Neutrophils in Spontaneous Apoptosis of Human Role of p38-Mitogen ...

Role of p38-Mitogen-Activated Protein Kinase in Spontaneous Apoptosis of Human Neutrophils1 Kazutetsu Aoshiba, Shuji Yasui, Mitsutoshi Hayashi, Jun Tamaoki, and Atsushi Nagai2 Neutrophils constitutively undergo apoptosis at both normal and inflamed sites: an important process that limits the toxic potential of the neutrophil. However, the signal pathway for neutrophil apoptosis is currently unknown. In this study, we evaluated the role of p38-mitogen-activated protein kinase (MAPK) in the spontaneous apoptosis of neutrophils in vitro. We found that p38-MAPK was constitutively tyrosine phosphorylated and activated during spontaneous apoptosis of neutrophils. Inhibition of p38-MAPK by SB203580 and an antisense oligonucleotide delayed apoptosis by approximately 24 h. The antioxidants catalase and N-acetylcysteine delayed neutrophil apoptosis, but failed to inhibit phosphorylation and activation of p38-MAPK. Granulocyte-macrophage CSF and anti-Fas Ab, which altered the rate of apoptosis, did not affect phosphorylation and activation of p38-MAPK. These results suggest that the constitutive phosphorylation and activation of p38-MAPK are involved in the program of spontaneous apoptosis in neutrophils. The Journal of Immunology, 1999, 162: 1692–1700.

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ature neutrophils have the shortest life span of the various leukocytes and die rapidly via apoptosis in vivo and in vitro, resulting in the demise of an entire population within 72 h (1, 2). The hallmark of neutrophil biology is their spontaneous induction of apoptosis. Rapid expression of apoptosis in neutrophils, and the subsequent engulfment of the apoptotic cells by phagocytes are important in the rapid resolution of inflammation (1, 2). This is necessary to avoid unwanted tissue damage caused by activated neutrophils (1, 2). Intriguing clues to the mechanism underlying expression of neutrophil apoptosis have begun to emerge from recent observations. For example, transient elevation of cytosolic-free Ca21 induced by low doses of calcium ionophores inhibits apoptosis in neutrophils (3). Conversely, intracellular ROS3 induce apoptosis in neutrophils as well as other cell types (4 – 6). Furthermore, the autocrine interaction of neutrophil Fas with soluble or cell-associated Fas ligand may cause spontaneous apoptosis that occurs in normal neutrophils (7, 8). However, the signal-transduction pathway mediating neutrophil apoptosis remains unclear. Recent studies indicate the involvement of tyrosine phosphorylation events in signaling pathways that results in neutrophil apoptosis. For example, treatment of neutrophils with inhibitors of tyrosine kinases and phosphatases alters the rate of neutrophil ap-

Department of Medicine, Chest Institute, Tokyo Women’s Medical College, Tokyo, Japan Received for publication February 19, 1998. Accepted for publication October 16, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a Grant-in Aid for Scientific Research (30147392) from the Ministry of Education, Science and Culture, Japan. 2 Address correspondence and reprint requests to Dr. Atsushi Nagai, Department of Medicine, Chest Institute, Tokyo Women’s Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. 3 Abbreviations used in this paper: ROS, reactive oxygen species; ERK, extracellular signal-regulated kinase; GM-CSF, granulocyte-macrophage colony-stimulating factor; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase; MAPK, mitogenactivated protein kinase; MAPKAP, mitogen-activated protein kinase-activated protein; MKK, mitogen-activated protein kinase kinase; MTT, 3-(4, 5-dimethylthiazole2-yl)-2,5-diphenyltetrazolium bromide.

Copyright © 1999 by The American Association of Immunologists

optosis (9). Furthermore, GM-CSF induces a rapid activation of Lyn, a Src family tyrosine kinase, whereas Lyn antisense treatment of neutrophils reverses the survival-promoting effect of GM-CSF (10). These studies suggest that neutrophils utilize a specific signaling pathway dependent on tyrosine phosphorylation and dephosphorylation to trigger their apoptotic protocol. p38-MAPK, a MAPK family member, is a serine/threonine kinase activated by phosphorylation of tyrosine and threonine residues (11). This kinase is phosphorylated and activated by many cellular stresses and inflammatory stimuli. To date, its physiologic roles have not been defined, but it seems to be involved in the regulation of important cellular responses such as apoptosis and inflammatory reactions. In neutrophils, p38-MAPK has been shown to be activated by agonistic stimulation with LPS, TNF-a, GM-CSF, platelet-activating factor, FMLP, and IL-8 (12–16). However, the functional significance of p38-MAPK in neutrophils is unknown. In this study, we present evidence that p38-MAPK plays an important role in spontaneous apoptosis in neutrophils. We found that p38-MAPK is continuously phosphorylated and activated during the program of spontaneous apoptosis. Inhibition of p38MAPK by SB203580 and specific antisense RNA delayed spontaneous apoptosis. These findings suggest that p38-MAPK activity is involved in spontaneous apoptosis in neutrophils.

