Anti-EBI3 (aa 129-229) polyclonal antibody

Members of the interleukin (IL)-12 family constitute subunits of IL-12, -23, and -27. These ILs represent pivotal mediators in the regulation of cell-mediated immune responses and in animal models of human inflammatory bowel disease. Recent work has suggested that intestinal endothelial cells might serve as a second line of defense in bacterial sensing of invading pathogens. The purpose of this study was to examine the production of IL-12 family members in intestinal endothelial cells (HIMEC). HIMEC were stimulated with proinflammatory agents (TNF-alpha, IFN-gamma, IL-1 beta) and microbial antigens [LPS, lipoteichoic acid, peptidoglycan, CpG-DNA, flagellin, poly(I:C)]. Expression of IL-12 family members and of Toll-like receptor (TLR)3 in HIMEC was assessed by real-time RT-PCR, immunostaining, flow cytometry, and immunoblot analysis. HIMEC display an induction of Epstein-Barr virusinduced gene 3 (EBI3), IL-12p35, and IL-23p19, whereas no expression of IL-12p40 and IL-27p28 was detectable. The strongest induction was induced by proinflammatory factors known to utilize the NF-kappa B pathway, and expression of EBI3 and IL-23p19 was diminished by an NF-kappa B inhibitor. HIMEC display regulated expression of TLR3. Adhesion and transmigration assays showed proinflammatory responses after HIMEC stimulation. HIMEC are capable of producing IL-12 family members as a response to microbial stimuli. The TLR3 agonist, poly( I: C), was shown to enhance leukocyte adhesion in vitro in HIMEC. Our data suggest that the intestinal microvasculature is responsive to ligands of TLR3 expressed on intestinal endothelial cells, thereby adding to the regulation of adaptive immunity and leukocyte recruitment.

EBI3 functions as the subunit of immune-regulatory cytokines, such as IL-27 and IL-35, by pairing with p28 and p35, respectively. We treated wild-type and EBI3-deficient mice with intratracheal administration of LPS and obtained bronchoalveolar lavage fluid (BALF) 24 h later. Although neutrophils were the predominant cells in BALF from both groups of mice, eosinophils were highly enriched and there was increased production of eosinophil-attracting chemokines CCL11 and CCL24 in BALF of EBI3-deficient mice. The bronchial epithelial cells and alveolar macrophages were the major producers of CCL11 and CCL24. Because no such increases in eosinophils were seen in BALF of p28/IL-27-deficient mice or WSX-1/IL-27Ra subunit-deficient mice upon intratracheal stimulation with LPS, we considered that the lack of IL-35 was responsible for the enhanced airway eosinophilia in EBI3-deficient mice. In vitro, IL-35 potently suppressed production of CCL11 and CCL24 by human lung epithelial cell lines treated with TNF-alpha and IL-1 beta. IL-35 also suppressed phosphorylation of STAT1 and STAT3 and induced suppressor of cytokine signaling 3. In vivo, rIL-35 dramatically reduced LPS-induced airway eosinophilia in EBI3-deficient mice, with concomitant reduction of CCL11 and CCL24, whereas neutralization of IL-35 significantly increased airway eosinophils in LPS-treated wild-type mice. Collectively, our results suggest that IL-35 negatively regulates airway eosinophilia, at least in part by reducing the production of CCL11 and CCL24.