* Department of Internal Medicine, University of Genova Medical School, Italy; and
Immunogenetic Department, National Institute of Cancer Research, Genova, Italy
Correspondence: Luciano Ottonello, M.D., Department of Internal Medicine and Medical Specialties, Viale Benedetto XV n.6, I-16132 Genova, Italy. E-mail: otto{at}unige.it
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Key Words: GM-CSF • interleukin • fMLP • CGD • inflammation
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Recent evidence suggests that neutrophil senescence, i.e., apoptosis, may represent a crucial step in the mechanisms that govern the resolution of neutrophilic inflammation [7 ]. Indeed, mature neutrophils undergo apoptosis spontaneously. This leads to the impairment of neutrophil functional responsiveness [8 ]. Furthermore, different from necrotic cells, apoptotic neutrophils maintain their membrane integrity, which in turn prevents extracellular leakage of histotoxic compounds [9 ]. Finally, senescent neutrophils undergo phenotypic modifications, leading to their recognition and uptake by macrophages without stimulating proinflammatory activities of phagocytosing cells [10 ]. Although neutrophils are terminally differentiated cells, there is evidence showing that their life span can be modulated [11 , 12 ]. In particular, soluble mediators, detectable in the inflammatory environment surrounding recruited neutrophils, are capable of modulating the cell survival retarding their apoptosis [11 12 13 14 15 16 ]. This suggests that during the acute phases of neutrophilic inflammatory diseases, the tissue burden of these cells can be very heavy as a consequence of an accelerated influx of recruited neutrophils, but also for delayed apoptotic process. On the contrary, full activation of neutrophils induced by phagocytosable particles, such as insoluble IC [17 , 18 ] or opsonized bacteria [19 ], results in the acceleration of apoptosis so that the life span of neutrophils is shortened. It is evident that at sites of inflammation, neutrophils are likely exposed to opposing forces capable of diverging effects on the cell survival. Furthermore, although recent studies have offered some clues about the molecular control of spontaneous neutrophil apoptosis and its modulation by cytokines [20 ], there is little information available regarding the intracellular pathway governing IC-dependent apoptosis. This is a particularly important issue, considering that the pharmacological manipulation of neutrophil apoptosis has been proposed as a possible intriguing approach to the therapy of neutrophilic inflammatory diseases [21 ].
To clarify the fate of neutrophils engaged in FcR-dependent effector functions at sites of inflammation and possibly the mechanisms regulating the survival of these cells, we investigated the effects of some proinflammatory, soluble mediators on spontaneous as well as IC-induced neutrophil apoptosis.
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Preparation of IC
IC were prepared by incubating human albumin and rabbit anti-human albumin IgG at fivefold Ag excess, as described previously [18
]. Albumin and anti-human albumin IgG were incubated for 2 h at 37°C and thereafter overnight at 4°C. Then, IC were centrifuged (500 g/min) for 10 min and resuspended in PBS. The amount of IC was determined by Lowry assay [22
].
Neutrophil isolation and culture
Heparinized (heparin 10 units/ml) venous blood was obtained from healthy volunteers after informed consent. Blood was also obtained from three patients with chronic granulomatous disease (CGD), all displaying a gp91phox X-linked form and all deficient in reduced nicotinamide adenine dinucleotide phosphate oxidase-derived oxidants, as tested by superoxide-anion generation and measured by the superoxide dismutase-inhibitable cytochrome c reduction, as described previously [23
]. Neutrophils were isolated by dextran sedimentation and subsequent centrifugation on a density gradient [24
]. Contaminating erythrocytes were removed by hypotonic lysis [24
]. Final cell suspensions (2x106/ml) always contained 97% or more viable cells. Then, neutrophils were incubated in tissue-culture tubes (17x100 mm; Falcon, Becton Dickinson, Oxnard CA) at 37°C in a 5% CO2 atmosphere (0.5 ml final volume). Experiments were carried out in the absence or presence of appropriate concentrations of IC, GM-CSF, IL-2, IL-6, IL-8, IL-15, and fMLP. At appropriate time points, cells were harvested and counted on a hemocytometer before subsequent assays.
