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International Immunology, Vol. 14, No. 10, pp. 1145-1153, October 2002
© 2002 Japanese Society for Immunology

Molecular basis of the synergistic production of IL-1 receptor antagonist by human neutrophils stimulated with IL-4 and IL-10

Luca Crepaldi1, Licia Silveri1, Federica Calzetti1, Cristina Pinardi1 and Marco A. Cassatella1

1 Department of Pathology, General Pathology Unit, University of Verona, Strada Le Grazie 4, 37134 Verona, Italy

Correspondence to: M. A. Cassatella; E-mail: marco.cassatella{at}univr.it
Transmitting editor: G. Trinchieri


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we report that the release of IL-1 receptor antagonist (IL-1ra) from IL-4-stimulated neutrophils is markedly enhanced in the presence of IL-10. We also show that up-regulation of IL-1ra release by IL-10 in IL-4-stimulated neutrophils takes place through IL-1ra mRNA stabilization and enhancement of IL-1ra de novo synthesis. Furthermore, we report that the ability of IL-10 to up-regulate IL-1ra mRNA expression in IL-4-treated neutrophils requires 5–6 h and it is preceded by the acquisition of the capacity to activate Stat3 tyrosine phosphorylation. This latter response to IL-10 was strictly dependent on the levels of expression of IL-10R1, which were in fact significantly increased by IL-4 in cultured neutrophils via a signaling pathway sensitive to the serine/threonine kinase inhibitor H-7. Collectively, our data emphasize the central role of IL-10R1 expression in regulating cell responsiveness to IL-10. In addition, the fact that IL-10 strongly up-regulates IL-1ra production in IL-4-activated neutrophils uncovers a novel mechanism whereby IL-10 and IL-4 cooperate to negatively modulate the inflammatory responses.

Keywords: IL-10R1, lipopolysaccharide, STAT3


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
IL-10 is a 18-kDa non-glycosylated polypeptide secreted by monocytes/macrophages, B lymphocytes, keratinocytes and subclasses of CD4+ T lymphocytes, which binds to a receptor composed of at least two subunits: the primary ligand-binding component, IL-10R1, and an accessory subunit, IL-10R2 (1). Both chains of the IL-10R have been classified as members of the class II subgroup of cytokine receptors, which, like other members of this family, interact with the Jak tyrosine kinase family: IL-10R1 associates with Jak1, while IL-10R2 associates with Tyk2 (2,3). IL-10 has been reported to activate the recruitment of Stat3 to the IL-10R, and the phosphorylation of Stat3 on tyrosine (2,3) and serine residues (4). In some types of cells Stat1 and Stat5 may also be tyrosine phosphorylated in response to IL-10 (1–3), but the mechanisms of their activation remain unclear. Upon phosphorylation, Stat1 and Stat3 (and Stat5) homo/heterodimerize and translocate to the nucleus where they bind to specific promoter sequences to modulate transcription (1,2).

IL-10 plays a fundamental role in the control of the inflammatory processes (1,3). The observations that IL-10 prevents the development of certain inflammatory and immune responses in vivo (1), including lipopolysaccharide (LPS)-induced lethality (5–7), and the analysis of IL-10 knockout mice that develop enterocolitis and exhibit dysregulated inflammatory responses (8,9), fully support the notion that IL-10 protects the host from overwhelming immune/inflammatory responses. Although the immunoregulatory mechanisms exerted by IL-10 appear complex and not fully defined yet, its anti-inflammatory activities are mainly achieved by inhibiting Th1 cell-mediated immune responses, as well as several functional responses of monocytes/macrophages and polymorphoneutrophils (PMN) (1,3,10). Among the responses regulated by IL-10 in the latter type of cells, the modulation of cytokine/chemokine production is certainly the most effective and, in the last years, it has been the focus of intensive investigation (10). The intracellular mechanisms whereby IL-10 inhibits pro-inflammatory cytokine production by activated phagocytes are still unknown, even though several studies reported that they are dependent on de novo protein synthesis (11,12). In this context, we have recently shown that IL-10 fails to induce Stat1 and Stat3 tyrosine phosphorylation (4), and binding of multimeric complexes containing both Stat1 and Stat3 to their target sequences in human neutrophils freshly isolated from the blood of normal donors (4,13). However, IL-10 becomes very effective in these activities if neutrophils are appropriately ‘primed’, e.g. with LPS (14). This occurs because the gene and surface expression of the IL-10R1, only present at low levels in circulating or freshly isolated PMN (13–16), is significantly up-regulated upon culture, reaching sufficient levels to confer IL-10 inducibility of responses such as Stat activation, modulation of cytokine production and enhanced suppressor of cytokine signaling (SOCS)-3 expression (14). Interestingly, responsiveness to IL-10 in terms of Stat3 tyrosine phosphorylation was observed also in neutrophils cultured for 4 h in the absence of LPS (14), suggesting that factors other than endotoxin may ‘prime’ PMN for IL-10 responsiveness.