Materials and Methods Reagents All reagents for cell culture were obtained from Life Technologies (Gaithersburg, MD). MTT, bisBenzimide (Hoechst33258), thymus DNA, genistein, N-acetylcysteine, catalase, leupeptin, aprotinin, PMSF, b-glycerophosphate, sodium vanadate, DTT, and polymyxin B sulfate were purchased from Sigma (St. Louis, MO). Recombinant human GM-CSF, antiFas Ab (CH-11), protein A plus G agarose, and PD98059 were purchased from Pharma Biotechnologie (Hannover, Germany), Medical and Biological Laboratories (Nagoya, Japan), Oncogene Research Product (Cambridge, MA), and Biomol Research Laboratories (Plymouth Meeting, PA), respectively. SB203580 was a generous gift of SmithKline Beecham Pharmaceuticals (King of Prussia, PA). The phosphorylated forms of the proteins p38-MAPK, ERK, and JNK were obtained from New England Biolabs (Beverly, MA). MAPKAP kinase-2-GST was purchased from Upstate Biotechnology (Lake Placid, NY). 0022-1767/99/$02.00

The Journal of Immunology Neutrophil preparation and cell culture Human neutrophils were isolated from EDTA-anticoagulated venous blood samples by dextran sedimentation and centrifugation on a Histopaque gradient (without endotoxin; Sigma), as previously described (17). Contaminating erythrocytes were removed by hypotonic water lysis. The isolated neutrophils were washed twice with PBS and resuspended in RPMI 1640 containing 5% heat-inactivated FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. To avoid accidental neutrophil activation and clumping, neutrophil preparations were carefully washed by centrifugation at 200 3 g and aspiration of the supernatant, followed by gentle resuspension with a pipet, and sudden changes in temperature were avoided (10). Endotoxin was not detected (,5 pg/ml) in Histopaque, culture medium, or FCS by a Limulus lysate test (Endospec-ST; Seikagaku, Tokyo, Japan). The purity of neutrophil populations was .95% on May-Gru¨nwald-Giemsa stain, and neutrophil viability was .98%, as determined by trypan blue dye exclusion. Less than 5% of neutrophil preparations showed a polarized shape, a sensitive marker for neutrophil activation (18). Unless otherwise stated, to assay for apoptosis, cell survival, and ROS generation, 105 cells were incubated in 200 ml medium in 96-well round-bottom plates (Becton Dickinson, Lincoln Park, NJ) at 37°C in a humidified incubator containing 5% CO2. For p38-MAPK assay and immunoblotting, 5 3 106 cells were suspended in 10 ml medium in a polypropylene tube (Becton Dickinson) and cultured as above. Normal human fetal lung fibroblasts (IMR-90; Clonetics, San Diego, CA) and human bronchial epithelial cells (16HBE) were cultured in DMEM or 1:1 Ham’s F-12/DMEM, respectively, supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin.

Antisense oligonucleotide treatment of cells Phosphorothiorate-modified antisense oligonucleotides specific for p38MAPK (59-GTCTTGTTCAGCTCCTGC-39) (19) as well as sense (59GCAGGAGCTGAACAAGAC-39) and scrambled (59-TGCTTAGTTCT CGTCCGC-39) oligonucleotides were synthesized. Neutrophils were incubated with 1 mM of each oligonucleotide in serum-free RPMI 1640 for 6 h before addition of 5% heat-inactivated FCS. After 24 h of culture, the cells were prepared for apoptosis assay and immunoblot analysis, as described below.

