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(Journal of Leukocyte Biology. 2003;73:145-154.)
© 2003 by Society for Leukocyte Biology

Adherence influences monocyte responsiveness to interleukin-10

Anne-France Petit-Bertron, Catherine Fitting, Jean-Marc Cavaillon and Minou Adib-Conquy

UP Cytokines & Inflammation, Institut Pasteur, Paris, France

Correspondence: Minou Adib-Conquy, UP Cytokines & Inflammation, Institut Pasteur, 28 rue Dr Roux, 75015 Paris, France.E-mail: madib{at}pasteur.fr


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ABSTRACT
 
We studied the effects of adherence on the properties of interleukin (IL)-10 on monocyte-enriched peripheral blood mononuclear cells. We found that the decrease of CD11b expression induced by IL-10 was enhanced by adherence. Toll-like receptor (TLR)2 and TLR4 mRNA, as well as TLR4 surface expression, were significantly up-regulated by IL-10 in adherent cells. The absence of adherence prevented the inhibitory effects of IL-10 on lipopolysaccharide-induced tumor necrosis factor (TNF) and granulocyte-colony stimulating factor production and increased IL-1ß production and soluble TNF receptor II release in IL-10-pretreated cells. Similarly, the absence of adherence amplified the enhancement of phagocytosis induced by IL-10. Tyk2 and signal transducer and activator of transcription 3 (STAT3) phosphorylation and suppressor of cytokine signaling 3 (SOCS3) expression were induced by IL-10 in both conditions, but a longer activation and/or expression were observed in adherent monocytes. Finally, heme oxygenase-1, an anti-inflammatory molecule, was induced by IL-10 in adherent monocytes, whereas its expression remained low in nonadherent cells. Altogether, these data illustrate that adherence modulates the properties and the anti-inflammatory effects of IL-10.

Key Words: inflammation • cytokines • endotoxin • monocytes/macrophages • human • Toll-like receptors • protein kinase • signal transduction • HLA-DR


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INTRODUCTION
 
Adherence is an important, initial step in the transition of a circulating monocyte to a tissue macrophage. During the process of extravasation, monocytes transiently adhere to capillary endothelium and subsequently to a variety of extracellular matrix components. These early interactions probably serve as modifiers of transcriptional activity and cellular reactivity. Numerous in vitro experiments have regularly reported that adherence selectively activates the expression of various genes. This was shown particularly for some cytokines, including interleukin (IL)-1{alpha} and IL-1ß [1 , 2 ], IL-8 [3 ], tumor necrosis factor (TNF) [4 ], and macrophage-colony stimulating factor (M-CSF) [5 ]; for growth factors, such as platelet-derived growth factor [6 , 7 ] and insulin-like growth factor-1 [7 ]; for proto-oncogens, such as c-fos and c-jun [6 ]; and for cell-surface markers, such as CD18 [7 ]. Similarly, adherence induces the release of ß-glucuronidase and cellular plasminogen activator [8 ], the generation of superoxide [8 ], and the expression of tissue-factor procoagulant activity [9 ]. Furthermore, adherence modifies monocyte responsiveness to activating signals. For example, formyl-Met-Leu-Phe-induced changes of the cytoplasmic calcium concentration were higher in monocytes selected by adherence and maintained adherent to plastic than in monocytes isolated by negative selection and kept nonadherent on Teflon® [10 ]. Although adherence is sufficient to induce high, steady-state levels of mRNA of different genes, the actual secretion of the mediators requires the exposure to a second signal such as endotoxin. This was particularly well-established for the release of IL-1ß and TNF [11 , 12 ]. In addition, lipopolysaccharide (LPS)-induced IL-8 production by monocytes is potentiated by adherence [13 ], LPS-induced IL-6 production is faster, and LPS-induced granulocyte (G)-CSF production is down-regulated in adherent cells [14 , 15 ]. Accordingly, the activation of some components involved in cellular signaling is modified by adherence. This has been recently established for the extracellular signal-regulated kinases (ERK) 1/2, members of the mitogen-activated protein kinase (MAPK) family classically associated with cell differentiation, and may be important in cytokine production [16 , 17 ]. Surprisingly, few investigations have addressed the influence of adherence on the responsiveness of monocytes to cytokine-induced signaling. It was reported that adherence potentiates the production of IL-8 upon stimulation of monocytes by TNF or IL-1 [13 ], but the effects of adherence on the reactivity of monocytes to deactivating cytokines have not been studied. We previously reported a priming effect of IL-10 on spontaneous interleukin-1 receptor antagonist production and on LPS-induced TNF and IL-6 production when adherence of monocytes was prevented by cultures of whole blood samples or by cultures of peripheral blood mononuclear cells (PBMC) on Teflon® [18 ]. TNF mRNA expression induced by LPS was decreased when the pretreatment of PBMC with IL-10 was performed on plastic, whereas this was not the case when the cells were precultured with IL-10 on Teflon®. Furthermore, nuclear factor (NF)-{kappa}B translocation following LPS activation was moderately increased after IL-10 pretreatment on Teflon®, whereas it was decreased on plastic. In the present study, we further characterized the modulatory properties of IL-10 on monocytes depending on adherent or nonadherent cell-culture conditions. We analyzed the effects of IL-10 and adherence on the secretion of other cytokines, on phagocytosis, on the expression of several surface molecules including Toll-like receptor (TLR)2 and TLR4, and on IL-10-induced intracellular signaling.


