas ligand, Bcl-2, granulocyte colony-stimulating factor, and p38 mitogen-activated protein kinase: Regulators of distinct cell death and survival pathways in granulocytes

A Villunger  1 , L A O'Reilly, N Holler, J Adams, A Strasser
Affiliations

Fas ligand, Bcl-2, granulocyte colony-stimulating factor, and p38 mitogen-activated protein kinase: Regulators of distinct cell death and survival pathways in granulocytes

A Villunger et al. J Exp Med. .

Abstract

The short life span of granulocytes, which limits many inflammatory responses, is thought to be influenced by the Bcl-2 protein family, death receptors such as CD95 (Fas/APO-1), stress-activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), and proinflammatory cytokines like granulocyte colony-stimulating factor (G-CSF). To clarify the roles of these various regulators in granulocyte survival, we have investigated the spontaneous apoptosis of granulocytes in culture and that induced by Fas ligand or chemotherapeutic drugs, using cells from normal, CD95-deficient lpr, or vav-bcl-2 transgenic mice. CD95-induced apoptosis, which required receptor aggregation by recombinant Fas ligand or the membrane-bound ligand, was unaffected by G-CSF treatment or Bcl-2 overexpression. Conversely, spontaneous and drug-induced apoptosis occurred normally in lpr granulocytes but were suppressed by G-CSF treatment or Bcl-2 overexpression. Although activation of p38 MAPK has been implicated in granulocyte death, their apoptosis actually was markedly accelerated by specific inhibitors of this kinase. These results suggest that G-CSF promotes granulocyte survival largely through the Bcl-2-controlled pathway, whereas CD95 regulates a distinct pathway to apoptosis that is not required for either their spontaneous or drug-induced death. Moreover, p38 MAPK signaling contributes to granulocyte survival rather than their apoptosis.

