The Role Of Intelectin In The Airway Inflammation In Asthma

Asthma is a common disease that the form of airway inflammation is chronic and non-specific nature, with excessive airway mucus secretion, airway hyperresponsiveness (AHR), serum IgE increased and airway eosinophil infiltration. Distinct chemokines expressing within the airway has led to the realization that there might be specific profiles of chemokines that mediate various stages of asthmatic disease. A large family of peptide chemotactic cytokine mediators have been identified that control leukocyte migration and activation. Chemokines have been divided primarily into two main groups on the basis of their sequence homology and the position of the first two cysteine residues, C-C and C-X-C [1]. Main cellular sources of chemokines within the asthmatic airway include alveolar macrophages and airway epithelial cells. As eosinophils are implicated in the pathophysiology of asthma, research has focused primarily on chemokines that have chemotactic activity for eosinophils. A lot of chemokines, such as RANTES /CCL5, MCP-4/CCL13 and MCP-3/CCL7, have been identified in the airways of asthmatics. These induce eosinophil recruitment through the CCR3 receptor, which expresses highly on eosinophils. In addition to recruiting and activating eosinophils[2,3], these same chemokines can affect other asthma-related leukocyte populations, such as T helper (TH)2-type lymphocytes and basophils. Recent studies have found that in the bronchoalveolar lavage fluid (BALF) the development of status asthmaticus was associated with significantly higher levels of MCP-1, CCL3 and CCL5 [4].Intelectin belongs to the lectins family, including Intelectin1and 2 which both are found to be expressed in paneth cells in mouse small intestine. Intelectin2 exhibits 91% sequence identity to Intelectin1. There are also two known intelectins in the human genome: Intelectin1 and Intelectin2. The mature hIntL is a secretory glycoprotein consisting of 295 amino acids and N-linked oligosaccharides, and its basic structural unit is a 120-kDa homotrimer in which 40-kDa polypeptides are bridged by disulfide bonds. Affinities of recombinant hIntL are revealed to D-pentoses and a D-galactofuranosyl residue in the presence of Ca2+, and recombinant hIntL can recognize the bacterial arabinogalactan of Nocardia containing D-galactofuranosyl residues. These results suggests that hIntL is a new type lectin recognizing galactofuranose, and that hIntL plays a role in recognizing bacteria-specific components in the host[5]. Furthermore, intelectin has also been shown to be upregulated in bronchial brushings and bronchoalveolar lavage fluid from asthmatics. We analyzed gene expression of intelectin was increased in IL-13–overexpressing mice , IL-13/Epi mice and ovalbumin allergic mice that developed mucus overproduction and airway hyper-reactivity with microarrays, which was further revealed that intelectin was associated with asthma[13]. And our previously study found that the expression of Intelectin1 and 2 is upregulated after sensitization and challenge with OVA in mouse lung, and Intelectin is expressed in airway mucous cells in a mouse allergic asthma model. A Th2 cytokine, IL-13, is a critical mediator of allergic airway disease[15,16]. Since Intelectin and the chemokines are produced by airway epithelial cells, we used MLE12 cells, a mouse lung epithelial cell line, to investigate the role of intelectin in IL-13-induced chemokine expression. IL-13 treatment increased MCP-1 and MCP-3 expression in MLE12 cells. Intelectin1 and 2 expression is also stimulated by IL-13 in MLE12 cells. Using RNA interference and galactose, an inhibitor of intelectin, we showed that intelectin is required for IL-13- induced MCP-1 and MCP-3 expression at both mRNA and protein levels. To our knowledge, this is the first study showing that intelectin mediates IL-13-induced upregulation of