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MATERIALS AND METHODS
 
Preparation of monocyte-enriched PBMC and culture conditions
PBMC were prepared from fresh blood samples of healthy donors drawn on citrate/phosphate/dextrose (Etablissement Français du Sang, Paris, France). Blood was diluted 1:2 in RPMI-1640 medium (Glutamax, Gibco-Life Technologies, Paisley, UK) and centrifuged over Ficoll (MSL, Eurobio, Les Ulis, France) for 20 min at 15°C and 600 g. Monocytes were prepared from the PBMC population by aggregation at 4°C, followed by rosetting with sheep red blood cells (Biomerieux, Marcy l’Etoile, France), as described previously [19 ]. Monocyte enrichment was assessed by CD14 expression analyzed by fluorescence-activated cell sorter (FACS), and monocyte content was 84.5 ± 1.2%. Monocyte-enriched PBMC were adjusted to 1 x 106 cells/ml in RPMI-1640 medium supplemented with antibiotics and 5% of a heat-inactivated pool of normal human serum. Monocyte-enriched PBMC were precultured in 24-well or 6-well multidish plates (Costar, Corning, NY) in the presence or absence of recombinant human (rh)IL-10 (10 ng/ml; Genzyme, Cambridge, MA) for 20 h at 37°C in a 5% CO2 incubator. At the end of the preculture, nonadherent cells were collected, each well was washed twice with RPMI-1640 medium, and fresh medium was added to plastic dishes to preserve adherent cell viability. The harvested, nonadherent cells were washed once and resuspended in RPMI-1640 medium supplemented with antibiotics, indomethacin (1 µg/ml), and 0.2% normal human serum and were plated in the same wells as during the preculture period after removal of the RPMI-1640 medium. Monocyte-enriched PBMC were futher cultured for a 20-h period at 37°C and 5% CO2 in the presence or absence of Escherichia coli LPS (0111: B4) at 1 µg/ml (Sigma Chemical Co., St. Louis, MO).

Monocyte-enriched PBMC were also precultured in Teflon® containers (PolyLabo, Strasbourg, France). At the end of the preculture period, the cells were washed, plated, and cultured in 24-well or 6-well multidishes for 20 h with or without LPS as describe above. At the end of the second culture period, the supernatants were harvested, centrifuged at 300 g for 10 min at 15°C, and kept at -20°C until cytokine assays were performed.

Cytokine measurements
Cytokines were measured in culture supernatants using specific in-house or commercial enzyme-linked immunosorbent assay (ELISA): TNF in-house ELISA [20 ], IL-1ß, and G-CSF (Duoset, R&D Systems, Minneapolis, MN) and soluble TNF receptor II (sTNFRII; ELISA kit, R&D Systems).

Phagocytosis
PBMC were precultured in the presence or absence of IL-10 (10 ng/ml for 20 h on plastic or Teflon®) as described above. For plastic, the cells were washed twice, resuspended in RPMI-1640 medium supplemented with 5% heat-inactivated normal human serum, and incubated in the same 24-well multidish plates for 1 h 30 min at 37°C. Cells cultured on Teflon® were washed twice and transferred to plastic 24-well multidish plates for 1 h 30 min at 37°C. After incubation, the adherent cells were washed twice, and incubated with fluorescein isothiocyanate (FITC)-coupled latex beads (Interfacial Dynamics Corp., Portland, OR; 50 beads/cell) in 250 µl RPMI-1640 medium supplemented with 2% heat-inactivated normal human serum at 37°C or 4°C for 1 h. The adherent cells were recovered after a 5-min (for 37°C) or 20-min (for 4°C) incubation in phosphate-buffered saline (PBS)–1% bovine serum albumin (BSA)–1.3 mM EDTA. They were washed and counted, and 1 x 105 cells were incubated with an anti-CD14 monoclonal antibody (mAb) coupled to phycoerythrin (PE) as described below. Data were collected on 10,000 cells with a FACScan analyzer (Becton Dickinson, San Jose, CA). The green fluorescence was analyzed after gating on CD14-positive cells. Results are expressed as percent of positive cells and as mean fluorescence ratio = mean fluorescence of sample (37°C)/mean fluorescence of control (4°C).