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Figures

Figure 1
Figure 1
Receptor multimerization is required for FasL-induced killing of granulocytes. CD95 expression was determined on sorted bone marrow–derived granulocytes from (a) wt and (b) lpr mice by indirect immunofluorescence staining and flow cytometric analysis. Control staining is shown by filled histograms. Specific staining for CD95 is shown by open histograms. (c) Sorted granulocytes from bone marrow of wt or lpr mice were cultured for 24 h in the absence or presence of 100 ng/ml soluble FasL or multimerized FasL or with Neuro2A cells expressing membrane-bound FasL. Treatment with M2 anti-FLAG mAb (500 ng/ml) with or without a FLAG–FADD-GST fusion protein (1 μg/ml) served as additional controls. Viability was assessed by trypan blue staining and analysis in a hemocytometer. Data shown represent arithmetic means ± SD of three independent experiments and animals. A statistically significant degree of cell killing was only observed in wt cells treated with either multimerized (P < 0.0003) or membrane-bound FasL (P < 0.009).
Figure 2
Figure 2
Bcl-2 and FasL regulate distinct pathways to apoptosis in granulocytes. Expression of transgene-encoded human Bcl-2 was determined on sorted bone marrow–derived granulocytes from (a) wt and (b) vav-bcl-2 transgenic mice by indirect immunofluorescence staining and flow cytometric analysis. The Bcl-2-100 Ab used recognizes specifically human but not mouse Bcl-2. Control staining is shown by filled histograms and specific staining for human Bcl-2 by open histograms. Granulocytes from bone marrow of (c) wt, (d) vav-bcl-2 transgenic, or (e) lpr mice were sorted and treated with graded concentrations of soluble FasL or FasL multimerized with M2 anti-FLAG mAb. Alternatively, granulocytes were cocultured with (f) control (neo) or FasL-expressing Neuro2A cells. Cell viability was determined after 24 h. In a kinetic analysis, sorted granulocytes were cultured in the (g) absence or (h) presence of 100 ng/ml multimerized FasL for 24, 48, or 72 h. Cell viability was assessed by propidium iodide staining and flow cytometric analysis. Data shown represent arithmetic means ± SD of four independent experiments and animals. There are no statistically significant differences between FasL-treated wt and bcl-2 transgenic cells regardless of time of treatment or the FasL concentration applied. Survival of granulocytes from vav-bcl-2 transgenic mice in simple culture medium was significantly better compared with that of granulocytes from wt or lpr mice (P < 0.006).
Figure 3
Figure 3
Bcl-2 inhibits spontaneous and drug-induced apoptosis in bone marrow and peritoneal exudate granulocytes. (a–c) Granulocytes from bone marrow or (d–f) peritoneal exudate of casein-injected (a and d) wt, (b and e) vav-bcl-2 transgenic, or (c and f) lpr mice were sorted and cultured with the anticancer drugs etoposide (10 μg/ml), cis-platin (50 μg/ml), doxorubicin (0.5 μg/ml), multimerized FasL (100 ng/ml), or were left untreated. Cell viability was determined at the indicated times as described in the legend to Fig. 2. Data shown represent arithmetic means ± SD of four independent experiments and four to seven animals. There was no statistically significant difference between FasL-treated wt or bcl-2 transgenic granulocytes, regardless of time point analyzed and origin of the cells (P > 0.05 or higher). Granulocytes from vav-bcl-2 transgenic mice died at significantly slower rates when cultured in simple medium or after drug treatment than granulocytes derived from wt or lpr mice, again regardless of their activation status (P < 0.03 or lower). No significant difference was observed between wt- and lpr-derived granulocytes after drug treatment (P > 0.13 or higher).
Figure 4
Figure 4
G-CSF inhibits spontaneous and drug-induced apoptosis of granulocytes. (a–e) Granulocytes were isolated from bone marrow (f–h) or peritoneal cavity of wt mice that had been injected with casein. Cells were cultured in the absence (open circles) or presence of 100 ng/ml human G-CSF (filled circles), which was added 30 min before the addition of death stimuli. Cells were cultured in (a and f) medium alone or (b and h) in the presence of 10 μg/ml etoposide, (c) 50 μg/ml cis-platin, (d) 0.5 μg/ml doxorubicin, or (e and g) 100 ng/ml cross-linked FasL. Cell viability was assessed as in the legend to Fig. 2. Data shown represent arithmetic means ± SD of three to four independent experiments and animals. G-CSF treatment significantly inhibited spontaneous and drug-induced apoptosis of resting wt granulocytes after treatment with etoposide or doxorubicin (P < 0.035 or lower) but had no statistically significant impact on cell death induced by cis-platin or FasL. In granulocytes from peritoneal exudate only spontaneous death was significantly delayed by G-CSF treatment (P < 0.004).
Figure 5
Figure 5
Expression of Bcl-2 family members in granulocytes. Immunoblot analysis of Bcl-2, Bcl-w, Bcl-XL, and Bax expression in extracts from freshly isolated granulocytes (0 h) or granulocytes after culture for 16 h in medium alone or medium supplemented with 100 ng/ml G-CSF. Filters had to be exposed between 10 min (Bax, Bcl-XL, and Bcl-w) and 60 min (Bcl-2) to detect signals specific for these Bcl-2 family members. Membranes were stripped and re-probed with an anti-Hsp70 Ab as a loading control.
Figure 7
Figure 7
Inhibition of p38 MAPK accelerates death of granulocytes. (a) Sorted granulocytes from bone marrow of wt mice were cultured in the absence or presence of human G-CSF (100 ng/ml), which was added 30 min before treatment with either DMSO (0.25%), the p38 MAPK inhibitors SB203580 or SB202190, or the MEK-1 inhibitor PD98059. Cell viability was assessed after 24 h in culture. Both p38 MAPK inhibitors significantly accelerated spontaneous apoptosis of resting wt granulocytes (P < 0.002 or lower). (b) Sorted granulocytes from bone marrow of wt mice were treated with G-CSF (100 ng/ml) for the time points indicated or were left untreated. p38 MAPK or ERK1 and ERK2 activity was determined by immunoblotting of cell extracts using an Ab specific for phosphorylated p38 MAPK or phosphorylated ERK1 and ERK2, respectively. Equal protein loading was confirmed by reprobing membranes with an Ab recognizing p38 MAPK or ERK1 and ERK2 irrespective of their status of phosphorylation. (c) Sorted granulocytes from bone marrow of wt mice were cultured in the absence or presence of human G-CSF (100 ng/ml), which was added 30 min before, simultaneously with, or 30 min after application of the p38 MAPK inhibitor SB203580 (SB). Cell viability was assessed after 48 h in culture. Data shown represent arithmetic means ± SD of three to five animals and two independent experiments.
Figure 6
Figure 6
Multimerized FasL enhances p38 MAPK activity in granulocytes. Sorted granulocytes from bone marrow of (a) wt and (b) lpr mice were treated with soluble or multimerized FasL (100 ng/ml), with etoposide (50 μg/ml), or were left untreated. (b) Spleen cells from wt mice stimulated with 10 μg/ml anisomycin or 100 ng/ml PMA plus 1 μg/ml ionomycin served as a positive control for p38 MAPK activation. p38 MAPK activity was determined by immunoblotting of cell extracts using an Ab specific for phosphorylated p38 MAPK. Equal protein loading was confirmed by reprobing membranes with an Ab recognizing p38 MAPK irrespective of its status of phosphorylation.
Figure 8
Figure 8
Cell death induced by inhibition of p38 MAPK is mediated by a Bcl-2–inhibitable and caspase-dependent apoptotic pathway. (a) Sorted granulocytes from bone marrow of wt, lpr, or vav-bcl-2 transgenic mice were cultured with either DMSO (0.25%) or the p38 MAPK inhibitors SB203580 or SB202190. Cell viability was assessed after 24 h in culture. (b) Sorted granulocytes from wt mice were cultured in the absence or presence of the caspase inhibitor zVADfmk (50 μM), which was added 30 min before treatment with either DMSO (0.25%) or the p38 MAPK inhibitor SB203580. Cell viability was assessed after 24 h in culture as described in the legend to Fig. 2. Data shown represent arithmetic means ± SD of two to four animals.
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Comment in

  • Cross-talk in cell death signaling.
    Roy S, Nicholson DW. Roy S, et al. J Exp Med. 2000 Oct 16;192(8):F21-5. J Exp Med. 2000. PMID: 11034612 Free PMC article. Review. No abstract available.

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