Neutrophil membrane integrity assay
Neutrophil viability measured as integrity of membrane was assessed according Dankberg and Persidsky [25
], as described previously [24
]. Briefly, cells (4x104/100 µl) harvested from culture tubes were mixed with 50 µl staining solution (2 µg/ml fluorescein diacetate, 4 µg/ml ethidium bromide in HBSS) and incubated for 10 min at room temperature. Thereafter, a drop of cell suspension was placed on a slide, sealed with a coverslip, and analyzed under ultraviolet light in a dark field illumination. Neutrophils with intact membrane (i.e., viable cells) appeared as green fluorescent cells, whereas neutrophils with damaged and ethidium bromide-permeable membrane (i.e., necrotic cells) displayed a fluorescent red nucleus.
Light microscopic assessment of neutrophil apoptosis
Cytocentrifuged cell preparations were fixed and stained with May-Grünwald-Giemsa. Thereafter, cytopreps were read blindly by two independent observers by oil-immersion light microscopic examination of at least 500 cells/slide (1000x magnifications). Cells showing apoptotic morphology were identified according to the typical criteria: cell shrinking, nuclear condensation and fragmentation, plasma membrane ruffling, and blebbing [9
], as described previously [24
].
Immunofluorescence flow cytometry of Annexin V-FITC binding
Immunofluorescence analysis of Annexin V binding was performed following the manufacturer’s instruction with minor changes, as described previously [18
]. Briefly, cells were washed and resuspended in 100 µl isotonic binding buffer. Then, Annexin V-FITC (3 µl) was added, and after incubation (15 min), cells were washed and resuspended in ice-cold PBS supplemented with 3% FCS and 0.1% sodium azide. Flow cytometry analysis was performed on an EPICS XL flow cytometer (Coulter, Hialeah, FL).
Flow cytometric assessment of neutrophil DNA content
Flow cytometric analysis of apoptotic nuclei was carried out according Nicoletti et al. [26
] with some changes, as described previously [18
]. Briefly, cells were washed and resuspended in 0.5 ml PBS, and the cell suspension was added by drops to 4.5 ml ice-cold, 80% ethanol while vortexing and put at -20°C for 24 h. Afterward, cells were washed twice, propidium iodide was added to a final concentration of 10 µg/ml, and the sample was analyzed by flow cytometry after an overnight incubation. Flow cytometric analysis was performed on an EPICS XL flow cytometer (Coulter). Briefly, living granulocytes were gated on the basis of physical properties (forward- vs. side-light-scatter), and at least 2000 living cells were analyzed for each sample.
Flow cytometric assessment of neutrophil-oxidative metabolism
Flow cytometric analysis of neutrophil-oxidative metabolism was carried out according to Bass et al. [27
], as described previously [18
]. Briefly, neutrophils, pretreated (15 min, 37°C) with DCFH-DA (5 µM), were incubated (2 h, 37°C) under the same conditions used for apoptosis assay. At the end of the incubation, the reaction was stopped by keeping the samples on ice until flow cytometric analysis was carried out using an EPICS XL flow cytometer (Coulter).
Immunocytochemistry
Bax protein expression was investigated by immunocytochemistry, as described by Dibbert et al. [28
], with slight modifications. Briefly, cytospins were prepared from 105 purified neutrophils, and then the spots were air-dried. After the rehydration in PBS, spots were submerged in peroxidase-quenching solution for 10 min to neutralize endogenous peroxidase activity. Then, the slides were incubated with the anti-human Bax polyclonal antibody (dilution 1:200 in PBS, 60 min, 24°C), followed by the incubation with the secondary biotinylated IgG antibody (Histostain SP, Zymed Laboratories, San Francisco, CA). After washing and subsequent incubation with the concentrate biotin-streptavidin-peroxidase (Histostain SP), slides were incubated at 24°C for 5 min with the peroxidase-substrate solution (Histostain SP), rinsed with PBS, and counterstained with hematoxylin. Then, cytospins were mounted in Eukitt, examined by light microscopy, and evaluated by image analysis. Controls were treated identically, except for omitting the primary antibody.
