Bit Na Kang

Allergen-Induced Airway Remodeling Is Impaired in Galectin-3–Deficient Mice

The role played by the β-galactoside–binding lectin galectin-3 (Gal-3) in airway remodeling, a characteristic feature of asthma that leads to airway dysfunction and poor clinical outcome in humans, was investigated in a murine model of chronic allergic airway inflammation. Wild-type (WT) and Gal-3 knockout (KO) mice were subjected to repetitive allergen challenge with OVA up to 12 wk, and bronchoalveolar lavage fluid (BALF) and lung tissue collected after the last challenge were evaluated for cellular features associated with airway remodeling. Compared to WT mice, chronic OVA challenge in Gal-3 KO mice resulted in diminished remodeling of the airways with significantly reduced mucus secretion, subepithelial fibrosis, smooth muscle thickness, and peribronchial angiogenesis. The higher degree of airway remodeling in WT mice was associated with higher Gal-3 expression in the BALF as well as lung tissue. Cell counts in BALF and lung immunohistology demonstrated that eosinophil infiltration in OVA-challenged Gal-3 KO mice was significantly reduced compared with that WT mice. Evaluation of cellular mediators associated with eosinophil recruitment and airway remodeling revealed that levels of eotaxin-1, IL-5, IL-13, found in inflammatory zone 1, and TGF-β were substantially lower in Gal-3 KO mice. Finally, leukocytes from Gal-3 KO mice demonstrated decreased trafficking (rolling) on vascular endothelial adhesion molecules compared with that of WT cells. Overall, these studies demonstrate that Gal-3 is an important lectin that promotes airway remodeling via airway recruitment of inflammatory cells, specifically eosinophils, and the development of a Th2 phenotype as well as increased expression of eosinophil-specific chemokines and profibrogenic and angiogenic mediators.

ORMDL3 promotes eosinophil trafficking and activation via regulation of integrins and CD48

ORM (yeast)-Like protein isoform 3 (ORMDL3) has recently been identified as a candidate gene for susceptibility to asthma; however the mechanisms by which it contributes to asthma pathogenesis are not well understood. Here we demonstrate a functional role for ORMDL3 in eosinophils in the context of allergic inflammation. Eosinophils recruited to the airways of allergen-challenged mice express ORMDL3. ORMDL3 expression in bone marrow eosinophils is localized in the endoplasmic reticulum and is induced by IL-3 and eotaxin-1. Over-expression of ORMDL3 in eosinophils causes increased rolling, distinct cytoskeletal rearrangement, ERK (1/2) phosphorylation and nuclear translocation of NF-κB. Knock-down of ORMDL3 significantly inhibits activation-induced cell shape changes, adhesion and recruitment to sites of inflammation in vivo, combined with reduced expression of CD49d and CD18. Additionally, ORMDL3 regulates IL-3-induced expression of CD48 and CD48-mediated eosinophil degranulation. These studies show that ORMDL3 regulates eosinophil trafficking, recruitment and degranulation, further elucidating a role for this molecule in allergic asthma and potentially other eosinophilic disorders.

Inactivation of heparan sulfate 2-O-sulfotransferase accentuates neutrophil infiltration during acute inflammation in mice

Neutrophil recruitment and extravasation at sites of inflammation provide a mechanism for host defense. We showed previously that heparan sulfate, a type of sulfated glycosaminoglycan, facilitates neutrophil recruitment based on the reduction of neutrophil infiltration in mice in which the overall sulfation of the chains was reduced by selective inactivation of N-acetylglucosamine N-deacetylase-N-sulfotransferase (Ndst1) in endothelial cells. Here we show that inactivation of uronyl 2-O-sulfotransferase in endothelial cells (Hs2st), an enzyme that acts downstream from Ndst1, results in enhanced neutrophil recruitment in several models of acute inflammation. Enhanced neutrophil infiltration resulted in part from reduced rolling velocity under flow both in vivo and in vitro, which correlated with stronger binding of neutrophil L-selectin to mutant endothelial cells. Hs2st-deficient endothelial cells also displayed a striking increase in binding of IL-8 and macrophage inflammatory protein-2. The enhanced binding of these mediators of neutrophil recruitment resulted from a change in heparan sulfate structure caused by increased N-sulfation and 6-O-sulfation of glucosamine units in response to the decrease in 2-O-sulfation of uronic acid residues. This gain-of-function phenotype provides formidable evidence demonstrating the importance of endothelial heparan sulfate in inflammation and suggests a novel enzyme target for enhancing the innate immune response.

