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(Journal of Leukocyte Biology. 2002;72:125-132.)
© 2002 by Society for Leukocyte Biology

Differential regulation of spontaneous and immune complex-induced neutrophil apoptosis by proinflammatory cytokines. Role of oxidants, Bax and caspase-3

Luciano Ottonello*, Guido Frumento{dagger}, Nicoletta Arduino*, Maria Bertolotto*, Patrizia Dapino*, Marina Mancini* and Franco Dallegri*

* Department of Internal Medicine, University of Genova Medical School, Italy; and
{dagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophil apoptosis represents a crucial step in the mechanisms governing the resolution of neutrophilic inflammation. Several soluble mediators of inflammation modulate neutrophil survival, retarding their apoptosis, whereas neutrophil activation by immune complexes (IC) results in the acceleration of apoptosis. To investigate neutrophil fate at the site of inflammation, we studied the effects of interleukin (IL)-2, IL-6, IL-8, IL-15, GM-CSF, and fMLP on spontaneous and IC-induced neutrophil apoptosis and the mechanisms regulating the survival of these cells. Spontaneous apoptosis was inhibited by GM-CSF, IL-6, and IL-15, but only GM-CSF overturned IC-induced apoptosis. No role of oxidants on the modulation of IC-dependent apoptosis was found. Indeed, fMLP or GM-CSF augmented the IC-dependent oxidative response, whereas the other compounds were ineffective. CGD neutrophils showed low levels of spontaneous apoptosis, but when exposed to IC, underwent a sharp increment of the apoptotic rate in a GM-CSF-inhibitable manner. Conversely, the expression of the proapoptotic protein Bax in 18-h aged neutrophils was down-regulated by GM-CSF, IL-6, and IL-15. Furthermore, IC induced a nearly threefold Bax up-regulation, which was completely reversed only by GM-CSF. Accordingly, the spontaneous activity of caspase-3 was inhibited by GM-CSF, IL-6, and IL-15. Furthermore, IC induced a sharp increment of enzymatic activity, and only GM-CSF inhibited the IC-dependent acceleration. Our results show that apoptosis of resting and IC-activated neutrophils is regulated differently, GM-CSF being the most potent neutrophil antiapoptotic factor. The results also unveil the existence of an oxidant-independent, Bax- and caspase-3-dependent, intracellular pathway regulating neutrophil apoptosis.

Key Words: GM-CSF • interleukin • fMLP • CGD • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Once recruited at inflamed sites, polymorphonuclear neutrophilic leukocytes (neutrophils) are involved in the inflammatory reaction against the invading microbial pathogens as the first-line host defense [1 ]. On the other hand, the inappropriate or exaggerated neutrophil activation can cause severe tissue damage during several diseases characterized by neutrophilic inflammation [2 ]. One of the most important mechanisms by which neutrophilic inflammation is generated and sustained is immune complex (IC) formation and/or deposition in tissue [2 , 3 ]. Indeed, the IC-induced, local generation of chemotactic factors and cytokines promotes neutrophil recruitment and creates an extracellular inflammatory milieu favoring the full cell activation [4 , 5 ]. This is triggered directly by IC-mediated cross-linking of neutrophil receptors for Fc fragments of immunoglobulin G (IgG; FcRs) [6 ]. Consequently, histotoxic compounds, such as oxidants and primary granule constituents, are secreted by activated neutrophils in the extracellular milieu [2 ], leading to the development of tissue injury.

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Medium and reagents
RPMI 1640 with 25 mM HEPES (Irvine Scientific, Santa Ana, CA), supplemented with 10% fetal calf serum (FCS; HyClone Eur. Ltd., Cramlington, NE), was used as incubation medium. Dulbecco’s phosphate-buffered saline (PBS) and Hanks’ balanced saline solution (HBSS) were from Irvine Scientific. Heparin was obtained from Roche (Milano, Italy). Ficoll-Hypaque was purchased from Seromed (Berlin, Germany). Fluorescein diacetate, ethidium bromide, propidium iodide, human albumin, rabbit anti-human albumin IgG, N-formyl-met-leu-phe (fMLP), leupeptin, and dithiothreitol (DTT) were from Sigma-Aldrich S.r.l. (Milan, Italy). An Annexin V-fluorescein isothiocyanate (FITC) kit was purchased from Boehringer Ingelheim (Heidelberg, Germany). Eukitt was from Kindler GmbH & Co. (Freiburg, Germany). The reagent 2',7'-dichlorofluorescein-diacetate (DCFH-DA) was from Molecular Probes (Eugene, OR). Human recombinant granulocyte macrophage-colony stimulating factor (GM-CSF) and human recombinant interleukin (IL)-6 were from Genzyme (Cambridge, MA). Human recombinant 72 amino acids IL-8 was from BioSource International (Camarillo, CA). Human recombinant IL-2 and human recombinant IL-15 were kindly donated by Prof. F. Indiveri (Department of Internal Medicine, University of Genova, Italy). Anti-human Bax polyclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). The biotin-streptavidin-amplified detection system was obtained from Biogenex Laboratories (S. Ramon, CA). Aprotinin and CHAPS were from ICN Biomedicals (Milano, Italy). Ac-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNa) was from Bachem AG (Bubendorf, Switzerland). Other reagent-grade compounds were used as obtained from commercial suppliers.

