Published online before print May 3, 2004
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chain
INRS-Institut Armand-Frappier, Université du Québec, Pointe-Claire, Canada
1Correspondence: INRS-Institut Armand-Frappier, 245 boul. Hymus, Pointe-Claire (PQ), Canada, H9R 1G6. E-mail: denis.girard{at}inrs-iaf.uquebec.ca
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, IL-2/15Rß (CD122), and 
c (CD132) subunits, the role of each receptor component has not been investigated in IL-15-induced human neutrophil responses. In the present study, fluorescein-activated cell sorter analysis revealed that the ability of IL-15 to enhance neutrophil phagocytosis is not a result of increased expression of IL-15R, CD122, or CD132 on the neutrophil cell surface. Pretreatment of neutrophils with specific antibodies to IL-15R, CD122, or CD132 was found to inhibit phagocytosis of opsonized-sheep red blood cells by nearly 40%, 21%, and 27%, respectively. As expected, pretreatment of neutrophils with anti-IL-2R (CD25) had no effect. Pretreatment of cells with the Syk inhibitor piceatannol was found to significantly inhibit the ability of IL-15 to enhance phagocytosis. In addition, IL-15 was found to induce tyrosine phosphorylation of Syk that was largely inhibited by pretreating cells with piceatannol. Moreover, we found that Syk kinase is physically associated with IL-15R. We conclude that IL-15R enhances neutrophil phagocytosis by a Syk-dependent mechanism and that the IL-15R chain plays a key role in mediating this response, at least by interacting with Syk kinase.
Key Words: inflammation • cytokine receptor • tyrosine kinases • CD122 • CD132
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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c (CD132), IL-2/IL-15Rß (CD122), and the more recently identified IL-15R [1
]. The c chain is shared by other receptors such as IL-2R, IL-4R, IL-7R, IL-9R, and IL-21 [2
3
4
5
]. In addition to the c chain, the IL-2R and IL-15R share the IL-2Rß subunit. This largely explains why these two cytokines possess some redundant biological actions [6
, 7
].
IL-15 is a human neutrophil agonist known to induce RNA and de novo protein synthesis, phagocytosis, and to delay apoptosis [8
]. Neutrophils are known to express a high-affinity IL-15R (composed of CD122, CD132, and IL-15R subunits) and an IL-2R of intermediate affinity, which is lacking one component, namely, the IL-2R (CD25) [9
10
11
]. 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-
B [12
]. In addition, we have reported that the plant lectin Viscum album agglutinin-I, which is a potent inhibitor of protein synthesis, did not alter the ability of IL-15 to enhance phagocytosis, suggesting that this biological activity of IL-15 is independent of the production of proteins [13
]. Inversely, this lectin was found to completely inhibit the ability of IL-15 to delay neutrophil apoptosis [13
]. Conversely, IL-15 was found to induce the production of IL-1R antagonist [14
]. Although it was previously demonstrated that IL-15 is a human neutrophil agonist, its mode of action remains to be elucidated. We have recently demonstrated that the antiapoptotic Mcl-1 protein is involved in IL-15-induced human neutrophil apoptosis and that IL-15 uses different cell-signaling pathways, as this cytokine activates Janus kinase-2, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase 1/2 (ERK1/2) but not signal transducer and activator of transcription-5a/b [15
]. The mechanism involved in IL-15-enhanced phagocytosis has not been investigated. It is of great importance to elucidate the mechanism of induction of phagocytosis by IL-15, as this cytokine, like IL-2, possesses interesting, potential therapeutic properties for humans, especially in advanced cancers [16
17
18
]. The use of IL-15 in cancer therapy offers a potential, beneficial effect, as it can enhance phagocytosis [8
], an important response that could, at least, partly prevent recurrent infections following cytokine treatment.