Transcriptional regulation of CD38 expression by tumor necrosis factor-α in human airway smooth muscle cells: role of NF-κB and sensitivity to glucocorticoids

The transmembrane glycoprotein CD38 catalyzes the synthesis of the calcium mobilizing molecule cyclic ADP‐ribose from NAD. In human airway smooth muscle (HASM) cells, the expression and function of CD38 are augmented by the inflammatory cytokine tumor necrosis factor‐alpha (TNF‐α), leading to increased intracellular calcium response to agonists. A glucocorticoid response element in the CD38 gene has been computationally described, providing evidence for transcriptional regulation of its expression. In the present study, we investigated the effects of dexamethasone, a glucocorticoid, on CD38 expression and ADP‐ribosyl cyclase activity in HASM cells stimulated with TNF‐α. In HASM cells, TNF‐α augmented CD38 expression and ADP‐ribosyl cyclase activity, which were attenuated by dexamethasone. TNF‐α increased NF‐κB expression and its activation, and dexamethasone partially reversed these effects. TNF‐α increased the expression of IκBα, and dexamethasone increased it further. An inhibitor of NF‐κB activation or transfection of cells with IκB mutants decreased TNF‐α‐induced CD38 expression. The results indicate that TNF‐α‐induced CD38 expression involves NF‐κB expression and its activation and dexamethasone inhibits CD38 expression through NF‐κB‐dependent and ‐independent mechanisms.— Kang, B.‐N., Tirumurugaan, K. G., Deshpande, D. A., Amrani, Y., Panettieri, R. A., Walseth, T. F., Kannan, M. S. Transcriptional regulation of CD38 expression by tumor necrosis factor‐α in human airway smooth muscle cells: role of NF‐κB and sensitivity to glucocorticoids. FASEB J. 20, E170–E179 (2006)

Deficiency of Endothelial Heparan Sulfates Attenuates Allergic Airway Inflammation

The effect of targeted inactivation of the gene encoding N-deacetylase/N-sulfotransferase-1 (Ndst1), a key enzyme involved in the biosynthesis of heparan sulfate (HS) chains, on the inflammatory response associated with allergic inflammation in a murine model of OVA-induced acute airway inflammation was investigated. OVA-exposed Ndst1f/fTekCre+ (mutant) mice deficient in endothelial and leukocyte Ndst1 demonstrated significantly decreased allergen-induced airway hyperresponsiveness and inflammation characterized by a significant reduction in airway recruitment of inflammatory cells (eosinophils, macrophages, neutrophils, and lymphocytes), diminished IL-5, IL-2, TGF-β1, and eotaxin levels, as well as decreased expression of TGF-β1 and the angiogenic protein FIZZ1 (found in inflammatory zone 1) in lung tissue compared with OVA-exposed Ndst1f/fTekCre− wild-type littermates. Furthermore, murine eosinophils demonstrated significantly decreased rolling on lung endothelial cells (ECs) from mutant mice compared with wild-type ECs under conditions of flow in vitro. Treatment of wild-type ECs, but not eosinophils, with anti-HS Abs significantly inhibited eosinophil rolling, mimicking that observed with Ndst1-deficient ECs. In vivo, trafficking of circulating leukocytes in lung microvessels of allergen-challenged Ndst1-deficient mice was significantly lower than that observed in corresponding WT littermates. Endothelial-expressed HS plays an important role in allergic airway inflammation through the regulation of recruitment of inflammatory cells to the airways by mediating interaction of leukocytes with the vascular endothelium. Furthermore, HS may also participate by sequestering and modulating the activity of allergic asthma-relevant mediators such as IL-5, IL-2, and TGF-β1.