Apoptosis assay Aliquots of neutrophils were cytospun on glass slides and dried. Slides were stained with May-Gru¨nwald-Giemsa. Apoptosis was assessed based on nuclear pyknosis or chromatin condensation together with cytoplasmic vacuolation on oil immersion microscopy (2). Three hundred cells were scored in each experiment to determine the percentage of apoptotic cells. Apoptosis was also evaluated by detection of cytosolic histone-bound DNA fragments. Briefly, 2 3 104 cells were incubated in a 96-well roundbottom plate for 12 h. The plate was centrifuged and cell pellets were analyzed for histone-bound DNA fragments using the cell detection ELISA kit (Cell Death Detection ELISAPLUS; Boeringer Mannheim, Indianapolis, IN), according to the manufacturer’s instruction. The principle of this test is based on the detection of mono- and oligonucleosomes in the cytoplasmic fractions of cell lysates by using biotinylated antihistone- and peroxidase-coupled anti-DNA Abs. The enrichment of mono- and oligonucleosomes released into the cytoplasm is calculated as absorbance of incubated cells/absorbance of freshly isolated cells.

Cell survival assay Cell survival was evaluated by both a colorimetric MTT assay and a fluorescence cellular DNA assay. For MTT assay, 0.5 mg/ml MTT was added to culture plates for the last 4 h of incubation. Then the plates were centrifuged and cell pellets were lysed in 200 ml DMSO and 25 ml Sorenson’s buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The OD was measured at 570 nm on a microplate immunoreader (Bio-Rad, Hercules, CA). For DNA assay, culture plates were centrifuged and cell pellets were lysed in 100 ml distilled water, followed by a cycle of freezing and thawing. Then, cell lysates were solubilized in 100 ml of TNE buffer (10 mM Tris, 1 mM EDTA, 2 M NaCl, pH 7.4) containing 10 mg/ml Hoechst33258. The fluorescence intensities were read at respective excitation and emission wavelengths of 350 and 460 nm on a Cytofluor II multiplate fluorometer (Perseptive Biosystems, Framigham, MA). DNA content was determined in comparison with reference curves generated with known amounts of thymus DNA.

Immunoblot analysis for protein phosphorylation Cell lysates were solubilized in RIPA buffer (150 mM NaCl, 50 mM TrisCl, pH 7.4, 0.5% Nonidet P-40, and 0.1% SDS) containing 10 mg/ml leu-

1693 peptin, 1 mM PMSF, 10 mg/ml aprotinin, and 1 mM sodium orthovanadate, and centrifuged at 10,000 3 g for 30 min at 4°C. The supernatant containing equivalent amounts of protein (50 mg) was fractionated by SDSPAGE, transferred to polyvinylidene difluoride membrane, and probed with Abs, all of which were used at a dilution of 1/1000. Primary Ab was detected by horseradish peroxidase-conjugated Ab (1:2500), which in turn was visualized using enhanced chemoluminescence (SuperSignal; Pierce, Rockford, IL). OD of positive bands was measured with the National Institute of Health Image software.

p38-MAPK assay Cell pellets (5 3 106 cells) were lysed in 500 ml of lysis buffer (150 mM NaCl, 20 mM Tris-Cl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 1% Triton-X, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM sodium orthovanadate, 1 mg/ml leupeptin, 1 mM PMSF) and centrifuged at 10,000 3 g for 30 min at 4°C. The supernatant containing equivalent amounts of protein (250 mg) was precleared with protein A plus G agarose and immunoprecipitated with 4 ml of anti-p38-MAPK Ab for 16 h at 4°C. The immunocomplex was captured by protein A plus G agarose for 4 h. Bead pellets were washed twice in lysis buffer and twice in kinase buffer (25 mM Tris, pH 7.5, 10 mM MgCl2, 5 mM b-glycerophosphate, 2 mM DTT, 100 mM sodium orthovanadate). The beads were incubated in 30 ml of kinase buffer containing 12.5 mg/ml MAPKAP kinase-2-GST, 30 mM ATP, and 10 mCi [g-32P]ATP for 20 min at 30°C. The reaction was terminated by the addition of 10 ml 53 SDS sample buffer and heating to 95°C for 5 min. Samples were resolved on a 12% acrylamide SDS-PAGE gel and subjected to autoradiography. The immunoprecipitated samples were also analyzed for p38-MAPK by immunoblotting to determine whether the same amount of p38-MAPK was immunoprecipitated. The OD of each band was determined, and the ratio of phosphorylated MAPKAP kinase-2-GST/immunoprecipitated p38-MAPK was plotted.

Immunocytochemistry for phosphorylation of p38-MAPK Venous blood was drawn and immediately smeared on a glass slide. Blood cells were fixed in 3% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with phosphospecific Ab to p38-MAPK (1:50) or control rabbit IgG. Primary Ab was detected by FITC-conjugated Ab. Cells were counterstained with propidium iodide and observed under fluorescence microscopy.

Statistics Results are presented as mean 6 SEM. Comparisons were made by Student’s t test or ANOVA with Scheffe’s correction where appropriate.

