Modulation of CLA, IL-12R, CD40L, and IL-2Rα expression and inhibition of IL-12- and IL-23-induced cytokine secretion by CNTO 1275

https://doi.org/10.1016/j.cellimm.2007.06.006Get rights and content

Abstract

Cytokines interleukin (IL)-12 and IL-23 are implicated in the pathogenesis of psoriasis. IL-12 causes differentiation of CD4+ T cells to interferon-γ (IFN-γ)-producing T helper 1 (Th1) cells, while IL-23 induces differentiation to IL-17-producing pathogenic Th17 cells. The effects of the monoclonal antibody to IL-12/23 p40 subunit (CNTO 1275) on IL-12 receptor (IL-12R) expression, markers associated with skin homing, activation, and cytokine secretion were investigated in vitro using human peripheral blood mononuclear cells (PBMCs) from healthy donors. PBMCs were activated in the presence or absence of recombinant human (rh) IL-12 or rhIL-23, with or without CNTO 1275. CNTO 1275 inhibited upregulation of CLA, IL-12R, IL-2Rα and CD40L expression and also inhibited IL-12- and IL-23-induced IFN-γ, IL-17A, tumor necrosis factor (TNF)-α, IL-2, and IL-10 secretion. Thus, the therapeutic effect of CNTO 1275 may be attributed to the IL-12/23 neutralization, resulting in decreased expression of skin homing and activation markers, and IL-12- and IL-23-induced cytokine secretion.

Introduction

Psoriasis is a chronic, T cell-mediated, inflammatory skin disease affecting approximately 1–3% of the world’s population [1], [2]. Both interleukin (IL)-121 and IL-23 are known to be involved in the pathogenesis of psoriasis [2], [3], [4], [5], as well as other autoimmune diseases [6], [7], [8]. IL-12 is composed of the subunits p35/p40, while IL-23 is composed of the subunits p19/p40. Both cytokines bind via the p40 subunit to the IL-12 receptor β1 (IL-12Rβ1) located on the surface of T and natural killer (NK) cells [6]. The unique subunits of IL-12 (p35) and IL-23 (p19) bind to distinct receptors, IL-12Rβ2 and IL-23R, respectively [9]. While IL-12Rβ1 subunit is the ligand binding subunit, IL-12Rβ2 is the intra-cellular signaling component of the IL-12R complex, but both subunits are necessary for high affinity binding to IL-12 [10]. Similar to IL-12Rβ2, IL-23R is not detected on resting naïve T cells, but is induced rapidly upon activation [11]. IL-12 and IL-23 receptor binding initiates the respective cytokine-signaling cascades of IL-12 and IL-23.

The role of IL-12 in psoriasis including T cell differentiation towards TH1 cytokine producing cells and facilitating T cell homing to the skin via induction of cutaneous lymphocyte antigen (CLA) has been well documented [3], [12], [13]. Less than 20% of peripheral blood T cells express CLA, while the majority of effector memory T cells in normal skin (under resting conditions) are CLA positive [14], [15]. About 85% of T cells infiltrating psoriatic lesions and <5% of T cells in extracutaneous sites express CLA [14], [16]. Evolving psoriatic lesions are reported to be enriched with CLA+ T cells preceding epidermal hyperproliferation. Also, a positive correlation was shown between disease severity and frequency of CLA expressing T cells in peripheral blood and lesions in psoriatic patients [13]. Several other activation (CD25) [17], co-stimulatory (CD40L) [18], and chemokine receptors (CXCR3) [19], [20] have also been implicated in the pathogenesis of psoriasis.