Glucocorticoid regulation of CD38 expression in human airway smooth muscle cells: role of dual specificity phosphatase 1

The enzymatic activity of CD38, ADP-ribosyl cyclase, synthesizes the calcium mobilizing molecule cyclic ADP-ribose from beta-NAD. In human airway smooth muscle (HASM) cells, CD38 expression is augmented by the inflammatory cytokine, TNF-alpha, causing increased intracellular calcium response to agonists. The transcriptional and posttranscriptional regulation of CD38 expression involves signaling through MAPKs and requires activation of NF-kappaB and activator protein-1 (AP-1). The cytokine-augmented CD38 expression is decreased by anti-inflammatory glucocorticoids due to inhibition of NF-kappaB activation and other mechanisms. In this study, we investigated glucocorticoid regulation of CD38 expression in HASM cells through the MKP-1. In HASM cells, dexamethasone and TNF-alpha induced MKP-1 expression (both mRNA and protein) rapidly. Dexamethasone decreased TNF-alpha-induced phosphorylation of the major MAPKs, i.e., ERK, p38, and JNK, and decreased the activation of NF-kappaB and AP-1. Dexamethasone also decreased CD38 expression induced by TNF-alpha, and part of this effect was attributable to decreased transcript stability. In cells transfected with MKP-1-specific small interfering RNAs (siRNAs), there was significant attenuation of MKP-1 expression and partial, but nonsignificant, reversal of dexamethasone inhibition of CD38 expression. These results indicate that regulation of CD38 expression in HASM cells by glucocorticoids involves decreased signaling through MAPKs and activation of transcription factors. The glucocorticoid effects on decreased CD38 expression and function result from regulation through transcription and transcript stability.

Regulation of Eosinophil Trafficking by SWAP-70 and Its Role in Allergic Airway Inflammation

Eosinophils are the predominant inflammatory cells recruited to allergic airways. In this article, we show that human and murine eosinophils express SWAP-70, an intracellular RAC-binding signaling protein, and examine its role in mediating eosinophil trafficking and pulmonary recruitment in a murine model of allergic airway inflammation. Compared with wild-type eosinophils, SWAP-70–deficient (Swap-70−/−) eosinophils revealed altered adhesive interactions within inflamed postcapillary venules under conditions of blood flow by intravital microscopy, exhibiting enhanced slow rolling but decreased firm adhesion. In static adhesion assays, Swap-70−/− eosinophils adhered poorly to VCAM-1 and ICAM-1 and exhibited inefficient leading edge and uropod formation. Adherent Swap-70−/− eosinophils failed to translocate RAC1 to leading edges and displayed aberrant cell surface localization/distribution of α4 and Mac-1. Chemokine-induced migration of Swap-70−/− eosinophils was significantly decreased, correlating with reduced intracellular calcium levels, defective actin polymerization/depolymerization, and altered cytoskeletal rearrangement. In vivo, recruitment of eosinophils to the lungs of allergen-challenged Swap-70−/− mice, compared with wild-type mice, was significantly reduced, along with considerable attenuation of airway inflammation, indicated by diminished IL-5, IL-13, and TNF-α levels; reduced mucus secretion; and improved airway function. These findings suggest that regulation of eosinophil trafficking and migration by SWAP-70 is important for the development of eosinophilic inflammation after allergen exposure.

Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-α and dexamethasone

BackgroundCD38 is expressed in human airway smooth muscle (HASM) cells, regulates intracellular calcium, and its expression is augmented by tumor necrosis factor alpha (TNF-α). CD38 has a role in airway hyperresponsiveness, a hallmark of asthma, since deficient mice develop attenuated airway hyperresponsiveness compared to wild-type mice following intranasal challenges with cytokines such as IL-13 and TNF-α. Regulation of CD38 expression in HASM cells involves the transcription factor NF-κB, and glucocorticoids inhibit this expression through NF-κB-dependent and -independent mechanisms. In this study, we determined whether the transcriptional regulation of CD38 expression in HASM cells involves response elements within the promoter region of this gene.MethodsWe cloned a putative 3 kb promoter fragment of the human cd38 gene into pGL3 basic vector in front of a luciferase reporter gene. Sequence analysis of the putative cd38 promoter region revealed one NF-κB and several AP-1 and glucocorticoid response element (GRE) motifs. HASM cells were transfected with the 3 kb promoter, a 1.8 kb truncated promoter that lacks the NF-κB and some of the AP-1 sites, or the promoter with mutations of the NF-κB and/or AP-1 sites. Using the electrophoretic mobility shift assays, we determined the binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-κB, AP-1, and GRE sites, and the specificity of this binding was confirmed by gel supershift analysis with appropriate antibodies.ResultsTNF-α induced a two-fold activation of the 3 kb promoter following its transfection into HASM cells. In cells transfected with the 1.8 kb promoter or promoter constructs lacking NF-κB and/or AP-1 sites or in the presence of dexamethasone, there was no induction in the presence of TNF-α. The binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-κB site and some of the six AP-1 sites was increased by TNF-α, and to some of the putative cd38 GREs by dexamethasone.ConclusionThe EMSA results and the cd38 promoter-reporter assays confirm the functional role of NF-κB, AP-1 and GREs in the cd38 promoter in the transcriptional regulation of CD38.

