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T cell Ig domain and mucin domain protein 1 (TIM-1) is a costimulatory molecule that regulates immune responses by modulating CD[4.sup.+] T cell effector differentiation. However, the function of TIM-1 on other immune cell populations is unknown. Here, we show that in vivo in mice, TIM-1 is predominantly expressed on B rather than T cells. Importantly, TIM-1 was expressed by a large majority of IL-10-expressing regulatory B cells in all major B cell subpopulations, including transitional, marginal zone, and follicular B cells, as well as the B cell population characterized as CD1[d.sup.hi]CD[5.sup.+]. A low-affinity TIM-1-specific antibody that normally promotes tolerance in mice, actually accelerated (T cell-mediated) immune responsiveness in the absence of B cells. TIM-[1.sup.+] B cells were highly enriched for IL-4 and IL-10 expression, promoted Th2 responses, and could directly transfer allograft tolerance. Both cytokine expression and number of TIM-[1.sup.+] regulatory B cells (Bregs) were induced by TIM-1-specific antibody, and this was dependent on IL-4 signaling. Thus, TIM-1 is an inclusive marker for IL-[10.sup.+] Bregs that can be induced by TIM-1 ligation. These findings suggest that TIM-1 may be a novel therapeutic target for modulating the immune response and provide insight into the signals involved in the generation and induction of Bregs., Introduction T cell Ig domain and mucin domain (TIM) proteins constitute a family of costimulatory molecules that play an important role in effector differentiation of CD[4.sup.+] cells (1). The 8 [...]

Neutrophils are potent contributors to the lung pathophysiological changes occurring in allergic airway inflammation, which typically involve T helper type 2 (Th2) cytokine overexpression. We have previously reported that equine pulmonary endothelial cells are activated by the Th2 cytokine IL-4 and express chemotactic factors for neutrophils after stimulation. We have further explored the possible mechanisms linking Th2-driven inflammation and neutrophilia by studying the effects of recombinant equine IL-4, a prototypical Th2 cytokine, on peripheral blood neutrophils (PBN) isolated from normal animals and from horses with asthmatic airway inflammation (equine heaves). We found that IL-4 induced morphological changes in PBN, dose- and time-dependent expression of IL-8 mRNA, as well as the release of chemotactic factors for neutrophils in culture supernatants. Also, IL-4 induced a mixed inflammatory response in PBN from control and asthmatic-animals with increased expression of proinflammatory IL-8 and TNF-[alpha] but a marked inhibition of IL-1[beta] IL-4 type I receptor (IL-4R[alpha]) and CD23 (Fc[member of]RII) expression were also upregulated by IL-4. Importantly, disease as well as chronic antigenic exposure modified gene expression by PBN. Finally, we found that activation of equine neutrophils with IL-4 involved STAT6 phosphorylation and p38 MAPK and phosphatidylinositol 3-kinase (PI3K); the pharmacological inhibitors, SB-203580 and LY-294002, respectively, significantly reversed IL-4-induced gene modulation in PBN. Overall, results from this study add to the link between Th2-driven inflammation and neutrophilia in the equine model and further extend the characterization of IL-4 effects on neutrophils. cytokines; allergy; equine asthma doi: 10.1152/ajplung.00135.2009.

Migration of airway epithelial cells (AEC) is an integral component of airway mucosal repair after injury. The inflammatory cytokine IL-4, abundant in chronic inflammatory airways diseases such as asthma, stimulates overproduction of mucins and secretion of chemokines from AEC; these actions enhance persistent airway inflammation. The effect of IL-4 on AEC migration and repair after injury, however, is not known. We examined migration in primary human AEC differentiated in air-liquid interface culture for 3 wk. Wounds were created by mechanical abrasion and followed to closure using digital microscopy. Concurrent treatment with IL-4 up to 10 ng/ml accelerated migration significantly in fully differentiated AEC. As expected, IL-4 treatment induced phosphorylation of the IL-4 receptor-associated protein STAT (signal transducer and activator of transcription)6, a transcription factor known to mediate several IL-4-induced AEC responses. Expressing a dominant negative STAT6 cDNA delivered by lentivirus infection, however, failed to block IL-4-stimulated migration. In contrast, decreasing expression of either insulin receptor substrate (IRS)-I or IRS-2 using a silencing hairpin RNA blocked IL-4-stimulated AEC migration completely. These data demonstrate that IL-4 can accelerate migration of differentiated AEC after injury. This reparative response does not require STAT6 activation, but rather requires IRS-1 and/or IRS-2. epithelium; interleukin-4; STAT6; IRS-1; IRS-2