FACS analysis of surface markers
PBMC were cultured in the presence or absence of IL-10 (10 ng/ml) for 20 h on plastic or Teflon® as described above. PBMC cultured in the Teflon® containers were recovered by centrifugation. For the plastic conditions, adherent cells were recovered after a 5-min incubation in PBS–1% BSA–1.3 mM EDTA, pooled with corresponding, nonadherent cells, and centrifuged. PBMC were counted, and 5 x 105 cells were used per sample. Double-staining was performed using an anti-CD14 mAb (MY4-RD2; Coulter-Immunotech, Miami, FL) coupled to PE and an anti-CD11a (25.3.1; Coulter-Immunotech), an anti-CD11b (BEAR1; Coulter-Immunotech), an anti-human leukocyte antigen (HLA)-DR (Immu-357; Coulter-Immunotech), an anti-CD62L (Dreg 56; Coulter-Immunotech), an anti-CD40 (5C3; PharMingen, San Diego, CA), an anti-TLR2 (a kind gift of Dr. Terje Espevik, Trondheim, Norway), or an anti-TLR4 (a kind gift of Dr. Kensuke Miyake, Saga, Japan) mAb coupled to FITC. A mouse immunoglobulin G (IgG)1–FITC (MOPC-2; Sigma Chemical Co.) and an IgG2b–PE (MOPC-141; Sigma Chemical Co.) were used as isotype controls. For IL-10R detection, we used IL-10 coupled to biotin followed by an incubation with streptavidin–FITC (detection kit, fluorokine, R&D Systems). Cells were incubated on ice with mAb in PBS–1% BSA for 30 min and washed in PBS–1% BSA, and data were collected on 10,000 cells with a FACScan analyzer (Becton Dickinson). The expression of the different molecules on monocytes was analyzed after gating on side- and forward-scatter and on CD14-positive cells. Results are expressed as percent of positive cells and as mean fluorescence intensity (MFI).

Isolation of RNA and reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA from PBMC was extracted using the RNA plus reagent (Bioprobe Systems, Montreuil-sous-Bois, France) and was treated with RNase-free DNase I. Reverse transcription was performed on 1 µg total RNA in 20 µl using the omniscript RT (Qiagen, Courtaboeuf, France) and an oligo-dT primer (Promega, Madison, WI). RT-PCR was performed on 2 µl cDNA using a Taq DNA polymerase (Qiagen) and primers specific for human TLR2, TLR4 [21 ], MD-2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The following sense (S) and antisense (AS) were used: TLR2, (S) 5'-GCCAAAGTCTTGATTGATTGG-3' and (AS) 5'-TTGAAGTTCTCCAGCTCCTG-3'; TLR4, (S) 5'-TGGATACGTTTCCTTATAAG-3' and (AS) 5'-GAAATGGAGGCACCCCTTC-3'; MD-2, (S) 5'-TTCCACCCTGTTTTCTTCCA-3' and (AS) 5'-TAGGTTGGTGTAGGATGACA-3'; and GAPDH, (S) 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and (AS) 5'-CATGTGGGCCATGAGGTCCACCAC-3'. The RT-PCR conditions were the following: 40 s at 95°C, 40 s at a gene-specific, annealing temperature, and 1 min at 72°C repeated 28 times. The annealing temperatures for each primer set were as follows: TLR2 and GAPDH 54°C, TLR4 50°C, and MD-2 58°C. RT-PCR products were separated on a 2% agarose gel containing ethidium bromide, and densitometric analysis was performed using the National Institutes of Health (NIH) Image software (Bethesda, MD). Values for TLR2, TLR4, and MD-2 were normalized against GAPDH. As negative control, RT-PCR was performed with 100 ng RNA instead of cDNA.

Immunoprecipitation and Western blot analysis of Tyk2
Four million monocyte-enriched cells were lysed with RIPA buffer [200 mM NaCl, 50 mM Tris-HCl, pH 8, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.03% sodium dodecyl sulfate (SDS), 2 mM EDTA, freshly added protease, and phosphatase inhibitors]. The insoluble material was removed by centrifugation at 12,000 g for 15 min at 4°C. The supernatants were incubated with an anti-Tyk2 antibody (R5–6, a kind gift of Dr. S. Pellegrini, Institut Pasteur, Paris) overnight at 4°C on a rotor. Protein A-sepharose (30 µl 50%; Amersham Biosciences, Orsay, France) was then added to the samples and incubated for an additional hour. The immunoprecipitates were washed three times with lysis buffer and once with PBS and were boiled for 5 min in SDS sample buffer. The samples were resolved on a 7% SDS-polyacrylamide gel electrophoresis (PAGE) gel and electroblotted to nitrocellulose sheets (Hybond C, Amersham Biosciences). Protein transfer was ascertained by ponceau red coloration. Membranes were then washed with PBS and blocked with PBS containing 0.1% Tween 20 (Sigma Chemical Co.) and 3% nonfat dry milk (PBS-T-MLK) for 1 h at room temperature. The membranes were washed and incubated with an antiphosphotyrosine antibody (clone 4G10; Euromedex, Mundolsheim, France) for 1 h at room temperature in PBS-T-MLK. The membranes were then washed and incubated with a peroxidase-labeled goat anti-mouse Ig (a kind gift of Dr S. Pellegrini) for 1 h at room temperature. After five washes, blots were developed using enhanced chemiluminescence-plus (Amersham Biosciences). The same membranes were then stripped and reprobed with an anti-Tyk2 antibody (clone T10–2; a kind gift of Dr. S. Pellegrini) and followed by the same peroxidase-labeled antibody. Densitometric analysis was performed on the Western blots using the NIH Image software.