Results Inhibition of p38-MAPK delays neutrophil apoptosis When cultured in DMEM containing 5% FCS, neutrophils underwent apoptosis constitutively, with 25.1% of cells apoptotic at 16 h and 94.3% apoptotic by 72 h, as assessed by light microscopy (Fig. 1). The constitutive rate of apoptosis in neutrophils shown in this work is within the range reported previously (2–5). Incubation of neutrophils with 50 mM of SB203580, a selective p38-MAPK inhibitor that has no inhibitory action on ERK and JNK (20), significantly inhibited apoptosis at each time point, with delayed onset of apoptosis by approximately 24 h (Fig. 1A). Genistein, a broad spectrum tyrosine kinase inhibitor, modestly inhibited apoptosis. In contrast, PD98059, an inhibitor of ERK kinase, had no effect on the rate of neutrophil apoptosis. Inhibition of apoptosis by SB203580 was dose dependent and was observed at a concentration as low as 5 mM, which is comparable with effective concentrations (1–25 mM) reported previously (12, 20 –25) (Fig. 1B). SB203580 also inhibited nucleosomal DNA fragmentation, as assessed by a cell death ELISA assay (Fig. 2), which detects cytosolic histone-bound DNA fragments formed in cells undergoing apoptosis. The inhibition of neutrophil apoptosis by SB203580 was supported by the fact that this compound prolonged survival of neutrophils, as assessed by an MTT assay (Fig. 3A), and by measurement of residual DNA in culture wells (Fig. 3B). In contrast to SB203580, the ERK inhibitor PD98059 did not affect neutrophil survival, consistent with the apoptosis data. These results

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FIGURE 2. Effect of SB203580 on nucleosomal DNA fragmentation in neutrophils. Neutrophils were cultured with or without 10 mM SB203580 for 12 h and analyzed for nucleosomal DNA fragmentation using a cell death ELISA kit. The enrichment of mono- and oligonucleosomes released into the cytoplasm is calculated as absorbance of cultured cells/absorbance of freshly isolated cells. Data represent mean 1 SEM of three experiments. pp, p , 0.01 versus control cells in medium alone.

FIGURE 1. Effects of SB203580, PD98059, and genistein on neutrophil apoptosis. Neutrophils were cultured for varying times (A) or 24 h (B) in RPMI 1640 containing 5% FCS with or without SB203580, PD98059, or genistein. Apoptotic cells were identified by morphology on light microscopy, and percentage of apoptosis was obtained from 300 counts per experiment. Data represent mean 6 SEM of six experiments. pp, p , 0.01 versus control cells in medium alone.

suggest that the pharmacologic inhibition of p38-MAPK, but not ERK, delays spontaneous apoptosis with resultant extension of survival in neutrophils. Although SB203580 has been shown to date to be specific for p38-MAPK (12, 20 –25), the rather high doses used for the long incubation time may result in nonspecific effects. Thus, to confirm the results described above, phosphorothiorate-modified antisense oligonucleotides were used to specifically deplete p38-MAPK. Treatment of neutrophils with the antisense oligonucleotides for 24 h reduced phosphorylated and total p38-MAPK in cell lysates by approximately 50% as compared with treatment with sense and scrambled oligonucleotides (Fig. 4A). When cells were treated with the antisense oligonucleotides, neutrophils exhibited a decreased level of apoptosis in comparison with cells treated with sense or scrambled oligonucleotides (Fig. 4B). These experiments suggest that p38-MAPK activity may play a role in spontaneous apoptosis in neutrophils. p38-MAPK is continuously phosphorylated and activated in neutrophils To assess p38-MAPK activity in neutrophils, we evaluated the phosphorylation and activation of p38-MAPK. Phosphorylation of p38-MAPK, ERK, and JNK was assessed by SDS-PAGE fraction-