In addition to cell surface expression markers, a complex network of stimulatory and inhibitory cytokines has been associated with psoriatic lesions with elevated levels of IL-12 (p40), IL-23, interferon-gamma (IFN-γ), IL-17A, tumor necrosis factor-alpha (TNF-α), IL-10, IL-5, IL-1, IL-2, IL-6, and IL-8 [3], [13], [21], [22], [23], [24]. Recently, IL-23 has also been reported as a key cytokine in the chronic inflammation associated with psoriasis mediated by the IL-23-induced pro-inflammatory cytokine, IL-17 [4], [25]. Also, Chan et al. showed that injection of IL-23 into the skin of mice promoted greater epidermal thickness than the injection of IL-12 [24]. Further, Zheng et al. indicated that IL-12 injections resulted in increased IFN-γ levels, whereas IL-23 increased the levels of IL-17A, IL-22, and other cytokines, but not IFN-γ[26]. Therefore, a therapy based on the neutralization of the common subunit of IL-12 and IL-23 (p40) should alter the downstream cytokine induction pathways leading to improved clinical efficacy in the treatment of psoriasis.

The first-in-human phase I [27] and phase II [28] clinical trials using the fully human IgG1 monoclonal antibody specific for the p40 subunit of IL-12 and IL-23, designated CNTO 1275 (Centocor, Inc., Malvern, PA), showed a significant improvement in psoriatic lesions in patients with moderate-to-severe psoriasis; CNTO 1275 binds to the IL-12/23 p40 subunit with high affinity, thereby blocking the IL-12Rβ1 receptor binding and preventing subsequent IL-12 and IL-23 receptor mediated signaling. The study presented here focused on the in vitro effects of CNTO 1275 on pro-inflammatory (IFN-γ, IL-17A, TNF-α) and anti-inflammatory (IL-10 and IL-5) cytokine secretion by polyclonal activators in the presence of exogenous IL-12 and IL-23. CNTO 1275 was shown to neutralize IL-12 and IL-23 in activated human peripheral blood mononuclear cells (PBMCs), which resulted in decreased expression of CLA, CD25, CD40L, and IL-12R, and inhibition of pro-inflammatory cytokine secretion. In the current study, we show that IL-12 and IL-23 exert differential cytokine secretion profiles and induction of T cell markers indicating that regardless of their individual contributions, targeting both cytokines may be a more effective therapeutic strategy. Notable clinical efficacy data from psoriasis phase I [27] and phase II [28] clinical studies using CNTO 1275 in psoriasis, supports this strategy.

Section snippets

Recombinant human IL-12 (rhIL-12)-induced upregulation of CLA expression on activated T cells is inhibited by CNTO1275

To determine the effects of CNTO 1275 on rhIL-12-induced expression of CLA, PBMCs were isolated from healthy donors and stimulated in vitro with either the mitogen phytohemaglutinin (PHA), anti-CD3 + anti-CD28 (α-CD3 + α-CD28), or the superantigen Staphylococcus Enterotoxin B (SEB), in the presence or absence of exogenous rhIL-12 or rhIL-23, and with or without CNTO 1275. Expression of CLA was increased by activation and further increased by the addition of exogenous rhIL-12, and inhibited by CNTO

Discussion

IL-12 and IL-23 are key immunoregulatory cytokines that coordinate innate and adaptive immune responses. IL-12 plays a central role in inducing and maintaining TH1 type immune responses, while IL-23 recently emerged as a late-stage effector cytokine responsible for maintaining chronic autoimmune inflammation through induction of IL-17 and direct activation of macrophages [29]. The concerted action and balanced expression of IL-12 and IL-23 are crucial to maintaining an effective cell-mediated

Study design

All procedures related to collection of blood from healthy volunteers were performed in accordance with the principles of the Declaration of Helsinki and followed all approved human study processes in effect at the time of the study. Written, informed consent was obtained from all healthy volunteers prior to any study procedures.

PBMC stimulation assay

PBMCs were isolated from peripheral whole blood collected from healthy volunteers using Vacutainer tubes with K3EDTA additive (Becton–Dickinson, Franklin Lakes, NJ) by

Acknowledgments

The authors thank Kristin Ruley, Ph.D. of Centocor, Inc. for editorial assistance and writing support in the preparation of this manuscript; Nathan Fried for technical support; Paul Tebbey, Ph.D. and Carrie Brodmerkel, Ph.D. of Centocor for critical review of the manuscript.

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