Whole-cell extracts and Western blot analysis of signal transducer and activator of transcription (STAT)3, suppressor of cytokine signaling (SOCS)3, and heme oxygenase (HO)-1
Whole-cell extracts were prepared at T0 (untreated cells) and after incubation of monocyte-enriched PBMC with IL-10 on Teflon® or plastic for 20 min, 3 h, and 5 h. Cells on Teflon® were harvested, centrifuged, and washed twice with PBS. Cells on plastic were also washed twice with PBS and harvested with a cell scraper in 100-µl extraction buffer, and whole-cell extracts were prepared as described previously [22 ]. Whole-cell extracts (6 µg) were subjected to SDS-PAGE and transferred onto nitrocellulose sheets (Hybond C, Amersham Biosciences). Protein transfer was ascertained by ponceau red coloration. Membranes were then washed with PBS and blocked with PBS-T and 0.5% gelatin for 1 h at room temperature. Antiphosphotyrosine-STAT3 (Cell Signaling, Beverly, MA) Western blot was performed according to the manufacturer’s instructions. The same membranes were then stripped and reprobed with anti-STAT3 (Cell Signaling). Western blot was also performed using an anti-SOCS3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or an anti-HO-1 antibody (StressGen, Victoria, Canada). Densitometric analysis was performed on the Western blots using the NIH Image software.

Statistical analysis
The results are given as means ± SEM. Statistical analysis was performed using the nonparametric Wilcoxon signed-rank test for Teflon® versus plastic conditions or presence versus absence of IL-10. The surveys of STAT3, SOCS3, or HO-1 levels after incubation with IL-10 were compared at T0, 20 min, 3 h, and 5 h using a Friedman test and the Statview software. P < 0.05 was considered the minimal level of significance.


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RESULTS
 
Effects of IL-10 on TNF, IL-1ß, and G-CSF production and on sTNFRII release by monocyte-enriched PBMC
We have previously reported that LPS-induced TNF production by human PBMC was inhibited by a 20-h preculture in the presence of rhIL-10 (10 ng/ml) on plastic, whereas on Teflon®, TNF production was increased [18 ]. In the present study, we analyzed the effects of adherence and IL-10 on the production of TNF as well as other inflammatory cytokines, such as IL-1ß or G-CSF in response to LPS by monocyte-enriched PBMC. As shown in Figure 1 , TNF production by monocyte-enriched PBMC was also inhibited by IL-10 on plastic but not on Teflon®. Similarly, preincubation with IL-10 inhibited G-CSF production significantly in response to LPS on plastic, whereas on the contrary, G-CSF production was increased on Teflon®. Surprisingly, IL-10 pretreatment failed to inhibit IL-1ß production in response to LPS on plastic. However, on Teflon®, IL-10 pretreatment was followed by a fivefold increase in IL-1ß production. We also analyzed the effects of IL-10 on the release of the sTNFRII. We observed that similar to IL-1ß, IL-10 pretreatment had no effect on the release of sTNFRII in response to a subsequent LPS stimulation on plastic. In contrast, it significantly increased the release of this soluble receptor when the pretreatment was performed on Teflon®.



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  		Figure 1. TNF, G-CSF, IL-1ß, and sTNFRII release in response to LPS by monocyte-enriched PBMC, precultured for 20 h in adhering (plastic) or nonadhering (Teflon®) conditions in the presence or absence of IL-10. The results are the mean of 11 experiments performed with different donors.*, P < 0.05 (absence vs. presence of IL-10); {lozenge}, P < 0.05 (plastic vs. Teflon®) using the Wilcoxon signed-rank test.

Effects of IL-10 on phagocytosis
It is well known that IL-10 favors the phagocytic activity of monocytes [23]. To determine if adherence could modulate the effects of IL-10 on this activity, we analyzed the phagocytosis of fluorescent latex beads by monocytes after incubation of PBMC with IL-10 on Teflon® or plastic. Figure 2A shows a representative FACScan analysis. The peaks in the left part of the histogram represent nonphagocytosing cells and those in the right part, the monocytes that have phagocytosed one or several latex beads. In the right part, the first peak corresponds to one phagocytosed bead, the second peak corresponds to two phagocytosed beads, and so on. As shown in Figure 2A , without IL-10, we observed some phagocytosis of latex beads when the cells were cultured at 37°C but not at 4°C, a temperature that does not allow phagocytosis. The data obtained at 4°C correspond to the attachment of the fluorescent latex beads onto the surface of the cells. Incubation with IL-10 increased the phagocytosis on plastic and Teflon®. Figure 2B shows that IL-10 pretreatment enhanced the number of cells undergoing phagocytosis (left) and the number of latex beads per monocyte (right), as shown by an increased MFI. These increases were found to be more pronounced on Teflon® and were significantly higher than on plastic.



