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Novel immunosuppressive properties of interleukin-6 in dendritic cells: inhibition of NF-B binding activity and CCR7 expression

Institute of Virology, University of Cologne, Cologne, Germany

¹Correspondence: Institute of Virology, University of Cologne, Fürst-Pückler-Str. 56, 50935 Cologne, Germany. E-mail: s.smola{at}uni-koeln.de

SPECIFIC AIMS

Interleukin-6 (IL-6) is produced during bacterial and viral infections and by various malignant tumors. Although characterized primarily as a proinflammatory cytokine, IL-6 has been proposed to have anti-inflammatory and immunosuppressive properties. The aim of the present study was to characterize the phenotype and determine the functional properties of dendritic cells (DC), central regulators of the immune response, under the influence of IL-6.

PRINCIPAL FINDINGS

1. IL-6 inhibits the chemotactic response toward CCR7 ligands in phenotypically mature dendritic cells
Peripheral blood monocytes were cultured with 100 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF) and 5 ng/mL IL-4 in the presence or absence of 100 ng/mL IL-6 for 8 days. Confirming previous studies, IL-6 impaired dendritic cell differentiation resulting in an intermediate phenotype with macrophage characteristics. However, activation of these cells with the maturation stimulus lipopolysaccharide (LPS) on day 6 not only repressed macrophage surface marker expression but consistently switched the cells to a typical DC morphology (IL-6-DC). The expression levels of markers for DC maturation like CD40 and CD80 were slightly increased; MHC class II, CD86, and DC-LAMP were significantly increased in IL-6-DC compared with control DC activated with LPS. CD83 induction showed donor-specific variability in IL-6-DC. It was not changed in 2 donors, slightly up-regulated in 3 donors, and inhibited by IL-6 in 11 other donors. Overall, however, CD83 was significantly (paired t test, 2-sided, P=0.004) down-regulated in IL-6-DC.

Upon maturation in vivo, DC acquire the capacity to leave the peripheral tissue through lymphatic vessels and migrate to the draining lymph node, where they encounter naive T cells. We therefore investigated the response of IL-6-DC to the CCR7-activating chemokines SLC (secondary lymphoid tissue chemokine) and ELC (EBI1 ligand chemokine). Both chemokines are involved in the lymph node homing process via stimulation of the CCR7 chemokine receptor. Stromal-derived factor-1 (SDF-1), a CXCR4 ligand, was used as a control. Monocytes were cultured and stimulated as above and tested on day 8 in in vitro migration assays. Migration of IL-6-DC to SLC and ELC was significantly reduced compared with control DC. SLC-induced chemotaxis was inhibited by up to 92% and ELC-induced chemotaxis by up to 89% in three different donors; migration toward SDF-1 was little affected by IL-6 treatment in LPS-matured DC.

2. IL-6 suppresses up-regulation of CCR7 in DC activated via TLR4-, TLR3-stimuli, or TNF-
The question arose as to whether IL-6-DC become less responsive to CCR7 ligands SLC and ELC due to altered receptor expression. In contrast to maturation-associated markers and CXCR4, CCR7 up-regulation was compromised in IL-6-DC (Fig. 1 ). Suppression of CCR7 expression took place at protein and mRNA levels not only with the standard IL-6 dose of 100 ng/mL (85% inhibition), but with as little as 5 ng/mL (67% inhibition). The IL-6 effect was strongest when the cytokine was added on the first day of culture. In DC of various donors, inhibition of CCR7 up-regulation by IL-6 added on the first day ranged from 53% to 100%. In all donors tested, CCR7 inhibition greatly exceeded the extent of CD83 inhibition.

View larger version (19K): [in this window] [in a new window]   Figure 1. IL-6 inhibits LPS-induced up-regulation of CCR7. Human blood monocytes were cultured with GM-CSF (100 ng/mL) and IL-4 (5 ng/mL) in the presence or absence of increasing doses of IL-6. On day 6 cells were stimulated with medium (upper panel) or LPS (250 ng/mL, lower panel). 48 h later, cells were stained with anti-CCR7 mAb (solid lines) or isotype-matched mAb (dotted lines) and analyzed by flow cytometry. Shown is 1 of 3 independent experiments.