In this work, we show that, among a number of typical neutrophil agonists tested, only IL-4 retains the ability to increase the gene and surface expression of IL-10R1 in neutrophils. Such increased IL-10R1 expression correlates well with the capacity of IL-10 to potently activate Stat3, as well as to synergize with IL-4 in inducing the production of IL-1ra in neutrophils. The fact that IL-10 up-regulates IL-1ra production in IL-4-activated PMN strengthens the notion that IL-10 is an important physiologic regulator of cytokine production from PMN, and emphasizes the potential role of IL-10 and IL-4 in negatively modulating the inflammatory responses.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell purification and culture conditions
Highly purified granulocytes (>98.5%), containing <4% eosinophils (n = 25), were isolated under endotoxin-free conditions from buffy coats of healthy donors, as previously described (17). Culture conditions slightly differed depending on the type of assay performed (14). For the Western blot experiments, PMN (5 x 106/ml) were suspended in ‘standard medium’ [RPMI 1640 medium (Biowhittaker, Walkersville, MD) supplemented with 10% low-endotoxin FBS (<0.05 EU/ml; Euroclone, Paignton, UK)], stimulated, distributed in either six- or 12-tissue culture well plates (Biowhittaker), and then cultured for 20 min at 37°C, 5% CO2 atmosphere, before cell disruption. Alternatively, neutrophils were cultured in the absence or presence of 10 ng/ml IL-4 (Peprotech, Piscataway, NJ), 100 ng/ml LPS (from Escherichia Coli serotype 026:B6; Sigma, St Louis, MO), 100 U/ml IFN-{gamma} (Hoffman-La Roche, Basel, Switzerland), 10 nM formyl-methionyl-leucyl-phenylalanine (fMLP; Sigma), 10 ng/ml granulocyte macrophage colony stimulating factor (GM-CSF; Peprotech), 50 ng/ml IL-8 (R & D Systems, Minneapolis, MN) or 50 ng/ml leptin for up to 5 h, in either standard culture medium or in serum-free medium on poly(2-hydroxyethyl methacrylate)-covered surfaces, to achieve non-adherent conditions (14), before stimulation for 20 min and cell lysis. The stimuli used were: 200 U/ml IL-10 (DNAX/Schering-Plough, Palo Alto, CA) (13) or 1000 U/ml granulocyte colony stimulating factor (G-CSF, Granulokine; Hoffmann-LaRoche) (14). In selected experiments, neutrophils were cultured with IL-4 or LPS for 4 h in the presence or in the absence of neutralizing anti-IL-4 mAb (kindly provided by Dr D’Ambrosio, Roche Milano Ricerche, Italy), 20 µg/ml cycloheximide (CHX; Sigma), 150 nM Wortmannin, 20 µM Ly294002, 10 µM SB203580 (Alexis Biochemicals, San Diego, CA), 10 µM PD98059 (Calbiochem, La Jolla, CA) or various concentrations of 1-(5'-isoquinolinesulfonyl)-2-methylpiperazine (H-7) (Alexis Biochemicals/Sigma), prior to stimulation with IL-10 or G-CSF and cell lysis. All the reagents used were of the highest available grade and were dissolved in clinical grade pyrogen-free water (14,17).

Immunoblots
For the direct detection of tyrosine-phosphorylated Stat3, detergent lysates were prepared from PMN according to the method previously described (4,14). Usually 35 µg of lysates prepared from PMN were electrophoresed and electroblotted. After blocking, membranes were incubated overnight at 4°C in the presence of the phospho-specific Stat3 antibody (Tyr705) (9131S; Cell Signaling Technology, Beverly, MA) diluted at 1:1000 in blocking buffer. After stripping, membranes were then re-probed with anti-Stat3 (C20, purchased from Santa Cruz Biotechnology, Santa Cruz, CA); 9132, Cell Signaling Technology) diluted 1:2000 in blocking buffer. Antibody binding was detected by using horseradish peroxidase-conjugated anti-rabbit IgG (1:5000 dilution in TBS/T) and revealed using the chemiluminescence system (ECL; Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instructions.