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  		Figure 2. Influence of adherence on the effects of IL-10 on the phagocytic capacity of monocytes. (A) One representative FACScan analysis of the phagocytosis of FITC-labeled latex beads by monocytes (CD14+ cells) derived from PBMC cultured for 20 h in the presence or absence of IL-10 (10 ng/ml) on plastic or Teflon®. Incubations with latex beads were performed at 37°C (black lines) or 4°C (gray lines) to distinguish between attachment and phagocytosis. (B) On the left panel, the percent of increase of phagocytosis, and on the right panel, the percent increase of MFI observed with IL-10. The results are the mean of five experiments performed with different donors. *, P < 0.05 (plastic vs. Teflon®) using the Wilcoxon signed-rank test.

Effects of IL-10 on cell-surface marker expression
We have shown that adherence is an important factor that modulates the effects of IL-10. Thus, we analyzed the impact of adherence on the modulation by IL-10 of the expression of several adhesion molecules (CD11a, CD11b, CD62L), of a costimulatory molecule (CD40), of HLA-DR, and finally of the IL-10R itself. Table 1 shows the percentage of positive cells for these markers. In the absence of IL-10, no differences were found between the two culture conditions for the expression of CD11a, CD62L, and IL-10R. In contrast, significant differences were found for the expression of CD11b, CD40, and HLA-DR. In the presence of IL-10, no modulation was observed for the expression of the IL-10R under either condition. The down-regulation of CD40 and the up-regulation of CD62L induced by IL-10 did not reach statistical significance, probably because heterogeneous results were obtained from one donor to another. However, MFI of CD62L was significantly decreased on plastic versus Teflon® after IL-10 incubation (P=0.027; data not shown). CD11a, CD11b, and HLA-DR surface expression were significantly decreased by IL-10 under both conditions. Nevertheless, we noticed that the down-regulation of CD11b induced by IL-10 was significantly stronger on plastic (-26%±6) than on Teflon® (-7%±3). No differences were found for the MFI of these markers (data not shown).


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  		Table 1. Cell-Surface Marker Expression on Monocytes (CD14-Positive Cells) Cultured for 24 h in Adhering (Plastic) or Nonadhering (Teflon®) Conditions in the Absence or Presence of IL-10 (10 ng/ml)

Effects of IL-10 on TLR
We investigated whether the effects of IL-10 on the production of TNF in response to LPS could be correlated with the modulation of the expression of TLR4 and MD-2, molecules involved in LPS-induced signaling. We monitored the expression of the mRNA of TLR4 and MD-2 by RT-PCR. As a control, we analyzed the expression of TLR2, a receptor that is involved in Gram-positive bacteria-induced signaling. Figure 3A shows one representative experiment and Figure 3B , the mean densitometric analysis of seven experiments. The expression of TLR4 and TLR2 mRNA increased in response to IL-10 on plastic but not on Teflon®. A similar result was observed with MD-2 but did not reach significance. We then analyzed the effects of IL-10 and adherence on the surface expression of TLR2 and TLR4 on monocytes. As shown in Figure 3C , 3IL-10 had no effect on TLR2 surface expression. In contrast, TLR4 expression was increased on monocytes cultured with IL-10 on plastic but not on Teflon®. However, in the absence of IL-10, the surface expression of TLR4 was significantly higher on monocytes cultured on Teflon® as compared with plastic.



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  		Figure 3. Modulation of the expression of TLR2, TLR4, and MD-2 by IL-10 after 20 h of culture on plastic (P) or Teflon® (T). A representative RT-PCR experiment is shown (A). (B) The densitometric analysis of TLR2, TLR4, and MD-2 normalized against GAPDH (mean±SEM of seven independent experiments). (C) The MFI of TLR2 and TLR4 expression on monocytes (CD14+ cells) analyzed by FACScan (mean±SEM of five different experiments). *, P < 0.05 (absence vs. presence of IL-10); {lozenge}, P < 0.05 (plastic vs. Teflon®) using the Wilcoxon signed-rank test.