As IL-15, but not IL-2, is known to enhance human neutrophil phagocytosis [8
, 19
] and as neutrophils express the specific IL-15R and the two chains common to IL-2R and IL-15R (CD122 and CD132) but not the IL-2R chain (CD25), we hypothesized that the IL-15R plays a key role in IL-15-enhanced neutrophil phagocytosis. In this study, we demonstrate that the ability of IL-15 to enhance phagocytosis is not related to an increase of cell-surface expression of IL-15R, CD122, or CD132. Inhibition experiments with specific antibodies reveal that IL-15R is an important component involved in IL-15-enhanced neutrophil phagocytosis and that IL-15 activates Syk when inducing its biological effect. Moreover, we found that Syk is associated with IL-15R.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(CD25, IgG1), and rat anti-human c (CD132, IgG2b) were purchased from PharMingen (Mississauga, Ontario, Canada). The anti-IL-15R (clone M161, IgG1) was kindly provided by Genmab (Utrecht, Netherlands), and for immunoprecipitation experiments, the goat anti-human IL-15R antibody was purchased from R&D Systems (Minneapolis, MN). Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG and F(ab')2 fragment-specific and FITC-conjugated affiniPure F(ab')2 donkey anti-rat IgG (H+L) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Tris-HCL, NaCl, Triton X-100, and sodium dodecyl sulfate (SDS) were purchased from Fisher Scientific (Whitby, Ontario, Canada). Orthovanadate, phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, and pepstatin were purchased from Sigma Chemical Co., and trypsin inhibitor was from Calbiochem (Pasadena, CA).
Neutrophil isolation
Cells were isolated from the venous blood of healthy volunteers by dextran sedimentation followed by centrifugation over Ficoll-Hypaque (Pharmacia Biotech, Quebec, Canada), as described previously [8
9
10
]. Blood donations were obtained from informed and consenting individuals according to 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, Swedesboro, NJ).
Cell-surface expression of IL-15R subunits
Following the incubation period, cells were suspended at 10 x 106 cells/ml, washed, and preincubated 30 min (4°C, light-protected) with 20% autologous serum to prevent nonspecific binding via Fc receptors. Cells (1x106 cells/ml) were then washed and incubated with the different antibodies (1 µg/mL) for 1 h (4°C, light-protected). After two additional washes, cells were incubated with FITC-conjugated secondary antibody (1:100). Cells were then washed and fixed with 0.5% paraformaldehyde. Flow cytometric analysis (10,000 events) was performed using a FACScan (Becton Dickinson, San Jose, CA).
Phagocytosis of sheep erythrocytes
Sheep red blood cells (SRBC) were opsonized with a final 1/200 dilution of rabbit IgG anti-SRBC antibody (Sigma Chemical Co.) followed by incubation for 45 min at 37°C as described previously [8
]. Prior to the phagocytosis assay, neutrophils were incubated 30 min with 1 µg/ml anti-CD25, -CD22, -CD132, or -IL-15R to evaluate a potential inhibitory effect. This was based on previous experiments performed with increasing concentrations of antibodies (0–10 µg/ml) incubated simultaneously or before addition of IL-15 (data not shown). Neutrophils (10x106 cells/ml in RPMI 1640), pretreated 30 min with buffer or IL-15 (250 ng/ml), were incubated with 50 x 106 opsonized SRBC for 45 min as above. The samples were centrifuged 200 g at 4°C for 10 min. Supernatants were discarded, and to eliminate noningested SRBC, osmotic shock was performed on the pellets by treating them with 300 µl H2O for 20 s followed immediately by the addition of 4.5 ml ice-cold phosphate-buffered saline (PBS; 1x). The samples were washed twice with ice-cold PBS, and the final pellets were suspended to a final concentration of 10 x 106 cells/ml. Duplicate cytocentrifuged preparations (Cyto-tek® centrifuge, Miles Scientific, Naperville, IN) were prepared in aliquots of 
200 µl and processed essentially as previously documented [8
]. Phagocytosis was measured as percentage of neutrophils ingesting at least one opsonized SRBC. IL-15 could be considered as a positive control in the present set of experiments, as we have previously reported that this cytokine can enhance neutrophil phagocytosis in a concentration-dependent manner [8
]. In some experiments, cells were pretreated 30 min (37°C) with increasing concentrations of piceatannol (10–100 µM).