RNA isolation, Northern blot analysis and ribonuclease protection assay (RPA)
Total RNA from PMN was extracted by the guanidinium isothiocyanate method, usually from 6–7 x 107 PMN per condition, and then analyzed by either Northern blotting (17) or by RPA (18). For Northern blot experiments, the filters were hybridized using IL-1ra and actin cDNA fragments (19), previously 32P-labeled using a Ready-to-go DNA labeling kit (Pharmacia, Uppsala, Sweden). For the RPA experiments, a RiboQuant Custom Human Template Set containing probes for IL-10R1, IL-10R2 and GAPDH was used according to the manufacturer’s instructions (BD PharMingen, La Jolla, CA). The extent of hybridization was quantitatively analyzed in an InstantImager (Packard Instruments, Meriden, CT) and plotted after actin normalization. Half-lives of IL-1ra mRNA, under the various experimental conditions, were calculated by regression analysis.

Extracellular staining of IL-10R1 and IL-10R2
Surface expression of IL-10R1 and IL-10R2 in PMN was analyzed by flow cytometry (14). mAb used in these assays were: 3B6 (anti-IL-10R1), 4B2.1 (anti-IL-10R2) (kindly provided by Dr K. Moore and Dr de Waal Malefyt, DNAX) and OKM1 (anti-CR3) (13). Irrelevant purified mouse IgG1 (Sigma) was used as control. Cell staining was performed using 10 µg/ml of the various mAb followed by a biotin-conjugated affinity-purified antibody [goat F(ab')2 anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) pre-adsorbed with human serum] and streptavidin–phycoerythrin (Becton Dickinson, Mountain View, CA) as fluorochrome. Cytofluorographic analyses (using at least 3 x 104 cells per sample) were performed on a FACScan (Becton Dickinson), using CellQuest software. Thresholds were set on control stains.

Cytokine determination
For the experiments aimed to determine IL-1ra production, neutrophils (4 x 106/ml) were suspended in standard culture medium and either pretreated with IL-10 before IL-4 addition or treated with IL-4 for up to 4 h before the addition of IL-10, and then cultured for up to 21 h in 24-tissue culture wells. In selected experiments, neutralizing mouse anti-hIL-10R1 (3B6, IgG1) and anti-hIL-10R2 mAb (1A8.3, IgG1) (kindly provided by Dr K. Moore and Dr de Waal Malefyt, DNAX) (14) were added to the neutrophil cultures 15 min before or after IL-10, as indicated. At predetermined times, cell-free supernatants were harvested and stored at –20°C. The corresponding pellets were washed in ice-cold PBS, snap-frozen in liquid nitrogen and stored at –70°C. Immediately before analysis, the cell pellets were thawed in PBS containing 0.5% NP-40, 5 mM EDTA, 1 mM PMSF, and 5 µg/ml leupeptin and pepstatin A, and then spun (14,000 g, 5 min) to remove cell debris (20). For the release of IL-8, neutrophils were suspended in standard culture medium, pretreated with 10 µM PD98059, 10 µM SB203580 for 15 min before IL-4 or LPS addition and then cultured for 4 h in 24-tissue culture wells (Biowhittaker). Antigenic IL-1ra and IL-8 were measured in the cell-free supernatants and in the cell pellets by using specific ELISA developed with antibodies purchased from commercial sources: Biosource International (Camarillo, CA) for IL-1ra and R & D System (Minneapolis, MN) for IL-8. The detection limits of these ELISA were 50 pg/ml for IL-1ra and 30 pg/ml for IL-8.

Respiratory burst activity
The assay of superoxide anion (O2.–) production, in response to 1 µM fMLP or 2 ng/ml phorbol myristate acetate (PMA), were performed essentially as previously described by Serra et al. (21).

