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Originally published online as doi:10.1189/jlb.1103585 on February 24, 2004

Published online before print February 24, 2004
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(Journal of Leukocyte Biology. 2004;75:893-900.)
© 2004 by Society for Leukocyte Biology

Interleukin-15 delays human neutrophil apoptosis by intracellular events and not via extracellular factors: role of Mcl-1 and decreased activity of caspase-3 and caspase-8

Amélie Bouchard, Claude Ratthé and Denis Girard1

INRS-Institut Armand-Frappier, Université du Québec, Pointe-Claire, Canada

1 Correspondence: INRS-Institut Armand-Frappier, 245 boul. Hymus, Pointe-Claire (PQ), Canada, H9R 1G6. E-mail: denis.girard{at}inrs-iaf.uquebec.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin-15 (IL-15) induces the de novo protein synthesis of intracellular polypeptides and delays neutrophil apoptosis by a mechanism that is still unclear. Herein, we investigated the potential antiapoptotic role of newly synthesized proteins released into the external milieu in IL-15-induced neutrophils. We found that IL-15 induces the de novo synthesis of an ~23-kDa protein, representing the predominant protein detected in the milieu, and identified it as IL-1 receptor antagonist (IL-1Ra) by Western blot and immunoprecipitation. We quantified IL-1Ra, IL-1{alpha}, and IL-1ß concentrations by enzyme-linked immunosorbent assay in intracellular and extracellular fractions from IL-15-induced neutrophils and found that IL-15 does not increase IL-1{alpha} or IL-1ß production but induces IL-1Ra release. Also, we demonstrated that IL-1Ra does not modulate apoptosis, even at a concentration 250 times greater than that measured in the external milieu. In contrast to granulocyte macrophage-colony stimulating factor, the supernatant harvested from IL-15-induced neutrophils was devoid of antiapoptotic activity. Addition of cycloheximide demonstrates that IL-15 delays apoptosis via de novo synthesis of intracellular proteins and that it increases myeloid cell differentiation factor-1 stability. We demonstrated also that IL-15 decreases the activity of caspase-3 and caspase-8, resulting in an inhibition of vimentin cleavage. Our results indicate that IL-15 can activate an anti-inflammatory loop, based on its ability to induce the synthesis of IL-1Ra by neutrophils. We conclude that IL-15 delays human neutrophil apoptosis by intracellular events and not via extracellular factors.

Key Words: cytokine • inflammation • de novo protein synthesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils are able to synthesize various proteins when appropriately activated [1 2 3 4 ]. Knowing the importance of neutrophils in the inflammatory process and because of their high cell turnover rate, it is not surprising that an increasing number of studies have focused on the cellular and molecular biology of neutrophil apoptosis [5 6 7 ].

Interleukin-15 (IL-15) is a proinflammatory cytokine suspected to be an important pathogenic factor in different human diseases, including inflammatory disorders [8 ]. In this regard, high concentrations of IL-15 have been detected in the synovial fluid and in synovial membrane cells from rheumatoid arthritis patients [9 10 11 12 ]. Human neutrophils are known to express the three IL-15 receptor (IL-15R) subunits on their surface, namely, IL-2/15Rß (CD122), {gamma}c (CD132), and IL-15R{alpha} [13 , 14 ]. We have previously documented that IL-15 is a neutrophil agonist [15 ]. This cytokine induces RNA synthesis, de novo intracellular protein synthesis, phagocytosis, and delays apoptosis. IL-15, unlike IL-2, was also found to induce the production of the potent neutrophil chemoattractant IL-8 and the activation of nuclear factor-{kappa}B [16 ]. In addition, it has been reported that IL-15 could not inhibit the ability of the plant lectin Viscum album agglutinin-I (VAA-I) to induce neutrophil apoptosis, and this was correlated with an inhibition of de novo protein synthesis induced by VAA-I [17 ]. In neutrophils, IL-15 is known to act by different cell-signaling pathways, as it activates Janus tyrosine kinase-2, p38 mitogen-activated protein kinase, and extracellular-regulated kinase-1/2 [18 ]. In addition to our observation that IL-15 delays human neutrophil apoptosis by preventing myeloid cell differentiation factor-1 (Mcl-1) degradation [18 ], others have found that IL-15 [as well as IL-6 and granulocyte macrophage-colony stimulating factor (GM-CSF)] down-regulates the expression of the proapoptotic Bax protein and the spontaneous activity of caspase-3 [19 ]. The ability of IL-15 to decrease the activity of other caspases is presently unknown.