ation of equivalent amounts (50 mg) of protein from each sample, followed by immunoblotting with phosphospecific Abs. In freshly isolated neutrophils (time 0), a significant level of p38-MAPK phosphorylation was detected (Fig. 5A). This basal phosphorylation level was sustained during a 24-h period, while neutrophils in parallel cultures increasingly underwent apoptosis with time (data not shown). In contrast to p38-MAPK, ERK and JNK showed no significant phosphorylation during the same period of culture (Fig. 5A). This was not due to the absence of ERK and JNK proteins because these proteins were detectable by immunoblotting with Abs to ERK and JNK. To determine whether phosphorylation of p38-MAPK is also seen in quiescent cultures of other cell types, we evaluated fibroblasts (IMR-90) and bronchial epithelial cells (16HBE). In contrast to neutrophils, fibroblasts and bronchial epithelial cells cultured in quiescent conditions showed no apparent phosphorylation of p38-MAPK (Fig. 5B). There was a possibility that the p38-MAPK phosphorylation seen in freshly isolated neutrophils was the result of accidental activation of neutrophils or endotoxin contamination during and after cell preparation because p38-MAPK has been shown to be phosphorylated following stimulation of neutrophils (12–14). However, this was unlikely for six reasons. First, less than 5% of neutrophil preparations showed polarization that reflects activation of neutrophils (18). Second, very few cells isolated exhibited spontaneous migration in a chemotaxis chamber (data not shown). Third, ERK, also known to be activated following stimulation of neutrophils (15, 26), was not significantly phosphorylated unless neutrophils were stimulated with PMA, an activator of protein kinase C (Fig. 5C). Stimulation of neutrophils with PMA modestly potentiated phosphorylation of p38-MAPK (Fig. 5C). Fourth, endotoxin was not detected (,5 pg/ml) in cell preparation reagents or culture medium by a Limulus lysate test. Fifth, inclusion of the endotoxin inhibitor polymyxin B (100 U/ml) in culture medium

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FIGURE 3. Effect of SB203580, PD98059, and genistein on neutrophil survival. Neutrophils were cultured for 24 h in RPMI 1640 containing 5% FCS with or without SB203580, PD98059, or genistein. Neutrophil survival was evaluated by MTT assay (A) or by measurements of DNA content per well (B). Data represent mean 1 SEM of six experiments. pp, p , 0.01 versus control cells in medium alone.

did not affect the phosphorylation of p38-MAPK (Fig. 5D). Sixth, to avoid endotoxin contamination and accidental cell activation during cell preparation, fresh blood smears made on glass slides were immunostained with a phosphospecific p38-MAPK Ab. A low but significant level of p38-MAPK phosphorylation was detectable in neutrophils in blood smears (Fig. 6). Activation of p38-MAPK was assessed by its ability to phosphorylate MAPKAP kinase-2-GST (27, 28). Consistent with the phosphorylation data, a basal activity of p38-MAPK that could be inhibited by SB203580 was detectable in freshly isolated neutrophils, and this activity lasted at 24 h of culture (Fig. 7). These experiments indicate that p38-MAPK in neutrophils is continuously phosphorylated and activated during the program of spontaneous apoptosis. Furthermore, a lack of phosphorylation in ERK and JNK confirms that p38-MAPK is selectively phosphorylated during spontaneous apoptosis.

FIGURE 4. Effect of p38-MAPK antisense (AS), sense (SS1), and scrambled (SS2) oligonucleotides on p38-MAPK expression and apoptosis in neutrophils. Neutrophils were incubated with 1 mM of each oligonucleotide in serum-free RPMI 1640 for 6 h before addition of 5% heat-inactivated FCS. After 24 h, the cells were assessed for p38-MAPK expression by immunoblot analysis (A) and apoptosis by morphology on light microscopy (B). A, Immunoblotting with control and phosphospecific Abs to p38MAPK was done as described in Fig. 5. A representative experiment of two performed is shown. The OD of each band was determined and plotted. Data represent mean 1 SEM. B, Data represent mean 1 SEM of three experiments. pp, p , 0.01 versus control cells in medium alone.

Phosphorylation and activation of p38-MAPK are not affected by a Fas agonist or GM-CSF We examined whether p38-MAPK activity was affected by Fas ligand, GM-CSF, and intracellular ROS, which are all known to affect the rate of neutrophil apoptosis (4 – 8, 29). The agonistic Fas

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FIGURE 5. Phosphorylation of p38-MAPK, ERK, and JNK in neutrophils, fibroblasts, and bronchial epithelial cells. A, Neutrophils were cultured for the indicated time, and cell lysates containing 50 mg protein were fractionated by SDS-PAGE and analyzed by immunoblotting with control Abs to p38-MAPK, ERK, or JNK and phosphospecific Abs to each protein. B, Neutrophils isolated freshly (PMN), human lung fibroblasts (IMR-90; FIB), and human bronchial epithelial cells (16HBE; BEC) were lysed, and cell lysates containing 50 mg protein were analyzed by immunoblotting for p38-MAPK. C, Neutrophils isolated freshly were stimulated with 1026 M PMA, and analyzed by immunoblotting for p38-MAPK and ERK. D, Neutrophils isolated freshly were suspended in medium with or without 100 U/ml polymyxin B (PolyB). The results are representative of three or four experiments. The OD of each band was determined, and the ratio of phospho/total protein was calculated. Data represent mean 1 SEM.