Statistical analysis
Data are expressed as means ± SEM. Statistical evaluation was performed by the Student’s t-test and considered significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Culture with IL-4 renders human neutrophils responsive to IL-10 in terms of Stat3 tyrosine phosphorylation
We have previously reported that IL-10 triggers a strong tyrosine phosphorylation of Stat3 in neutrophils preincubated for 4 h in the presence of LPS, but not in freshly isolated PMN (14). Figure 1 shows that, in addition to LPS, incubation with IL-4 for 4 h also induces a remarkable neutrophil responsiveness to IL-10 in terms of Stat3 tyrosine phosphorylation, whereas other typical PMN agonists, including IL-8, fMLP, IFN-{gamma}, GM-CSF and leptin (not shown), are ineffective. Characterization of the effect of IL-4 revealed that (i) it is specific, since it is abrogated by neutralizing anti-IL-4 mAb (Fig. 1A); (ii) it is unaffected by the absence of serum in the culture medium (Fig. 1B), as in the case of LPS (14); (iii) it is dose dependent, although it does occur within a limited concentration range (Fig. 1C); and (iv) it requires at least 3–4 h to be fully apparent (not shown). In addition, tyrosine phosphorylation of Stat3 in response to IL-10 is already evident by 5 min, reaches a maximum at 30 min and is still detectable at 60 min (not shown). Importantly, Stat3 tyrosine phosphorylation in response to IL-10, but not to G-CSF, was consistently abrogated if IL-4-treated neutrophils were cultured in the presence of the protein synthesis inhibitor, CHX (Fig. 1D). Taken together, these data prove that IL-4 is very effective in rendering neutrophils responsive to IL-10 in terms of Stat3 tyrosine phosphorylation and that its effect relies on de novo protein synthesis, similarly to that previously shown using LPS (14).



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  		Fig. 1. Characterization of IL-10-mediated Stat3 tyrosine phosphorylation in neutrophils cultured with IL-4. (A) PMN, cultured for 4 h with or without 10 ng/ml IL-4 in the presence or the absence of 10 µg/ml neutralizing anti-IL-4 mAb, were stimulated with 200 U/ml IL-10 for 20 min before lysis. (B) PMN, cultured for 4 h either in standard or in serum-free medium with or without IL-4 or 100 ng/ml LPS, were stimulated with IL-10 for 20 min before lysis. (C) PMN were cultured for 4 h with various concentrations of IL-4 and then stimulated with IL-10 for 20 min before lysis. (D) PMN, cultured for 4 h with or without IL-4 in the presence or absence of 20 µg/ml CHX, were stimulated with IL-10 or 1000 U/ml G-CSF for 20 min before lysis. As a positive control, freshly isolated PMN were also stimulated with G-CSF. Then 35 µg of lysate was loaded on the gels and immunoblots performed using antibodies specific for tyrosine-phosphorylated forms of Stat3. Subsequent re-blotting with the indicated anti-Stat3 antibodies was carried out to ensure that similar amounts of material were loaded on each lane. The data for each panel are representative of at least two independent experiments.

 
IL-4 up-regulates IL-10R1 mRNA and protein expression in neutrophils
Because IL-10R1 expression is known to play a critical role in IL-10-mediated responses (14,22,23), we investigated whether a possible up-regulation of IL-10R1 mRNA and surface expression might be involved in the IL-4-mediated induction of IL-10 responsiveness. Figure 2 shows that culture of PMN for up to 180 min in medium containing IL-4 significantly increases the gene expression of IL-10R1, at levels comparable (but slightly lower) to those induced by LPS. By contrast, IL-10R2 mRNA levels show only minimal variation between freshly isolated and cultured PMN (Fig. 2), consistent with previous results (14). Indirect immunofluorescence flow cytometry (FACS) analysis (Fig. 3A and B) revealed that surface expression of IL-10R1 also becomes significantly up-regulated in neutrophils cultured for 4 h, whereas surface expression of IL-10R2 or CR3 (CD11b/CD18) (not shown) does not. However, the difference in IL-10R1 surface expression between cells cultured for 4 h in medium alone or in the presence of IL-4 does not seem proportional to the marked changes in IL-10R1 gene expression under the same conditions. The latter observations perhaps indicate that although the proportion of neutrophils expressing IL-10R1 slightly increases after 4 h of exposure to IL-4 in culture, those cells expressing IL-10R1 do express it more strongly. Taken together, our data demonstrate that IL-10R1 expression is markedly increased in IL-4-cultured neutrophils.



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  		Fig. 2. Up-regulation of IL-10R1 mRNA accumulation in IL-4-treated neutrophils. PMN, either freshly isolated (as indicated by ‘0’) or cultured for the times indicated in the presence or the absence of IL-4 or LPS, were lysed for RNA extraction and processed for RPA analysis. Total RNA (5 µg) was loaded on each gel lane. This experiment is representative of three.