Protein extracts and Western blot analysis
Bax protein expression was investigated by Western blot analysis, as described by Airoldi et al. [29
], with slight modifications. After incubation, neutrophils were incubated for 30 min on ice with lysis buffer containing 20 mM HEPES, 150 mM NaCl, 10% glycerol, 0.25% Nonidet P-40, 1 mM ethylenediaminetetraacetate (EDTA), 2.5 mM DTT, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na3VO4. During this time interval, cells were subjected to vortex mixing every 5 min. Thereafter, lysates were centrifuged at 12,000 rpm for 15 min at 4°C, and supernatants were quantitated by the bicinchoninic acid kit assay (Pierce, Rockford, IL). Equal amounts of protein (20 µg) were loaded on 12% sodium dodecyl sulfate-polyacrylamide gel and boiled 3 min before application. Gel was blotted onto Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech Italia, Cologno Monzese, Italy) overnight at 10 V. Blots were blocked with 5% (v/v) notfat powdered milk, followed by incubation with the rabbit anti-human Bax polyclonal antibody. After three washings in 0.5% Tween 20 in PBS, blots were incubated for 1 h with goat anti-rabbit Ig conjugated with horseradish peroxide (Sigma-Aldrich S.r.l.). Detection was performed by enhanced chemiluminescence (Amersham Pharmacia Biotech Italia).
Image analysis
Image analysis was performed by the Leica Q500 MC image analysis system (Leica, Cambridge, UK). For each sample, we randomly analyzed 100 cells and quantitated, with a PC computer, the optical density of the signals. The video image was digitized for image analysis at 256 grey levels. Imported data were analyzed quantitatively by Q500 MC Software-Qwin (Leica). The operator using the cursor randomly selected single cells. Then, the positive area was estimated automatically. Constant optical threshold and filter combination were used.
Caspase-3 assay
The assay was performed as described previously [18
]. After the appropriate incubation time, neutrophils (106) were washed in cold PBS and resuspended in 50 µl 50 mM NaCl, 2 mM MgCl2, 5 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid, 2 mg/ml leupeptin, 2 mg/ml aprotinin, 10 mM Hepes, pH 7.4. After 20 min incubation on ice, cells were lysed by freezing and thawing in liquid nitrogen. The cell lysate was spun (14.000 g, 4°C, 15 min), and the supernatant was removed and diluted to 200 µl in the assay buffer consisting of 25 µM Hepes, pH 7.4, 0.1% CHAPS, 10% glycerol, 1 mM EDTA, and 5 mM DTT, supplemented with 50 µM caspase-3 substrate Ac-DEVD-pNa. Then, the enzymatic activity was determined spectrophotometrically (Titertek TwinReader Plus, Flow Lab, Ltd., Irvine, Scotland) for 60 min at 405 nm assuming an extinction coefficient of 8.8 x 103 M-1 cm-1.
Statistical analysis
Data were expressed as mean ± 1 SD. Differences were determined by one-way or repeated measures analysis of variance with Bonferroni’s post-test using GraphPad InStat version 3.05 for Windows 95, GraphPad Software (San Diego, CA). Differences were accepted as significant when P < 0.05.
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As shown in Figure 1A , a significant reduction in the percentage of spontaneous apoptosis was observed in the presence of GM-CSF (10 ng/ml), IL-6 (10 ng/ml), and IL-15 (10 ng/ml). The three cytokines displayed their inhibitory activity in a dose-dependent manner (data not shown). On the contrary, IL-2 (1000 U/ml), IL-8 (10 ng/ml), and fMLP (0.1 nM) were ineffective, also when used at 100-fold higher concentrations (data not shown). Conversely, as shown in Figure 1B , among the six proinflammatory compounds tested, only GM-CSF was found capable of slowing the rate of neutrophil apoptosis in the presence of IC, whereas the other cytokines and chemokines were ineffective, even when tested at 100-fold higher concentrations (not shown). These findings were confirmed by flow cytometric evaluation of propidium iodide incorporation (Fig. 2A ) and Annexin V binding (Fig. 2B) , both considered reliable markers of cell apoptosis [26 , 30 ].