In a recent study, IL-15 was found to induce the simultaneous secretion of IL-1ß and its natural inhibitors IL-1R agonist (IL-1Ra) and soluble IL-1RII by human neutrophils isolated from normal and tumor-bearing hosts [20 ]. IL-15 was also found to induce IL-ß and IL-1Ra secretion by neutrophils from healthy controls but not by neutrophils from cancer patients. However, a priming effect of IL-15 on IL-1ß production by lipopolysaccharide (LPS)-stimulated cells was noted in patients with oral cavity cancers [20 ].

The present study was conducted to better understand the mechanism by which IL-15 delays human neutrophil apoptosis. We identified the predominant protein detected in the supernatant of IL-15-induced neutrophils as IL-1Ra. Unfortunately, we found that this protein is not involved in IL-15-induced suppression of neutrophil apoptosis. In fact, unlike the supernatant from GM-CSF-induced cells, which delayed neutrophil apoptosis, the supernatant harvested from IL-15-induced neutrophils was devoid of activity. This suggests the existence of a specific set of proteins (probably intracellular) that were involved in this process. We found that IL-15 decreases the activity of not only caspase-3 but also caspase-8.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals, agonists, and antibodies
IL-15 and GM-CSF were obtained from PeproTech Inc. (Rocky Hill, NJ). LPS, cycloheximide (CHX), and the monoclonal antibodies (mAb) to human cytoskeletal vimentin (clone Vim 13.2) and vinculin (clone Vin-11-5) were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human (rh)IL-1Ra and the goat anti-human IL-1Ra polyclonal antibody were purchased from R&D Systems (Minneapolis, MN). IL-1{alpha}, IL-ß, and IL-1Ra enzyme-linked immunosorbent assay (ELISA) kits were obtained from Medicorp (Montreal, Qc, Canada). The Mcl-1 antibody (RC13 clone) was purchased from BioSource (Montreal, Qc, Canada). Fluorescein isothiocyanate (FITC)–Annexin-V and FITC–mouse anti-human CD16 mAb were purchased from BD PharMingen Canada (Mississauga, ONT). Horseradish peroxidase (HRP)-labeled rabbit anti-goat and goat anti-mouse immunoglobulin (Ig)M+IgG) secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Monoclonal anti-caspase-8 (clone 5F7) was purchased from Upstate Biotechnology (Lake Placid, NY), and the rabbit anticaspase-3 polyclonal antibody was purchased from BD PharMingen Canada.

Neutrophil isolation
Cells were isolated from venous blood of healthy volunteers by dextran sedimentation followed by centrifugation over Ficoll-Paque (Pharmacia Biotech Inc., Qc, Canada), as described previously [15 , 17 , 21 ]. Blood donations were obtained from informed and consenting individuals, according to our institutionally approved procedures. Cell viability (>98%) was monitored by Trypan blue exclusion, and the purity (>98%) was verified by cytology from cytocentrifuged preparations colored by the Hema 3 stain set (Biochemical Sciences Inc., Swedesboro, NJ).

Metabolic labeling and de novo protein synthesis assay
Cells (10x106 cells/ml in RPMI 1640 supplemented with 1% autologous serum) were metabolically labeled with 125 µCi Redivue Pro-Mix L-[35S] in vitro cell-labeling mix (Amersham BioSciences Inc., Baie d’Urfé, Qc, Canada) in the presence or absence of agonists, as indicated in the figure legends, for 22 h, as previously published [15 , 17 ]. Cell lysates were prepared, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. An equivalent of 0.5 x 106 cells was loaded per well (to compare de novo protein synthesis) [15 , 17 ]. After electrophoresis, gels were stained with Coomassie blue (to verify equivalent loading), dried, and exposed to Kodak film X OMAT-RA at –80°C for 1–3 days.