 


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  		Fig. 3. IL-4 up-regulates IL-10R1 surface expression neutrophils. (A) Neutrophils, in whole blood or cultured for 4 h in serum-free medium in the presence or IL-4, were stained with anti-IL-10R1, anti-IL-10R2, anti-CR3 and control mAb, before indirect immunofluorescence analysis. The experiment depicted is representative of four. (B) Bars depict MFI ± SEM from four independent experiments performed as described above. MFI intensity was calculated by subtracting the MFI of the IgG1-stained PMN from the corresponding IL-10R-stained cells. Asterisks denote MFI significantly different from that of circulating PMN (*P < 0.05; **P < 0.01).

 
Involvement of H-7-sensitive pathways in IL-4- and LPS-induced neutrophil responsiveness to IL-10 and IL-10R1 up-regulation
IL-4-, as well as LPS-, dependent responses are mediated through various signaling pathways, including activation of phosphatidylinositol-3' kinase (PI3 kinase), p38 mitogen-activated protein kinase (p38 MAPK) and p42/44 extracellular signal-regulated protein kinase (ERK) MAPK, and serine/threonine protein kinases (24,25). To start investigating if, and which one(s) of, these signaling pathways could play a role in the IL-4- (and LPS)-induced PMN responsiveness to IL-10, we assessed the effects of inhibitors of PI3 kinase (Wortmannin and Ly294002) (26,27), ERK and p38 MAPK (PD98059 and SB203580 respectively) (28,29), and serine/threonine kinases (H-7) (30). For this purpose, neutrophils were cultured for 4 h with IL-4 or LPS in the presence or the absence of the various inhibitors, and then stimulated with IL-10 or G-CSF for 20 min. Under these conditions, Wortmannin, Ly294002, PD98059 and SB203580 did not influence Stat3 tyrosine phosphorylation responses to IL-10, even though PI3 kinase inhibitors completely suppressed the ability of fMLP to trigger O2.– release (31) and ERK/p38 MAPK inhibitors dramatically reduced the ability of LPS to elicit the secretion of IL-8 (32) (data not shown). In sharp contrast, tyrosine phosphorylation of Stat3 in response to IL-10 (Fig. 4A and B), but not to G-CSF (Fig. 4B), was completely inhibited by the presence of 20 µM H-7 in the neutrophil cultures. However, if H-7 was added at the end of the 4-h incubation period 30 min prior to IL-10 stimulation, then the IL-10-induced tyrosine phosphorylation of Stat3 in either LPS- (Fig. 4C) or IL-4-cultured neutrophils (data not shown) displayed resistance to the drug, whereas the ability of PMA to trigger O2.– release was, as expected (33), completely suppressed (data not shown). Importantly, Fig. 4(D) demonstrates that in LPS-treated PMN, as well as in IL-4-treated cells (not shown), H-7 dramatically suppresses the induction of surface IL-10R1. Altogether, the data indicate that H-7 does not directly interfere with the signaling events triggered by the IL-10 receptor, but rather prevents the specific ability of both IL-4 and LPS to up-regulate IL-10R1 expression and therefore render PMN responsive to IL-10.



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  		Fig. 4. Effect of H-7 on the induction of neutrophil responsiveness to IL-10 by IL-4 and LPS. (A) PMN, cultured for 4 h with or without IL-4 or LPS in the presence or the absence of various concentrations of H-7, were stimulated with IL-10 for 20 min before lysis. (B) PMN, cultured for 4 h with or without LPS in the presence or the absence of 20 µM H-7, were stimulated with IL-10 or G-CSF for 20 min. (C) PMN were cultured with LPS for 4 h before addition of 20 µM H-7: after 30 min, cells were stimulated with IL-10 for 20 min. The data for each panel are representative of at least two independent experiments. (D) PMN, after a 4-h culture in serum-free medium with or without LPS, in the presence or absence of 20 µM H-7, were stained with anti-IL-10R1, anti-IL-10R2, control mAb and analyzed by FACS. This experiment is representative of three.

 
IL-10 synergistically enhances the production of IL-1ra in IL-4-treated neutrophils
To identify a possible biological implication of our observations, we investigated whether IL-10 could influence the well-established capacity of IL-4 to directly induce the release of IL-1ra by PMN (34,35). As shown in Fig. 5, IL-4-elicited IL-1ra extracellular production was strongly enhanced in PMN co-stimulated with IL-10, this enhancing effect of IL-10 being already remarkable after 7 h and maximal at 21 h of incubation (62 ± 20%, n = 6). As reported earlier (19,35), IL-10 consistently fails to exert any direct effect upon the basal IL-1ra release measured in unstimulated cells, even after 21 h (Fig. 5). Interestingly, the synergism between IL-4 and IL-10 on IL-1ra release (measured after 21 h of culture) did not significantly vary in its extent even if IL-10 was added to the cells 3.5 h after IL-4 (dashed line of Fig. 5).