MCP-1 and MCP-3, but the mechanism is not yet known.So in this study using RNA interference to downregulate Intelectin1 and 2 expression in OVA-induced asthmatic mouse, we will observe the change of inflammation in asthma and MCP-1 and MCP-3 expression. We also use IL-13- stimulated MLE12 cell to find the path way by which intelectin mediates IL-13-induced upregulation of MCP-1 and MCP-3. This research is divied into two parts:PartⅠThe Role of Intelectin in Airway Inflammation and the Expression of MCP-1 and MCP-3 in AsthmaPurpose: To observe the role of Intelectin in airway inflammation in asthma and the expression of MCP-1 and MCP-3.Method: Mice were divided into three groups: (1) normal mice + negative control plasmid group, (2) asthmatic mice + negative control plasmid group, (3) asthmatic mice + Intelectin shRNA group. We instilled the transfection reagents and plasmids into mice'noses. Real time PCR was applied to detect the expression of Intelectin1 and 2, MCP-1 and MCP-3 mRNA. Immunohistochemistry was applied to detect the expression of Intelectin in lung. We observd the change of airway inflammation in mouse lung with inflammation score and the number of eosinophils in BAL fluid. Result: Compared with OVA group, Intelectin1, 2 mRNA of OVA + shRNA group respectively decreased by 72% and 59%, P < 0.05; MCP-1, MCP-3 mRNA of OVA + shRNA group decreased remarkably, P < 0.05; the quantity of eosinophile granulocyte in BALF of OVA + shRNA group decreased by 76%, P < 0.05. The inflammation score, OVA group exceeded control goup, P < 0.01, OVA + shRNA group were much less than OVA group, P < 0.05.Conclution: Histological analysis revealed that MCP-1 and MCP-3 expression was downregulated after intelectin shRNA transfection, and inflammatory cell infiltration around conducting airways was ameliorated, the number of eosinophile granulocyte was also reduced .Key words: asthma; Intelectin1, 2; shRNA; MCP-1 and MCP-3PartⅡThe Role and Mechanism of Intelectin in IL-13-Induced MCP-1 and MCP-3 Expression in MLE12 CellsPurpose: To find the role and mechanism of Intelectin affecting Inflammatory mediators MCP-1 and MCP-3 in MLE12 cells.Method: We used IL-13 to stimulate the MLE 12 cell with Intelectin shRNA transfection, the cells were divided into three groups: (1) normal + negative control plasmid group; (2) IL-13 + negative control plasmid group; (3) IL-13 + Intelectin shRNA group. Real time PCR was applied to detect the expression of Intelectin1 and 2, MCP-1 and MCP-3 mRNA. Protein expression of ERK 1/2 and P-ERK 1/2 were measured by Western blotting. We used IL-13 and PD98059 to stimulate the MLE 12 cell, the cells were divided into three groups: (1) normal group; (2) IL-13 group; (3) IL-13 + PD98059 group. Real time PCR was applied to detect the expression of MCP-1 and MCP-3 mRNA.Result: Compared with IL-13 group, Intelectin1 and -2, MCP-1 and MCP-3 mRNA of IL-13 + shRNA group were declined, P < 0.05. MCP-1 and MCP-3 mRNA of IL-13 + PD98059 group were declined, P < 0.01. The expression of P-ERK 1/2 of IL-13 group was much more than control group, but IL-13 + shRNA group was less than IL-13 group.Conclution: We found inhibiting Intelectin could reduce the expression of MCP-1 and MCP-3. We also found that PD 98059, an inhibitor of ERK 1/2, blocked IL-13-induced MCP-1 and MCP-3 expression in MLE 12 cells. Furthermore, IL-13 stimulation increased ERK 1/2 phosphorylation, and Intelectin shRNA transfection inhibited IL-13-induced ERK 1/2 phosphorylation. These data indicated that the ERK 1/2 pathway mediated IL-13-induced MCP-1 and MCP-3 expression, and that Intelectin expression was required for IL-13-induced ERK 1/2 activation in mouse lung epithelial cells. Therefore, Intelectin may participate in IL-13-induced MCP-1 and MCP-3 expression, at least in part, through contributing to ERK 1/2 activation. Key words: Intelectin; PD98059; shRNA; MLE 12 cell; MCP-1; MCP-3; ERK1/2; P-ERK1/2...