Western blot
Neutrophils (40x106 cells/mL RPMI-Hepes-P/S) were stimulated from 15 s to 30 min with the indicated agonists. Whole cell lysates were prepared as described previously [8
]. Proteins (250x105 cells/well) were separated on 7.5% SDS-polyacrylamide gels and transferred to nitrocellulose. Membranes were blocked for 1 h at room temperature in Tris-buffered saline (TBS)–Tween + 5% nonfat dry milk (Carnation, Don Mills, Ontario, Canada). After washing, the antiphosphospecific Syk antibody (#2711, New England Biolabs, Beverly, MA) or the antibody directed against the nonphosphorylated form of Syk (SC-1240, clone 4D10, Santa Cruz Biotechnology, Santa Cruz, CA) was added at a final dilution of 1:1000 or 1:400, respectively, in TBS–Tween + 5% bovine serum albumin (BSA) or TBS–Tween + 5% nonfat dry milk. The membranes were kept overnight at 4°C. Membranes were then washed with TBS–Tween and incubated for 1 h at room temperature with a goat anti-rabbit horseradish peroxidase (HRP) secondary antibody (Jackson ImmunoResearch Laboratories) at 1:20,000 in TBS–Tween + 5% BSA or a goat anti-mouse HRP secondary antibody (Jackson ImmunoResearch Laboratories) at 1:20,000 in TBS–Tween + 5% nonfat dry milk followed by washes. The Syk protein expression was revealed with an enhanced chemiluminescence Western blotting detection system as previously published and quantified using a Fluor-S multi-imager (Bio-Rad, Hercules, CA) and the Multi-Analyst version 1.1 program (Bio-Rad) [15
].
Immunoprecipitation
Neutrophils (40x106 cells/ml) were untreated or stimulated with IL-15 for the indicated time, centrifuged, and lysed in 400 µl nondenaturing lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Triton X-100, 0.01% SDS, 1 mM orthovanadate, 1 mM PMSF, 10 µg/ml trypsin inhibitor, and 10 µg/ml aprotinin, leupeptin, and pepstatin) for 1 h on ice and sonicated three times. The lysates and supernatants were precleared using 40 µl protein G-Sepharose (Amersham Biosciences, Little Chalfont, UK). After 1 h, brief centrifugation followed to remove the Sepharose beads, and the samples were incubated with 2 µg/ml goat anti-human IL-15R at 4°C with gentle agitation overnight at 4°C. Protein G-Sepharose (40 µl) was then added for a further 3-h incubation. The solid matrix was collected and washed three times with lysis buffer before suspending it in 35 µl sample buffer and heating to 100°C for 5 min. Immunoprecipitates were electrophoresed on a 7.5% SDS-polyacrylamide gel, transferred on a nitrocellulose membrane, incubated with the anti-Syk antibody followed by two washes with TBS–Tween, and then incubated with the goat anti-mouse HRP secondary antibody, according to protocols described previously.
Statistical analysis
Statistical analysis was performed with SigmaStat for Windows Version 2.0 with a one-way ANOVA or Student’s t-test. Statistical significance was established at P < 0.05.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, CD122, or CD132, CD122, and CD132 on their surface, we decided to investigate the potential increase in expression of one or more of these IL-15R components. As illustrated in Figure 1
, treatment with IL-15 for 30 min (time-point corresponding to the phagocytosis assay) or 24 h did not increase the expression of any IL-15R subunit. The diminution of expression of CD122 and IL-15R subunits after 30 min of treatment corresponds with the internalization of IL-15R following IL-15 binding [20
]. Of note, we have performed time-course experiments and have never observed a significant modulation of any chain after 15 min, 30 min, 2 h, 4 h, 6 h, and 24 h (data not shown). This indicates that IL-15 does not increase the ability of neutrophils to stimulate phagocytosis by up-regulating IL-15R components.
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Figure 1. IL-15 does not modulate the expression of IL-2/15Rß, c, or IL-15R on human neutrophil cell surface. Freshly isolated human neutrophils were incubated for 30 min or 24 h in the presence or absence (Ctrl) of 250 ng/ml IL-15 or 1 µg/ml lipopolysaccharide, and cell-surface expression of IL-2/15Rß, c, and IL-15R was measured by flow cytometry as described in Materials and Methods. Results are means ± SEM (n=4). Inset, Representative data plotted in the bar graph. MFI, Mean fluorescence intensity.