Western blot
Freshly isolated neutrophils (300 µl of a 10x106 cells/ml suspension) were incubated in RPMI 1640–HEPES–penicillin–streptomycin supplemented with 10% fetal calf serum (FCS) for 21 h at 37°C with buffer, GM-CSF (65 ng/ml), or IL-15 (250 ng/ml) [15 ]. Cells were harvested and centrifuged to collect the cells and the supernatant, to which an equivalent volume of 2x Laemmli sample buffer was added. An equivalent of 0.5 x 106 cells was loaded onto 15% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). Nonspecific sites were blocked with 2% skim milk in Tris-buffered saline (TBS)–Tween (25 mM Tris-HCl, pH 7.8, 190 mM NaCl, 0.15% Tween 20) overnight at 4°C. The goat anti-IL-1Ra polyclonal antibody (1 µg/ml) was added for 1 h at room temperature followed by washes with TBS–Tween. The membrane was then incubated with a HRP-labeled rabbit anti-goat secondary antibody at 1:10,000 in TBS–Tween + 2% nonfat dry milk for 45 min at room temperature followed by washes. The IL-1Ra protein was revealed with the enhanced chemiluminescence Western blotting detection system (Amersham Biosciences Inc.). Protein loading was verified by staining the membranes with Coomasie blue at the end of the experiment. In some experiments, the primary antibodies were the antivimentin (1:1000), anti-vinculin (1:200), or anti-Mcl-1 (1 µg/ml), as described above.

Immunoprecipitation of radiolabeled IL-1Ra
Metabolically labeled neutrophils (8x106) were centrifuged to separate the cells from the medium. Pellets were washed twice with phosphate-buffered saline (PBS) and lysed in 160 µl nondenaturant lysis buffer (10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1% deoxycholic acid, 1% Triton X-100, 0.01% SDS) for 30 min on ice. The lysates and supernatants were precleared using 20 µl protein G-Sepharose (Amersham Biosciences Inc.). After 1 h, brief centrifugation followed to remove the Sepharose beads, and the samples were incubated with 2 µg/ml goat anti-IL-1Ra with gentle agitation for 3 h at 4°C. Protein G-Sepharose (15 µl) was then added for an additional 1-h incubation. The solid matrix was collected and washed three times with PBS before suspending it in 30 µl sample buffer and heating to 100°C for 5 min. Labeled proteins were resolved by gel electrophoresis in a 15% acrylamide/bis-acrylamide gel. After electrophoresis, gels were stained with Coomassie blue (to verify equivalent loading), dried, and exposed with Kodak film X OMAT-RA at –80°C for 1–3 days.

Cytokine production
Freshly isolated human neutrophils (10x106 cells/ml in RMPI 1640 supplemented with 5% FCS) were stimulated with buffer, 1 µg/ml LPS, 100 or 250 ng/ml IL-15 for 24 h, as described previously [22 , 23 ]. After incubation, cells and supernatants were harvested and centrifuged. Then, supernatants were transferred into corresponding tubes, and pellets were washed twice. Both fractions (intracellular and extracellular) were conserved at –80°C. Cell pellets were gently sonicated just before quantification. Intracellular and extracellular concentrations of IL-1Ra, IL-1{alpha}, and IL-1ß were measured using commercially available ELISA kits, according to the manufacturer’s instructions no more than 2 weeks after harvesting. Cytokine concentrations were measured in parallel using eight blood donors.

Assessment of neutrophil apoptosis
Freshly isolated human neutrophils (10x106 cells/ml in RPMI 1640 supplemented with 10% FCS) were incubated for 24 h in the presence or absence of neutrophil agonists, as indicated under figure legends. Apoptosis was evaluated by the three following methods as previously published [15 , 18 ].

For assessment of apoptosis by FITC–Annexin-V staining, cells were washed twice with cold PBS and then resuspended in 100 µL 1x binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). The cell suspension was incubated for 15 min at room temperature and light-protected, after the addition of 2 µL FITC–Annexin-V (Medicorp). A volume of 400 µL 1x binding buffer was added to each tube before fluorescein-activated cell sorter (FACS) analysis. Flow cytometric analysis (10,000 events) was performed using a FACScan (Becton Dickinson, San Jose, CA).

