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Originally published online as doi:10.1189/jlb.0306209 on January 30, 2007

Published online before print January 30, 2007
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(Journal of Leukocyte Biology. 2007;81:1287-1296.)
© 2007 by Society for Leukocyte Biology

Molecular mechanisms involved in interleukin-4-induced human neutrophils: expression and regulation of suppressor of cytokine signaling

Claude Ratthé, Martin Pelletier, Sonia Chiasson and Denis Girard1

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

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


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ABSTRACT
 
Interleukin-4 (IL-4) is a CD132-dependent cytokine known to activate the Jak-STAT pathway in different cells and cell lines. Although IL-4 has been demonstrated previously to be an agonist in human neutrophils, its capacity to activate different cell signaling pathways in these cells has never been investigated. Two types of IL-4 receptor (IL-4R) exist: the Type I (CD132/IL-4R{alpha} heterodimer) and the Type II (IL-4R{alpha}/IL-13R{alpha}1 heterodimer). In a previous study, we demonstrated that neutrophils express the Type I receptor. Herein, using flow cytometry, we demonstrated that neutrophils, unlike U-937 cells, do not express IL-13R{alpha}1 and IL-13R{alpha}2 and confirmed the expression of CD132 and IL-4R{alpha} on their surface. We also demonstrated that IL-4 induced phosphorylation of Syk, p38, Erk-1/2, JNK, Jak-1, Jak-2, STAT6, and STAT1 and that treatment of cells with the inhibitors piceatannol, SB203580, PD98059, or AG490 reversed the ability of IL-4 to delay neutrophil apoptosis. Using RT-PCR, we demonstrated for the first time that neutrophils express mRNA for all suppressor of cytokine signaling (SOCS) members, namely SOCS1–7 and cytokine-inducible Src homology 2 protein. It is interesting that IL-4 increased expression of SOCS3 at the mRNA and protein levels. The effect of IL-4 on SOCS3 protein expression was increased markedly when the proteasome inhibitor MG132 was added to the cultures, but this was inhibited by cycloheximide, suggesting that SOCS3 is de novo-synthesized in response to IL-4. We conclude that neutrophils express only the Type I IL-4R on their surface and that IL-4 signals via different cell signaling pathways, including the Jak/STAT/SOCS pathway.

Key Words: inflammation • cytokine • receptor • Jak • STAT • SOCS3 • IL-4 • SOCS


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INTRODUCTION
 
Suppressors of cytokine signaling (SOCS) proteins represent a family of eight members, designated SOCS1–7 and cytokine-inducible Src homology 2 protein (CIS). They were discovered a few years ago as negative regulators of the Jak/STAT pathway but have been linked to other signaling kinases recently, including Syk [1 ]. As more studies about SOCS have emerged, it has become clear that these proteins play other roles in cells. In this regard, it was demonstrated recently that SOCS functions were disturbed in certain types of cancers and inflammatory diseases [2 ]. For example, SOCS3 expression was increased in patients suffering from Crohn’s disease, inflammatory arthritis, and ulcerative colitis [3 4 5 6 ]. SOCS proteins can inhibit cell signaling by two major mechanisms: First, they bind to Jaks and to the cytokine receptors and block subsequent signaling protein activation; second, they redirect their bound proteins to proteasomal degradation via the SOCS box. SOCS proteins are expressed constitutively in cells or induced by cytokines, and some of them, such as SOCS3, are known to be degraded rapidly by the proteasome [7 ].

Interleukin-4 (IL-4) is a member of the CD132 ({gamma}c)-dependent cytokine family, which also includes IL-2, IL-7, IL-9, IL-15, and IL-21. We have demonstrated previously that human neutrophils express complete functional receptors for IL-4 (IL-4R{alpha} and CD132, known as the IL-4R Type I) [8 , 9 ] and for IL-15 (IL-15R{alpha}, IL-2/15Rß, and CD132 chains) [10 , 11 ]. To date, the expression of the IL-13R{alpha}1 in neutrophils, which together with IL-4R{alpha} constitutes the Type II IL-4R [12 ], is unknown. In contrast to IL-4R{alpha} and IL-15R{alpha}, human neutrophils do not express IL-2R{alpha}, IL-7R{alpha}, IL-9R{alpha}, and IL-21R{alpha} components (see ref. [13 ] for a review). IL-4 and IL-15 exert several effects on neutrophil cell physiology. Both can delay neutrophil apoptosis, increase phagocytosis of opsonized SRBCs, and induce RNA and de novo protein synthesis [9 10 11 , 13 ]. More recently, IL-15 was found to enhance human neutrophil phagocytosis by a Syk-dependent mechanism, and the importance of the IL-15R{alpha} chain in IL-15-induced neutrophils has been demonstrated [14 ]. Also, we have shown that IL-15 delayed human neutrophil apoptosis by intracellular events and not via extracellular factors [15 ]. In this latter study, IL-15 was found to prevent the loss of the antiapoptotic protein Mcl-1 and to decrease the activity of caspase-3 and caspase-8. Although IL-15 is known to induce phosphorylation of Jak-2, p38 MAPK, Erk-1/2, and Syk [14 , 16 ], the direct cell signaling events involved in IL-4-induced neutrophils have never been studied. However, in one study, IL-4-stimulated neutrophils were found to release the IL-1R{alpha} in response to IL-10, and this was correlated with tyrosine phosphorylation of STAT3, which was strictly dependent on the level of IL-10R1 expression [17 ]. Different studies performed in a variety of cells other than neutrophils have demonstrated that IL-4 activates at least the Jak/STAT pathways [18 , 19 ].