Ab CH-11 (100 ng/ml) shortened neutrophil survival and promoted apoptosis (Fig. 8). In contrast, GM-CSF (100 ng/ml) and the antioxidants catalase (500 U/ml) and N-acetylcysteine (500 mg/ml) extended survival and inhibited apoptosis (Fig. 8). However, none of these reagents or H2O2 significantly affected p38-MAPK phosphorylation and activation in neutrophils (Fig. 9, A and B). These results indicate that p38-MAPK function in neutrophils is independent of signaling pathways triggered by Fas ligand, GM-CSF, and ROS.

Discussion It is now clear that neutrophil apoptosis is a novel part of homeostasis and the inflammatory response (1). Unlike many other cell types, neutrophils undergo spontaneous apoptosis without any need for apparent external inductive stimuli. However, no signaltransduction pathways have been shown to mediate spontaneous neutrophil apoptosis to date. The present study demonstrates that the p38-MAPK pathway is involved in spontaneous apoptosis in

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FIGURE 6. Immunocytochemical detection of p38-MAPK phosphorylation in neutrophils in blood smear samples. Blood smears made on glass slides were immunostained with a phosphospecific p38-MAPK Ab (A) or control rabbit IgG (C), followed by FITC-conjugated secondary Ab. Cells were counterstained with propidium iodide to identify cell nuclei (B and D). Note that a neutrophil shows a low but significant level of cytoplasmic and nuclear staining for p38-MAPK phosphorylation (A).

neutrophils. To our knowledge, this is the first study to demonstrate that a specific kinase is involved in signaling pathways that lead to spontaneous apoptosis in neutrophils.

FIGURE 8. Effect of agonistic anti-Fas Ab, GM-CSF, and antioxidants on neutrophil survival and apoptosis. Neutrophils were cultured for 24 h with or without anti-Fas Ab (CH-11; 100 ng/ml), GM-CSF (50 ng/ml), catalase (500 U/ml), and N-acetylcysteine (500 mg/ml). Survival of neutrophils (A) was evaluated by a MTT assay (n 5 6), and percentage of apoptosis (B) by light microscopy (n 5 6). Data represent mean 1 SEM of six experiments. p, p , 0.05; pp, p , 0.01 versus control cells in medium alone.

FIGURE 7. Activation of p38-MAPK in neutrophils. Neutrophils were cultured for the indicated time. p38-MAPK was immunoprecipitated from cell lysates containing 250 mg protein, and p38-MAPK activity was measured in an immune complex protein kinase assay using [g-32P]ATP and MAPKAP kinase-2-GST as substrates. Phosphorylated MAPKAP kinase2-GST was detected after SDS-PAGE by autoradiography. The SB203580 lane represents a sample prepared from freshly isolated neutrophils and assayed in the presence of 20 mM SB203580. The immunoprecipitated samples were also analyzed for p38-MAPK by immunoblotting. The results are representative of three experiments. The OD of each band was determined, and the ratio of phosphorylated MAPKAP kinase-2-GST/immunoprecipitated p38-MAPK was plotted. Data represent mean 1 SEM.

Recent studies suggest the importance of tyrosine phosphorylation and dephosphorylation events in signaling pathways that result in neutrophil apoptosis. For example, a study showed that increased tyrosine-phosphorylated proteins were detected in GMCSF-treated neutrophils that showed increased survival, while genistein, a broad tyrosine kinase inhibitor, abrogated the survivalpromoting effect of GM-CSF (9). Another study indicated that the tyrosine kinase lyn is specifically involved in GM-CSF-mediated signaling that promotes neutrophil survival (10). These studies indicate that tyrosine phosphorylation of certain proteins is necessary for the inhibition of apoptosis by GM-CSF. On the other hand, the present study indicates that tyrosine phosphorylation events are also important for the induction of apoptosis that occurs spontaneously. Our data suggest that continuous activation of p38MAPK is involved in spontaneous apoptosis in neutrophils. Endotoxin contamination during and after cell preparation is a constant problem that may prime neutrophils and confuse results. Although endotoxin has been shown to activate p38-MAPK in

