Paradoxical priming effects of IL-10 on cytokine production
Minou Adib-Conquy,
Anne-France Petit,
Christelle Marie,
Catherine Fitting and
Jean-Marc Cavaillon
Unité d'Immuno-Allergie, Institut Pasteur, 28 rue Dr Roux, 75015 Paris, France
Correspondence to:
J.-M. Cavaillon
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Abstract
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IL-10 is a well-known immunosuppressive and/or anti-inflammatory cytokine. However, we report in vitro experimental studies in which IL-10 primed leukocytes and led to an enhanced production of tumor necrosis factor (TNF) upon further stimulation by lipopolysaccharide (LPS). Monocytes and peripheral blood mononuclear cells (PBMC) prepared from whole blood maintained for 20 h at 37°C in the presence of recombinant human IL-10 had an enhanced capacity to produce TNF in response to LPS. In addition to TNF, LPS-induced IL-6 and spontaneous IL-1ra production were also enhanced. When isolated PBMC were first cultured for 20 h in the presence of IL-10 on Teflon to prevent adherence, washed to remove IL-10 and then further cultured in plastic dishes for an additional 20 h in the presence of LPS or IL-1ß, an enhanced release of TNF was observed. This was not the case when PBMC were pre-cultured in plastic multidishes in the presence of IL-10. TNF mRNA expression induced by LPS was decreased when the pre-treatment of PBMC with IL-10 was performed on plastic, whereas this was not the case when cells were pre-cultured with IL-10 on Teflon. Furthermore, NF
B translocation following LPS activation was higher after IL-10 pre-treatment on Teflon than on plastic. Interestingly, an enhanced frequency of CD16 and CD68+ cells among the CD14+ cells was observed in the presence of IL-10, independently of the pre-culture conditions of the PBMC. Altogether, these results indicate that the IL-10-induced up-regulation of cytokine production depends on the prevention of monocyte adherence by red cells in the whole blood assays or by cultures of PBMC on Teflon. In contrast, the adherence parameter has no effect on the IL-10-induced modulation of some monocyte surface markers.
Keywords: cytokine production, IL-10
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Introduction
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IL-10 has been identified as a cytokine synthesis inhibitory factor capable of suppressing the production of Th1 cytokines including IL-2 and IFN-
(14). In addition, IL-10 is capable of inhibiting the production of most cytokines produced by monocytes/macrophages (5,6) and neutrophils (PMN) (79). Thus, IL-10 is known to be an immunosuppressive as well as a potent anti-inflammatory mediator. While many in vitro experimental models have established the anti-inflammatory properties of IL-10, several in vivo situations suggested that in some circumstances, IL-10 could act as a pro-inflammatory and/or an immunostimulatory cytokine. This was observed in graft rejection (10-12), ocular inflammation (13), auto-immune diseases (14,15) or anti-tumor immunity (16). Indeed, a very clear pro-inflammatory activity was shown following the implant of adenocarcinoma-transfected cells expressing IL-10 (17). Furthermore, during phase I trials of IL-10 in healthy humans, some unexpected results emerged including fever and leukocytosis (18,19).
Some reports have shown that IL-10 could also favor certain aspects of the inflammatory response. Thus, IL-10 induces E-selectin expression on small and large blood vessel endothelial cells (20). IL-10 may behave differently depending on the nature of the target cells. For example, a consistent or even enhanced production of IL-8 has been reported by dendritic cells (21) and endothelial cells (22), while suppression of IL-8 production has been described in monocytes and PMN (59). Similarly, IL-10 inhibits IL-6 release by lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC), whereas it does not interfere with cytokine production by activated human endothelial cells (23). Furthermore, IL-10 can repress the production of NO by macrophages or keratinocytes (24,25), does not modify its release by mesangial cells (26), and even enhances the production of NO by bone marrow-derived macrophages and osteoclasts (27,28). The inhibitory capacity of IL-10 may also depend on the nature of the triggering agent. For example, IL-10 represses the LPS-induced IL-8 production by neutrophils while it does not when PMN are activated by tumor necrosis factor (TNF)-
(9). Furthermore, IL-10 could act differently depending on the state of maturation and/or activation of the target cells or depending on the sequence of signaling. So far, this has been essentially demonstrated for IL-4 (2931), but a study on blood and sputum neutrophils also showed a differential responsiveness of these cells to IL-10 (32). In the present study we demonstrate that monocytes and PBMC prepared from blood samples maintained overnight in the presence of IL-10 had an enhanced capacity to produce TNF-
upon stimulation. This finding could be mimicked by pre-incubating PBMC with IL-10 as long as adherence was prevented by pre-cultures on Teflon. To further analyze the underlying mechanisms of the observed phenomenon, TNF mRNA expression and NF
B translocation have been investigated.