Although several studies have been conducted regarding the role of SOCS as a negative regulator of cytokines, only a few investigating the role of SOCS proteins in activated neutrophils have been reported in the literature [20 21 22 23 24 25 26 27 ]. Among these, it was observed that G-CSF and GM-CSF induced SOCS3 in human neutrophils [20 , 22 , 23 , 27 ]. Although IL-4 was reported to induce SOCS3 in murine B cells [25 ], there are presently no studies investigating the role of SOCS in IL-4-induced neutrophils. In particular, despite the fact that SOCS3 appears to be an important molecule in neutrophil cell physiology [20 21 22 23 ], its role has never been investigated in IL-4-induced neutrophils. Thus, we were interested in studying the cell signaling events that occur in IL-4-induced human neutrophil cells. In addition, although the presence of CIS, SOCS1, SOCS2, and SOCS3 has been studied in myeloid 32D cells [26 ], the expression of the various SOCS has never been investigated systematically in human neutrophils.

In the present study, we demonstrated the IL-4 induced phosphorylation of Syk, p38, Erk-1/2, Jak-1, Jak-2, STAT1, and STAT6 in human neutrophils. In addition, we demonstrated that the ability of IL-4 to delay neutrophil apoptosis was reversed by the inhibitors piceatannol, SB203580, PD98059, or AG490. Using RT-PCR, we demonstrated that human neutrophils express all CIS and SOCS1–7 and that IL-4 up-regulated CIS, SOCS1, SOCS2, and SOCS3 at the mRNA level. At the protein level, IL-4 was found to induce SOCS3, which appeared to have a rapid turnover and to be newly synthesized. These results provide the first evidence that IL-4 signals through different cell signaling pathways and that it activates the Jak/STAT/SOCS pathway in human neutrophils.


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MATERIALS AND METHODS
 
Chemicals and agonists
G-CSF, GM-CSF, and IFN-{gamma} were purchased from PeproTech Inc. (Rocky Hill, NJ, USA). IL-2 and IL-4 were purchased from R&D Systems Inc. (Minneapolis, MN, USA). The Jak-2/Jak-3 STAT1, -3, -5a, and -5b inhibitor tyrphostin B42 (or AG490), the MAPK/ERK inhibitor PD98059, the p38 MAPK inhibitor SB203580, the protein synthesis inhibitor cycloheximide (CHX), and the proteasome inhibitor MG132 were purchased from Sigma-Aldrich (St. Louis, MO, USA). When applicable, the final concentration of DMSO in each treatment never exceeded 1%.

Neutrophil isolation
Cells were isolated from venous blood of healthy volunteers by dextran sedimentation followed by centrifugation over Ficoll-Hypaque (Amersham Pharmacia Biotech Inc., Baie d’Urfé, Québec, Canada), as described previously [9 10 11 ]. 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 stained using the Hema 3 stain set (Biochemical Sciences Inc., Swedesboro, NJ, USA), according to the manufacturer’s protocol.

Cell surface expression of IL-4R subunits
Neutrophils (10x106 cells/ml) were washed and preincubated 30 min (4°C, light-protected) with 20% autologous serum to prevent nonspecific binding via FcRs. Cells (1x106 cells/ml) were then washed and incubated with antihuman IL-4R{alpha} (Genzyme, Cambridge, MA, USA), CD132 (TUGh4, BD Biosciences, San Jose, CA, USA), IL-13R{alpha}1 (B-K19, Diaclone, Stamford, CT, USA), or IL-13R{alpha}2 (B-D13, Diaclone; each at 10 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. In parallel, U-937 cells (CRL-1593.2TM) from American Type Culture Collection (Manassas, VA, USA) were used as positive controls for IL-13R{alpha}2 [28 ]. Flow cytometric analysis (10,000 events) was performed using a FACScan (Becton Dickinson, San Diego, CA, USA).

Phosphorylation events
Neutrophils (40x106 cells/ml in RPMI 1640) were incubated for the indicated periods of time at 37°C with buffer or the indicated agonist in a final volume of 120 µl. Reactions were stopped by adding 125 µl 2x Laemmli’s sample buffer, as we have described previously [14 , 16 ]. Aliquots corresponding to 1 x 106 cells were loaded onto 10% SDS-PAGE and transferred from gel to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Nonspecific sites were blocked with 1% BSA in TBS-Tween (25 mM Tris-HCl, pH 7.8, 190 mM NaCl, 0.15% Tween-20) for 1 h at room temperature. After washing, the membranes were incubated with monoclonal antiphosphotyrosine UB 05-321 [1:4000; United Biomedical Inc. (UBI), Hauppauge, NY, USA) for 1 h at room temperature. Membranes were then washed and incubated with a HRP-conjugated goat antimouse IgG + IgM (1:10,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 h at room temperature in fresh blocking solution. Membranes were washed three times with TBS-Tween, and phosphorylated bands were revealed with the ECL Western blotting detection system (Amersham Pharmacia Biotech Inc.). Protein loading was verified by staining the membranes with Coomassie blue at the end of the experiments.