Allergies involve a state of immediate hypersensitivity to antigens, including food proteins. The mechanism underlying the initiation and development of allergic responses involves IL-4 that directly induces the differentiation of committed effector Th2 lymphocytes. Although it is clear that Th2 responses play a pivotal role in the development of allergic responses, it remains unclear which mechanisms are involved in the development of the intestinal damages observed in food allergy. Accordingly, this work aimed to study the role of Th2/IL-4-dependent responses in the development of food allergy and intestinal pathology. C57BL/6 wild-type (WT) and IL-[4.sup.-/-] mice were sensitized with peanut proteins, challenged with peanut seeds, and followed for the development of food allergy and intestinal inflammation. Results demonstrated that exposure to peanut seeds led to weight loss in WT but not in IL-[4.sup.-/-] mice that preserved gut integrity with no signs of mucosal inflammation. These animals presented increased levels of IgG2a in sera, suggesting a role for allergic antibodies in the pathogenesis of WT animals. Most importantly, results also showed that lack of IL-4 modulated gut mucosal response in food allergy through diminished expression of TNF-[alpha] mRNA, increased Th1 IFN-[gamma] IL-12p40, regulatory cytokines, and Foxp3, demonstrating their relevance in the control of allergic inflammatory processes, especially in the intestine. Finally, this study highlighted some of the complex mechanisms involved in the pathogenesis of allergic responses to food antigens in the gut, thereby providing valuable tools for directing novel therapeutic or preventive strategies to the control of allergic enteropathy. interleukin-4; peanut; Th2 response

Byline: Tadaatsu Imaizumi (1), Masaharu Hatakeyama (1), Kageaki Taima (2), Akira Ishikawa (1), Koji Yamashita (1), Hidemi Yoshida (1), Kei Satoh (1) Keywords: double-stranded RNA; vascular endothelial cells; epithelial neutrophil activating peptide-78; interleukin-4 Abstract: Epithelial neutophil activating peptide-78 (ENA-78)/CXCL-5 is a member of CXC chemokines. ENA-78 was originally described as a factor produced by epithelial cells only. But other types of cells including vascular endothelial cells also produce it. ENA-78 production by endothelial cells may be important for the regulation of neutrophil activation in inflammatory reactions. Polyinosinic-polycytidylic acid (poly IC) is a synthetic double-stranded RNA, which mimics the viral infection when applied to cells and affects the expression of various genes related to inflammatory reactions. In the present study, we examined the effect of poly IC on the expression of ENA-78 in human umbilical vein endothelial cells (HUVEC). HUVEC in culture were treated with poly IC and the expression of ENA-78 mRNA and protein were analyzed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Poly IC induced ENA-78 expression in time- and concentration-dependent manners. Th2-type cytokine IL-4 partially inhibited the induction of ENA-78 by poly IC. 2-Aminopurine, an inhibitor of dsRNA-dependent kinase, suppressed the induction of ENA-78 by poly IC. ENA-78 may be involved in the inflammatory reactions elicited by viral infection in endothelial cells. Author Affiliation: (1) Department of Vascular Biology, Institute of Brain Science, Hirosaki University School of Medicine, Hirosaki, Japan (2) The Second Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki, Japan Article History: Registration Date: 20/12/2004

