Serum IgA augments adhesiveness of cultured lung microvascular endothelial cells and suppresses angiogenesis.

Immunoglobulin A (IgA) is important in local immunity and is also abundant in the blood. This study aimed to evaluate the effects of serum IgA on cultured lung microvascular endothelial cells (HMVEC-Ls), which are involved in the pathogenesis of inflammatory lung diseases. Serum IgA induced adhesion molecules and inflammatory cytokine production from HMVEC-Ls, and enhanced adhesion of peripheral blood mononuclear cells to HMVEC-Ls. In contrast, migration, proliferation, and tube formation of HMVEC-Ls were significantly suppressed by serum IgA. Experiments with siRNAs and western blotting revealed that two known IgA receptors, ß1,4-galactosyltransferase 1 (b4GALT1) and asialoglycoprotein receptor 1 (ASGR1), and mitogen-activated protein kinase and nuclear factor-kappa B pathways were partly involved in serum IgA-induced cytokine production by HMVEC-Ls. Collectively, serum IgA enhanced cytokine production and adhesiveness of HMVEC-L, with b4GALT1 and ASGR1 partially being involved, and suppressed angiogenesis. Thus, serum IgA may be targeted to treat inflammatory lung diseases.

Leptin-producing monocytes in the airway submucosa may contribute to asthma pathogenesis.

BACKGROUND: Obesity leads to an increase in the incidence and severity of asthma. Adipokines, such as leptin, secreted by adipocytes induce systemic inflammation, causing airway inflammation. We previously reported that leptin activates both inflammatory and structural cells, including lung fibroblasts. However, little is known about the differential leptin expression and responsiveness to leptin in asthmatic individuals and healthy controls (HC). In this study, we investigated the expression and origin of leptin in asthmatic airways. We also compared the effect of leptin on asthmatic and HC fibroblasts. METHODS: Lung specimens from asthmatic and non-asthmatic patients were analyzed by immunohistochemical staining using anti-leptin and anti-CD163 antibodies. Leptin mRNA and protein levels in human monocytes were detected by real-time PCR and western blotting and ELISA, respectively. We used flow cytometry to analyze asthmatic and HC lung fibroblasts for leptin receptor (Ob-R) expression. Further, we determined cytokine levels using cytometric bead array and ELISA and intracellular phosphorylation of specific signaling molecules using western blotting. RESULTS: Asthma specimens displayed accumulation of leptin-positive inflammatory cells, which were also positive for CD163, a high-affinity scavenger receptor expressed by monocytes and macrophages. Leptin expression was observed at both transcript and protein levels in human blood-derived monocytes. No significant differences were observed between asthmatic and HC lung fibroblasts in Ob-R expression, cytokine production, and intracellular phosphorylation of p38 mitogen-activated protein kinase. CONCLUSIONS: Our findings reveal similar responsiveness of control and asthmatic fibroblasts to leptin. However, the accumulation of inflammatory leptin-producing monocytes in the airway may contribute to the pathogenesis of asthma.

Immunoglobulin A promotes IL-6 and IL-8 production, proliferation, and migration by the human bronchial smooth muscle cells.

Immunoglobulin A (IgA) is important in biological defense, mainly in the mucosal area, and plays pathogenic roles in various diseases by activating both inflammatory and structural cells. The current study aimed to validate the effects of IgA on the human bronchial smooth muscle cell (BSMC), which plays a major role in airway inflammation and remodeling. Serum IgA induced interleukin (IL)-6 and IL-8 production at both mRNA and protein levels, and enhanced cell proliferation and migration by the BSMCs. The synthetic phenotype markers were regulated and the contractile phenotype markers were downregulated by serum IgA. Mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and nuclear factor-κB pathways were involved in IgA-induced IL-6 and IL-8 production. The BSMCs expressed transferrin receptor (TfR), and TfR siRNA transfection inhibited IL-6 and IL-8 production by serum IgA. In summary, serum IgA is a potent activator of the BSMCs at least partially via TfR.

Identification of ANXA2 on epithelial cells as a new receptor for secretory IgA using immunoprecipitation and mass spectrometry.