CD16 expression is known to be down-regulated in apoptotic neutrophils, and the level of CD16 expression was measured to evaluate the apoptotic rate according to previous procedures [24 ]. After the incubation, cells were washed, suspended at a concentration of 1.5 x 106 cells/ml, and preincubated for 30 min (4°C, light-protected) with 20% autologous serum to prevent nonspecific binding via Fc receptors. Cells were then washed and incubated with 2 µl FITC–mouse anti-human CD16 mAb (BD PharMingen Canada) for 30 min at 4°C in the dark before FACS analysis.

For assessment of apoptosis by cytology, cytocentrifuged preparations of neutrophils (with ~200 µl) were performed using a Cyto-tek® centrifuge (Miles Scientific, Naperville, IL) and processed essentially as previously documented [15 , 21 ]. Cells were examined by light microscopy at 400x final magnification, and apoptotic neutrophils were defined as cells containing one or more characteristic, darkly stained pyknotic nuclei. An ocular containing a 10 x 10 square grill was used to count at least five different fields (>100 cells) for the assessment of apoptotic cells. Results were expressed as percentage of apoptotic cells.

Statistical analysis
Statistical analysis was performed with SigmaStat for Windows version 2.0 with a one-way ANOVA. Statistical significance was established at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of IL-15-induced de novo protein synthesis released in the supernatant
The role of protein synthesis during neutrophil apoptosis is still obscure. Knowing that IL-15 delays human neutrophil apoptosis and induces de novo protein synthesis of several unidentified, intracellular polypeptides [15 ], we decided to study the expression of newly synthesized polypeptides in the external milieu from IL-15-induced neutrophils. Curiously, only a few proteins were detected by SDS-PAGE (Fig. 1A ). It is interesting that one predominant protein of ~23 kDa was detected, and it comigrated with the major protein detected in GM-CSF-induced cells but at a weaker intensity (Fig. 1A) . Of note, although the corresponding Coomassie-stained gel reveals the presence of equal amount of proteins loaded (left), the effect of CHX can be clearly observed, where very few radioactive polypeptides were detected (right).



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  		Figure 1. IL-15 induces de novo protein synthesis of a neutrophil protein identified as IL-1Ra. Human neutrophils were metabolically labeled (A and C) or not (panel B) as described in Materials and Methods and incubated for 24 h in the presence or absence of stimuli. Extracellular fractions (supernatants) were prepared as described in Materials and Methods and then run on 15% SDS-PAGE. Ctrl, Unstimulated cells; GM, 65 ng/ml GM-CSF; CHX, 10 µg/ml CHX; and IL-15, 250 ng/ml IL-15. Note the very weak signal obtained after CHX treatment on the film (35S; A) when compared with the total protein loaded in the corresponding lane (Coomassie blue). In other experiments, supernatants were harvested, prepared, and immunoblotting (B), or immunoprecipitation (IP; C) was performed with an anti-human IL-1Ra antibody as described in Materials and Methods. Results are from one representative experiment of at least three. mw, Molecular weight.

 
Identification of the ~23-kDa protein as IL-1Ra
It has been previously reported that GM-CSF, LPS, and tumor necrosis factor {alpha} (TNF-{alpha}) induce de novo neutrophil protein synthesis of IL-1Ra released into the milieu [25 ]. As the molecular mass of IL-1Ra is ~23 kDa, we suspected that the protein synthesized in response to IL-15 was IL-1Ra. We performed Western blots with the collected supernatants from unstimulated as well as from GM-CSF- or IL-15-induced neutrophils, using a commercially available, neutralizing, anti-human, IL-1Ra antibody known to be suitable for Western blot and ELISA experiments. As illustrated in Figure 1B , IL-1Ra expression was markedly increased in the supernatants from GM-CSF- or IL-15-induced neutrophil cells when compared with control cells. However, the intensity of the signal was weaker with IL-15 as compared with GM-CSF, corresponding with the intensity of the bands observed in Figure 1A . Our results strongly suggested that this protein was in fact IL-1Ra. To further demonstrate this, we then decided to investigate de novo synthesis of IL-1Ra. To do so, cells were metabolically labeled and stimulated, and supernatants were collected after 24 h. Immunoprecipitation was performed using the same anti-IL-1Ra antibody. As illustrated in Figure 1C , the ~23 kDa detected from the supernatant was effectively IL-1Ra. Again, the intensity was weaker with IL-15 when compared with GM-CSF. Collectively, these data demonstrated that the major protein that is de novo-synthesized and released into the supernatant of IL-15-induced neutrophils is IL-1Ra. We also performed immunoprecipitation with the intracellular fraction following metabolic labeling or not and found that the levels of IL-1Ra remained similar regardless of whether cells were incubated with buffer, GM-CSF, or IL-15 (data not shown).

