Expression and function of the inducible costimulator ligand B7-H2 in human airway smooth muscle cells.

BACKGROUND: B7-H2 is a ligand for the inducible costimulator (ICOS). The aim of this study was to examine the expression and function of B7-H2 in human airway smooth muscle (ASM) cells and compare them with those of CD40 or OX40 ligand (OX40L). METHODS: Expression of B7-H2, CD40 and OX40L in ASM cells and their respective counterparts in T cells was analyzed by RT-PCR or flow cytometry. The modulating effect of polyinosinic-polycytidylic acid (poly I:C) on expression of B7-H2, CD40 and OX40L was also examined. The function of these three molecules was evaluated by virtue of adhesion of anti-CD3-activated T cells, IL-6 and IL-8 production and DNA synthesis. RESULTS: ASM cells constitutively expressed B7-H2, CD40 and OX40L that mediated adhesion of activated T cells expressing ICOS, CD40L and OX40. ASM cells responded to poly I:C with upregulated expression of B7-H2, CD40 and OX40L and displayed enhanced adhesion of activated T cells. Functional analysis performed on untreated ASM cells showed that engagement of B7-H2 with ICOS-Ig clearly induced DNA synthesis, whereas that of CD40 or OX40L with trimeric CD40L or OX40-Ig greatly increased IL-6 and IL-8 production. These responses were enhanced in poly I:C-treated ASM cells. CONCLUSIONS: The data demonstrate that ASM cells express functionally active B7-H2, CD40 and OX40L and suggest that B7-H2-dependent signaling may play an active role in a proliferative response rather than in cytokine and chemokine production. In addition, the modulation of B7-H2, CD40 and OX40L expression and function by poly I:C may have important implications for the function of virus-infected ASM cells.

Ligation of Toll-like receptor 3 differentially regulates M2 and M3 muscarinic receptor expression and function in human airway smooth muscle cells.

BACKGROUND: Viral infection causes asthma exacerbations and airway hyperreactivity. Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA) of viral or synthetic origin in a fashion different from protein kinase R (PKR). The aim of this study was to examine the expression and function of TLR3 in human airway smooth muscle (ASM) cells. METHODS: Expression of TLR3 and muscarinic receptor (MR), histamine receptor (HR), and cysteinyl leukotriene receptor (CysLTR) subtypes was analyzed by quantitative real-time PCR, flow cytometry, or Western blotting. It was assessed whether ASM cells respond to polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of dsRNA, with alterations in M2R, M3R, H1R, and CysLT1R expression. The function of these subtypes was evaluated by cholinergic regulation of forskolin-stimulated cyclic AMP accumulation or by mobilization of intracellular calcium upon stimulation. RESULTS: ASM cells expressed TLR3 and PKR, and intracellular TLR3 expression was demonstrated. Poly I:C caused decreased M2R and increased M3R expression, without affecting H1R and CysLT1R expression. Poly I:C-treated cells showed decreased cholinergic inhibition of forskolin-stimulated cyclic AMP accumulation and enhanced calcium flux in response to acetylcholine, but not to histamine and LTD4. These modulating effects of poly I:C were reversed by chloroquine, but not by 2-aminopurine. CONCLUSIONS: The data indicate that poly I:C internalized by ASM cells differentially regulates M2R and M3R expression and function by interacting with TLR3 rather than with PKR, suggesting that these changes may contribute to airway hyperreactivity.

Familial Churg-Strauss syndrome in two sisters.

Churg-Strauss syndrome (CSS) is an uncommon systemic vasculitis with an increase in the number of eosinophils in the peripheral blood and tissues. Its pathogenesis is unknown, and there is no evidence that genetic factors influence susceptibility to this disease. We present a case of familial CSS in two sisters with atopic-type bronchial asthma and negative perinuclear anti-neutrophil cytoplasmic antibody results. We investigated the human leukocyte antigen typing of the sisters and their six living siblings but found no evidence for heritability of CSS. To our knowledge, this is the first report of familial CSS.

Induction and activation of the aryl hydrocarbon receptor by IL-4 in B cells.

It is widely known that IL-4 and IL-13 act on various kinds of cells, including B cells, resulting in enhancement of proliferation, class switching to IgE and expression of several surface proteins. These functions are important for the recognition of the various antigens in B cells and are known to be involved in the pathogenesis of allergic diseases. However, it has not been known whether IL-4/IL-13 is involved in the metabolism of various kinds of xenobiotics including 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), and it remains undetermined whether TCDD, an environmental pollutant, influences IgE production in B cells, exaggerating allergic reactions. We identified IL-4- or IL-13-inducible genes in a human Burkitt lymphoma cell line, DND-39, using microarray technology, in which the AHR gene was included. The AHR gene product, the aryl hydrocarbon receptor (AhR), was induced by IL-4 in both mouse and human B cells in a STAT6-dependent manner. IL-4 alone had the ability to translocate the induced AhR to the nuclei. TCDD, a ligand for AhR, rapidly degraded the induced AhR by the proteasomal pathway, although IL-4-activated AhR sustained its expression. AhR activated by IL-4 caused expression of a xenobiotic-metabolizing gene, CYP1A1, and TCDD synergistically acted on the induction of this gene by IL-4. However, the induction of AhR had no effect on IgE synthesis or CD23 expression. These results indicate that the metabolism of xenobiotics would be a novel biological function of IL-4 and IL-13 in B cells, whereas TCDD is not involved in IgE synthesis in B cells.

Differential effect of IL-4 and IL-13 on the expression of recombination-activating genes in mature B cells from human peripheral blood.