Syk, p38, Erk-1/2, Jak-1, Jak-2, STAT1, and STAT6 phosphorylations
Neutrophils were isolated and incubated as above at 37°C with buffer or the indicated agonist in a final volume of 25 µl. Reactions were stopped by adding 10 µl 4x Laemmeli’s sample buffer, as we described previously [14 15 16 ]. Samples corresponding to 1 x 106 cells were loaded onto 10% SDS-PAGE and transferred from gel to nitrocellulose membranes (Amersham Pharmacia Biotech Inc.) or PVDF membranes (Millipore). Nonspecific sites were blocked with 5% nonfat dried milk (Carnation, Don Mills, Ontario, Canada) in TBS-Tween, and Western blots were performed as described previously [14 15 16 ]. Polyclonal antiphospho-Syk (Tyr525/526) antibody (1:1000; Cell Signaling Technology, Beverly, MA, USA) and HRP-conjugated goat antirabbit IgG + IgM (1:20,000), diluted in 5% BSA, were used. Membranes were stripped for 30 min at 55°C with stripping buffer (100 mM 2-ME, 2% SDS, 62.5 mM Tris, pH 6.7), washed, and reprobed with an anti-Syk antibody (1:800; Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by a HRP-conjugated goat antimouse IgG + IgM (1:20,000; Jackson ImmunoResearch Laboratories, Inc.). Syk protein expression was revealed with an ECL. In other experiments, the phosphorylation of p38, Erk-1/2, Jak-1, Jak-2, STAT1, and STAT6 was studied, essentially as described previously, using the following antibodies [16 ]: anti-p38[pTpY180/182] (BioSource International, Camarillo, CA, USA), the antiphosphospecific ERK-1/2 MAPK (BioSource International), the specific antibody against the nonphosphorylated form of ERK-1/2 (UBI), the rabbit polyclonal anti-JNK1 and -2/stress-activated protein kinase[pTpY183/185]-phosphospecific antibody (Biosource International), the rabbit polyclonal anti-phosphorylated (p)-Jak-1[pYpY1022/1023] (Biosource International), the rabbit polyclonal anti-Jak-2[pYpY1007/1008] (BioSource International), the anti-STAT6 (Tyr 641; Santa Cruz Biotechnology), and the antiphospho-STAT1 (Tyr701; 58D6) rabbit mAb (Cell Signaling Technology).

Assessment of neutrophil apoptosis
Freshly isolated human neutrophils (10x106 cells/ml in RPMI 1640 supplemented with 10% autologous serum) were preincubated for 60 min in 24-well plates with the transduction signal inhibitors and then incubated for 23 h in the presence or absence of agonists. Inhibitors remained in the culture. Apoptosis was evaluated by cytology as published previously [14 15 16 ]. Briefly, cytocentrifuged preparations of neutrophils (200 µl) were performed using a Cyto-tek® centrifuge (Miles Scientific, Naperville, IL, USA) and processed essentially as documented previously [10 ]. Cells were examined by light microscopy at 400x final magnification, and apoptotic neutrophils were defined as cells containing one or more characteristic, darkly stained, pycnotic nuclei. Results were expressed as percentage of apoptotic cells.

RT-PCR
Neutrophils [10x106 cells/ml in RPMI-HEPES-penicillin and streptomycin (P/S) containing 10% FCS] were stimulated for 1 to 3 h at 37°C with HBSS, 50 ng/ml G-CSF, 65 ng/ml GM-CSF, or 250 ng/ml IL-2, IL-4, or IL-6. Cells were harvested and washed three times with HBSS, and RNA extraction was performed using the Absolutely RNA® miniprep kit (Stratagene, La Jolla, CA, USA), according to the manufacturer’s protocol. The RT reaction was performed using 0.5 µg RNA and 200 U Moloney murine leukemia virus enzyme in the following PCR buffer reaction: 5 mM MgCl2, 1 mM deoxy (d)-unspecified nucleoside 5'-triphosphate (dATP, d-cytidine 5'-triphosphate, d-guanosine 5'-triphosphate, d-TTP, d-thymidine 5'triphosphate), 40 U RNase inhibitor, 0.03 U random hexamers. PCR reactions were performed using the newly made cDNA, 0.5 U Taq polymerase, and the following primers: upstream 5'-GATCTGCTGTGCATAGCCAA-3', downstream 5'-ACAAAGGGCTGCACCAGTTT-3' for CIS; upstream 5'-GAGAGCTTCGACTGCCTCTT-3', downstream 5'-AGGTAGGAGGTGCGAGTTCA-3' for SOCS1; upstream 5'-GATAAGCGGACAGGTCCAGA-3', downstream 5'-AAGAAGGCAAGGCATTCTGA-3' for SOCS2; upstream 5'-CTCAAGACCTTCAGCTCCAA-3', downstream 5'-TTCTCATAGGAGTCCAGGTG-3' for SOCS3; upstream 5'-CTTAGATCATTCCTGTGGGC-3', downstream 5'-ATGCCACCTAAAGGCTAAATC-3' for SOCS4; upstream 5'-AATTGTGCCACAGAAATCCCT-3', downstream 5'-AGCATCCAATGAACTCTGGG-3' for SOCS5; upstream 5'-GGACTCACTGGCACAGAAGC-3', downstream 5'-TTCAGAGTCCCTGATTGAATGC-3' for SOCS6; upstream 5'-AAATATAGTTCCCCGTCCCC-3', downstream 5'-CAGGAATGACAAATGATCCG-3' for SOCS7; and upstream 5'-TCCATGACAACTTTGGTATCGTGG-3', downstream 5'-GTCGTCGTTGAAGTCAGAGGAGAC-3' for the GAPDH control. Samples were loaded onto a 1.5% agarose gel containing 0.006% ethidium bromide. The bands were visualized using the Bio-Rad Bioscan with a UV lamp, and the results were analyzed using the Multi-Analyst program (Bio-Rad, Hercules, CA, USA).