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Methods
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Whole blood pre-culture
Whole blood samples from healthy donors (Etablissement de transfusion sanguine de l'Assistance Publique, Paris, France), collected on CPD (citrate/phosphate/dextrose), were maintained in culture flasks (75 cm2; Costar, Cambridge, MA) in the absence or presence of recombinant human IL-10 (10 ng/ml) (DNAX, Palo Alto, CA/Genzyme, Cambridge, MA) for 20 h at 37°C in a 5% CO2 incubator. At the end of this pre-culture period, whole blood samples were diluted 1:3 in RPMI 1640 medium (Glutamax; Gibco Life Technologies, Paisley, UK) and layered on Ficoll-Hypaque (MSL; Eurobio, Les Ulis, France). The ratio was 2 volumes of blood to 1 volume of MSL. After centrifugation for 30 min at 15°C and 600 g, PBMC were washed twice in RPMI 1640 medium. PBMC (6x106 PBMC/ml or adjusted to 106 non-specific esterase+ cells/ml) were suspended in RPMI 1640 medium supplemented with antibiotics (100 IU/ml penicillin; 100 µg/ml streptomycin), 0.2% heat-inactivated normal human serum (a pool of sera from healthy volunteers) and 1 µg/ml indomethacin, and incubated in 24-well multidish plates (Costar) (0.5 ml/well). In the case of adherent cells, non-adherent cells were removed after a 2 h incubation at 37°C and fresh complete medium was added. PBMC and adherent cells were incubated for 20 h in the presence or absence of various stimuli. Stimuli used were Escherichia coli 0111:B4 LPS (2 µg/ml; Sigma, St Louis, MO), Neisseria meningitidis LPS (2 µg/ml; a kind gift of Dr M. Caroff), recombinant human IL-1ß (20 ng/ml; Rhône Poulenc, France) and Staphylococcus aureus Cowan I (200 µg/ml; Pansorbin, Calbiochem, La Jolla, CA). At the end of the culture period, supernatants were harvested, centrifuged for 10 min at 15°C and 300 g, and kept at 30°C until cytokine assessments.
PBMC pre-culture
PBMC were prepared from freshly sampled blood of healthy donors, diluted 1:2 in RPMI 1640 medium and centrifuged over Ficoll for 20 min at 15°C and 600 g. After washings, PBMC were adjusted to 6x106 cells/ml in RPMI 1640 medium supplemented with antibiotics and 5% (unless otherwise mentioned) heat-inactivated normal human serum or autologous plasma. PBMC were pre-cultured in 24-well multidish plates (0.5 ml/well) in the absence or presence of recombinant human IL-10 (10 ng/ml) for 20 h at 37°C in a 5% CO2 incubator. At the end of the pre-culture, PBMC were collected, each well was washed twice with 1 ml RPMI 1640 medium and 1 ml of fresh medium was added to plastic dishes to preserve adherent cell viability. The harvested non-adherent cells were washed once and resuspended in 0.5 ml RPMI 1640 medium supplemented with antibiotics, indomethacin and 0.2% normal human serum, and plated in the same wells as during the pre-culture period after removal of the RPMI 1640 medium. PBMC were further cultured for a 20 h period at 37°C and 5% CO2 in the presence or absence of various cell activators.
PBMC were also pre-cultured in Teflon containers (2 ml/container) (PolyLabo, Strasbourg, France). At the end of the pre-culture period, PBMC were harvested and Teflon containers washed. PBMC were resuspended, plated and cultured in 24-well multidishes as described above. At the end of the second culture period, the supernatants were harvested, centrifuged for 10 min at 15°C and 300 g, and kept at 30°C until cytokine assays were performed.
Cytokine measurements
Cytokines were measured using specific in-house or commercial ELISA: TNF-
(33), IL-6 (34), IL-1ra and IL-10 (R & D Systems, Abingdon, UK).