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ROLE OF p38-MAPK IN SPONTANEOUS APOPTOSIS OF NEUTROPHILS neutrophils (13, 14), we could not obtain any evidence favoring the possibility that p38-MAPK phosphorylation and activation seen in freshly isolated neutrophils were due to endotoxin contamination or accidental neutrophil activation. However, although a relationship between apoptosis and p38-MAPK is clearly demonstrated, our results may neither exclude contamination by undetectable levels of endotoxin, nor rule out the necessity of a certain degree of neutrophil activation for constitutive activation of p38-MAPK. It should also be emphasized that because cell preparation procedures induce some degree of neutrophil activation (18), extrapolation of in vitro data to in vivo situations requires caution. Nevertheless, additional evidence for a close relationship between neutrophil apoptosis and p38-MAPK is provided by a recent paper published after our original submission of this manuscript (30). Frasch et al. have reported that SK & F 86002, a specific p38-MAPK inhibitor, suppressed neutrophil apoptosis induced by stress stimuli such as UV, hyperosmolarity, and sphingosine (30). In contrast to us, they observed no inhibition of spontaneous apoptosis by the p38-MAPK inhibitor. However, there are several methodologic differences between their study and our own. First, they used a different p38-MAPK inhibitor (SK & F 86002) than we used (SB203580). Second, they assessed the effect of the p38MAPK inhibitor on spontaneous apoptosis only at one time point (24 h). Third, they assessed apoptosis by fluorometric measurement of DNA content in propidium iodide-stained cells by flow cytometry. It should be noted that in our experiments, when compared with morphologic (Fig. 1B) and MTT-based assays (Fig. 3A), the fluorometric measurement of DNA content (Fig. 3B) detected a lower degree of inhibition of apoptosis by the same dose (10 mM) of p38-MAPK inhibitor. It is likely that the different methods of assessing apoptosis detect various stages of apoptosis. Fourth, neutrophils isolated in their study may not have exhibited basal phosphorylation and activation of p38-MAPK. However, in accordance with our own, their figures show low but significant levels of basal phosphorylation and activation of p38-MAPK at time 0 and 90 min of incubation. Recent studies suggest that spontaneous neutrophil apoptosis is elicited by the interaction between the constitutively expressed Fas and Fas ligand molecules in neutrophils (7, 8, 31). In the present study, we found that an agonistic Fas Ab, which promoted neutrophil apoptosis, did not affect the phosphorylation and activation of p38-MAPK. This finding is consistent with a recent report that the incubation of neutrophils with anti-Fas Ab did not affect the tyrosine phosphorylation or tyrosine phosphatase activity of the cells (31) nor the phosphorylation and activation of p38-MAPK (30). In Jurkat T lymphocytes, however, Fas has been shown to activate p38-MAPK (32, 33). Although we cannot explain these differences, our data suggest that p38-MAPK works independent of or in parallel with Fas-Fas ligand systems in neutrophils.

FIGURE 9. Effect of agonistic anti-Fas Ab, GM-CSF, and antioxidants, and H2O2 on p38-MAPK phosphorylation and activation. Neutrophils were cultured for 6 h with or without anti-Fas Ab (CH-11; 100 ng/ml; Fas), GM-CSF (50 ng/ml; GM-CSF), catalase (500 U/ml; Cat), N-acetylcysteine (500 mg/ml; NAC), and H2O2 (1, 10, and 100 mM). Cell lysates were analyzed for p38-MAPK phosphorylation by immunoblotting (A, B) and for p38-MAPK activation by immune complex protein kinase assay (C). The results are representative of three experiments. The OD of each band was determined, and the ratio of phosphorylated MAPKAP kinase-2-GST/ imunoprecipitated p38-MAPK was plotted. Data represent mean 1 SEM. We also found that catalase up to 1600 U/ml and N-acetylcysteine up to 5 mM did not affect p38-MAPK phosphorylation (data not shown).