Secretory immunoglobulin A plays an important role in the protection against exogenous pathogens and antigens, but it has also been reported to have pathogenic potential. We previously found that secretory immunoglobulin A accumulated in the peripheral lungs during idiopathic pulmonary fibrosis and that transferrin receptor/CD71 was partially involved in secretory immunoglobulin A-induced inflammatory cytokine production in A549 cells. This study aimed to identify the receptor responsible for the induction of cytokine production by secretory immunoglobulin A-stimulated airway epithelial cells. To this end, immunoprecipitation followed by time-of-flight mass spectrometry and peptide mass fingerprinting were performed and Annexin A2 was detected as a novel receptor for secretory immunoglobulin A. Enzyme-linked immunosorbent assay demonstrated binding of secretory immunoglobulin A to Annexin A2, and flow cytometry showed robust expression of Annexin A2 on the surface of BEAS-2B cells, A549 cells, and normal human bronchial/tracheal epithelial cells. Experiments in A549 cells using Annexin A2 small interfering RNA and neutralizing antibodies suggested that Annexin A2 was partially involved in the production of interleukin-8/CXCL8 and C-C motif chemokine ligand 2/monocyte chemoattractant protein-1 induced by secretory immunoglobulin A. Immunohistochemistry using lung sections revealed clear expression of Annexin A2 on airway epithelial cells, although the staining remained equivalent in idiopathic pulmonary fibrosis, asthma, and healthy control lungs. In conclusion, we identified that Annexin A2 expressed in airway epithelial cells is a novel receptor for secretory immunoglobulin A, which is involved in cytokine synthesis.

Leptin enhances cytokine/chemokine production by normal lung fibroblasts by binding to leptin receptor.

BACKGROUND: Obesity is a known risk and exacerbation factor for bronchial asthma. Leptin is an adipokine secreted by adipocytes and enhances energy consumption. Earlier studies have shown that leptin also activates inflammatory cells and structural cells, including airway epithelial cells, thereby exacerbating inflammation. However, little is known about leptin's effect on normal human lung fibroblasts (NHLFs), which are deeply involved in airway remodeling in asthma. This study aimed to elucidate the direct effect of leptin on NHLFs. METHODS: NHLFs were co-cultured with leptin, and production of cytokines/chemokines was analyzed with real-time PCR and cytometric bead arrays (CBA). Expression of alpha smooth muscle actin (α-SMA) in the lysate of NHLFs stimulated with leptin was assessed by western blotting. Expression of leptin receptor (Ob-R) was analyzed by real-time PCR and flow cytometry. NHLFs were transfected with Ob-R small interference ribonucleic acid (siRNA) by electroporation and used for experiments. RESULTS: Leptin enhanced production of CCL11/Eotaxin, monocyte chemoattractant protein-1 (CCL2/MCP-1), CXCL8/IL-8, interferon gamma-induced protein 10 (CXCL10/IP-10) and IL-6 by NHLFs at both the protein and messenger ribonucleic acid (mRNA) levels. Leptin also slightly, but significantly, elevated expression of α-SMA. We found robust Ob-R expression on cell surfaces, and transfection with Ob-R siRNA suppressed the enhanced production of CCL11/Eotaxin, CXCL10/IP-10 and IL-6 by leptin, although not completely. CONCLUSIONS: These findings indicate that leptin may contribute to worsening of asthma in obese patients by enhancing production of inflammatory mediators by binding to Ob-R and accelerating myofibroblast differentiation.

Epithelial-mesenchymal transition promotes reactivity of human lung adenocarcinoma A549 cells to CpG ODN.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is reported to promote airway remodeling in asthmatics, which is the main histological change that causes complex and severe symptoms in asthmatics. However, little is known about whether EMT also plays a role in acute exacerbations of asthma evoked by respiratory tract infections. METHODS: A human lung adenocarcinoma line, A549, was incubated with TGF-ß1 at 10 ng/ml to induce EMT. Then the cells were stimulated with CpG ODN. Expression of surface and intracellular molecules was analyzed by flow cytometry. IL-6, IL-8 and MCP-1 in the culture supernatant were measured by Cytometric Bead Assay, and the expression of mRNA was quantitated by real-time PCR. CpG ODN uptake was analyzed by flow cytometry. RESULTS: The culture supernatant levels of IL-6, IL-8 and MCP-1 and the expression of mRNA for these cytokines in CpG ODN-stimulated A549 cells that had undergone EMT was significantly higher compared to those that had not. Addition of ODN H154, a TLR9-inhibiting DNA, significantly suppressed the CpG ODN-induced production of those cytokines. However, flow cytometry found the level of TLR9 expression to be slightly lower in A549 cells that had undergone EMT compared to those that had not. On the other hand, CpG ODN uptake was increased in cells that had undergone EMT. CONCLUSIONS: EMT induction of A549 cells enhanced CpG ODN uptake and CpG ODN-induced production of IL-6, IL-8 and MCP-1. These results suggest that EMT plays an important role in exacerbation in asthmatics with airway remodeling by enhancing sensitivity to extrinsic pathogens.