Apoptosis assay
PBMC apoptosis was assessed according to the percentage of cells containing hypodiploid DNA by using the technique described by Nicoletti et al. (35). After 20 h of pre-culture of blood, PBMC were prepared and cells were centrifuged at 200 g for 10 min and washed in PBS. The cell pellets were gently resuspended in hypotonic fluorochrome solution (50 µg/ml propidium iodide, 0.1% sodium citrate, 0.1% Triton X-100) and stored at 4°C in the dark overnight before the flow-cytometric analysis using a FACScan flow cytometer (Becton Dickinson Immunocytometry System, San Jose, CA). The red fluorescence of propidium iodide in individual nuclei, the forward scatter and the side scatter were simultaneously measured. Cell debris was excluded from acquisition by raising the forward scatter threshold. Apoptotic nuclei were easily distinguishable from residual debris by the high side scatter value due to the condensation of nuclear chromatin. A total of 10,000 events was collected and analyzed using the software Lysys II program. Apoptotic nuclei were distinguished by their hypodiploid DNA content from the diploid DNA content of normal nuclei.
Electrophoretic mobility shift assay (EMSA)
Nuclear protein extractions were carried out as described (36). PBMC were washed once with PBS, adherent cells were harvested with a cell scraper, added to non-adherent cells and suspended in buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and 0.1% NP-40) supplemented with protease inhibitors. The protease inhibitors included: 0.5 mM PMSF, 25 µg/ml aprotinin, 10 µg/ml chymostatin, 2 µg/ml antipain, 8 µg/ml pepstatin, 10 µg/ml leupeptin, 0.1 mg/ml
1-antitrypsin and 0.5 mM 3,4-dichloroisocoumarin (all from Sigma). Cells were incubated for 10 min at 4°C and then centrifuged for 2 min at 10,000 r.p.m. The pellet was suspended in buffer C and incubated for 20 min at 4°C. Buffer C contained 20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT and protease inhibitors. Cells were then centrifuged for 10 min at 14,000 r.p.m., the supernatant corresponding to the nuclear extract was harvested and kept at 80°C. A double-stranded oligonucleotide containing the NF
B motif (promega) was end-labeled with T4 kinase in the presence of [
-32P]ATP. Extracts (4 µg) were incubated in the binding buffer for 15 min at room temperature (4% Ficoll, 20 mM HEPES, pH 7, 35 mM NaCl, 60 mM KCl, 0.01% NP-40, 2 mM DTT, 0.1 mg/ml BSA and 1.5 µg/µl salmon sperm DNA). After 15 min, the radiolabeled nucleotide was added (100,000 c.p.m.) and the mixture was again incubated for 15 min at room temperature. EMSA was performed in a 5% acrylamide gel in 0.5xTBE, and gels were dried and subjected to autoradiography. The NF
B complexes were quantified using a PhosphorImager and the ImageQuant software (Molecular Dynamics). The composition of the NF
B complexes was assessed by supershift assays using specific antibodies (anti-p50 and anti-p65; Santa Cruz Biotechnology, Santa Cruz, CA). The upper EMSA band was identified as a p50p65 complex, while the second one was identified to be a p50 homodimer (data not shown).
RNA dot-blot
Total RNA from PBMC was extracted using the RNA PLUS reagent (Bioprobe Systems, Montreuil sous bois, France). Several dilutions of each sample were put onto Gene Screen Plus membranes (Dupont, Boston, MA) using a dot-blot apparatus (Minifold I; Schleicher & Schuell, Dassel, Germany) and hybridized with 32P-labeled TNF-
and ß-actin probes kindly provided by Dr N. Haeffner-Cavaillon (INSERM U430, Hôpital Broussais). The human TNF-
and the mouse ß-actin cDNA probes were 1.18 and 0.97 kb PstI fragments respectively. The transfer and the hybridization conditions were those recommended by the manufacturer for Gene Screen membranes. After hybridization, the membranes were washed and the radioactivity was quantified using a PhosphorImager and the ImageQuant software.