Effects of IL-10 on the activation of Tyk2 and STAT3 and on the induction of SOCS3
IL-10 signals through the Janus tyrosine kinase (Jak)-STAT pathway. IL-10 induces the phosphorylation and activation of Jak1, Tyk2, and STAT3. These activation pathways result in SOCS3 transcription, which is one of the inhibitory factors of the Jak-STAT pathway. To ensure that we specifically addressed cellular signaling within monocytes, the experiments were performed with monocyte-enriched PBMC. We ascertained that the IL-10 pretreatment was unable to down-regulate the production of TNF by monocyte-enriched PBMC cultured on Teflon®, whereas it inhibited it on plastic. We analyzed the effect of adherence on the kinetics of Tyk2 and STAT3 activation and on SOCS3 induction by IL-10. As shown in Figure 4A and 4B , IL-10 induced the phosphorylation of Tyk2 after 5 min of incubation in both conditions. Tyk2 phosphorylation persisted after 15 min of incubation on Teflon® and decreased afterward. On plastic, the phosphorylation of Tyk2 induced by IL-10 increased until 45 min and remained high after 90 min. In parallel, we investigated the effect of adherence itself on the phosphorylation of Tyk2. As shown in Figure 4C , in the absence of IL-10, a transient activation of Tyk2 was induced by adherence: Tyk2 phosphorylation was maximal at 15 min, decreased at 45 min, and remained diminished at 90 min, whereas in the presence of IL-10, a plateau was observed between 15 and 90 min. Thus, on plastic, Tyk2 phosphorylation resulted from both signals induced by adherence and IL-10.



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  		Figure 4. Kinetics of Tyk2 phosphorylation induced by IL-10 on plastic or Teflon®. (A) A representative experiment and (B) the ratio between the phosphorylated form and total Tyk2 analyzed by densitometry (mean±SEM of three independent experiments). (C) The ratio between the phosphorylated form and total Tyk2 in monocytes cultured on plastic in the presence or absence of IL-10 (one representative of two additional experiments).

IL-10 induced STAT3 activation with a maximum of phosphorylation after 20 min in both conditions (Fig. 5 ). However, on Teflon®, STAT3 phosphorylation was down-regulated at 3 h and remained low at 5 h, whereas on plastic, STAT3 activation persisted at 3 h, and high levels of phosphorylated STAT3 were still detected 5 h after addition of IL-10. Indeed, at 3 h and 5 h, significantly higher amounts of STAT3 were detected in monocytes cultured on plastic than in those cultured on Teflon®.



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  		Figure 5. Kinetics of STAT3 phosphorylation induced by IL-10. The left part of the figure shows a representative experiment and the right part, the ratio between the phosphorylated form and total STAT3 analyzed by densitometry (mean±SEM of five independent experiments). A significant difference was seen between the values at T0 and those at 20 min (20mn; 20'), 3 h, and 5 h (Friedman test, P=0.02 on Teflon® and P=0.008 on plastic). {lozenge}, P < 0.05 (plastic vs. Teflon®) using the Wilcoxon signed-rank test.

In addition, we analyzed the expression of SOCS3 after incubation of monocytes with IL-10 in adherent or nonadherent conditions (Fig. 6 ). SOCS3 was detected after 3 h on plastic and Teflon®. Its expression decreased after 5 h on Teflon® but not on plastic. The amounts of SOCS3 found on Teflon® at 5 h were significantly lower than those expressed at 3 h in the same culture conditions. Furthermore, after 5 h of incubation with IL-10, significantly higher amounts of SOCS3 were found in monocytes cultured on plastic as compared with Teflon®. In the absence of IL-10, adherence alone had no effect on STAT3 activation and SOCS3 expression (data not shown).



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  		Figure 6. Kinetics of SOCS3 induction by IL-10. The upper part of the figure shows a representative experiment, and the lower part shows the mean ± SEM of the densitometric analysis of five independent experiments. A significant difference was seen between the values at T0 and those at 20 min (20'), 3 h, and 5 h (Friedman test, P=0.03 on Teflon® and P=0.007 on plastic). *, P < 0.05 (3 h vs. 5 h); {lozenge}, P < 0.05 (plastic vs. Teflon®) using the Wilcoxon signed-rank test.

Effects of IL-10 on HO-1 expression
Finally, we analyzed the effects of adherence on the induction by IL-10 of HO-1, a molecule that has been shown to mediate the anti-inflammatory effect of IL-10 in mice [24 ]. As shown in Figure 7 , HO-1 was not found in unstimulated monocyte-enriched PBMC. Incubation with IL-10 induced HO-1 on plastic after 20 min, and its expression continued to increase at 3 h and 5 h. In contrast, in the absence of adherence, IL-10 was a poor inducer of HO-1, and even after 5 h, low levels of this molecule were detected on Teflon®.



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  		Figure 7. Kinetics of HO-1 induction by IL-10. The upper part of the figure shows a representative experiment and the lower part, the mean ± SEM of the densitometric analysis of four independent experiments. A significant difference was seen between the values at T0 and those at 20 min (20'), 3 h, and 5 h on plastic (Friedman test, P=0.017) but not on Teflon®.