Leptin enhances ICAM-1 expression, induces migration and cytokine synthesis, and prolongs survival of human airway epithelial cells.

There is rising interest in how obesity affects respiratory diseases, since epidemiological findings indicate a strong relationship between the two conditions. Leptin is a potent adipokine produced mainly by adipocytes. It regulates energy storage and expenditure and also induces inflammation. Previous studies have shown that leptin is able to activate inflammatory cells such as lymphocytes and granulocytes, but little is known about its effect on lung structural cells. The present study investigated the effects of leptin on human airway epithelial cells by using human primary airway epithelial cells and a human airway epithelial cell line, BEAS-2B. Flow cytometry showed enhanced ICAM-1 expression by both of those cells in response to leptin, and that effect was abrogated by dexamethasone or NF-κB inhibitor. Flow cytometry and quantitative PCR showed that airway epithelial cells expressed leptin receptor (Ob-R), whose expression level was downregulated by leptin itself. Multiplex cytokine analysis demonstrated enhanced production of CCL11, G-CSF, VEGF, and IL-6 by BEAS-2B cells stimulated with leptin. Furthermore, transfection of Ob-R small interference RNA decreased the effect of leptin on CCL11 production as assessed by quantitative PCR. Finally, leptin induced migration of primary airway epithelial cells toward leptin, suppressed BEAS-2B apoptosis induced with TNF-α and IFN-γ, and enhanced proliferation of primary airway epithelial cells. In summary, leptin was able to directly activate human airway epithelial cells by binding to Ob-R and by NF-κB activation, resulting in upregulation of ICAM-1 expression, induction of CCL11, VEGF, G-CSF, and IL-6 synthesis, induction of migration, inhibition of apoptosis, and enhancement of proliferation.

Alcohol-related injury to peribiliary glands is a cause of peribiliary cysts: based on analysis of clinical and autopsy cases.

BACKGROUND AND GOAL: Peribiliary cysts, which are known to be associated with various hepatobiliary diseases including alcoholic liver disease, have been reported to originate in the peribiliary glands along the biliary tree. The causal relationship between the peribiliary cysts and alcohol-related hepatic and pancreatic disease were examined in this study. METHODS AND RESULTS: Peribiliary cysts were surveyed in the radiologic reports of out-patients and in-patients at our hospital (between 2007 and 2011), and a total of 31 patients with peribiliary cysts were found; 9 patients were associated with alcoholic liver disease and 2 patients with alcoholic pancreatitis. Among 202 consecutive autopsy cases with a history of heavy drinking (chronic alcoholics) at our Department (between 1990 and 2011), peribiliary cysts were found in 29 cases (14%), and the frequency of these cysts was correlated with the degree of alcohol-related hepatic fibrosis. Interestingly, peribiliary cysts were frequently associated with adenitis of the peribiliary glands (72%), and peribiliary adenitis and cyst formation correlated well with the degree of pancreatic fibrosis. CONCLUSIONS: These results suggest that peribiliary cysts are more likely to occur in chronic alcoholics. The frequent association of peribiliary cysts with the degree of alcohol-related hepatic fibrosis suggests the involvement of the hepatic fibrogenetic process in peribiliary cyst formation. The frequent association of peribiliary adenitis and cyst formation with the degree of pancreatic fibrosis in chronic alcoholics suggests the involvement of alcoholic injuries in the pancreas, resulting in progressive fibrosis, and peribiliary glands, resulting in adenitis and cyst formation.