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  		Fig. 5. IL-10 synergistically enhances the production of IL-1ra in IL-4-treated neutrophils. (A) PMN were either pretreated with IL-10 before IL-4 addition or treated with IL-4 for 3.5 h before IL-10 addition and then cultured for up to 21 h. Cell-free supernatants were then collected at the indicated time points and the levels of IL-1ra determined by ELISA. The experiment depicted is representative of three.

 
Effect of IL-10 on the IL-1ra mRNA expression, synthesis and secretion in IL-4-stimulated neutrophils
That IL-10 could modulate the production of IL-1ra in response to IL-4 prompted us to determine whether its action might involve changes in IL-1ra mRNA accumulation. Figure 6(A) shows that IL-4 up-regulates the steady-state levels of IL-1ra mRNA in a time-dependent manner, with a peak at 3–4 h, followed by a return to near-baseline levels by 21 h. Co-incubation of IL-4-treated PMN with IL-10 had no significant effect on IL-4-elicited enhancement of IL-1ra mRNA accumulation up to 4 h, while, at later time points, the amounts of IL-1ra mRNA transcripts were maintained at much higher levels by IL-10 (Fig. 6A). To elucidate the mechanisms whereby IL-10 affects the accumulation of IL-1ra transcripts in IL-4-treated PMN, we examined the influence of IL-10 on IL-1ra mRNA stability (19). PMN were thus stimulated with IL-4 for 7 h, in the presence or absence of IL-10, and then treated with actinomycin D to block the formation of additional transcripts. At increasing intervals thereafter, the cultures were processed for Northern analysis, and changes in the amount of IL-1ra and actin mRNA were quantitated by InstantImager scanning. Figure 6(B) shows that IL-10 prolongs the half-life of IL-1ra mRNA relative to IL-4-only-treated cells (163 versus 80 min respectively). In contrast, IL-10 did not affect the stability of actin mRNA isolated from IL-4-treated neutrophils (not shown).



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  		Fig. 6. Effect of IL-10 on the IL-1ra mRNA expression, synthesis and secretion in IL-4-stimulated neutrophils. (A) PMN were incubated for the times indicated with or without IL-4, in the presence or the absence of IL-10, before total RNA extraction and Northern blot analysis for IL-1ra mRNA expression. The panel shows a quantitative representation of IL-1ra mRNA accumulation plotted after actin normalization by InstantImager analysis. The experiment depicted is representative of three. (B) Quantitative representation of IL-1ra mRNA decay. PMN were cultured with IL-4 in the presence or the absence of IL-10. After 7 h, actinomycin D (10 µg/ml) was added and the cells were further cultured as indicated. Total RNA was extracted and analyzed by Northern blot for IL-1ra mRNA expression. The extent of hybridization was quantified and half-lives were calculated by regression analysis. The experiment depicted is representative of two. (C) PMN were incubated with or without IL-4 for 21 h, in the presence or absence of IL-10. The cell-free supernatants and the corresponding pellets were harvested and the levels of IL-1ra determined by ELISA. The mean values ± SEM of the total production of IL-1ra, depicted as cell-associated and released, from three independent experiments are shown.