FACS analysis
Cells were cultured in the absence or the presence of IL-10 (10 ng/ml) for 20 h, using the whole blood assay, as well as isolated PBMC on plastic or Teflon as previously described. At the end of the pre-incubation, PBMC were isolated from whole blood using Ficoll Hypaque, PBMC cultured in the Teflon containers were recovered by centrifugation. For the plastic condition, adherent cells were recovered after a 5 min incubation in PBS/BSA 1%/EDTA 1.3 mM, pooled with corresponding non-adherent cells and centrifuged. PBMC were washed with PBS/BSA 1%, counted and 1x106 cells were used per sample. Double staining was performed using an anti-CD14 mAb (MY4-RD1; Coulter, Miami, FL) coupled to phycoerythrin (PE) and an anti-CD16 mAb (3G8; Immunotech, Marseille, France) or an anti-CD68 mAb (KP1; Dako, Glostrup, Denmark) coupled to FITC. Mouse IgG1FITC (MOPC-21; Sigma) and IgG2bPE (MOPC-141; Sigma) were used as isotypic controls. Cells were incubated on ice with mAb in PBS/BSA for 30 min, washed with PBS/BSA and data were collected on 10,000 cells in a FACScan analyzer (Becton Dickinson). The monocyte population was analyzed after gating on side and forward scatters. Results are expressed as percent of positive cells and as mean fluorescence ratio (MFR = mean fluorescence of sample/mean fluorescence of control).
Statistical analysis
Statistical analysis has been performed using the non-parametric Wilcoxon signed-rank test.
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Results
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Reactivity of adherent mononuclear cells isolated from IL-10 pretreated blood samples
In a first set of experiments, LPS-induced TNF-
production by human monocytes was investigated with adherent cells prepared from blood samples maintained in culture for 20 h either in the absence or in the presence of recombinant human IL-10. The results depicted in Fig. 1
indicate that increased amounts of TNF were detected in the supernatants of monocytes prepared from IL-10 pre-treated blood samples as compared to adherent cells derived from blood maintained for 20 h in the absence of IL-10. Fourteen different donors were studied and the observation was highly reproducible (p < 0.001). The minimum timing of IL-10 pre-incubation in the whole blood samples leading to the enhanced TNF production was 14 h (data not shown). While this long period suggests that protein synthesis might be involved in the observed phenomenon, preliminary experiments to demonstrate it with cycloheximide failed due to a long-lasting effect of the drug which later on interfered with the cytokine synthesis induced upon activation (data not shown). A wide range of TNF levels was observed consistent with previous findings due to the wide genetic diversity seen with human cells (37). The mean TNF increase of 3.3-fold was not due to a difference in the number of apoptotic cells since a similar total number of PBMC and a similar number of apoptotic PBMC (11 ± 4%) were recovered from blood samples maintained for 20 h at 37°C in the absence or presence of IL-10. However, we noticed an enhanced proportion (42 ± 13%) of adhering monocytes recovered after incubation onto plastic of PBMC prepared from the blood samples supplemented with IL-10.
Reactivity of PBMC isolated from IL-10 pretreated blood samples
Although the observed enhancement of adherent cells was far below the enhancement of the TNF production, it could reflect a selection of a highly reactive subpopulation. So, we decided to pursue the analysis without selecting the adherent cells. As shown in Fig. 2A
, similar enhancement of TNF production was observed when whole PBMC were cultured and activated for an additional 20 h after being prepared from blood in which increasing amounts of IL-10 had been added. The enhancement was independent of the bacterial origin of the LPS as well as when IL-1 was employed as a triggering signal. The presence of low (0.2%) or high (2%) concentrations of normal human serum or the addition of indomethacin influenced the amounts of produced TNF
but did not modify the IL-10 induced effect (Fig. 2B
). Fourteen new different experiments have been performed with PBMC and a fixed amount of recombinant human IL-10 (10 ng/ml). The mean enhancement of Escherichia coli LPS-induced TNF production by PBMC derived from blood maintained for 20 h in the presence of IL-10 was 3.4 ± 0.9-fold (P = 0.016). Although high LPS concentrations (12 µg/ml) have been used throughout the study, the amplificatory activity of IL-10 was also observed with lower concentrations of LPS (1100 ng/ml) (Table 1
). The mean enhancements were 2.1 ± 0.4-, 2.6 ± 0.5- and 3.4 ± 1.1-fold for 1, 10 and 100 ng/ml respectively. A weaker amplificatory activity was found when S. aureus Cowan was used as TNF-
inducer (1.3 ± 0.2-fold).
