Elsevier

Cellular Immunology

Volume 215, Issue 2, February 2002, Pages 162-172
Cellular Immunology

IL-10 alters DC function via modulation of cell surface molecules resulting in impaired T-cell responses

https://doi.org/10.1016/S0008-8749(02)00007-2Get rights and content

Abstract

IL-10 is a potent inhibitor of T-cell activation and has tolerizing effects on these cells. These effects are primarily mediated via modulation of antigen presenting cell function. Here, it is demonstrated that IL-10 completely inhibits LPS-induced DC maturation, resulting in altered DC–T-cell interactions and reduced T-cell responses. IL-10 inhibited LPS-induced upregulation of costimulatory molecules, MHC Class II, and the secretion of IL-12, TNF-α, IL-6, and IL-1β by DCs, although it upregulated the SLAM (CD150) expression at both the mRNA and protein levels. IL-10 pre-treated DC did not respond to subsequent LPS activation and its stimulatory ability for allogeneic and antigen-specific T-cells was severely impaired. Importantly, T-cells derived from co-cultures with Ag-pulsed, IL-10-treated DC were impaired in their responses to subsequent Ag-specific restimulation. Transwell and DC-derived plasma membrane experiments indicated that the capacity of IL-10-treated DC to induce T-cell unresponsiveness results from alterations in the cell surface molecules rather than modulation of cytokine secretion.

Introduction

Dendritic cells are professional antigen presenting cells, being potent initiators of primary immune responses [1], [2]. Immature DC1 precursors, present in non-lymphoid tissues, are efficient in antigen capture [3] and subsequently travel via blood, or lymph, to lymphoid organs. During antigen capture and processing, mature DCs express large amounts of peptide–MHC complexes and accessory molecules on their surface that are necessary for naı̈ve T-cell activation [2], [4], [5]. Products released by pathogens, e.g., LPS and the local production of TNF-α or IL-1 are also mediators of DC maturation and trigger migration of DCs towards the T-cell areas of lymphoid organs [6]. LPS released from Gram-negative bacteria, has been demonstrated to induce the migration of DCs from non-lymphoid tissues such as heart and kidney to lymph nodes [7]. Furthermore, systemic administration of LPS results in the recruitment of DC precursors into lymphoid tissues [7]. These DC precursors migrate to the lymphoid tissues of mice that had received heart allografts from LPS-treated mice [8].

In mouse models, IL-10 is effective in protecting mice from LPS-induced death [9], [10], [11], [12], [13]. Although IL-10 can directly inhibit T-cell activation, through inhibition of IL-2 production, its anti-inflammatory role is mainly attributed to its de-activating effects on human monocytes and macrophages [14], [15], [16]. IL-10 down-regulates expression of HLA-DR and CD86 on human peripheral blood dendritic cells [17] and inhibits the transport of peptide-loaded MHC Class II molecules to the cell surface on monocytes [18]. Functionally, IL-10 inhibits the capacity of peripheral blood DC to stimulate alloreactive T-cells [17]. In addition, differentiation of monocyte-derived DC with GM-CSF and IL-4 can be effectively inhibited by IL-10 [19]. It has been demonstrated that IL-10 has the ability to inhibit the development of fully mature DCs induced by TNF-α [20]. DCs pre-cultured with TNF-α + IL-10 were shown to induce a state of alloantigen-specific anergy in CD4+ T cells and peptide-specific anergy in a peptide-specific CD8+ T-cell line [21]. Yet, it is unclear whether IL-10 acts mainly via changing the cell-surface phenotype or the cytokines produced by the APCs.

In the present study, it is shown that IL-10 inhibits the LPS-induced activation of DC and that the reduced stimulatory capacity of these cells when pre-treated with IL-10 + LPS is a consequence of the changes in the cell surface molecules. Although IL-10 inhibited IL-12 and TNF-α production by DCs, this lack of cytokine production was not crucial for the T-cell non-responsiveness. These data indicate that changes of cell surface molecules, rather than inhibition of cytokine production, are the major mechanisms of IL-10 modulation of DC and indirectly the T-lymphocyte function. Interestingly, despite the IL-10 downregulatory properties on most DC surface molecules, this cytokine enhanced the expression of SLAM (CD150) on these cells.

Section snippets

Medium and reagents

The medium used was RPMI-1640, supplemented with 2 mM l-glutamine, 50μg/ml streptomycin, 50 U/ml penicillin, and 10% heat-inactivated FCS (Gibco, Paisley, Scotland). Recombinant human GM-CSF, IL-2, and IL-4 were provided in-house. Recombinant human IL-10 was purchased from Pharmingen (San Diego, CA) and recombinant TNF-α was from R&D Systems (Minneapolis, MN). LPS and PHA were purchased from Sigma–Aldrich (St.Louis, MO). Tetanus toxoid (TT) was purchased from LIST Biological Laboratories

IL-10 inhibits the LPS activation of DC and results in T-cell unresponsiveness

To test whether IL-10 treatment of DCs would alter their stimulatory capacity for Ag-specific T-cells and the ability of the latter to respond to subsequent stimuli, DCs were treated with LPS or LPS + IL-10, in the presence or absence of the TT. After extensive washing, these DCs were used to stimulate an autologous CD4+ tetanus-specific T-cell clone. As expected, DCs pulsed in the presence of LPS induced optimal proliferation of the T-cell clone. By contrast, T-cell proliferation was strongly

Discussion

LPS is a potent activation stimulus and induces the terminal maturation of immature monocyte-derived DC [23]. The data presented here demonstrate that IL-10 is very effective in inhibiting the LPS activation of DC and that these cells exhibit a severely reduced stimulatory capacity to both antigen-specific and allogeneic T-cells. The effect was specific to IL-10, as the inclusion of a neutralizing anti-IL-10 antibody prevented its inhibitory activity.

The precise mechanisms by which IL-10 is

Acknowledgements

We wish to thank Dr. Elisabeth Kroemer for performing regular elutriations to enrich monocytes and Dr. Christoph Schwaerzler for his expertise in intracellular cytokine staining. This work was supported by the Novartis Research Institute.

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