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, CD122, or CD132 was found to inhibit phagocytosis of opsonized SRBC by nearly 40%, 21%, and 27%, respectively. This indicates that IL-15R is highly involved in IL-15-induced biological activity. Preincubation with an anti-CD25 antibody did not block the ability of IL-15 to enhance phagocytosis, consistent with the fact that human neutrophils do not express the IL-2R chain on their cell surface [8
, 11
]. In addition, we have added a mixture of anti-CD22 + anti-CD132 + anti-IL-15R or another mixture consisting of anti-CD122 + anti-CD132 + anti-CD25 (all antibodies at 1 µg/ml) in two separate experiments. The basal level of phagocytosis was 23.6 ± 0.8 (mean±SEM), and IL-15 enhances this to 39 ± 0.5. When the first mixture was added, we observed a level of phagocytosis of 28.6 ± 1.2 and of 32.8 ± 1.5 for the second mixture. When neutrophils were pretreated with a mixture one or two and then incubated with IL-15, the values were 30.5 ± 1.1 and 38.5 ± 2.5.
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Figure 2. Role of the IL-15R components in IL-15-enhanced neutrophil phagocytosis. Prior to the phagocytosis assay, neutrophils were incubated in the presence (+) or absence (–) of 1 µg/ml anti-CD25, -CD22, -CD132, or -IL-15R and were then stimulated with (+) or without (–) 250 ng/ml IL-15. Phagocytosis of opsonized SRBC was evaluated as described in Materials and Methods. Results are means ± SEM (n 4). *, P < 0.05, versus control by Student’s t-test.
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Figure 3. Inhibition of IL-15-enhanced neutrophil phagocytosis by piceatannol. Prior to the phagocytosis assay, neutrophils were incubated in the presence of piceatannol at the indicated concentrations and were then stimulated with buffer (Ctrl) or 250 ng/ml IL-15. Phagocytosis of opsonized SRBC was evaluated as described in Materials and Methods. (A) Two separate concentration-dependent experiments (exp #1 and exp #2) were performed. Arrows indicate that the best concentration of picetannol to inhibit IL-15 effect is 10 µM. (B) Results are means ± SEM (n4). *, P< 0.05, versus control (Ctrl) by ANOVA.
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Figure 4. IL-15 induces Syk phosphorylation. Neutrophils were stimulated with 250 ng/ml IL-15 for the indicated periods of time. (A) The membrane was probed with an antiphosphospecific Syk (P-Syk) antibody (upper panel) as described in Materials and Methods, stripped, and probed with an anti-Syk (Syk) antibody indicating equivalent loading. Results are from one representative experiment out of four. mw, Molecular weight.
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Figure 5. Effect of piceatannol on IL-15-induced Syk phosphorylation. Cells were pretreated with the Syk inhibitor piceatannol (10 µM; right panels), stimulated with IL-15, and Western blots were performed as in Figure 4
. (Upper panels) Phosphorylated Syk (P-Syk). (Lower panels) Unphosphorylated Syk (Syk). Results are from one representative experiment out of four.
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in human neutrophils subunit could induce cell signaling through association with Syk in human B cells [25
], we hypothesized that Syk is associated with IL-15R in human neutrophils. As illustrated in Figure 6A
, Syk was detected following immunoprecipitation with the anti-IL-15R antibody. Furthermore, the association was increased following activation of cells by IL-15, suggesting that even if Syk is constitutively activated or nonspecifically activated (as a result of cell-isolation procedures), its level of activation is further increased by IL-15 as well as is its association with IL-15R. Immunoprecipitation was also done with the resting supernatant fraction (S in Fig. 6A
) as a technical control indicating that the level of Syk remaining in this fraction is equivalent whether or not cells were stimulated with IL-15. In addition, Syk was found to be phosphorylated when associated with IL-15R (Fig. 6B)
. Although the signal was not very strong, we can possibly attribute this to rapid dephosphorylation.