To investigate whether IL-6-mediated inhibition of CCR7 induction is limited to toll-like receptor 4 (TLR4)-activated DC, DC were stimulated with poly (I:C) (200 µg/mL) activating TLR3, tumor necrosis factor (TNF- (5000 U/mL), or CD40 ligand (paraformaldehyde-fixed BHK_CD40L cells) on day 6. Similar to TLR4-matured DC, CCR7 up-regulation was abolished in TLR3-matured DC and greatly reduced in TNF--matured DC, while CD40L-induced CCR7 expression levels were least affected by IL-6.

3. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) is not suppressed by IL-6
Various signaling pathways have been implicated in CCR7 regulation in different cell types and in DC maturation. To elucidate which cellular responses to LPS were altered by IL-6, IL-6-treated DC and controls were stimulated with LPS for different intervals and assessed for activation of the p38 MAPK using phosphorylation-specific antibodies (Abs). Unexpectedly, the signal detected in IL-6-DC was even stronger than in the controls, although the p38 kinase itself was equally expressed. We concluded that IL-6 enhanced rather than suppressed p38 MAPK activation.

4. IL-6 stimulation impairs LPS-induced NF-B binding activity and NF-B-dependent cytokine or chemokine production
In addition to the inhibition of CCR7 induction, we observed that in IL-6-DC production levels of the NF-B-dependent factors interferon- inducible protein-10 (IP-10) and TNF- were reduced. Application of the IB kinase inhibitor N-acetylcysteine or CAPE (caffeic acid phenylethyl ester) not only suppressed LPS-induced NF-B activation but also potently inhibited CCR7 up-regulation in the same cells, suggesting NF-B involvement. To test whether LPS-induced NF-B activation is negatively regulated by IL-6, cells were analyzed for nuclear factor-B (NF-B) binding activity (Fig. 2 ). Supershift analyses identified the composition of NF-B complexes after 2 and 4 h of LPS stimulation. In normal immature DC, only one NF-B complex (complex III) was found by EMSA, containing NF-B subunits p65 and p50. LPS activation on day 6 led to a strong induction of NF-B binding activity. LPS stimulation not only enhanced the binding activity of complex III, but induced binding of c-Rel (complex II) after 1 h and RelB (complex I), most prominent at 4 h of stimulation. In IL-6-DC, NF-B binding patterns were extensively disturbed. In IL-6-treated cells, a novel complex appeared (complex IV) consisting of the NF-B subunit p50, further increased by LPS stimulation at early time points. In contrast, complexes I and II were hardly visible, and complex III was activated less than in controls. Thus, at early time points IL-6-DC supported induction of the transcriptionally inactive p50-p50 homodimers. Later, IL-6 strongly suppressed the formation of active NF-B p65 homo- and heterodimers and almost prevented the formation of c-Rel and RelB NF-B complexes. As judged from Western blot analysis of nuclear and cytoplasmic extracts, nuclear p65, p50, and c-Rel were reduced by IL-6 stimulation; nuclear RelB was not suppressed. This indicated that for RelB, IL-6 only led to inhibition of its DNA binding. In the case of p65 and c-Rel, inhibition of nuclear translocation might have contributed to the lower binding observed in Fig. 2 .

View larger version (74K): [in this window] [in a new window]   Figure 2. IL-6 modulates LPS-induced NF-B binding activity and subunit composition. Human blood monocytes from 4 different donors were cultured with GM-CSF (100 ng/mL) and IL-4 (5 ng/mL) in the absence (lanes 1, 3, 5, 7, 9, 11, and 13) or presence of IL-6 (100 ng/mL, lanes 2, 4, 6, 8, 10, 12, and 14). On day 6 they were stimulated with medium (lanes 1, 2) or LPS (250 ng/mL, lanes 3–14). Only results of 2 h LPS stimulation are shown for donors 2–4 (right panel). Nuclear extracts were prepared and analyzed by EMSA using a 32P-labeled NF-B-specific probe. 4 different complexes (I–IV) were shifted.