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Table 1. TNF- production (pg/ml), upon activation by increasing amounts of E. coli LPS, by PBMC isolated from blood samples maintained for 20 h at 37°C in the presence or absence of IL-10 (10 ng/ml)
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To assess whether the enhancing effect of the IL-10 pre-treatment was specific for TNF, we also investigated its role on IL-6, IL-1ra and IL-10 production. As shown in Table 2
, the pre-culture of whole blood in the presence of IL-10 significantly increased the E. coli LPS-induced IL-6 production by PBMC (P = 0.03) and to a lesser extent that induced by N. meningitidis LPS (P = 0.07). Such IL-10 pre-treatment greatly increased the spontaneous IL-1ra production as well as the LPS-induced IL-1ra. The S. aureus Cowan-induced IL-6 and IL-1ra productions were not significantly enhanced. IL-10 production was not affected.
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Table 2. IL-6, IL-10 and IL-1ra production (pg/ml) by PBMC derived from blood maintained for 20 h in the absence or presence of IL-10 (10 ng/ml)a
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Reactivity of PBMC following an IL-10 pretreatment in Teflon containers
We and others have demonstrated that pre-incubation of monocytes with IL-10 led to a reduction of TNF production upon LPS stimulation (33,38). The converse obtained with pre-incubation of whole blood with IL-10 suggested that either compounds present in whole blood or experimental parameters may later on affect the responsiveness of the cells. Autologous plasma, presence of platelets and adherence of the cells were considered. Further experiments were performed with freshly isolated PBMC which were first maintained in the presence or absence of IL-10 in the presence of various amounts of autologous plasma. To prevent adherence, PBMC were pre-cultured for 20 h in Teflon containers before being transferred in the classical plastic 24-well multidishes and further cultured for 20 h in the presence of various stimuli. As shown in Fig. 3
, the presence of IL-10 in such pre-cultures resulted in cells which were capable of producing higher levels of TNF than cells pre-cultured in the absence of IL-10, independent of the type of activating signal (i.e. LPS, S. aureus Cowan and IL-1). Similar results were obtained with pre-incubation of the PBMC in the presence of platelet-free autologous plasma or a pool of heat-inactivated normal human serum (data not shown).

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Fig. 3. TNF- production by human PBMC pre-cultured for 20 h in Teflon containers in the absence (light shaded) or in the presence (heavy shaded) of recombinant human IL-10 (10 ng/ml) and in the presence of 5% (left panel) or 50% (right panel) autologous plasma. PBMC were further cultured in plastic 24-well multidish plates in the presence or absence of either E. coli (E.c.) LPS (2 µg/ml), N. meningitidis (N.m.) LPS (2 µg/ml), S. aureus Cowan (200 µg/ml) or IL-1ß (20 ng/ml). The experiment is representative of three.
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Reactivity of PBMC following IL-10 pretreatment either in Teflon containers or in plastic dishes
To ascertain that the lack of adherence was responsible of the priming effect of IL-10 during the pretreatment period, we compared pre-culture conditions either on plastic or on Teflon. As shown in Table 3
, the experimental design had a profound effect on the capacity of the PBMC from the same donor to subsequently release TNF upon activation by LPS. In agreement with previous reports (33,38), when adherence was allowed, IL-10 renders the PBMC hyporeactive to a further stimulation, whereas the opposite observation was observed when adherence was prevented during the pretreatment period.
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Table 3. TNF production (pg/ml) by PBMC derived from pre-cultures (020 h) performed in the presence or absence of IL-10 (10 ng/ml) either on plastic dishes or on Teflon
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TNF-
mRNA expression
The paradoxical effect of IL-10 was also analyzed at the TNF mRNA level (Fig. 4
). We analyzed the expression of TNF-
mRNA by the Northern RNA dot-blot technique and found that IL-10 pre-treatment had different effects on cells depending whether they had been cultured on Teflon or on plastic. On plastic, IL-10 pre-treatment had a significant inhibitory effect on LPS-induced TNF-
transcription when compared to LPS-activated PBMC which had not been pre-cultured with IL-10. In contrast, the IL-10 pre-treatment on Teflon did not induce a down-regulation of TNF mRNA, the mRNA expression of LPS-activated PBMC being identical independently of the IL-10 pre-treatment.