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Figure 6. Association of Syk with IL-15R. After isolation, neutrophils were stimulated with buffer (Ctrl) or 250 ng/ml IL-15 for 5 min. Lysates were immunoprecipitated (IP; +) or not (–) with anti-IL-15R, and Syk kinase was detected using the anti-Syk antibody as described in Materials and Methods. S, The resting supernatant of the corresponding fraction obtained after the detergent solubilized neutrophils to indicate that the different level of Syk observed in lanes 2 and 5 is not a result of a difference in loading. Results are from one representative experiment out of three. The densitometry analysis from the three experiments indicated an intensity of 2.7 ± 0.02, 1.8 ± 0.05, 4.2 ± 0.4, 2 ± 0.2, and 5.4 ± 0.4 for lanes 1–5, respectively. (B) IL-15R was immunoprecipitated and then immunoblotted with antiphospho-Syk antibody (P-Syk). The same membrane was stripped and immunoblotted with the anti-Syk antibody (Syk). Results have been confirmed in one other experiment.pic10, piceatannol 10 µm.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Despite the fact that IL-15 is a proinflammatory cytokine and that neutrophils could be activated by it, there is little information concerning its mode of action on these cells. We have recently demonstrated that IL-15 delays neutrophil apoptosis by preventing the loss of expression of the antiapoptotic Mcl-1 protein and via several kinases including Jak-2, p38, and ERK1/2 [15
]. Prior to the present study, no data were available concerning the participation of the different IL-15R subunits in IL-15-induced neutrophil responses as well as the mechanism by which IL-15 enhances the ability of neutrophils to exert phagocytosis. Here, data obtained from inhibitory experiments with antibodies against IL-15R subunits indicate that the IL-15R plays a key role in phagocytosis, as the anti-IL-15R antibody largely inhibited the biological activity of IL-15. We do not rule out the possibility that CD122 and/or CD132 are involved in the process. However, it was recently reported that IL-15R could directly signal in lymphoid cells without the requirement of the CD122 and CD132 [25
]. In fact, our present results confirm our hypothesis that IL-15R is preferentially involved in IL-15-enhanced neutrophil phagocytosis, as IL-2 was previously reported to be unable to enhance such function as a result of the lack of expression of the IL-2R component necessary to the functional, high-affinity IL-2R. In contrast, IL-15 can bind with good affinity to cells expressing only the IL-15R subunit [38
], and neutrophils are known to express all three IL-15R components [8
9
10
11
].
The important role of Syk in the cell-signaling cascade during neutrophil phagocytosis induced by different agonists is well established [23
, 24
]. However, its role in IL-15-enhanced neutrophil phagocytosis is not known. To the best of our knowledge, this study provides the first evidence that IL-15 enhances neutrophil phagocytosis of opsonized SRBC by activating the protein tyrosine kinase Syk. This is supported by the fact that treatment with the Syk inhibitor piceatannol inhibited the ability of IL-15 to enhance neutrophil phagocytosis. In addition, IL-15 induced tyrosine phosphorylation of Syk. Moreover, pretreatment of cells with piceatannol markedly inhibited Syk phosphorylation induced by IL-15. In one study, Syk was found to be physically associated with IL-15R in Raji cells, and IL-15 was found to recruit this kinase for mediating its effect [25
]. In the present study, our results suggest that Syk is physically associated with IL-15R and phosphorylated during such association in neutrophils, demonstrating the important role of Syk in IL-15-induced biological functions in other cells. However, we do not rule out the possibility that Syk could also be associated with the receptor through another bound protein. Like others, we observed a basal level of phosphorylation of Syk in control cells [38
, 39
]. However, it is not clear if this corresponds to a constitutive activation of Syk in neutrophils or if this is a result of nonspecific activation following cell isolation. Of note, in some studies, pretreatment of neutrophils with di-isopropyl fluorophosphates (DFP) was found to eliminate [40
, 41
] or drastically decrease [42
] the basal level of phosphorylated Syk. In our hands, the basal level was variable from donor to donor after cell isolation. Nevertheless, Syk phosphorylation was always increased by IL-15. In fact, we believe that doing experiments with neutrophils without addition of DFP before activation with IL-15 represents a more physiological model, knowing that during inflammation, neutrophils are already activated by several agents including surrounding cytokines.
Collectively, results from this study indicate that the ability of IL-15 to enhance phagocytosis is not related to an increase of cell-surface receptor components. Rather, based on the use of piceatannol and induction of Syk phosphorylation, we can conclude that IL-15 mediates its effect by activating Syk kinase, which is associated with the IL-15R chain. These data may provide new, clinical perspectives in future IL-15 therapy.
Received June 28, 2003; accepted March 17, 2004.
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