SOCS proteins expression
Neutrophils (10x106 cells/ml in RPMI-HEPES-P/S containing 10% FCS) were stimulated for 2–8 h at 37°C with agonists as described above and were pretreated or not with inhibitors. Cells were washed twice using HBSS, and proteins were denatured using 1x Laemmeli’s sample buffer as described previously [14 15 16 ]. Samples corresponding to 0.5 x 106 cells were loaded onto 10% or 15% SDS-PAGE and transferred from gel to nitrocellulose membranes (Amershem Pharmacia Biotech Inc.). Nonspecific sites were locked with 5% BSA (Sigma-Aldrich) in TBS-Tween (25 mM Tris-HCl, pH 7.8, 190 mM NaCl, 0.15% Tween-20) for 1 h at room temperature. After washing with TBS-Tween, the membranes were incubated overnight at 4°C with a polyclonal antibody against one of the SOCS family members. The following clones (H-80 for CIS, H-93 for SOCS1, H-74 for SOCS2, and H-103 for SOCS3), from Santa Cruz Biotechnology Inc., were used at a final dilution of 1:1000 in TBS-Tween containing 5% BSA. Membranes were washed and incubated for 1 h at room temperature with a HRP-conjugated goat antirabbit IgG + IgM (1:20,000; Jackson ImmunoResearch Laboratories Inc.), diluted in TBS-Tween containing 5% BSA.

In some experiments, SOCS3 was immunoprecipitated, and its ubiquitination was assessed by immunoblotting using the antiubiquitin antibody (Santa Cruz Biotechnology Inc.). Membranes were washed with TBS-Tween, and bands were revealed using the ECL Western blotting detection system (Amersham Pharmacia Biotech Inc.).

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


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RESULTS
 
Human neutrophils express the IL-4R Type I but not Type II
Two types of IL-4R exist: Type I, which is composed of CD132 and IL-4R{alpha}, and Type II, composed of IL-4R{alpha} and IL-13R{alpha}1 [28 , 29 ]. Herein, as illustrated in Figure 1 , we confirmed our previous results demonstrating that human neutrophils express CD132 and IL-4R{alpha} at their cell surface [9 10 11 ] and show for the first time that these cells do not express IL-13R{alpha}1 and IL-13R{alpha}2. The latter subunit is a component specific for IL-13 binding [28 , 29 ]. The antibodies were suitable for cell surface binding, as a good signal was observed in U937 cells (Fig. 1) , which was used as positive control.


Figure 1
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  		Figure 1. Human neutrophils express only the Type I IL-4Rs. Freshly isolated human neutrophils or U937 cells were used to study cell surface expression of CD132, IL-4R{alpha}, IL-13R{alpha}1, and IL-13R{alpha}2 by flow cytometry as described in Materials and Methods. Each antibody was used from 1 to 10 µg/ml (open area), and for simplicity, only the results obtained with the highest concentration are shown; filled area, appropriated, corresponding isotypic control. Results are from one representative experiment out of ≥3.

IL-4 induces phosphorylation events in human neutrophils
Although we and others [9 , 17 ] reported that IL-4 is a human neutrophil agonist, phosphorylation events occurring in response to this cytokine in neutrophils have never been investigated. To elucidate this and as human neutrophils express the high-affinity IL-4R, we first treated freshly isolated human neutrophils with IL-4 and tested the ability of this cytokine to induce tyrosine phosphorylation events by Western blot over time. As illustrated in Figure 2A , IL-4 induced tyrosine phosphorylation of different proteins as well as GM-CSF used as a positive control. As we reported previously that IL-15, another neutrophil agonist belonging to the CD132-dependent cytokine family, induced phosphorylation of Syk, p38, Erk-1/2, Jak-2, but not STAT5, we next investigated the effect of IL-4 on different key proteins. IL-4 induced a rapid phosphorylation of p38 and Erk-1/2 (Fig. 2B) . The intensity of the signal of p38 and Erk-1/2 phosphorylation decreased after a few minutes but remained elevated for up to 30 min when compared with control cells. In addition to p38 and Erk-1/2, we demonstrated that IL-4 induced the phosphorylation of the p54 JNK isoform (Fig. 2B , bottom panel). As expected, GM-CSF did not activate p54 JNK in human neutrophils [30 ]. As illustrated in Figure 2C , IL-4 also induced the phosphorylation of Syk tyrosine kinase, similar to our previous observation with IL-15 [14 ].


Figure 2
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  		Figure 2. IL-4 induces the phosphorylation events in human neutrophils. Freshly isolated human neutrophils (10x106 cells/ml) were incubated with buffer (C), 65 ng/ml GM-CSF (GM) for 15 s (0.25 min), or 250 ng/ml IL-4 for 0.25–30 min, and the expression of tyrosine-phosphorylated proteins (A); p38, Erk-1/2, and JNK (B); or Syk (C) was assessed by Western blot as described in Materials and Methods. Results are from one representative experiment out of at least three. For Erk-1/2, p38, and Syk, the membranes were stripped and probed with antibody directed against the corresponding nonphosphorylated form of the protein. Aliquots from the same cell lysate used for Erk-1/2 were also used for JNK2 (p54 isoform). Molecular weights are indicated on the left.

Although IL-4, like all other CD132-dependent cytokines, is known to activate Jak-3, as Jak-3 is constitutively associated with CD132 [19 ], its ability to activate other Jaks is less well established. This is true, especially for neutrophils, as this has never been investigated. Here, we demonstrated that IL-4 induces the phosphorylation of Jak-1 and Jak-2 (Fig. 3A ) after 5 and 15 min. However, the response was increased after 15 min for Jak-2 but decreased (but still higher vs. control) for Jak-1. Unlike IL-4, we could not detect any effects by the other CD132-dependent cytokines, namely IL-2, IL-7, and IL-9 (Fig. 3A) .


