Intracellular JNK, p38 MAPK and NF-κB regulate IL-25 induced release of cytokines and chemokines from costimulated T helper lymphocytes

Abstract

Novel Th2 cytokine IL-25 has been shown to be elevated in allergic inflammation. We investigated the intracellular mechanisms regulating IL-25-induced Th2 cytokines and chemokines from human Th lymphocytes upon costimulation by anti-CD3 and anti-CD28 antibodies. Cytokines, chemokines, and phosphorylated p38 mitogen activated protein kinases (MAPK), c-Jun amino-terminal kinase (JNK) and extracellular signal-regulated protein kinase were analyzed by bead-based array using flow cytometry. Nuclear factor (NF)-κB and total MAPK were assessed by electrophoretic mobility shift assay and Western blot, respectively. IL-25 could synergistically induce the release of Th2 cytokines IL-4, IL-5 and IL-10, inflammatory cytokine IL-6, Th1 related chemokines CXCL9 and CXCL10, and chemokine CCL5 from anti-CD3 and anti-CD28 antibodies costimulated Th cells, especially memory Th cells. Costimulation could also upregulate the cell surface expression of IL-25 receptor on Th cells. Costimulation with or without IL-25 treatment could activate JNK, p38 MAPK and NF-κB. The upregulation of costimulation-induced IL-25 receptors and release of cytokines and chemokines from IL-25 treated costimulated Th cells were differentially regulated by intracellular JNK, p38 MAPK and NF-κB activity. Therefore, the optimal activation of Th cells by IL-25 for the release of Th2 cytokines and chemokines requires the CD3 and CD28 mediated costimulation of Th cells via the upregulation of IL-25 receptors and the activation of intracellular signaling pathways. This mechanistic study shows that IL-25 and CD28 costimulation can play pathophysiological roles by inducing inflammation and hyperresponsiveness through the production of both Th2 cytokines and chemokines from memory Th cells.

Introduction

Optimal activation of T lymphocytes requires the engagement of the T cell receptor (TCR)/CD3 complex with a peptide-major histocompatibility complex (MHC), as well as the costimulatory interaction between the B7 family ligands [B7-1 (CD80), B7-2 (CD86), and B7RP-1] on antigen presenting cells and their corresponding receptors [CD28, cytotoxic T lymphocyte antigen 4 (CTLA-4), and inducible costimulator (ICOS)] on T cells [1], [2]. CD28 functions during the initial events of T cell activation, while other costimulatory receptors, such as ICOS mediate later phase of the immune response [3], [4]. Upon T cell costimulation, a variety of cytokines and chemokines can be induced by various Th1/2 cytokines for the subsequent inflammation. The production of Th2 cytokines interleukin (IL)-4 and IL-5 causes eosinophilia and elevated serum IgE concentration in allergic inflammation, and Th1 cytokines, such as interferon (IFN)-γ and tumor necrosis factor (TNF)-α can activate the cell-mediated inflammation [5], [6]. T cell activation by costimulatory interaction, particularly CD28, plays an important role in the immunopathogenesis of inflammation, such as allergic inflammation and infection [7], [8], [9]. Therefore, allergen-activated CD3 and ligation of costimulatory receptor CD28 coordinate positive signaling for the initiation, clonal expansion and effector function of allergen-activated T cells in allergic inflammation.

Th2 cytokines and certain chemokines play essential roles in the pathogenesis of allergic asthma [10]. IL-25 (IL-17E) is a novel Th2 proinflammatory cytokine belonging to a newly discovered member of the IL-17 cytokine family with receptor homology IL-17 receptor homology 1 (Rh1) [11], [12]. IL-25 is secreted by CD4+ activated memory (CD45 + RO+) T cells [13]. Intranasal administration of IL-25 has been shown to induce the production of Th2 cytokines and the expression of chemokine eotaxin mRNA in the murine lung [14], [15], resulting in Th2-like response marked by increased serum IgE, IgG₁ and IgA concentrations, eosinophilia in blood, bronchoalveolar lavage and lung tissue, and the development of pathological changes including eosinophilic infiltrates, epithelial cell hyperplasia/hypertrophy, increased mucus secretion and airway hyperreactivity in mice [14], [15]. Ikeda et al. [16] reported that bone marrow derived mast cells could generate large amounts of IL-25 when the cells were challenged by IgE cross-linking, thereby indicating that mast cells may affect Th2 immune response through the production of IL-25 [16]. Therefore, IL-25 plays an important role in provoking allergic inflammation, especially in IgE-dependent atopic diseases and eosinophil-mediated late phase allergic reactions [17], [18].

Signal transduction studies using Jurkat T cell lines have demonstrated that costimulation of CD3 and CD28 can activate TCR signaling networks via phosphorylation of phospholipase C-γ/Src homology 2 domain-containing transforming protein 1/Grap2/Vav-1 and downstream components including mitogen activated protein kinases (MAPK) [19]. Our previous results suggested that IL-25-induced release of cytokines and chemokines from eosinophils is mediated by the combined activation of MAPK and nuclear factor (NF)-κB pathways [17]. However, the optimal activation of CD28 costimulated Th cells by IL-25 has not been studied yet. In an attempt to elucidate the detailed mechanisms by which Th2-related IL-25 induces the release of Th cytokines and chemokines from costimulated Th cells, the expression of IL-25 receptor and intracellular MAPK and NF-κB activities upon activation were investigated in the present study.