 
Since the potentiation of IL-1ra release in IL-4-treated neutrophils by IL-10 was accompanied by a corresponding change in IL-1ra mRNA expression, we investigated whether IL-10 might also enhance the synthesis or secretion of IL-1ra. To this end, we measured, in parallel, both the IL-1ra immunoreactivity which remained cell-associated and that which was released by the cells. Figure 6(C) shows that following incubation of neutrophils in the presence of IL-4, the total amount of IL-1ra protein was higher than that of untreated cells, in keeping with the effect of IL-4 on IL-1ra mRNA accumulation. Addition of IL-10, either at the same time of IL-4 or after 4 h, dramatically enhanced the total synthesis of IL-1ra, this effect being already evident after 7 h (not shown) and maximal at 21 h (Fig. 6C). Interestingly, the percentages of secreted IL-1ra by neutrophils stimulated with IL-4 alone or with IL-4 in combination with IL-10 were substantially similar (Fig. 6C), suggesting that IL-10 enhances only the synthesis, but not the secretion of IL-1ra.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The findings presented in this study establish, for the first time, that IL-10 and IL-4 synergize in inducing the production of IL-1ra by human neutrophils. In fact, whereas the spontaneous release of IL-1ra into the culture supernatants by unstimulated PMN was not significantly affected by the presence of IL-10 (19,35), the release of IL-1ra protein determined by IL-4-stimulated neutrophils was greatly potentiated by IL-10. Such an enhancing effect of IL-10 required at least 6–7 h to be significant and, if measured after an overnight culture, it did not substantially change in its extent even though IL-10 was added 4 h after IL-4. The latter observations suggested that IL-4-treated neutrophils require some time before becoming susceptible to the regulatory effects of IL-10 and were consistent with the fact that a fully functional IL-10 receptor complex is not present in freshly isolated PMN (14). In this regard, we (14), and others (22,23), have recently shown that IL-10R1 expression plays a critical role in determining whether cells fully respond to IL-10 or not. Herein, we demonstrate that, in a 4-h period, IL-4 significantly up-regulates IL-10R1 mRNA and surface expression, and, in parallel, enhances the responsiveness of neutrophils to IL-10 in terms of Stat3 tyrosine phosphorylation (14). That the increase of surface IL-10R1 is necessary to initiate the synergism between IL-10 and IL-4 was demonstrated not only by the kinetics of its appearance, which strictly correlated with the ability of IL-10 to potentiate the production of IL-1ra induced by IL-4, but also by the effect of neutralizing anti-IL-10R1 and anti-IL-10R2 antibodies, which both displayed a dramatic suppression on the IL-10-mediated enhancement of IL-1ra release, even if added to the cell cultures up to 4–5 h after IL-10 (not shown). However, because the acquisition of responsiveness to IL-10 in terms of Stat3 tyrosine phosphorylation was dependent on de novo protein synthesis, at present we cannot exclude that, in addition to IL-10R1, other unidentified polypeptides contributing to such responsiveness are also synthesized in IL-4-treated neutrophils.

The observations described in this work are strongly reminiscent of the results recently obtained with neutrophils incubated with LPS (14), which are also induced to synthesize and express higher levels of IL-10R1 than freshly isolated cells, and as a result become fully responsive to IL-10 (as assessed by Stat3 tyrosine phosphorylation, SOCS-3 expression and modulation of cytokine production) (14). On the basis of these similarities, we would tend to speculate that both IL-4 and LPS utilize the same molecular cascade(s) to render neutrophils responsive to IL-10. Further evidence supporting this consideration derives from the experiments performed with pharmacological inhibitors of signal transduction pathways that are known to be implicated in either IL-4- or LPS-mediated gene responses (24,25,36), including Wortmannin and Ly294002 (inhibitors of PI3 kinase activation), PD98059 and SB203580 (blocking p38 MAPK and ERK MAPK activation respectively), and H-7 (a broad inhibitor of serine/threonine protein kinases). We found that both IL-4- and LPS-induced neutrophil responsiveness to IL-10 (as revealed by activation of Stat3 tyrosine phosphorylation) displayed an identical sensitivity to these drugs, in the sense that it was completely prevented only by H-7, and unaffected by Wortmannin, Ly294002, PD98059 and SB203580. Since the suppressive effect of H-7 was not caused by its direct effect of the IL-10R-induced immediate signaling events, but rather by a strong inhibition of IL-10R1 up-regulation, our data suggest that H-7 interferes with the IL-4- and LPS-induced signaling cascade(s) controlling the expression of the IL-10R1 gene. In this respect, the literature in this field reports many examples documenting that gene regulation by both IL-4 and LPS may be dependent on H-7-sensitive pathways. For instance, it has been shown that H-7 inhibits IL-4-regulated expression of CD38 (37), CD23 (38,39) or cytochrome P4502E1 (CYP2E1) (40). Similarly, in other studies, the regulatory signals initiated by LPS to modulate the gene expression of IL-1{alpha}/ß and tumor necrosis factor (TNF)-{alpha} (41), tissue factor (42), type IV collagenases/gelatinases (43), and lysozyme (44) have been shown to be negatively influenced by H-7. We did not explore the nature of the specific signal-transduction molecule(s) that is/are affected by H-7 in our IL-4- or LPS-treated neutrophils (a likely candidate might be protein kinase C), but certainly the identification of the target(s) for the inhibitory effect of H-7 will provide an important insight into the signaling regulating IL-10R1 transcription.