5. Endogenous IL-10 partially contributes to CCR7 suppression in IL-6-DC
Suppression of NF-B binding activity was reminiscent of the effects of IL-10. We determined IL-10 levels in supernatants of control DC and IL-6-DC at various times. Immature control DC did not produce detectable IL-10 levels. When DC were cultured in the presence of IL-6 (from the first day of culture), IL-10 was present in culture supernatants at low levels on day 2 and at higher levels (2 ng/mL) on day 6 of culture. Neutralizing anti-IL-10 mAbs (10 µg/mL) reversed the inhibitory IL-6 effect on CCR7 expression from 67% to 39.8% as calculated from corresponding MFIs. In control experiments, neutralizing mAbs completely blocked CCR7 suppression mediated by 5 ng/mL of exogenous IL-10 applied from the first day of culture. Thus, effects of an IL-10 dose higher than that produced after IL-6 stimulation were neutralized but the IL-6 effect itself was only partially blocked. When DC were treated with IL-6 from day 4 only, CCR7 was still partially suppressed but no IL-10 was detectable. Therefore, the CCR7 suppression occurring in these experiments appeared to be IL-10 independent.

6. IL-6 inhibits the capacity of DC to stimulate allogeneic T cell proliferation
MLRs were performed using DC pretreated with medium or IL-6 and activated with LPS vs. medium. Under all conditions tested, IL-6 strongly suppressed the capacity of DC to induce the proliferation of cocultured naive allogeneic responder T helper lymphocytes.

CONCLUSIONS AND SIGNIFICANCE

Dendritic cells play a central role in the initiation and coordination of the immune response to infectious agents and tumors. Their function is tightly controlled by the local environment via cytokine and chemokine signals.

Here we show that the cytokine IL-6 vigorously impairs CCR7 expression and chemotaxis in DC, which is required to recruit these cells to lymphoid tissues in vivo via a chemotactic gradient of SLC and ELC. Moreover, IL-6 inhibited their capacity to produce TNF- and IP-10 as well as to induce allogeneic T cell proliferation. The functional defect was paralleled by a strong IL-6-mediated inhibition of CCR7 up-regulation at protein and mRNA levels after LPS stimulation while expression levels of other maturation-associated molecules remained unchanged or were even up-regulated in IL-6-DC. Only expression of the surface molecule CD83 was partially reduced in several donors. These data indicated that IL-6 largely dissociates the regulation of CCR7 and most other maturation-associated molecules. This result was surprising, since CCR7 regulation is thought to be firmly linked to maturation and to be coregulated with other maturation markers.

IL-6-mediated suppression of CCR7 expression was not restricted to LPS-matured DC. It was observed when DC were activated with a TLR3 stimulus mimicking double-stranded RNA or TNF-.

At which regulatory step did IL-6 signals interfere with the signaling pathways induced by TLR stimuli and TNF-? In IL-6-DC, LPS-induced binding activities of NF-B subunits important for functional DC maturation, including c-Rel, RelB, and the p65 subunit, were greatly reduced whereas phosphorylation of the p38 MAPK was enhanced. These results indicate that CCR7 expression apparently is sensitive to NF-B activity, an observation already been made in Hodgkin’s lymphoma cells. Moreover, our data show that CCR7 suppression is mediated in part via autocrine IL-10, itself able to impair NF-B activation. This suggests that IL-6, a cytokine labeled as proinflammatory, can engage a classical anti-inflammatory cytokine and result in immunosuppression.

From our data we propose a model in which exogenous IL-6, in concert with autocrine IL-10, can lead to the generation of phenotypically mature but functionally impaired DC (Fig. 3 ). This functional impairment is associated with disturbed NF-B activation, suppression of CCR7 up-regulation and chemotaxis, as well as cytokine production and stimulation of T helper cell proliferation.

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic diagram of DC development under the influence of IL-6.

As IL-6 is expressed in response to various proinflammatory stimuli in vivo, the mechanisms described here might contribute to an IL-6-mediated negative feedback down-regulating the immune response initiated by pathogens or in persistent infections. Moreover, they may play a role in malignant diseases where high IL-6 production is correlated with immune failure or a negative prognosis of disease progression.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0969fje;

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This Article Full Text (PDF) All Versions of this Article: 18/12/1439 03-0969fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by HEGDE, S. Articles by SMOLA-HESS, S. Search for Related Content PubMed PubMed Citation Articles by HEGDE, S. Articles by SMOLA-HESS, S.