Quantification of IL-1Ra, IL-1{alpha}, and IL-1ß in the intracellular and extracellular fractions from IL-15-induced neutrophils
Because of the importance of IL-1Ra in the general physiology of neutrophils in response to IL-1{alpha}/ß [26 ], we determined the concentration of this natural inhibitor found in the intracellular and extracellular fractions of IL-15-induced neutrophils. As illustrated in Figure 2 , the levels of IL-1Ra found in the intracellular fractions were similar, whether cells were stimulated with GM-CSF, IL-15, or even with LPS. It is interesting that the levels of IL-1Ra were significantly increased in the extracellular fractions from cells stimulated with GM-CSF or LPS and IL-15 at 250 but not 100 ng/ml. When plotting the results from both fractions, it became clear that the agonists induced the release of IL-1Ra into the external milieu (Fig. 2) .



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  		Figure 2. Quantification of IL-1Ra in the intracellular and extracellular fractions from IL-15-induced neutrophils. Neutrophils were incubated for 24 h in the presence of buffer (C), LPS, GM-CSF (GM), or IL-15 (100 or 250 ng/ml), and intra- and extracellular fractions were prepared for quantification of IL-1Ra, IL-1{alpha}, and IL-1ß by ELISA, as described in Materials and Methods. Results are mean ± SEM (from eight different blood donors). *, P < 0.05, by ANOVA.

 
In parallel with the quantification of IL-1Ra, the concentrations of IL-1{alpha} and IL-1ß were also quantified. As illustrated in Figure 2 , IL-15, unlike GM-CSF or LPS, does not increase the production of IL-1{alpha} significantly in both fractions. It is interesting that GM-CSF was found to significantly increase IL-1{alpha} levels in the intracellular but not the extracellular fractions. LPS markedly increased the levels of IL-1{alpha} in both fractions, suggesting that the cells release a higher amount of this cytokine over time. IL-15 did not significantly increase the levels of IL-1ß in both fractions, in contrast to GM-CSF and LPS (Fig. 2) .

The production of neutrophil IL-1Ra by IL-15 is not linked with its ability to delay apoptosis
The direct role of IL-1Ra on neutrophil apoptosis has never been reported. As IL-1Ra is the major product found in the supernatant of IL-15-induced neutrophils under experimental conditions in which IL-15 delays apoptosis, we were interested in studying its potential role during this biological process. Neutrophil apoptosis was evaluated by flow cytometry (FITC–Annexin-V binding and monitoring the CD16 expression) and by cytology. As illustrated in Figure 3 , addition of IL-1Ra in the culture did not alter the neutrophil apoptotic rate, even at a concentration 250 times greater than that found in the extracellular milieu [500 ng/ml vs. 2 ng/ml (2000 pg/ml in Fig. 2 ), respectively]. This was not a result of cell unresponsiveness, as GM-CSF and IL-15, as expected, were found to delay neutrophil apoptosis [15 ].



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  		Figure 3. IL-1Ra does not modulate the apoptotic rate of human neutrophils. Freshly isolated human neutrophils (10x106 cells/ml in RPMI 1640 supplemented with 10% FCS) were incubated for 24 h in the presence of buffer (Ctrl), GM-CSF (GM), IL-15, or increasing concentrations of IL-1Ra (10, 100, or 500 ng/ml), and apoptosis was evaluated by flow cytometry (FITC–Annexin-V and FITC–CD16) and by cytology as described in Materials and Methods. Results are mean ± SEM (n>=3). *, P < 0.05, by ANOVA.

 
IL-15-treated, neutrophil-conditioned medium does not possess biological activity for delaying neutrophil apoptosis
Because of our interest in understanding the mechanism involved in IL-15-induced suppression of neutrophil apoptosis and knowing that the major product IL-1Ra is not involved, we next investigated the role of the overall, collected supernatant on neutrophil apoptosis. As illustrated in Figure 4 , when cells were incubated in the presence of 5, 10, 20, 50, or even 100% of IL-15-treated, neutrophil-conditioned medium, the apoptotic rate remained similar to the basal apoptotic rate. However, the supernatant collected from GM-CSF-induced cells delayed apoptosis. This suggested that IL-15 does not modulate human neutrophil apoptosis via the release of a factor in the supernatant.