Figure 3
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  		Figure 3. IL-4 induces the JAK/STAT pathway in human neutrophils. (A) Neutrophils were incubated for 5 or 15 min with buffer, 65 ng/ml GM-CSF, 100 ng/ml G-CSF (G), or 250 ng/ml IL-2, IL-4, IL-7, or IL-9, and activation of Jak-1 and Jak-2 was assessed by Western blot, as described in Materials and Methods. (B and C) Neutrophils were incubated for 30 min in the presence or absence of 65 ng/ml GM-CSF, 100 ng/ml G-CSF, 250 ng/ml IL-4, 250 ng/ml IL-15, 10 ng/ml IFN-{gamma}, or 50 ng/ml IFN-{gamma}. The phosphorylated forms of STAT6 (B) and STAT1 (C) were detected by Western blot, as described in Materials and Methods. The membranes were stripped and stained with an antibody directed against the cytoskeletal vinculin protein to verify equivalent protein loading. Results are from one representative experiment out of at least two others. (C) The two arrows illustrate two degradation products of STAT1, which are reproducibly recognized by the antibody.

As we were interested in studying the expression and regulation of SOCS in human neutrophils and as these negative regulators are known to be activated by cytokines, inducing the Jak/STAT pathway, including all CD132-dependent cytokines, we then investigate whether IL-4 could induce STATs, knowing that it induces several Jaks. IL-4 is known to activate STAT6 specifically [8 , 18 ]. Recently, STAT1 was reported to be activated by IL-4 in multiple cell types, so we examined IL-4 activation of STAT6 and STAT1 in human neutrophils. As illustrated in Figure 3B , IL-4 induced STAT6 phosphorylation, demonstrating that neutrophils respond like other cells to IL-4 [8 , 18 ]. It is interesting that IL-15 was also found to activate STAT6 but not G-CSF, GM-CSF, or IFN-{gamma}. As illustrated in Figure 3C , IL-4 induced STAT1 but not as strongly as G-CSF or GM-CSF after 5 and 15 min. However, the response appeared to increase after 30 min, a period of time when STAT1 phosphorylation starts to decline after activation with G-CSF or GM-CSF, suggesting that the response induced by IL-4 is less rapid. It is interesting that IL-15, another CD132-dependent cytokine, also activated STAT1 with similar kinetics to IL-4.

Inhibition of IL-4 induced suppression of human neutrophil apoptosis
We have documented previously that IL-4 is a human neutrophil agonist and that among the responses it modulates, IL-4 delays neutrophil apoptosis [9 ]. As we found here that IL-4 activated several intracellular cell-signaling molecules, we decided to investigate the possibility of reversing the ability of IL-4 to delay neutrophil apoptosis using signal transduction inhibitors, which were added in the culture 60 min prior to the addition with the agonist. As illustrated in Figure 4 , using the MAPK/ERK inhibitor PD98059, we found that unlike G-CSF and GM-CSF, the ability of IL-4 to delay neutrophil apoptosis was reversible when the inhibitor was used at a concentration of 1 µM (Fig. 4B) . Using a higher concentration of this inhibitor (10 or 50 µM) did not result in a greater inhibitory effect of IL-4. The ability of G-CSF and GM-CSF to delay neutrophil apoptosis was not inhibited significantly at any concentrations of PD98059 tested. Use of the p38 inhibitor SB203580 at a concentration of 1, 10, or 20 µM did not cause an inhibition in G-CSF- or GM-CSF-induced cells, and at 20 µM, the effect of IL-4 on neutrophil apoptosis was reversed (Fig. 4A) . As illustrated in Figure 4C , the inhibitor AG490 (initially considered to be a specific inhibitor of Jak-2 but now known to inhibit Jak-2/Jak-3 STAT1, -3, -and 5a/b, and MAPK) [31 , 32 ] was found to reverse the ability of IL-4 to delay neutrophil apoptosis at a concentration as low as 1 µM, whereas a concentration of 10 µM and 100 µM was required to inhibit the effects of G-CSF and GM-CSF, respectively. When the Syk inhibitor, piceatannol, was used at a concentration of 1 and 10 µM [14 ], the effects of IL-4 were abolished (Fig. 4D) . In contrast, G-CSF and GM-CSF were still able to inhibit neutrophil cell apoptosis in the same culture conditions, suggesting that Syk is not (or is less) involved in the ability of G-CSF and GM-CSF to inhibit neutrophil apoptosis. The PI-3K inhibitor Wortmanin, as well as LY294002, abolished the ability of IL-4 to delay neutrophil apoptosis (Fig. 4E and 4F) .


Figure 4
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  		Figure 4. The ability of IL-4 to delay human neutrophil apoptosis is reversed by the Jak-2/Jak-3 STAT1, -3, and -5a/b inhibitor AG490 and the Syk inhibitor piceatannol. Cells (10x106/ml) were preincubated for 60 min with the indicated concentrations of inhibitors and were then incubated for 23 h in the presence of buffer (Ctrl), 100 ng/ml G-CSF, 65 ng/ml GM-CSF, or 250 ng/ml IL-4. Apoptosis was assessed by cytology as described in Materials and Methods. *, P < 0.05, versus Ctrl by ANOVA.