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DISCUSSION
 
IL-10 is widely recognized as a potent anti-inflammatory and immunosuppressive cytokine. Nevertheless, it has been shown that IL-10 stimulates 10.7% of the 1300 genes tested in macrophages [25 ], and its action on monocytes promotes their maturation to macrophages [26 ]. Indeed, IL-10 has divergent properties on monocytes. For example, IL-10 increases the surface expression of CD16 and CD64 [27 , 28 ], whereas it decreases that of CD23 and B7 [29 30 31 ]. IL-10 increases phagocytosis [23 ] and up-regulates the production of monocyte chemoattractant protein-1 (MCP-1) [32 ] and of the chemoattractant S100A8 protein [33 ] but inhibits the production of IL-1, IL-6, IL-8, TNF, GM-CSF, and G-CSF [34 ].

We had previously shown that PBMC, first cultured in the presence of IL-10 on Teflon® to prevent adherence, when further cultured in plastic dishes in the presence of LPS or IL-1ß, released enhanced levels of TNF, whereas this was not the case when PBMC were precultured in plastic multidishes in the presence of IL-10 [18 ]. These results correlated with differential TNF mRNA expression following LPS activation and higher NF-{kappa}B translocation in cells pretreated by IL-10 in the absence of adherence. We previously investigated the expression of CD16 and CD68 on the CD14-positive cells. An enhanced frequency of CD16- and CD68-positive cells was observed in the presence of IL-10, independently of adherence. In the present study, we have enlarged the panel of cell-surface markers and focus our attention on adhesion and costimulatory molecules. Adherence itself enhanced the expression of HLA-DR and CD40 and decreased that of CD11b. As already reported [28 ], IL-10 decreased the expression of HLA-DR, and adherence did not influence this property. Only the IL-10-induced reduction of CD11b expression was more markedly observed with adherent cells than in the absence of adherence.

IL-10 pretreatment differently affected the LPS-induced production of TNF, G-CSF, and IL-1ß and the release of sTNFR by monocytes, depending on the presence or absence of adherence. On plastic, pretreatment with IL-10 led to a reduced production of TNF and G-CSF and an enhanced release of sTNFR, similar to the data obtained when IL-10 is added simultaneously with LPS [34 , 35 ]. However, in contrast to the down-regulation of LPS-induced IL-1ß production by simultaneous addition of IL-10 [34 ], pretreatment with IL-10 led to an enhanced IL-1ß production. On Teflon®, in the absence of adherence, pretreatment with IL-10 never led to a reduced production of cytokines, and LPS-induced IL-1ß production was far higher than that obtained when IL-10 pretreatment occurred on plastic. These results illustrate that several parameters may modify the signals delivered by IL-10. The effect of IL-10 differs depending on the nature of the produced cytokines. For example, it was shown that IL-10 reduced LPS-induced macrophage-inflammatory protein-1ß (MIP-1ß) production but did not modify the MIP-1{alpha} production [36 ]. The timing is another important parameter: It has been reported that simultaneous addition of IL-10 with T cell activators reduced IL-2 production by T cell clones, whereas an IL-10 pretreatment enhanced this production [37 ]. Finally, the nature of the studied cells influences the regulation of cytokine production: IL-10 inhibited LPS-induced MCP-1 production by human monocytes, whereas it enhanced this production by alveolar macrophages [32 ]. The increased cytokine production induced by IL-10 has also been reported in vivo in humans, in patients with Crohn’s disease [38 ], or in volunteers injected with LPS [39 ]. This may parallel the proinflammatory effects of IL-10 observed in LPS-induced uveitis [40 ] and in many in vivo animal models [41 42 43 ].

We had previously shown that the priming effect of IL-10 on Teflon® was independent of the expression of CD14, the cell-surface LPS-binding molecule, which was increased after IL-10 pretreatment on Teflon® and plastic [18 ]. In the present study, we showed that IL-10 and adherence significantly enhanced the expression of TLR2 and TLR4 mRNA, and it was not the case in the absence of adherence. There was no direct correlation between the modulation of mRNA expression and surface expression, as we failed to detect any significant changes of TLR2 surface expression by IL-10, regardless of the culture conditions. In contrast, pretreatment by IL-10 of adhering monocytes led to an enhanced surface expression of TLR4. This observation reflects the capacity of cytokines to modulate surface expression of TLR4 on monocytes: Interferon-{gamma}, IL-2, and M-CSF up-regulate, and IL-4 down-regulates TLR4 expression [44 , 45 ], whereas GM-CSF has minimal effect [46 ]. Thus, the different reactivity of monocytes in response to LPS after IL-10 pretreatment is not linked to the level of surface expression of TLR4. This result is reminiscent of other studies that failed to demonstrate a link between the level of TLR4 expression and the intensity of response to LPS [47 , 48 ]. Furthermore, TLR4 needs CD11b/CD18 for optimal activation of LPS-inducible genes [49 ], as demonstrated by the fact that macrophages derived from CD11b/CD18-deficient mice had a lower activation of MAPK and NF-{kappa}B in response to LPS than macrophages from normal mice. In this study, we found that CD11b expression was strongly down-regulated by IL-10 on plastic but only moderately on Teflon®. Accordingly, the higher expression of CD11b may contribute to the priming effect of IL-10 in nonadhering conditions.