The p110δ subunit of PI3K regulates bone marrow-derived eosinophil trafficking and airway eosinophilia in allergen-challenged mice

Trafficking and recruitment of eosinophils during allergic airway inflammation is mediated by the phosphatidylinositol 3-kinase (PI3K) family of signaling molecules. The role played by the p110δ subunit of PI3K (PI3K p110δ) in regulating eosinophil trafficking and recruitment was investigated using a selective pharmacological inhibitor (IC87114). Treatment with the PI3K p110δ inhibitor significantly reduced murine bone marrow-derived eosinophil (BM-Eos) adhesion to VCAM-1 as well as ICAM-1 and inhibited activation-induced changes in cell morphology associated with reduced Mac-1 expression and aberrant cell surface localization/distribution of Mac-1 and α4. Infused BM-Eos demonstrated significantly decreased rolling and adhesion in inflamed cremaster muscle microvessels of mice treated with IC87114 compared with vehicle-treated mice. Furthermore, inhibition of PI3K p110δ significantly attenuated eotaxin-1-induced BM-Eos migration and prevented eotaxin-1-induced changes in the cytoskeleton and cell morphology. Knockdown of PI3K p110δ with siRNA in BM-Eos resulted in reduced rolling, adhesion, and migration, as well as inhibition of activation-induced changes in cell morphology, validating its role in regulating trafficking and migration. Finally, in a mouse model of cockroach antigen-induced allergic airway inflammation, oral administration of the PI3K p110δ inhibitor significantly inhibited airway eosinophil recruitment, resulting in attenuation of airway hyperresponsiveness in response to methacholine, reduced mucus secretion, and expression of proinflammatory molecules (found in inflammatory zone-1 and intelectin-1). Overall, these findings indicate the important role played by PI3K p110δ in mediating BM-Eos trafficking and migration by regulating adhesion molecule expression and localization/distribution as well as promoting changes in cell morphology that favor recruitment during inflammation.

N-Glycans Differentially Regulate Eosinophil and Neutrophil Recruitment during Allergic Airway Inflammation

Background: Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment. Results: Deficiency of MGAT5 causes attenuation of allergen-induced eosinophilia and Th2 cytokines but increases neutrophilic inflammation and airway hyperreactivity. Conclusion: Recruitment of eosinophils and neutrophils is differentially regulated by MGAT5-modified N-glycans during airway inflammation. Significance: This study demonstrates a significant role for N-glycans in the development of allergic airway inflammation and asthma. Allergic airway inflammation, including asthma, is usually characterized by the predominant recruitment of eosinophils. However, neutrophilia is also prominent during severe exacerbations. Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment during inflammation. Here, the involvement of UDP-N-acetylglucosamine:α-6-d-mannoside β1,6-N-acetylglucosaminyltransferase V (MGAT5)-modified N-glycans in eosinophil and neutrophil recruitment during allergic airway inflammation was investigated. Allergen-challenged Mgat5-deficient (Mgat5−/−) mice exhibited significantly attenuated airway eosinophilia and inflammation (decreased Th2 cytokines, mucus production) compared with WT counterparts, attributable to decreased rolling, adhesion, and survival of Mgat5−/− eosinophils. Interestingly, allergen-challenged Mgat5−/− mice developed airway neutrophilia and increased airway reactivity with persistent elevated levels of proinflammatory cytokines (IL-17A, TNFα, IFNγ)). This increased neutrophil recruitment was also observed in LPS- and thioglycollate (TG)-induced inflammation in Mgat5−/− mice. Furthermore, there was significantly increased recruitment of infused Mgat5−/− neutrophils compared with WT neutrophils in the peritoneal cavity of TG-exposed WT mice. Mgat5−/− neutrophils demonstrated enhanced adhesion to P-selectin as well as increased migration toward keratinocyte-derived chemokine compared with WT neutrophils in vitro along with increased calcium mobilization upon activation and expression of elevated levels of CXCR2, which may contribute to the increased neutrophil recruitment. These data indicate an important role for MGAT5-modified N-glycans in differential regulation of eosinophil and neutrophil recruitment during allergic airway inflammation.