NF
B translocation
We analyzed the possible effects of IL-10 pre-treatment on the nuclear translocation of NF
B, which is strongly activated by LPS. As shown in Fig. 5
, the NF
B factor was differently modulated depending on whether the IL-10 pre-treated PBMC were cultured on Teflon or on plastic. Following IL-10 pre-treatment on plastic, LPS-stimulated PBMC expressed less NF
B in their nucleus cells than untreated cells. In contrast, on Teflon, IL-10 pre-treatment led to an increased translocation of NF
B into the nucleus following cell activation by LPS. These modifications were observed at both levels of the homo- and heterodimer. However, while the difference between Teflon and plastic was found to be statistically significant for the p50p65 dimer (P = 0.04), it was not the case for the inactive complex p50p50 (P = 0.14) (Fig. 5B
).
Effect of IL-10 pre-treatment on the expression of CD14, CD16 and CD68 by monocytes
As shown in Table 4
, independently of the culture condition (Teflon versus plastic), IL-10 enhanced the percent of CD14+ cells expressing CD16 or CD68 markers. When IL-10 was acting on whole blood, the modulation of CD16 was also observed while that of CD68 was absent. For CD14, the MFR values were increased after IL-10 pre-treatment on both Teflon and plastic, whereas for whole blood the effect was less pronounced (Table 4
). IL-10 did not change the MRF for CD16 and CD68; the values were very low for the latter marker.
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Discussion
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While IL-10 has been widely described as an anti-inflammatory cytokine capable of repressing the cytokine release by many cell types including monocytes/macrophages, IL-10 also possesses certain triggering activities, particularly on B lymphocytes (39,40), CD8+ T lymphocytes (41) and endothelial cells (20,22). Its effects on monocytes/macrophages are variable depending on the parameters analyzed. For example, it has been reported that IL-10 diminishes MHC class II, CD54 (ICAM-1), and TNFR I and II expression, has no effect on CD32 (Fc
RII), CD16 (Fc
RIII) and CD11b (CR3) expression, but enhances CD64 (Fc
RI) and MHC class I expression by human monocytes (4245). The data may depend on the level of cell maturation and/or activation since IL-10 was reported to enhance CD23 (Fc
RII) on a U937 monocytic cell line while it diminished its expression on freshly isolated human monocytes (44). Interestingly, IL-10 was shown to be capable of enhancing direct as well as Fc
R- and CR3-mediated phagocytosis (42,44,46) as well as antibody-dependent cellular cytotoxicity (43). However, the results obtained depend greatly on the experimental protocols used. For example, IL-10 does not modify CD11b expression when acting on isolated monocytes (42), whereas the adhesion molecule expression is enhanced on monocytes prepared from blood samples incubated with IL-10 (47). In most studies in which the analysis of cytokine release by human monocytes has been addressed, the investigations were performed using cells cultured in plastic dishes throughout the experiments. It is worth noting that adherence itself is capable of activating cells as seen by the enhancement of basal levels of cytokine mRNA (4850) and cytokine expression (51,52). When experiments are performed with whole blood assays, the presence of rapidly sedimenting erythrocytes prevents monocytes from adhering to plastic dishes. As described by Krause et al. (52), the whole blood culture system is the best model for reproducing the in vivo picture of circulating non-adherent blood monocytes before they leave the vasculature. Under such conditions, when monocytes were prepared from blood maintained for 20 h in the presence of IL-10, the cells had enhanced adherence properties and a greater capacity to release TNF-
upon activation by low or high concentrations of LPS as compared to cells prepared from blood cultured in the absence of IL-10. Similar findings were obtained with PBMC derived from blood maintained in the absence or presence of IL-10. While the effects of plasma and platelets were shown not to contribute to the observed phenomenon, we investigated the effects of adherence. When PBMC maintained during the pre-culture period for 20 h in the presence of IL-10 were first cultured on plastic or on Teflon, a significant difference was observed. In the presence of IL-10, a reduced TNF-
production was found among cells pre-cultured on plastic as we previously reported (33), whereas enhanced TNF-
production was detected when cells were derived from Teflon containers. The phenomenon was not specific to LPS stimulation, suggesting that the modulation of CD14 expression by IL-10 (44) is not sufficient to explain the observation.