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Figure 1. Effect of six chemokines and cytokines on neutrophil apoptosis: morphological evaluation. (A) Neutrophils (2x106/ml) were incubated for 18 h in the absence (open bars) or presence (solid bars) of GM-CSF (10 ng/ml), IL-2 (1000 U/ml), IL-6 (10 ng/ml), IL-8 (10 ng/ml), IL-15 (10 ng/ml), and fMLP (0.1 nM). Then, apoptosis was evaluated morphologically on cytopreps stained with May-Grünwald-Giemsa. Results are expressed as the mean ± 1 SD from five to six experiments. Apoptosis in the absence vs. presence of GM-CSF = P < 0.0001; IL-2 = not significant (N.S.); IL-6 = P < 0.01; IL-8 = N.S.; IL-15 = P < 0.001; fMLP = N.S. One-way analysis of variance. (B) Neutrophils (2x106/ml) were incubated for 12 h with 25 µg/ml IC in the absence (open bars) or presence (solid bars) of GM-CSF (10 ng/ml), IL-2 (1000 U/ml), IL-6 (10 ng/ml), IL-8 (10 ng/ml), IL-15 (10 ng/ml), and fMLP (0.1 nM). Then, apoptosis was evaluated morphologically on cytopreps stained with May-Grünwald-Giemsa. Results are expressed as the mean ± 1 SD from four to nine experiments. Apoptosis in the absence vs. presence of GM-CSF = P < 0.0001; IL-2 = N.S.; IL-6 = N.S.; IL-8 = N.S.; IL-15 = N.S.; fMLP = N.S. One-way analysis of variance. Spontaneous apoptosis (i.e., in the absence of IC treatment): 25.2 ± 9.9, mean ± 1 SD; n = 14.
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Figure 2. Effect of six chemokines and cytokines on neutrophil apoptosis: flow cytometric analysis. (A) Annexin V-FITC binding. Upper line: Neutrophils (2x106/ml) were incubated for 18 h in the absence (Nil) or presence of GM-CSF (10 ng/ml), IL-2 (1000 U/ml), IL-6 (10 ng/ml), IL-8 (10 ng/ml), IL-15 (10 ng/ml), and fMLP (0.1 nM). Lower line: Neutrophils (2x106/ml) were incubated for 12 h with 25 µg/ml IC in the absence (IC) or presence of GM-CSF (10 ng/ml), IL-2 (1000 U/ml), IL-6 (10 ng/ml), IL-8 (10 ng/ml), IL-15 (10 ng/ml), and fMLP (0.1 nM). Then, after 15 min incubation with Annexin V-FITC, flow cytometry analysis was conducted. A representative experiment of the two performed is shown. (B) Propidium iodide (PI) staining. Upper line: Neutrophils (2x106/ml) were incubated for 18 h in the absence (Nil) or presence of GM-CSF (10 ng/ml), IL-2 (1000 U/ml), IL-6 (10 ng/ml), IL-8 (10 ng/ml), IL-15 (10 ng/ml), and fMLP (0.1 nM). Lower line: Neutrophils (2x106/ml) were incubated for 12 h with 25 µg/ml IC in the absence (IC) or presence of GM-CSF (10 ng/ml), IL-2 (1000 U/ml), IL-6 (10 ng/ml), IL-8 (10 ng/ml), IL-15 (10 ng/ml), and fMLP (0.1 nM). Propidium iodide incorporation was evaluated after cold ethanol fixation and overnight incubation with propidium iodide, the appropriate mediator. A representative experiment of the two performed is shown.
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Figure 3. Effect of six chemokines and cytokines on neutrophil-oxidative metabolism triggered by IC. Neutrophils were pretreated with 5 µM DCFH-DA for 5 min and incubated (2 h) in the absence or presence of IC (25 µg/ml) and the appropriate mediator. Thereafter, mean fluorescence intensity (MFI) was examined by flow cytometry analysis. Results are expressed as the mean ± 1 SD; n = 3. GM-CSF versus IC: P < 0.05; IL-2 versus IC: N.S.; IL-6 versus IC: N.S.; IL-8 versus IC: N.S.; IL-15 vs. IC: N.S.; fMLP versus IC: P < 0.001. Repeated measures analysis of variance.
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Figure 4. Effect of IC and GM-CSF on neutrophil apoptosis from three CGD patients. After isolation from peripheral blood, neutrophils (2x106/ml) were incubated (18 h) with medium (Nil), 25 µg/ml IC (IC), or 25 µg/ml IC plus 10 ng/ml GM-CSF (IC + GM-CSF). Then, apoptosis was evaluated morphologically on cytopreps stained with May-Grünwald-Giemsa. Results are expressed as the mean ± 1 SD; n = 3. Nil versus IC: P < 0.001; IC versus IC + GM-CSF: P < 0.001. Repeated measures analysis of variance.
