Negative regulation of IL-17 production by OX40/OX40L interaction

https://doi.org/10.1016/j.cellimm.2008.04.010Get rights and content

Abstract

The T-cell cytokine IL-17 is implicated in multiple inflammatory diseases through its induction of several pro-inflammatory cytokines and chemokines in a broad range of cell targets. Production of IL-17 defines the Th17 subset of helper T-cells associated with protection against microorganisms, a profile best characterized in the murine system. Multiple regulators of Th17 cell differentiation and IL-17 production are reported, but the impact of OX40L is not described. OX40 ligand (OX40L) is an early-stage activator of T-cells through its interaction with CD134 (OX40) that is up-regulated on antigen challenged T-cells. Here, we show that OX40L suppresses IL-17 production by PHA-stimulated human PBMC and purified CD4 and CD8 cells. In agreement with prior reports, OX40L signaling through CD134 increased IFNγ and IL-4, both of which are reported to inhibit the production of IL-17. OX40L suppression of IL-17 was completely reversed by a neutralizing IFNγ antibody while there was no effect with a neutralizing IL-4 antibody. Moreover, OX40L also suppressed IL-17 in the presence of IL-23, an established inducer of IL-17 and differentiation factor for Th17 cells. Presuming mediation by IFNγ, we evaluated expression of this cytokine in the presence of OX40L and IL-23. Surprisingly, IL-23 also induced IFNγ by PHA-stimulated T-cells and this effect was enhanced in the presence of OX40L. Addition of the IFNγ antibody not only reversed the OX40L suppression of IL-17 in the presence of IL-23, it markedly enhanced the level of IL-17. These results further establish IFNγ as a primary modulator of IL-17 production in the human cells, much as in the murine system.

Introduction

OX40 (CD134) is a TNFR family member that is primarily expressed on activated T lymphocytes [1]. The ligand of OX40 (OX40L) is present on activated antigen presenting cells (APC) [2], endothelium [3], and activated T-cells [4]. The engagement of OX40 with OX40 ligand (OX40L) delivers a strong co-stimulatory signal to newly activated effector T-cells and enhances both Th1 and Th2 responses [5]. Consistent with this stimulatory function, OX40 or OX40L-deficient mice show impaired production of both Th1 and Th2 cytokines [6], [7], [8], [9].

The interleukin-17 family of cytokines consists of six members: IL-17 (also designated as IL-17A) and IL-17 B-F [42]. IL-17 is secreted mainly by activated CD4 and CD8 T-cells [10] and within the CD4 population its expression defines the Th17 subset [11], [12]. In the mouse, differentiation of Th17 cells in vivo requires IL-23, but this cytokine alone does not induce their differentiation in vitro from CD4 cells [13], [14], [15]. Rather, this differentiation is regulated by IL-6 and TGF-β[16], [17], which recently was shown in the murine system to involve an autocrine loop of IL-21 and IL-23R expression [18], [19], [20], [34], [35]. A quite different profile is emerging for human cells where IL-23 alone may be sufficient to differentiate CD4+ T-cells into Th17 cells [41] IL-17 induces the production of multiple pro-inflammatory cytokines/chemokines from various cell types and also plays a crucial role in neutrophil mediated immune responses [22], [23]. IL-17 is implicated in the development of rheumatoid arthritis [24], osteoarthritis [25], asthma [26], inflammatory bowel disease [22], [27], and multiple sclerosis [22], [28].

Although both OX40 and IL-17 are associated with T-cell activation, the potential impact of OX40 signaling on IL-17 production has not been elucidated. In this study we demonstrated that ligation of OX40 on human T-cells with OX40L inhibits IL-17 production and that the inhibition is through up-regulation of IFNγ.

Section snippets

Purification of CD4+ and CD8+ T-cells

T-cells were isolated from normal human peripheral blood mononuclear cells (PBMC) using Pan T-cell isolation kit II (Miltenyi Biotec, CA) according to the manufacture’s instructions. CD4 and CD8 T-cells were then separated from purified T-cells by positively selecting CD4 T-cells (CD4 isolation kit, Miltenyi Biotec, CA). The purity of CD4+ and CD8+ T-cells was >92% and >95%, respectively, as analyzed by flow cytometry.

Cytokine production assay

Effect of OX40L on PHA induced IL-17 production in PBMC, or the purified

OX40L down-regulates PHA induced IL-17 expression

Addition of PHA to human PBMC stimulated the production of IL-17 and co-addition of soluble human OX40L reduced IL-17 production in a dose dependent manner (Fig. 1A). The specificity of this inhibition was shown by its reversal with addition of a neutralizing OX40L antibody (Fig. 1B). Inhibition of IL-17 production through ligation of OX40 was somewhat unexpected since OX40 is an early marker of T-cell activation and is implicated in the function and survival of these T-cells. To determine

Discussion

IL-17 is produced by both activated CD4 and CD8 cells but its expression by a specific population of helper T-cells defined as the Th17 subset, which is best characterized functionally in the murine system. IL-6 and TGB-β are sufficient to drive Th17 differentiation and IL-17 production from naïve murine T-cells in vitro[16], [17], [21]. IL-23 is also a potent stimulator of murine IL-17 in vitro[13]. It is not required exogenously for differentiation of Th17 cells in culture, but may function

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    These authors contributed equally to this article.

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