We examined the expression of recombination-activating genes (RAG-1 and RAG-2) and activation-induced cytidine deaminase (AID) by mature human blood B cells stimulated with anti-CD40 in the presence of IL-4 or IL-13. IL-4 was an effective cofactor for RAG-1 and RAG-2 expression, whereas IL-13 was not. In addition, IL-4-dependent RAG expression combined with AID and IgE expression allowed predominant expression of newly rearranged lambda light chains on IgE+ cells generated from kappa+ cells. Although the magnitudes of IL-4- and IL-13-dependent AID and IgE expression were related to expression levels of binding subunits of the IL-4 and IL-13 receptors, IL-13 was ineffective for light chain replacement in the induced IgE+ cells due to the failure in RAG expression. Our studies using mature blood B cells indicate that IL-4-responsive cells, unlike IL-13-responsive cells, undergo lambda gene rearrangement leading to replacement in parallel with RAG expression and suggest that this replacement may contribute to the regulation of affinity maturation of IgE antibodies.

Reciprocal regulation of thymus and activation-regulated chemokine/macrophage-derived chemokine production by interleukin (IL)-4/IL-13 and interferon-gamma in HaCaT keratinocytes is mediated by alternations in E-cadherin distribution.

Keratinocytes produce many cytochemokines that are involved in the pathogenesis of skin disorders. In particular, the CC chemokines thymus and activation-regulated chemokine (TARC)/macrophage-derived chemokine (MDC) play an important role in the infiltration of Th2 cells. This study was undertaken to examine the regulatory effects of interleukin (IL)-4, IL-13, and interferon (IFN)-gamma on TARC/MDC production in the human keratinocyte cell line HaCaT. HaCaT cells spontaneously secrete TARC and MDC. The production of TARC/MDC was downregulated by IL-4/IL-13, whereas it was upregulated by IFN-gamma. To explore these regulatory mechanisms, we investigated the capacity of cytokines to regulate expression of several adhesion molecules that may affect TARC/MDC production. Of the adhesion molecules examined, the constitutive surface expression of E-cadherin was downregulated by IL-4/IL-13, but was upregulated by IFN-gamma. Moreover, disruption of the homophilic adherence of E-cadherin by anti-E-cadherin antibody or calcium chelation abolished the production of TARC/MDC. We further examined the distribution of the adherens junction complex composed of E-cadherin, alpha-catenin, beta-catenin, and gamma-catenin. IL-4/IL-13 decreased the levels of membrane staining for adherens junction proteins, whereas IFN-gamma increased membrane staining. Taken together, these results suggest that IL-4/IL-13 and IFN-gamma induce alternations in the distribution of adherens junctions in a different fashion and thereby contribute to the reciprocal regulation of TARC/MDC production.

Differential regulation of thymus- and activation-regulated chemokine induced by IL-4, IL-13, TNF-alpha and IFN-gamma in human keratinocyte and fibroblast.

The CC chemokine thymus- and activation-regulated chemokine (TARC/CCL17) acts on CC chemokine receptor 4 (CCR4), which is known to be selectively expressed in Th2 cells. In order to compare the regulatory profiles of TARC production by tumor necrosis factor-alpha (TNF-alpha), IFN-gamma, interleukin-4 (IL-4) and IL-13 in keratinocytes and fibroblasts, HaCaT cells, a human keratinocyte cell line, and NG1RGB cells, a human skin fibroblast cell line, were used. The expression of TARC protein was measured using enzyme-linked immunosorbent assay (ELISA), and the mRNA level was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The spontaneous expression of TARC protein and mRNA levels were augmented by TNF-alpha and IFN-gamma and were inhibited by IL-4 and IL-13 in the keratinocytes. The fibroblasts expressed the TARC protein and mRNA only in the presence of IL-4+TNF-alpha or IL-13+TNF-alpha stimulation. IFN-gamma further enhanced the IL-4+TNF-alpha or IL-13+TNF-alpha-induced TARC production in the fibroblasts. Thus, TNF-alpha and IFN-gamma -induced TARC production was differentially regulated by IL-4 and IL-13 in human keratinocytes and fibroblasts.

CD40-mediated tumor necrosis factor receptor-associated factor 3 signaling upregulates IL-4-induced germline Cepsilon transcription in a human B cell line.

Induction of germline C epsilon transcription in B cells by IL-4, which is a critical initiating step for IgE class switching, is enhanced by CD40 engagement. Although signaling by CD40 is initiated by the binding of tumor necrosis factor receptor-associated factor (TRAF) family members to its cytoplasmic domain, whether those TRAF family proteins mediate enhancement of germline Cepsilon transcription is not evident. We report here that CD40-induced TRAF3-dependent activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) is involved in the upregulation of IL-4-driven germline C epsilon transcription in a human Burkitt's lymphoma B cell line, DG75. Among the six known TRAF proteins, TRAF2, 3, 5, and 6 associated with CD40 in an unstimulated state, and the levels of these four proteins were unaffected by anti-CD40 stimulation. Antisense oligodeoxynucleotide (ODN) for TRAF3 inhibited CD40-induced activation of MEK1-ERK pathway by decreasing expression of TRAF3 protein, but antisense ODNs for TRAF2, 5, and 6 were ineffective. Furthermore, CD40-mediated enhancement of IL-4-driven germline C epsilon transcription was inhibited by antisense ODN for TRAF3 and by a MEK1 inhibitor, PD98059. These results suggest that in DG75 cells, TRAF3-induced MEK1 activation may be involved in CD40-mediated upregulation of IL-4-driven germline C epsilon transcription.