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  		Figure 4. IL-15 does not modulate neutrophil apoptosis via the release of molecule(s) in the external milieu. Freshly isolated human neutrophils (10x106 cells/ml in RPMI 1640 supplemented with 5, 10, 20, 50, or 100% of IL-15- or GM-CSF-conditioned medium) were incubated for 24 h, and apoptosis was evaluated by cytology. Results are means ± SEM (n=4). *, P < 0.05, by ANOVA.

 
IL-15 delays human neutrophil apoptosis by a protein synthesis-dependent mechanism
Although we have previously suggested that IL-15 delays human neutrophil apoptosis via de novo protein synthesis of intracellular polypeptides [15 ], this has not been directly investigated. As IL-15-treated, neutrophil-conditioned medium is devoid of activity for suppression of apoptosis (this report) and as we recently found that IL-15 prevented the loss of expression of the intracellular, antiapoptotic Mcl-1 protein, we investigated the role of protein synthesis dependency by treating cells in the presence of the potent protein synthesis inhibitor CHX. As illustrated in Figure 5A , IL-15 did not delay neutrophil apoptosis when CHX was added to the culture. As expected, the antiapoptotic effect of GM-CSF was also inhibited by CHX. To further demonstrate the importance of intracellular protein synthesis in IL-15-induced suppression of human neutrophil apoptosis, we monitored the expression of Mcl-1 with or without CHX in the culture. As illustrated in Figure 5B , the addition of CHX decreased the level of expression of Mcl-1. This finding supports the suggested de novo synthesis of this antiapoptotic factor following reduced availability of Mcl-1, which was demonstrated using antisense oligonucleotides [27 ].



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  		Figure 5. Involvement of de novo protein synthesis in IL-15-induced suppression of neutrophil apoptosis. (A) Neutrophils (10x106 cells/ml) were treated with buffer (Ctrl), 65 ng/ml GM-CSF, or 250 ng/ml IL-15 in the presence (+) or absence (–) of 10 µg/ml CHX, and apoptosis was assessed by cytology as described in Materials and Methods. Results are mean ± SEM (n=3). *, P < 0.05, by ANOVA. (B) Expression of the antiapoptotic Mcl-1 protein was studied by Western blot as described in Materials and Methods. Results are representative of five different experiments. GM, GM-CSF.

 
Recently, based on enzymatic activity determined spectrophotometrically, the spontaneous activity of caspase-3 was found to be decreased by different cytokines including GM-CSF and IL-15 [19 ]. In the present study, we investigated the ability of IL-15 to decrease the activity of not only caspase-3 but also caspase-8, as measured by decreased expression of procaspase-3 and procaspase-8 proteins. As illustrated in Figure 6A and 6B , we confirm, using a different approach, that IL-15 effectively decreases caspase-3 activity (as well as that of G-CSF and GM-CSF) and demonstrate that IL-15 (and GM-CSF) decreases caspase-8 activity. Addition of CHX to the culture indicates that G-CSF and GM-CSF but not IL-15 maintain procaspase-8 stability when compared with control cells, whereas the stability of procaspase-3 was less affected by these cytokines. As both caspases were found to cleave the cytoskeletal protein vimentin [28 ] and that vimentin is known to be cleaved by a caspase-dependent mechanism during neutrophil apoptosis [21 ], we then decided to examine whether IL-15 prevents or decreases vimentin cleavage. As shown in Figure 6C , IL-15 (and GM-CSF) was found to prevent vimentin degradation, and as for procaspases, we cannot state that de novo protein synthesis of vimentin occurs, as the level of expression of vimentin in fresh cells was higher (data not shown). The observed differences were not a result of protein loading, as the expression of another cytoskeletal protein, vinculin, was consistent in all conditions. This is in agreement with our previous results, indicating that vinculin is not cleaved in apoptotic human neutrophils [21 ].