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Figure 5. Effects of some proinflammatory mediators on the expression of the proapoptotic protein Bax. (A) Neutrophils (2x106/ml) were incubated for 18 h in the absence (Nil) or presence of GM-CSF (10 ng/ml), IL-6 (10 ng/ml), and IL-15 (10 ng/ml). Then, Bax expression was determined by immunocytochemistry. Results are expressed as the mean ± 1 SD; n = 3. Nil versus GM-CSF: P < 0.001; Nil versus IL-6: P < 0.01; Nil versus IL-15: P < 0.001. Repeated measures analysis of variance. (B) Neutrophils (2x106/ml) were incubated for 12 h with 25 µg/ml IC in the absence (IC) or presence of GM-CSF (10 ng/ml), IL-6 (10 ng/ml), and IL-15 (10 ng/ml). Then, Bax expression was determined by immunocytochemistry. Results are expressed as the mean ± 1 SD; n = 3. IC vs. GM-CSF: P < 0.01; IC versus IL-6: P = N.S.; IC vs. IL-15: P = N.S. Repeated measures analysis of variance. (C) Neutrophils were incubated in the same conditions as (B). Then, Bax expression was determined by Western blotting. 1, Nil; 2, IC; 3, IC + GM-CSF; 4, IC + IL-6; 5, IC + IL-15.
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Figure 6. Effects of some proinflammatory mediators on caspase-3 activity. (A) Neutrophils (2x106/ml) were incubated for 18 h in the absence (Nil) or presence of GM-CSF (10 ng/ml), IL-6 (10 ng/ml), and IL-15 (10 ng/ml). Then, caspase-3 activity was determined spectrophotometrically on whole-cell lysates. Results are expressed as the mean ± 1 SD; n = 3. Nil vs. GM-CSF: P < 0.01; Nil versus IL-6: P < 0.05; Nil vs. IL-15: P < 0.01. Repeated measures analysis of variance. (B) Neutrophils (2x106/ml) were incubated for 12 h with 25 µg/ml IC in the absence (IC) or presence of GM-CSF (10 ng/ml), IL-6 (10 ng/ml), and IL-15 (10 ng/ml). Then, caspase-3 activity was determined spectrophotometrically on whole-cell lysates. Results are expressed as the mean ± 1 SD; n = 3. IC versus GM-CSF: P < 0.001; IC vs. IL-6: P = N.S.; IC versus IL-15: P = N.S. Repeated measures analysis of variance.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is generally accepted that neutrophils express proteins belonging to the Bcl-2 family, but data regarding the role of these proteins in the regulation of neutrophil apoptosis are contradictory [20 , 35 36 37 38 ]. In particular, contrasting data have been described regarding GM-CSF-mediated regulation of Bax expression during spontaneous neutrophil apoptosis. In fact, some authors cannot demonstrate down-regulation of Bax expression by agents delaying neutrophil apoptosis, including GM-CSF [38 ]. On the contrary, other authors showed a well-detectable, GM-CSF-dependent suppression of Bax in neutrophils [35 , 36 , 39 ]. In the present work, we found that GM-CSF as well as IL-6 and IL-15 inhibit constitutive Bax expression and spontaneous apoptosis. Most important, here we present the first evidence that neutrophil FcR triggering by IC results in a dramatic up-regulation of Bax protein expression, which strictly correlate with the IC-dependent neutrophil apoptosis induction. Furthermore, we demonstrate that GM-CSF, but not IL-6 and IL-15, is capable of overriding IC-dependent acceleration of neutrophil apoptosis by acting on the Bax levels. Consistent with the present findings, neutrophils from individuals with neutrophilic lung diseases, characterized by high levels of GM-CSF and IC, expressed little or no Bax protein, whereas bronchoalveolar lavage from the same patients can delay apoptosis of, and reduce Bax expression in, normal neutrophils [35 ].