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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of some proinflammatory mediators on neutrophil apoptosis
In agreement with our previous observations [18 ], preliminary data demonstrated that the rate of spontaneous neutrophil apoptosis is increased sharply by coincubation with insoluble human albumin/antialbumin IC in a dose- and time-dependent manner (data not shown). Based on these preliminary experiments, we studied apoptosis of neutrophils from healthy volunteers in the absence or presence of six proinflammatory mediators under two experimental conditions, spontaneous apoptosis after 18 h incubation and neutrophil apoptosis after 12 h incubation with 25 µg IC. These time points were chosen to observe inhibitions and accelerations of apoptosis. Apoptosis was evaluated by the light microscopic examination of the typical morphological changes in four to nine independent experiments, each using cells from a single donor preparation. In both conditions, more than 98% viable cells capable of excluding ethidium bromide were detected in all experiments.

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.

 
Relationship between oxidants and apoptosis in neutrophils exposed to proinflammatory mediators
It is well known that neutrophils exposed to insoluble IC undergo a sustained oxidative response [31 ]. In contrast, several recent studies suggest the involvement of oxidants in neutrophil apoptosis [32 33 34 ]. To investigate whether oxidants are involved in GM-CSF modulation of IC-triggered apoptosis, neutrophil-oxidative burst stimulated by 25 µg/ml IC was studied in the absence or presence of the six proinflammatory mediators. The oxidative status of neutrophils was assessed measuring the intracellular hydrogen peroxide levels by flow cytometry. As shown in Figure 3 , neutrophils exposed to IC produced significant amounts of hydrogen peroxide. Coincubation of neutrophils with fMLP, and at a lesser degree with GM-CSF, resulted in the augmentation of the IC-dependent oxidative response, whereas the other compounds did not affect IC-dependent oxidative burst.



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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.

 
To further investigate possible linkages between neutrophil-oxidative status and GM-CSF-dependent inhibition of IC-triggered apoptosis, experiments were performed using neutrophils purified from patients affected by CGD, congenitally incapable of producing oxidants in response to soluble as well as particulate stimuli. Indeed, it is well known that neutrophils from CGD patients display a slower rate of spontaneous apoptosis because of their defective oxidative metabolism [31 ]. Nevertheless, although after 18 h incubation CGD neutrophils showed a very low level of spontaneous apoptosis, they underwent a sharp increment in the apoptosis rate when exposed to 25 µg/mg IC (Fig. 4 ). Finally, the coincubation with GM-CSF reversed the IC-induced acceleration of cell senescence (Fig. 4) .



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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.

 
Effects of some proinflammatory mediators on the expression of the proapoptotic protein Bax
Recent studies suggest that the balance between proapoptotic and antiapoptotic members of the Bcl-2 family is crucial for the regulation of neutrophil apoptosis [35 36 37 38 ]. We thus focused on Bax expression, a constituent of the Bcl-2 family acting as an apoptosis agonist [35 36 37 38 ]. We performed immunocytochemical analysis of the protein expression on neutrophils maintained under the same conditions of the apoptosis assay. As shown in Figure 5A , Bax was clearly expressed on 18 h- aged neutrophils. Furthermore, coincubation with GM-CSF, IL-6, or IL-15 induced a well-detectable down-regulation of the protein expression. More strikingly, when neutrophils were incubated with IC, they underwent an almost threefold up-regulation of Bax. Finally, GM-CSF, but not IL-6 and IL-15, reversed the IC-induced increment of Bax expression completely (Fig. 5B) . These last findings were confirmed in Western blot analysis (Fig. 5C) .



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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.

 
Effects of some proinflammatory mediators on caspase-3 activity
It has been shown that the proapoptotic activity of Bax is mediated by the release of cytochrome c from mitochondria, with subsequent proteolytic cleavage of procaspase-3 and activation of the execution phase of apoptosis [36 ]. Thus, we studied the enzymatic activity in cell lysate from cells incubated in the same experimental conditions of apoptosis and Bax assay. Neutrophils displayed a time-dependent increase in caspase-3 activity (not shown). After 18 h, the enzymatic activity was found able to be significantly inhibited by GM-CSF, IL-6, and IL-15 (Fig. 6A ). Furthermore, in agreement with our recent findings [18 ], IC induced a sharp increment of proteolytic activity of caspase-3 detected after 12-h incubation (Fig. 6B) . Once again, in these conditions, only GM-CSF among the three cytokines under investigation was found capable of inhibiting the acceleration of caspase-3 activity (Fig. 6B) .



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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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here we studied the regulation of the survival of neutrophils engaged in FcR-dependent effector function. Toward this end, we compared the effects of six proinflammatory compounds on apoptosis of resting, as well as IC-triggered, neutrophils. The results show that GM-CSF, IL-6, and IL-15 retard spontaneous neutrophil apoptosis efficiently; neutrophils engaged in FcR-mediated effector functions undergo accelerated senescence; and among the three cytokines effective for spontaneous neutrophil survival, only GM-CSF is capable of overriding the proapoptotic properties of insoluble IC. Thus, our results suggest that apoptosis of resting and phagocytosing neutrophils is regulated differently, GM-CSF being the most potent compound endowed with neutrophil antiapoptotic properties. Also, among the cytokines and chemokines studied, GM-CSF appears to be unique in its capacity to prevent spontaneous and IC-induced apoptotic processes.

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.


   ACKNOWLEDGEMENTS
 
This work is supported by a grant from Programma di Ricerca Scientifica di Interesse Nazionale MM06118858_003 to F. D.

Received November 28, 2001; revised February 8, 2002; accepted February 11, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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