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  		Figure 6. IL-15 inhibits the activity of caspase-3 and caspase-8, resulting in a decrease ability to cleave vimentin. Neutrophils (10x106 cells/ml) were treated (as described in Fig. 5 ) in the presence (+) or absence (–) of 10 µg/ml CHX, and the protein expression of procaspase-8 and procaspase-3 (A) or vimentin and vinculin (C) was studied by Western blot as described in Materials and Methods. Results are from one representative experiment out of three. (B) The level of expression of procaspase-8 was quantified by densitometry (n=4) using Fluor-S MultiImager (Bio-Rad, Hercules, CA) and the Multi-Analyst version 1.1 program (Bio-Rad). Note that the two procaspase-8 bands were not always separated as in A. We have considered this for the densitometry analysis by cumulating the density of each band, as they were always altered similarly. Ctrl and C, Control; GM, GM-CSF.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
IL-15 is a proinflammatory cytokine known as a general inhibitor of apoptosis. Despite this, its mode of action is still unclear and needs to be further elucidated. Under normal conditions, IL-15 production is under strict control [29 30 31 ], suggesting that unchecked release of this cytokine is not suitable for an organism. In this regard, elevated concentrations of IL-15 have been observed in several inflammatory disorders, including active sarcoidosis, chronic hepatitis C, active ulcerative colitis, and rheumatoid arthritis [8 9 10 11 12 ]. For example, up to 1200 pg/ml IL-15 was detected in the synovial fluid of rheumatoid arthritis patients [12 ].

Several inhibitors of apoptosis are thought to act via de novo protein synthesis, as addition of CHX to the culture medium reverses this effect [32 33 34 ]. Addition of this drug was reported to block TNF-{alpha}-mediated, antiapoptotic signaling in neutrophils [35 ]. Also, dexamethasone-induced suppression of neutrophil apoptosis was found to require continuous stimulation of new protein synthesis, based on the addition of CHX or actinomycin D [32 , 36 ]. Prevention of apoptosis by GM-CSF was associated with induction of RNA and protein synthesis in neutrophils, as addition of actinomycin D or CHX reversed the effect [37 ]. Even induction of MM46 mouse mammary carcinoma cell apoptosis by calprotectin was markedly inhibited by actinomycin D or CHX [38 ], suggesting that not only suppressors but also activators of apoptosis require de novo protein synthesis.

Knowing that IL-15 induces de novo intracellular protein synthesis in neutrophils [15 ] and because of the importance in understanding how IL-15 delays apoptosis, we have hypothesized that this cytokine could act via the production and release of survival factor(s) in the milieu. A major, newly synthesized polypeptide of ~23 kDa was detected in the supernatant of IL-15-induced neutrophils and identified as IL-1Ra. Despite the fact that IL-1Ra is the only as-yet identified protein known to be de novo-synthesized and released into the external milieu in response to GM-CSF, LPS, TNF-{alpha} [25 ], or IL-15 (this report), its direct role in neutrophil apoptosis has never been studied. We found that incubation of freshly isolated human neutrophils with rhIL-1Ra did not alter the apoptotic rate. It is important to mention that IL-15 did not significantly increase IL-1Ra levels at a concentration of 100 ng/ml, a concentration known to significantly delay neutrophil apoptosis (ref. [15 ] and this report). This indicates that the mode of action of IL-15 for delaying neutrophil apoptosis is via an IL-1Ra-independent mechanism. Moreover, we can conclude that IL-15 delays this response by IL-1{alpha}- and IL-ß-independent mechanisms, as IL-15 does not increase the production of intracellular and extracellular IL-1{alpha} and IL-ß. Our results contrast those reported in a unique study demonstrating that IL-15 can increase IL-1ß secretion by human neutrophils isolated from normal and tumor-bearing patients [20 ]. The discrepancies between these two studies may be related to the different experimental conditions used. Based on this, we believe that the major difference observed concerning the IL-1ß production in response to IL-15 resides in the fact that Jablonska et al. [20 ] added 10% autologous serum to the culture, and in contrast to IL-15, they did not verify the level of IL-1ß present in the different sera [20 ]. In one study, GM-CSF and LPS were reported to delay neutrophil apoptosis via an IL-1ß-dependent mechanism, as addition of an antisense oligonucleotide for IL-1ß, a blocking anti-IL-1ß antibody, or a preincubation with IL-1Ra was found to reverse the suppression of apoptosis [39 ]. In contrast to this study, preincubation with increasing concentrations of IL-1Ra failed to reverse the effect of IL-15 (data not shown), agreeing with the inability of IL-15 to increase IL-1ß production. Results from our experiments conducted with IL-15-treated, neutrophil-conditioned medium indicate that in contrast to GM-CSF, IL-15 does not delay apoptosis by the release of survival factors into the milieu. This presents the possibility that intracellular proteins are involved. In this respect, we recently demonstrated that IL-15 increases the expression of the antiapoptotic Mcl-1 protein, a member of the Bcl-2 family of proteins [18 ]. As we did not study de novo synthesis of Mcl-1 in these experiments, it was not clear whether the synthesis of Mcl-1 was increased or rather if IL-15 prevents its loss by an unknown mechanism, as speculated by others in different systems [7 , 40 , 41 ]. However, as Mcl-1 possesses a very short half-life and is subject to rapid turnover [40 ], induction of its synthesis by IL-15 is plausible; this possibility was confirmed in the present study. GM-CSF was recently found to act via the regulation of Mcl-1 [41 ], indicating that unlike IL-15, the mode of action of GM-CSF might involve intra- and extracellular proteins, which inhibit neutrophil apoptosis. It is interesting that we have recently demonstrated that IL-15, in contrast to GM-CSF, does not activate tyrosine phosphorylation of signal transducer and activator of transcription-5a/b [18 ], attesting to their different modes of action. The possibility that GM-CSF-treated neutrophil-conditioned medium delays apoptosis via free, available, unbound GM-CSF is minimized by the fact that in contrast to the total fluid diluted from 1/1 (100%) to 1/20 (5%), which inhibits apoptosis, GM-CSF did not suppress apoptosis when diluted to a concentration that corresponds to 20% of the fluid (equivalent to 13 ng/ml; data not shown).