Expression and regulation of SOCS in human neutrophils
As we established here that IL-4 activated the JAK/STAT pathway in human neutrophils, a pathway that is highly regulated by SOCS, and as there had been no study reporting a systematic analysis of SOCS expression in human neutrophils, we decided to use RT-PCR to investigate the expression of all known SOCS in these cells. As illustrated in Figure 5 , neutrophils constitutively expressed mRNA for SOCS1, SOCS4, SOCS5, SOCS6, and SOCS7. Neutrophils also expressed CIS and SOCS2, but this was observed after cell activation. For SOCS3, the results differ from experiment to experiment. Its expression ranged from undetectable to weakly detectable but was never expressed strongly, as were SOCS4–7. The above results indicate that human neutrophils express mRNA of all SOCS. CIS mRNA expression was induced in neutrophils in response to GM-CSF and IL-4, but not to G-CSF, IL-2, or IL-6. SOCS1 expression was markedly expressed by IL-4, G-CSF, and GM-CSF. SOCS2 mRNA was induced by GM-CSF and slightly, but reproducibly, by IL-4. SOCS3 mRNA expression was increased by IL-4 but not as strongly as GM-CSF and G-CSF. Although we found that SOCS6 mRNA expression was increased by GM-CSF, G-CSF, and IL-6, it was not modulated by IL-4. Of note, mRNA expression of SOCS4, SOCS5, and SOCS7 was not modulated by IL-4 nor by any of the cytokines tested, namely, G-CSF, GM-CSF, IL-2, and IL-6. In summary, IL-4 increased expression of CIS, SOCS1, SOCS2, and SOCS3 but not SOCS4–7.


Figure 5
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  		Figure 5. mRNA expression of SOCS family members in human neutrophils. Freshly isolated human neutrophils (1x107 cells/ml) were incubated with or without cytokines (65 ng/ml GM-CSF, 250 ng/ml IL-4, 250 ng/ml IL-2, or 50 ng/ml G-CSF) for 1 h at 37°C. Total RNA was purified from neutrophils, stimulated with buffer or the indicated cytokines, and subjected to semiquantitative RT-PCR analysis with oligonucleotide primers specific for each gene (CIS, SOCS1–7), as described in Materials and Methods. Results are from one representative experiment out of at least three. GAPDH signal is indicated to illustrate loading equivalence.

Expression of SOCS proteins in IL-4-induced neutrophils
The above results suggested that SOCS4–7 were less important than CIS, SOCS1, SOCS2, and SOCS3 in regulating IL-4-induced neutrophil responses, and this was in agreement with the general observations of cytokine signaling in hematopoietic cells [2 , 22 , 23 , 33 34 35 ]. Thus, we investigated the potential effect of IL-4 on the expression of these four negative regulators of cytokine signaling at the protein level. When neutrophils were stimulated for a period of 2, 4, 6, or 8 h with IL-4, the protein expression of CIS, SOCS1, and SOCS2 remained stable when compared with control cells, indicating an absence of correlation between IL-4-inducible mRNA and protein expression (Fig. 6A ). It is interesting that this observation was not exclusive to IL-4, as we did not observe any modulation of CIS, SOCS1, and SOCS2 with G-CSF, GM-CSF, and IL-2, suggesting that these proteins were degraded rapidly. To investigate this, cells were pretreated with the proteasome inhibitor MG132, known to increase the stability of several proteins, including SOCS. Unfortunately, this procedure yielded no significant results for CIS, SOCS1, and SOCS2 (data not shown). In contrast, SOCS3 protein expression was induced after 4 h of stimulation with IL-4, albeit with less potency than G-CSF and GM-CSF. Moreover, in this case, pretreatment with MG132 was efficient (Fig. 6B) . The increased SOCS3 expression was observed as soon as after 2 h of stimulation with G-CSF or GM-CSF but not IL-4, suggesting that the response to IL-4 was not as rapid as that of the other cytokines (data not shown). SOCS3 expression started to decline after 8 h of stimulation with G-CSF, GM-CSF, and IL-4, probably as a result of a loss of MG132 effects, a reversible inhibitor.


Figure 6
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  		Figure 6. Regulation of CIS, SOCS1, SOCS2, and SOCS3 protein expression in IL-4-induced neutrophils. Freshly isolated human neutrophils (1x107 cells/ml) were preincubated (A and B) or not (Ctrl, B) with 50 µM MG132 (proteasome inhibitor) for 1 h and then incubated for 0, 2, 4, 6, or 8 h [only results from 8 h (A) or 4 h (B) are illustrated] with the indicated cytokine. Cells were lysed before performing Western blot experiments as indicated in Materials and Methods. (C) CHX was added to the culture as described in Materials and Methods. (D) A total of 2 x 106 cells was used for the immunoprecipitation (IP) of SOCS3. Western blotting (WB) was then performed with an antiubiquitin antibody (anti-ubi). Results are from one representative experiment out of three (A–C) or two (D).

As SOCS proteins are subject to rapid degradation via the proteasome and as we observed that addition of MG132 to the culture allowed us to improve the detection of SOCS3, we decided to determine whether its increased expression observed in response to IL-4 was related to de novo protein synthesis. To do so, cells were pretreated with CHX, a potent protein synthesis inhibitor, in the presence or absence of MG132 and then stimulated with IL-4 or other cytokines. As illustrated in Figure 6C , treatment of cells with CHX reversed the ability of all tested cytokines to increase SOCS3 protein expression, indicating that de novo synthesis of SOCS3 occurred in response to these cytokines. Treatment with CHX reversed the ability of MG132 to stabilize SOCS3 expression, leading to the inability to detect cytokine-inducible SOCS3 expression. Treatment with CHX did not alter the constant level of CIS and SOCS2 protein expression observed, regardless of whether cells were stimulated with the cytokines G-CSF, GM-CSF, IL-2, or IL-4, indicating that CHX did not systematically decrease the basal level expression of CIS/SOCS, demonstrating the importance of de novo protein synthesis for SOCS3 regulation. Ubiquitination of SOCS3 has been reported recently to be an important step in explaining why SOCS3 is unstable and degraded rapidly by the proteasome [36 ]. Although this was not performed in human neutrophils but rather in Ba/F3 cells, we asked whether this could also occur in neutrophils. As illustrated in Figure 6D , immunoprecipitation of SOCS3 followed by immunoblotting with an antiubiquitin antibody revealed that SOCS3 was ubiquitinated effectively. Mono- as well as polyubiquitination were observed, indicating that SOCS3 protein expression is better observed in the presence of a proteasome inhibitor.