In this study, we also investigated the molecular mechanisms underlying the effects of IL-10 on IL-1ra gene expression in IL-4-cultured neutrophils. In agreement with previous observations (34), IL-4 proved to be a late inducer of IL-1ra production in neutrophils, insofar as IL-1ra mRNA levels were only up-regulated after 2–3 h of stimulation and the secreted protein only became detectable at later time points. We found that the potentiation of IL-1ra release by IL-10 observed in IL-4-stimulated neutrophils was accompanied by a maintenance of high levels of IL-1ra mRNA transcripts and by a marked enhancement of IL-4-induced IL-1ra formation. In addition, we found that IL-10 increased the stability of IL-1ra mRNA, but did not alter the levels of IL-1ra secretion. While our results do not exclude an effect of IL-10 at the level of IL-1ra gene transcription, our data are in agreement with previous observations demonstrating that IL-10 markedly potentiates the mRNA and extracellular yield of IL-1ra in LPS- and in TNF-{alpha}-stimulated neutrophils respectively via a prolongation of IL-1ra mRNA half-life (19) and via an increased IL-1ra novel synthesis (35). Whether IL-10-induced activation of Stat3 tyrosine phosphorylation in IL-4-cultured neutrophils is necessary for initiating the signaling cascade that leads to the up-regulation of IL-1ra mRNA accumulation or synthesis remains, however, to be determined. Our preliminary experiments indicate that TNF-{alpha}, in a 4-h culture period, also renders neutrophils potently responsive to IL-10 in terms of Stat3 tyrosine phosphorylation. On the other hand, it is intriguing that, despite the fact that IL-10 activates Stat3 tyrosine phosphorylation and DNA-binding activities even in neutrophils cultured for 4 h under cytokine-free conditions (14), IL-1ra mRNA expression or production is never induced (14,35), therefore arguing against the suggestion for a role of Stat3 tyrosine phosphorylation in regulating IL-1ra gene expression. Further studies are therefore necessary to clarify this issue.

IL-4 and IL-10 are cytokines with potent anti-inflammatory activities, and represent major immunoregulatory molecules playing a central role in Th2-mediated immunity (45). Although both IL-4 and IL-10 have been reported to modulate several PMN responses, including phagocytosis (10,46), respiratory burst activity (10,46), production of arachidonic acid metabolites (10,47,48), chemotaxis and degranulation (46), cytoskeletal rearrangements (49), and apoptosis (10,49), they modulate neutrophil activation primarily by the inhibition of pro-inflammatory cytokine and chemokine production (10,50). In addition, IL-4 has been shown to directly stimulate the synthesis of several cytokine inhibitors by neutrophils, such as IL-1ra, and the soluble IL-1 receptor type II, a receptor that acts as a decoy for IL-1 (51). The findings reported in this study on the ability of IL-4 to also prepare neutrophils to fully respond to IL-10, emphasize the peculiar characteristic of IL-4 to act in concert with specific cytokine inhibitors and soluble cytokine receptors in order to down-regulate the inflammatory response. Our data also support the view that a combination of anti-inflammatory cytokines could be more effective in up-regulating the mechanisms that physiologically limit the extent of inflammatory reactions. The use of IL-4 in combination with IL-10 may thus have a more relevant therapeutic benefit for those diseases characterized by massive neutrophil infiltration, e.g. rheumatoid arthritis or inflammatory bowel diseases (52).


   Acknowledgements
 
We would like to thank Dr K. W. Moore and Dr R. de Waal Malefyt (DNAX/Schering-Plough, Palo Alto, CA) for kindly providing us with IL-10, and anti-hIL-10R1 and anti-hIL-10R2 mAb, and Dr C. Laudanna for his invaluable criticisms and suggestions. This work was supported by grants from MURST (COFIN and 60% funds), Fondazione Cassa di Risparmio di Verona, AIRC and ‘Consorzio per lo Studio e lo Sviluppo degli Studi Universitari di Verona’.


   Abbreviations
 
CHX—cycloheximide

ERK—extracellular signal-regulated protein kinase

FMLP—formyl-methionyl-leucyl-phenylalanine

IL-1ra—IL-1 receptor agonist

G-CSF—granulocyte colony stimulating factor

GM-CSF—granulocyte macrophage colony stimulating factor

LPS—lipopolysaccharide

MAPK—mitogen-activated protein kinase

PI3 kinase—phosphatidylinositol-3' kinase

PMA—phorbol myristate acetate

PMN—polymorphoneutrophil

RPA—ribonuclease protection assay

SOCS—suppressor of cytokine signaling

TNF—tumor necrosis factor


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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