Among the various activities modulated by IL-10, we investigated its effect on phagocytosis and the influence of adherence. We confirmed previous observations that IL-10 enhances phagocytosis [23 , 50 ]. We further demonstrated that the effects of IL-10 were significantly more pronounced when cells had been treated in the absence of adherence. To extrapolate our in vitro data, we may suggest that the enhancing effect of IL-10 on phagocytosis is more efficient in the blood compartment than within tissues. To illustrate our hypothesis, it is worth mentioning the observation by Koedel et al. [51 ], who showed that systemic but not intrathecally administration of IL-10 had beneficial effects on Streptococcus pneumoniae-induced meningitis. IL-10 has been shown to be beneficial or deleterious in various infectious models. Its ambiguous role may reflect the capacity of IL-10 to enhance phagocytosis, associated with the ability to diminish microbicidal activity [52 ]. Furthermore, the role of IL-10 may vary throughout the time course of infection. For instance, IL-10 impaired early resistance to Listeria monocytogenes but favored complete clearance [53 ].

Finally, we analyzed the effect of adherence on IL-10-induced signaling. The IL-10R is constituted by two chains (IL-10R1 and IL-10R2) [54 ]. IL-10 interacts with its receptor as a homodimer, binding to two adjoining IL-10R1 molecules. This interaction leads to the phosphorylation of the tyrosine kinase Jak1, recruited by the IL-10R1 chain, and Tyk2, recruited by the IL-10R2 chain [55 ]. As a consequence, two tyrosine residues of the IL-10R1 chain are phosphorylated, and together with their flanking peptides, they serve as temporary docking sites for a latent cytosolic transcription factor STAT3. Following phosphorylation and homodimerization, STAT3 translocates to the nucleus where it binds to a specific promoter gene present in the gene of SOCS3 [56 ]. It was reported that adherence alone is sufficient to induce significant activation of ERK and transcription factors AP-1 [16 ] and STAT1 [57 ]. In the present study, we demonstrated that adherence influences the kinetics of Tyk2 and STAT3 phosphorylation and SOCS3 production induced by IL-10. Adherence itself did not enhance the activation and/or production of STAT3 and SOCS3 but allowed a longer expression of these factors in response to IL-10. In contrast, adherence alone induced Tyk2 phosphorylation and also modified its kinetics of activation in response to IL-10. The longer activation of Tyk2 following cumulative effects of adherence and IL-10 could explain as a consequence the longer activation of STAT3 and the longer expression of SOCS3. The reduced anti-inflammatory capacities of IL-10 on nonadherent monocytes may also be linked to the expression of HO-1. Indeed, recently, a role of HO-1 in the anti-inflammatory action of IL-10 has been demonstrated in mice [24 ]. HO-1 was shown to mediate the suppression of LPS-induced TNF production in mouse macrophages, and this effect was STAT3- and SOCS3-independent. We found that HO-1 was also induced by IL-10 in human monocytes cultured on plastic, whereas its expression remained low in the absence of adherence. Thus, the low induction of HO-1 by IL-10 on Teflon® may contribute to the absence of inhibition of LPS-induced TNF and G-CSF production. Our results further suggest that the initiation of different signaling pathways by adherence and IL-10 may result in global signaling, different from that induced by IL-10 alone, as previously shown for adherence and LPS [17 ].

In conclusion, as noted by Moore and colleagues [58 ]: "IL-10 can effect very different outcomes depending on timing, dose, and location of expression; in some scenarios the expected immunosuppressive activities are observed, while in others IL-10 enhances immune or inflammatory response", as elegantly illustrated throughout their review. Indeed, some undesired, proinflammatory effects were observed when IL-10 was injected to healthy volunteers, alone or in combination with LPS [39, 59] . In the present report, we demonstrate that adherence is one of the events that modulates the properties of IL-10.


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ACKNOWLEDGEMENTS
 
Part of this work and A-F. P-B. were supported by a grant from the "Association Vaincre la Mucoviscidose." The authors gratefully acknowledge Prof. Kensuke Miyake (Saga, Japan) and Dr. Terje Espevik (Trondheim, Norway) for the kind gifts of anti-TLR4 and anti-TLR2 mAb, respectively; Dr. Jerome Pugin (Geneva, Switzerland) for MD-2 primer sequences; and Dr. Sandra Pellegrini (Institut Pasteur, Paris, France) for the kind gift of anti-Tyk2 antibodies and valuable advice on immunoprecipitation experiments.

Received August 7, 2002; revised October 3, 2002; accepted October 13, 2002.


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N. Beaufort, D. Leduc, J.-C. Rousselle, V. Magdolen, T. Luther, A. Namane, M. Chignard, and D. Pidard
Proteolytic Regulation of the Urokinase Receptor/CD87 on Monocytic Cells by Neutrophil Elastase and Cathepsin G
J. Immunol., January 1, 2004; 172(1): 540 - 549.
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