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DISCUSSION
 
We have demonstrated previously that IL-4 [9 ] and IL-13 [37 ] are two human neutrophil agonists. Although we have reported the presence of CD132 and IL-4R{alpha}, corresponding to the Type I IL-4R in neutrophils [8 , 12 , 18 ], it was not known prior to the present study if these cells express a functional Type II IL-4R composed of IL-4R{alpha} and IL-13R{alpha}1. Herein, we demonstrated that IL-13R{alpha}1 was not expressed on the cell surface of neutrophils, indicating that these cells respond to IL-4 via IL-4R Type I, which is specific to IL-4 and not IL-13 [8 , 12 ]. It is interesting that we also demonstrated that these cells do not express IL-13R{alpha}2, a component acting as a nonsignaling "decoy" receptor [12 , 38 ]. Thus in neutrophils, IL-4 uses the Type I IL-4R to mediate biological activities [9 , 13 ].

IL-4 is known particularly to be involved in the regulation of IgE and IgG1 production by B cells and to favor the Th2 differentiation of Th cells and to inhibit the differentiation toward the Th1 phenotype [39 ]. Such inhibition is a result, in part, of the capacity of IL-4 to induce the production of SOCS3, which prevents the IL-12-induced Th1 differentiation [39 ]. Accordingly, IL-4 is known to be implicated in the development of asthma, where it activates Th2 cells. Neutrophils have also been linked to asthma, where they are in an activated state. In contrast to eosinophils, the number of neutrophils at inflammatory sites was not found to be increased. However, an increased number of neutrophils are observed in cases of severe asthma, suggesting that these cells may have a role to play in the severity of the disease. These observations, together with the facts that human neutrophils expressed the Type I IL-4R and that IL-4 induced suppression of neutrophil apoptosis [9 ], attest to the importance of studying the interaction between IL-4 and neutrophils.

In this study, we demonstrated that IL-4 induced tyrosine phopshorylation events in human neutrophils, as well as the phosphorylation of p38, Erk-1/2, JNK, Syk, Jak-1, Jak-2, at least STAT6, and to a lesser extend, STAT1. This indicates that several cell signaling pathways are activated by IL-4 in human neutrophils. Although the role of p38, Erk-1/2, JNK, and Jaks is known to be somewhat involved in the regulation of agent-induced suppression of neutrophil apoptosis [15 , 16 , 40 ], the role of Syk kinase is less well understood. Rather, this protein is well recognized for its role in neutrophil phagocytosis [41 ]. It is interesting that IL-4 was found to enhance the ability of human neutrophils to ingest opsonized SRBC [9 ], agreeing with the phosphorylation of Syk observed in the present study. However, as we have demonstrated previously that Syk activation also occurred in neutrophils in response to another CD132-dependent cytokine, namely IL-15 [14 ], and that inhibition of Syk reversed the ability of IL-15 to delay apoptosis, we performed experiments with different signal transduction inhibitors, including piceatannol, a Syk-specific inhibitor, to elucidate the mechanism involved in IL-4 activation of neutrophils. This latter inhibitor, at the low concentrations we tested, allowed us to demonstrate that IL-4, at least partly, delayed neutrophil apoptosis by a Syk-dependent mechanism. It is interesting that it was recently demonstrated that an IgM antineutrophil cytoplasm antibody, with specificity for myeloperoxidase, was found to inhibit apoptosis by a Syk-dependent signaling cascade [42 ]. In the present study, the use of other inhibitors demonstrated that the signaling cascade involved in IL-4-induced suppression of neutrophil apoptosis is complex. It is interesting that as we observed an activation of Erk-1/2 after IL-4 stimulation and that at least in B cells, Syk activation leads to phosphorylation of phospholipase C-{gamma}2, resulting in downstream activation of Erk, it is tempting to speculate that this also occurs in neutrophils. As mentioned above, inhibition experiments with the specific Syk inhibitor, piceatannol, support this. Conversely, we also observed an inhibition of IL-4-induced suppression of apoptosis with two specific inhibitors of PI-3K, Wortmanin and LY294002. Thus, to further complicate the signaling events involved in IL-4-induced neutrophils, these latter results suggest that Syk activation also increased Akt activity in response to IL-4, as activation of Syk is known to phosphorylate PI-3K, leading to increased Akt activity [43 , 44 ].