Caspase-3 is considered among the major requirements for the execution phase of apoptosis. One of the pathways driving to the activation of procaspase-3 is represented by Bax insertion into mitochondria [40 ]. This induces the release of cytochrome c, which in turn triggers the activation of caspase-3 and the execution phase of apoptosis [40 ]. Furthermore, it has been demonstrated recently that caspase-3 activity is necessary for the appropriate insertion of Bax in mitochondria, suggesting an amplificatory loop between caspase-3 and Bax activities [36 ]. In accord with this view, in our experimental conditions the enzymatic activity of caspase-3 correlated with expression of Bax protein, confirming different regulatory pathways for spontaneous and IC-mediated neutrophil apoptosis.
GM-CSF had been identified 25 years ago as a hematopoietic growth factor stimulating proliferation and maturation of myeloid progenitors and inducing the differentiation of several cell lineages, primarily neutrophils, eosinophils, and monocytes [41 ]. Furthermore, in vitro and ex vivo observations demonstrated that GM-CSF increases the functional activities of several mature effector cells, including neutrophils [42 43 44 45 ]. More recently, GM-CSF was found capable of increasing neutrophil survival [11 ], also in the presence of some proapoptotic events, such as Fas ligation or ultraviolet exposure [46 , 47 ]. These studies demonstrated GM-CSF capacity of overriding the FcR-mediated acceleration of neutrophil apoptosis extends the concept of GM-CSF as a crucial stimulator of myeloid progenitors and as a powerful priming agent. In fact, by means of its ability to increase the number and the life span of neutrophils engaged in FcR-dependent effector activities, GM-CSF can be seen as a useful tool for incrementing the antimicrobial activities of neutrophils at sites of tissue infections. Nevertheless, during some IC-mediated diseases characterized by neutrophilic inflammation, GM-CSF-induced prolongation of the survival of improperly activated neutrophils can result in long-lasting release of phagocyte-derived, harmful products capable of producing extensive tissue damage.
It is well known that neutrophils, upon activation by phagocytosable stimuli such as insoluble IC, mount a respiratory burst with the production of huge amounts of oxidants [31 ], which in turn have been implicated as major proapoptotic mediators [32 33 34 ]. Furthermore, it has been suggested recently that in certain cell lines, GM-CSF signal transduction is mediated by intracellular oxidants [48 ]. Consequently, this latter study shows that inhibitory activity of GM-CSF toward IC-induced neutrophil apoptosis may be related to an interference of the cytokine with the neutrophil-oxidative status. Nevertheless, our results do not support this hypothesis. In fact, no correlation was found between the oxidative status and the rate of apoptosis of IC-stimulated neutrophils incubated in the absence or presence of the six cytokines and chemokines. Furthermore, neutrophils from CGD patients constitutively incapable of producing oxidants displayed a GM-CSF-inhibitable acceleration of the apoptosis rate when exposed to IC. In other words, IC can induce a threefold increment of neutrophil apoptosis in oxidant-free conditions as well, such as the intracellular milieu of CGD neutrophils, whereas the rate of neutrophil apoptosis can be slowed by GM-CSF also in the presence of high levels of intracellular oxidants, i.e., in IC-stimulated normal neutrophils in the presence of GM-CSF.
In conclusion, the present data confirm and extend our previous findings, which have uncovered the existence of an oxidant-independent, FcR-dependent, intracellular pathway regulating neutrophil apoptosis via modulation of caspase-3 activation. Indeed, here we show that FcR-dependent activation induces Bax up-regulation, which in turn results in caspase-3 activation and subsequent acceleration of neutrophil apoptosis. Furthermore, we provide evidence that GM-CSF, but not several other proinflammatory mediators including IL-6 and IL-15, is capable of inhibiting the FcR-dependent death signal by acting on the aforementioned oxidant-independent pathway.
Received November 28, 2001; revised February 8, 2002; accepted February 11, 2002.
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R Gallin, J. I. Goldstein, I. M. Snyderman, R. eds. Inflammation: Basic Principles and Clinical Correlates ,497-510 Raven New York. 
-mediated apoptosis of human neutrophils Kidney Int. 53,84-91[Medline]RIII and acquire annexin V binding sites during apoptosis in vitro Blood 85,532-540 receptor-specific mechanisms J. Immunol. 145,3026-3032[Abstract]-mediated regulation of apoptosis of human neutrophils via caspase-3 Blood ,3106-3115RII (CD32), CD11b-CD18 integrins and CD66b glycoproteins Blood 93,3505-3511This article has been cited by other articles:
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