Byline: Ivone Martins Cipriano (1), Mario Mariano (1), Edna Freymuller (2), Celia Regina Whitaker Carneiro (1) Keywords: epithelioid cells; granuloma; macrophages; IL-4; GM-CSF Abstract: Epithelioid cells (ECs) found in granulomas are thought to derive from mononuclear phagocytes. Although GM-CSF and/or IL-4 are known to promote cell differentiation their role in the development of ECs has never been demonstrated. Here we showed that mouse macrophages treated exclusively with recombinant IL-4 (rIL-4) differentiate into epithelioid-like cells. Macrophages cultivated with rIL-4 presented a fried-egg shape, and ultrastructural studies revealed membrane interdigitations, cytoplasmic vesicles, prominent Golgi complex, and rough endoplasmic reticulum. Compared with controls, rIL-4 treated cells displayed increased expression of MHC class II molecules and of Migration Inhibitory Factor-Related Protein-14. Whereas mannose receptor-mediated phagocytosis was increased, FcI3-receptor mediated phagocytosis and the production of nitric oxide were decreased in treated cultures. All these features overlap those reported for ECs from granulomatous lesions. In conclusion, treatment of mouse peritoneal macrophages with rIL-4 drives their in vitro differentiation to an epithelioid phenotype and provides a tool to investigate the biology of ECs. Author Affiliation: (1) Department of Microbiology, Immunology and Parasitology, Federal University of Sao Paulo, Sao Paulo, Brazil (2) Electron Microscopy Center, Federal University of Sao Paulo, Sao Paulo, Brazil Article History: Registration Date: 20/10/2004

Byline: K. Noguchi (1), M. Shitashige (2), H. Watanabe (1), S. Murota (2), I. Ishikawa (1) Abstract: The purpose of the present study was to investigate the involvement of cyclooxygease-1 (COX-1) and cyclooxygenase-2 (COX-2) in prostaglandin (PG) production by human periodontal ligament (PDL) fibroblasts stimulated with a proinflammatory cytokine, inerleukin-1[beta] (IL-1[beta]), and to examine the effect of interleukin-4 (IL-4), a Th2 cytokine, and interferon-I3 (IFN-I3), a Th1 cytokine, on PG production by the cells. IL-1[beta]-stimulated PDL fibroblasts produced prostaglandin E.sub.2 (PGE.sub.2) in a time-dependent manner. Indomethacin, a non-selective COX-1/COX-2 inhibitor, and NS-398, a selective COX-2 inhibitor, completely inhibited PGE.sub.2 production by IL-1[beta]-stimulated cells. Northern blot analysis showed that COX-2 mRNA was detected in IL-1[beta]-stimulated PDL cells, although not detected in unstimulated cells, while expression of COX-1 mRNA was in the same extent in both the cells. Dexamethasone inhibited COX-2 mRNA expression, COX activity and PGE.sub.2 production in IL-1[beta]-stimulated cells. IL-4 and IFN-I3 suppressed PGE.sub.2 production by IL-1[beta]-stimulated PDL fibroblasts, but COX activity enhanced by IL-1[beta] treatment was significantly inhibited by IL-4, not by IFN-I3. Northern blot analysis showed that IL-4 depressed COX-2 mRNA expression with no effect on COX-1 mRNA expression. On the other hand, IFN-I3 had no effect on expression of COX-1 and -2 mRNA. These data suggest that COX-2 is primarily responsible for PGE.sub.2 production by IL-1[beta]-stimulated human PDL fibroblasts and that IL-4 inhibited PGE.sub.2 production by IL-1[beta]-stimulated PDL fibroblasts through down-regulation of COX-2 expression, while IFN-I3 suppressed the PGE.sub.2 production with no effect on COX-2 expression. Author Affiliation: (1) Department of Periodontology, Graduate School, Faculty of Dentistry, Tokyo Medical and Dental University, 1--5--45, Yushima, Bunkyo-ku, Tokyo, 113--8549, Japan (2) Department of Physiological Chemistry, Graduate School, Faculty of Dentistry, Tokyo Medical and Dental University, 1--5--45, Yushima, Bunkyo-ku, Tokyo, 113--8549, Japan Article History: Registration Date: 14/10/2004