All CD132-dependent cytokines, IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, are known to activate the Jak/STAT pathway in hematopoietic cells. In addition, as Jak-3 is constitutively associated with CD132, all of these cytokines are known to activate Jak-3 [19 ]. Herein, for the first time, we have demonstrated that IL-4 activated the Jak/STAT pathway in human neutrophils. Jak-1 and Jak-2 were phosphorylated by IL-4 and also STAT1 and STAT6. It is interesting that activation of Jak-1 and Jak-2 was observed recently in IL-4-induced murine tight skin/+ fibroblasts [45 ], corroborating our present data. Moreover, almost 10 years ago, Murata et al. [46 ] reported that receptors for IL-4 do not associate with CD132 and that IL-4 induced Jak-2 phosphorylation in human HT-29 and WiDr colon carcinoma cells. Recently, in neutrophils, we also demonstrated the importance of Jak-2 in response to another CD132-dependent cytokine, IL-15 [16 ]. In fact, this cytokine was found to activate p38, Erk-1/2, Jak-2, but not STAT5a/b. Others [47 , 48 ] also have reported the importance of Jak-2 in neutrophils in response to GM-CSF. It is interesting that although IL-4 is typically known to activate the Jak/STAT pathway in a variety of cells, it is only recently that activation of p38 MAPK by IL-4 has been reported, and this was observed in B cells [25 ]. Thus, we show here for the first time that IL-4 activates p38 in human neutrophils and that the p38 inhibitor (SB203580) partially reversed the biological activity of IL-4. Also, we found that IL-4 induced STAT6 (and STAT1) phosphorylation. Knowing the importance of CIS and SOCS in regulating the Jak-STAT pathway and having established that IL-4 induced the Jak-STAT pathway in human neutrophils, it was logical to investigate the role of these regulators in response to IL-4.

Herein, we are the first to investigate a systematic expression of SOCS in human neutrophils. By RT-PCR, we found that neutrophils express mRNA for CIS and SOCS1–7. Using a similar approach, Stevenson et al. [26 ] found that promyelocytic HL-60 cells also expressed CIS and SOCS1–7. They also reported that human neutrophils expressed SOCS1 at mRNA and protein levels and that fMLP and IL-8 increased SOCS1 expression in these cells. However, they were not able to observe a modulation of SOCS3 mRNA, because as they mentioned (data not shown), the basal level of SOCS3 mRNA was too high to observe a clear modulation in response to fMLP or IL-8. In contrast, we did not observe a high basal level of mRNA SOCS3 expression. In fact, we failed to detect a high basal level of SOCS3 mRNA. However, we clearly observed a high signal in response to GM-CSF and a weaker but detectable signal in response to IL-4. Hörtner et al. [20 ] observed a weak basal level of SOCS3, which was increased highly in response to G-CSF in human neutrophils. Similarly, we observed a weak but clear basal level of SOCS1 mRNA, which was up-regulated by G-CSF (data not shown). We further report here that GM-CSF or IL-4 also up-regulated SOCS3 mRNA expression. An increased expression of SOCS3 mRNA [21 ] and protein [27 ] was observed in neutrophils in response to IL-10, particularly when cells were pretreated with LPS. Presently, as there are few studies dealing with the regulation of SOCS in human neutrophils, it is difficult to explain why one team reported a high basal level of SOCS3 mRNA level expression, and three others reported different degrees of such expression. It is tempting to speculate that this may have been a result of different experimental conditions. Future studies in this area will certainly help to understand this better. Nevertheless, our present study reveals that human neutrophils express mRNA for CIS and SOCS1–7 and that according to what is observed normally in other hematopoietic cells, the expression of CIS, SOCS1, SOCS2, and SOCS3 is regulated by cytokines but not SOCS4, SOCS5, SOCS6, and SOCS7 [19 ]. Why we observed a modulation of SOCS6 in response to G-CSF, IL-2 and in neutrophils is unclear and requires further investigation.

Although we demonstrated that human neutrophils express CIS and SOCS1–7 at the mRNA and protein levels, there is not a systematic relationship between the up-regulation of mRNA and protein expression. However, it is clear that SOCS3, actually one of the most studied SOCS proteins, is an important target of IL-4 activation in neutrophils. We also found that this protein was constitutively expressed, but inhibition of the proteasome chymotrypsin activity by addition of MG132 yielded better results. This indicates that SOCS3 is also a target to IL-4, and its activation is modest without the presence of MG132. This is in agreement with others who have reported that the proteasome plays a major role in the rapid degradation of SOCS3 [7 , 36 ]. Use of CHX indicated that SOCS3 was synthesized de novo in response to IL-4 and the other tested cytokines. Moreover, the same results were obtained when cells were treated with CHX and MG132. Of note, treatment with MG132 yielded better results, not only for IL-4 but also for G-CSF or GM-CSF. All of the above results show that SOCS3 is unstable in neutrophils and is degraded rapidly by the proteasome. Concurrently, we demonstrated that SOCS3 was polyubiquitinated in response to IL-4. It seems, however, that there is a certain portion of the amount of the protein in neutrophils, which is expressed constitutively.

In summary, this study is the first to demonstrate that human neutrophils express only the Type I IL-4R. In addition, we demonstrate that IL-4 induced several kinases in these cells, namely, p38, Erk-1/2, JNK, and Syk, and that it activated the Jak-STAT pathway. Also, this is the first study to report that human neutrophils express all SOCS at mRNA levels and that IL-4 induces SOCS3 protein expression, which is ubiquitinated and depends on de novo protein synthesis.


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ACKNOWLEDGEMENTS
 
This study was supported partly by the Canadian Institutes of Health Research (CIHR; MOP-89534) and Fonds de la Recherche en Santé du Québec (FRSQ). C. R. and M. P. hold a Ph.D. Canada Graduate Scholarships Doctoral Award, and D. G. is a Scholar from FRSQ. We thank Mary Gregory for reading this manuscript.

Received March 15, 2006; revised December 5, 2006; accepted December 23, 2006.


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