Indeed, our results suggest that the up-regulation of CD14 expression by IL-10 does not explain its paradoxical effect on TNF-
production since a similar modulation was observed on plastic and Teflon. The analysis of the phenotype of PBMC in terms of CD16 and CD68 revealed that IL-10 increased the frequency of CD14+ CD16+ monocytes, in accordance with another report (53). The important point was that IL-10 acted on the expression of the tested molecules in the same way on Teflon and plastic. The IL-10 pre-treatment also increased the percent of CD14+ cells expressing the CD68 marker. Indeed, in vitro experiments on monocytes have shown that IL-10 in the presence of granulocyte macrophage colony stimulating factor and IL-13 induced their maturation to macrophages from day 4 of culture (53). Thus, there was a discrepancy between the capacity of IL-10 to modulate some cell surface markers which are not influenced by adherence and the capacity of IL-10 to modulate a later cytokine production which is influenced by adherence.
To further understand the mechanisms involved in this observation performed at the protein level, we investigated whether the various conditions of pre-cultures with IL-10 may also differently affect the TNF mRNA expression. This was the case and the IL-10 pre-treatment on Teflon did not modify the LPS-induced mRNA expression as compared to untreated cells, whereas similar IL-10 pre-treatment on plastic led to a further decreased expression of TNF mRNA. The observations at the transcriptional level did not totally parallel those obtained at the protein level, and analysis of TNF mRNA half-life and kinetics would require further studies. Nevertheless, the LPS-induced mRNA expression following IL-10 pre-culture on Teflon was significantly higher than that obtained following IL-10 pre-treatment of plastic (P = 0.03). Thus we wanted to know whether the observed different modulations of the cell reactivity by IL-10 were upstream the mRNA transduction. For that purpose we analyzed the translocation of NF
B, one of the main nuclear factor that regulates the transcription of numerous genes of cytokines, including TNF-
, IL-1ß, IL-6 and IL-8 (54). The NF
B family is composed of various members, p50 (NF
B1), p52 (NF
B2), p65 (RelA), RelB and c-Rel, which can form homo- and heterodimers. In most cells, the complex that is commonly found is the p50p65 heterodimer which is a potent transactivator, while it is generally admitted that the p50p50 homodimer is not (55). In plastic culture conditions, IL-10 showed an inhibitory activity towards NF
B translocation. However, when IL-10 pre-treatment of PBMC was performed in the absence of adherence, i.e. on Teflon, the nuclear expression of NF
B was moderately enhanced. The p50p65 NF
B heterodimer expression induced by LPS was significantly lower in the cells cultured in plastic than in Teflon. There are few in vitro studies in the literature dealing with the effects of IL-10 on NF
B activation in human PBMC and monocytes showing contradictory results (56,57). Both experiments were carried out in classical culture conditions on plastic; Wang et al. (56) found that IL-10 inhibited the LPS-induced NF
B activity, while Dokter et al. (57) found that it did not. Our results for PBMC cultured on plastic are similar to those of Wang et al. and in addition we show that in the absence of adherence the effect of IL-10 can be completely reversed.
An in vivo model in which an enhanced TNF-
production was observed following IL-10 treatment of mice in a model of multiple organ dysfunction syndrome induced by i.p. injection of zymosan further supports our findings (58). The sequence of the signals delivered to the cells as well as the nature of the responding cells are crucial for the quality of the cell reactivity. This has been well illustrated when studying the modulatory properties of IL-4 (2931) or of cortisol (59).
Our experiments clearly indicate that adherence is an important co-factor which greatly influences the further behavior of the monocytes/macrophages in response to IL-10. These data suggest that depending on the compartment, circulating blood versus tissues, IL-10 may differentially influence the responsiveness of monocytes/macrophages. Finally, these results should be kept in mind when IL-10 is considered as a therapeutic agent in human inflammatory conditions.
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Acknowledgments
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The authors thank Dr P. Poncet for his valuable help for the FACS analysis and K. W. Moore (DNAX) for his kind gift of IL-10. This work was presented in part at the 37th ICAAC Meeting, Toronto, September 1997, abstract symposium S13.
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Abbreviations
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EMSA | electrophoretic mobility shift assay |
LPS | lipopolysaccharide |
MRF | mean fluorescence ratio |
PBMC | peripheral blood mononuclear cells |
PE | phycoerythrin |
PMN | polymorphoneutrophils |
TNF | tumor necrosis factor |
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Notes
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Transmitting editor: L. du Pasquier
Received 9 September 1998,
accepted 19 January 1999.
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