In this study, we demonstrated for the first time that IL-15 delays human neutrophil apoptosis by inhibiting caspase-8 activity. This, in addition to inhibition of caspase-3 activity, results in a decrease of vimentin cleavage, an intracellular event. In addition to Mcl-1, we found that vimentin (but not vinculin) expression was decreased by CHX treatment. This suggests that de novo synthesis of vimentin is potentially related to the ability of IL-15 to delay neutrophil apoptosis and that not all proteins are de novo-synthesized in response to IL-15. However, the level of expression of vimentin was never greater than the one observed in fresh cells, suggesting that only minimal protein synthesis could occur. It is interesting that it was recently found that vimentin is de novo-synthesized and released into the milieu by activated human macrophages [42 ]. This remains to be investigated in human neutrophils. However, if this were true, the concentration of vimentin released would be insufficient (or inactive), as we were unable to delay neutrophil apoptosis when cells were incubated in the presence of IL-15-treated, neutrophil-conditioned medium. The importance of cytoskeletal proteins in neutrophil apoptosis was recently demonstrated in gelsolin knockout mice [43 ]. Neutrophils isolated from these animals were found to be more refractory to induction of apoptosis by TNF-{alpha} and CHX when compared with neutrophils isolated from wild-type animals [43 ].

As previously mentioned, identification of proteins that are de novo-synthesized from neutrophils has not been the object of numerous studies. In fact, except for IL-1Ra, only actin [15 , 44 ] and fibronectin [2 ] have been identified as being synthesized in response to an agent that suppresses human neutrophil apoptosis. Numerous studies indicate that agent-induced neutrophils can produce/release different cytokines that can modulate apoptotic rate [1 , 45 46 47 48 ]; however, none of these studies have demonstrated de novo synthesis of a particular cytokine following metabolic cell labeling.

Although IL-15 is frequently presented as a proinflammatory cytokine, data from the present study suggest that IL-15 might also induce an anti-inflammatory loop by increasing IL-1Ra production by neutrophils.


   ACKNOWLEDGEMENTS
 
This study was supported by Canadian Institutes of Health Research (MOP-89534) and Fonds de la Recherche en Santé du Québec (FRSQ). A. B. holds an M.Sc. Studentship Award from FRSA, C. R. holds a Ph.D. Bourse d’études supérieures du Canada award, and D. G. is a Scholar from FRSQ. We thank Mary Gregory for reading this manuscript.

Received November 24, 2003; revised January 6, 2004; accepted January 14, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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