The Journal of Immunology Our finding that p38-MAPK phosphorylation and activation were not affected by stimulation of neutrophils with the Fas agonist or GM-CSF raises the question of how p38-MAPK in neutrophils is continuously activated without any need for apparent external stimuli. We initially hypothesized that ROS generated intracellularly may contribute to continuous phosphorylation and activation of p38-MAPK. This hypothesis was based on the following evidence. First, previous investigators showed that ERK, another MAPK family member, is activated following treatment of neutrophils with the oxidizing agent diamide and H2O2 (34). Second, we have found recently that ROS act as an activator of p38-MAPK in fibroblasts depleted of thiols.4 Third, like other aerobic cells, neutrophils constitutively generate ROS within cells and undergo apoptosis in response to ROS accumulation (4, 5, 35). In support of this, we observed that peroxides, as detected by CDCFH (6carboxy-2979-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester)) fluorescence, are accumulated within neutrophils cultured in quiescent conditions (unpublished data). Fourth, we have found in the present study that the antioxidants catalase and N-acetylcysteine inhibited spontaneous apoptosis and extended survival of neutrophils. Taken together, these lines of evidence confirm that ROS play an important role in spontaneous apoptosis in neutrophils. However, the lack of effect of these antioxidants and extragenous H2O2 on P38-MAPK phosphorylation and activation we observed suggests that ROS generation does not lie upstream of p38-MAPK activation. In other mammalian cell types such as COS-1, COS-7, and HeLa cells, the small GTP-binding proteins Rac and Cdc42 (36, 37) and different MAPK kinases (MKK-3, MKK-4, MKK-6) (38, 39) have been implicated as upstream regulators of p38-MAPK. In neutrophils, however, the upstream regulators of p38-MAPK signaling remain ill defined. In this respect, a recent report suggested that activation of p38-MAPK by LPS in neutrophils uses MKK-3, but not Raf, mitogen-activated protein/ERK-1, or mitogen-activated protein/ERK-2 (14). Other reports showed that activation of p38-MAPK by chemoattractants is mediated through a pathway involving phosphatidylinositol 3-kinase, protein kinase C, and Ca21 (15, 16). In turn, changes in Ca21 have been shown to affect the constitutive rate of neutrophil apoptosis (3). To date, intracellular events preceding p38-MAPK phosphorylation and activation in neutrophils have not been defined. Further studies will be needed to determine what mediates the continuous phosphorylation and activation of p38-MAPK that lead to apoptosis in neutrophils. Activation of p38-MAPK has been implicated in extracellular stress-induced apoptosis in other cell types such as neuronal cells and lymphocytes (40, 41). However, the mechanisms by which p38-MAPK activation participates in apoptosis are unknown. In our findings in neutrophils, inhibition of p38-MAPK activity by SB203580 and by antisense oligonucleotides delayed apoptosis, but could not abolish its expression. In addition, the phosphorylation and activation of p38-MAPK were detected even in freshly isolated neutrophils that had not yet expressed the morphologic features of apoptosis (i.e., at time 0). These findings suggest that p38-MAPK activation in neutrophils is not required for the execution of apoptosis, but rather is required for the constitutive activation of other necessary components of a cell death pathway. It should also be emphasized that SB203580 effectively inhibited p38-MAPK activity, yet only inhibited apoptosis by 50%. This suggests a partial role of p38-MAPK in the program of spontaneous apoptosis in neutrophils. Nevertheless, the continuous phos4 K. Aoshiba, S. Yasui, K. Nishimura, and A. Nagai. Thiol depletion induces apoptosis through an ordered cell death pathway in cultured lung fibroblasts. Submitted for publication.

1699 phorylation and activation of p38-MAPK presented in this work may reflect the constitutively expressed apoptotic program in neutrophils, which rapidly die via apoptosis in vitro and in vivo (1, 2). The functional role of p38-MAPK in neutrophils has not previously been demonstrated. Several reports documented that p38MAPK is activated following stimulation of neutrophils with various agonists such as LPS, TNF-a, GM-CSF, platelet-activating factor, FMLP, IL-8, and protein kinase C (12–16). However, many of these agonists activate ERK as well (15, 26, 42), indicating multiple MAPK activation by inflammatory stimuli. Our findings suggest that the activation of p38-MAPK may serve specifically to limit the longevity of neutrophil survival following activation. Indeed, it seems clear that the constitutive rate of apoptosis in neutrophils is the major determinant of their survival and functional longevity at inflamed sites (1). Although elevation of intracellular Ca21 (3) or cAMP (43) by agonistic stimuli has been shown to inhibit neutrophil apoptosis, the effect of inflammatory mediators such as FMLP, C5a, IL-1, and IL-8 on neutrophil apoptosis remains controversial (34, 44, 45). Based on our findings, activation of p38-MAPK by inflammatory mediators may activate an apoptotic program to counteract the antiapoptotic effects of inflammatory signals. This may be important to limit the toxic potential of neutrophils. The validity of this hypothesis remains to be determined. In summary, our data suggest that p38-MAPK plays a role in driving spontaneous apoptosis of neutrophils. Clarification of this role perhaps awaits further investigations of its regulators and targets. Definition of the p38-MAPK-mediated pathway will provide clues to the complex mechanism underlying neutrophil apoptosis.

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