| Objectives:Airway epithelial cell is the first barrier which forms the initial defense to inhaled air, pathogen and other substances. Under normal circumstances, injurious chemicals, particulates, and biological insults can be removed effectively by structural integrity and functional homeostasis of airway epithelial cells. However, disruption or functional breakdown of airway epithelial cell could induce or intensify many lung diseases. The integrity and structural adhesion of airway epithelial cells rely on the expression of adhesion molecule families. Integrinβ4 is a structural adhesion molecule that mediates the stable adhesion of epithelial cells to the basal membrane through hemidesmosomes architecture. The distinguishing structural feature of integrinβ4, i.e., the typical cytoplasmic domain, is different from any other member of integrin families in both size and structure. This feature of cytoplasmic domain indicated the possible complex signaling pathway and multiple functions of integrinβ4. Progress has been made in identifying specific regions and motives within the integrinβ4 cytoplasmic domain that mediate the cytoskeleton interactions and signaling properties of integrinβ4, but much remains to be determined. The precise physiologic function of integrinβ4 on airway epithelial cell was almost unknown. Our previous work identified genes that were differentially expressed in the peripheral leukocytes of asthma patients by using cDNA microarray analysis. The results demonstrated that integrinβ4 was significantly down-regulated in asthma patients, which indicated the possible relationship between integrinβ4 and asthma.Therefore, this study focused on:(1) The role of integrinβ4 to wound repair and anti-oxidation ability on RTE;(2) The expression of integrinβ4 on the mucus of asthma patients and the possible parthenogenesis of asthma by down regulation of integrinβ4;(3) The correlation between integrinβ4 expression and AHR after ozone stress;(4) The role of integrinβ4 to the antigen presentation ability of airway epithelial cells and the derivation of T cells.Contents:1. The role of integrinβ4 to wound repair and anti-oxidation ability on RTEBoth 16HBE14o- and RTE cells were stressed by ozone exposure for 30 min. A significant increase of integrinβ4 expression was observed by western blotting and flow cytometry detection after ozone stress. An OVA-challenged asthma model was constructed, the expression of integrin β4 on airway mucus was down regulated and the content of oxygen DCF in the OVA stressed airway epithelial cells was increased. These results indicated that integrinβ4 may engage in the regulation of anti-oxidation ability. Meanwhile, we detect integrinβ4 expression after mechanical injure. The results showed that expression of integrinβ4 was increased significantly 12h after mechanical injure both in 16HBE14o- and RTE. In the OVA-challenged model, immunohistochemistry results showed that bronchi walls and pulmonary alveoli were destroyed and unregular, which suggested the possible role of integrinβ4 in the wound repair processes of airway epithelial cells.To accurately identify the role of integrinβ4 on airway epithelial cells, we constructed integrinβ4 overexpression vector and integrinβ4 silence vector. The wound repair ability was assessed by wound repair index. The proliferation was detected by MTT assay. Our results showed that would repair of RTE cells were inhibited in integrinβ4 silence group. In contrast, cells with high expression of integrinβ4 showed increased repair ability. Also, we found that that proliferation of RTE cells was inhibited after integrinβ4 was silenced and promoted after integrinβ4 was overexpressed. The anti-oxidation ability was assessed by T-AOC assay and the content of SOD, GSH-Px, catalse was detected using commercial assay kits. We found that upregulation of integrinβ4 promoted the antioxidation ability of RTE cells and integrinβ4 silence inhibited. Meanwhile, integrinβ4 overexpression can significantly increase the expression of all three enzymes, while integrinβ4 silence cells showed a decreased regulation.Accordingly, these results suggested that expression of integrinβ4 on airway epithelial cells was regulated by lots of outer stimulation. Besides adhesion, integrinβ4 involves in regulating wound repair and anti-oxidation ability on airway epithelial cells. These new physiological functions of integrinβ4 may be of value in studying the pathogenesis of many airway diseases.2. The expression of integrinβ4 on the mucus of asthma patients and the possible parthenogenesis of asthma by down regulation of integrinβ4Our previous work identified genes that were differentially expressed in the peripheral leukocytes of asthma patients by using cDNA microarray analysis. The results demonstrated that integrinβ4 was significantly down-regulated in asthma patients, and three variation sites were acquired in 5 flanking region, which was correlated with down expression of integrinβ4. Here, we further detected the expression of integrinβ4 on the airway emucus of asthma patients. And the relationship between imbalanced expression of integrinβ4 and dysfunction of the airway epithelial cells on asthma patients was investigated. By immnohistochemistry, we detected that in both extracellular and cytoplasmic domains, integrinβ4 expression decreased significantly on the airway mucus of asthma patients. To explore the relationship between reduced expression of integrinβ4 and the pathological phenotypes of the airway epithelial cells on asthma airway, the specific integrinβ4 siRNAs was designed and screened. Then, a lentivirus vector was constructed and transfected into 16HBE14o-. Upon silencing of integrinβ4,16HBE14o-showed reduced proliferation ability, cell cycle analysis also revealed G1 phase arrest, the wound repair ability of airway epithelial cells was blocked and more early and late apoptosis signal was induced.In summary, these results provided interesting evidences that integrinβ4 was down-regulated on the airway mucus of asthma patients. The down-regulation of integrinβ4 on airway epithelia cells appeared to be correlated with asthma pathogenesis and phenotype.3) Involvement of integrinβ4 in the ozone stress-induced AHRAHR is one of the main clinical manifestations of asthma and allergic rhinitis. The second part of this study demonstrated that integrinβ4 was down-regulated on the airway mucus of asthma patients. In addition, decreased expression of integrinβ4 has a close relationship to the proliferation and wound repair of airway epithelial cells. It is becoming increasingly clear that, after ozone stress, disruption of structural integrity, and shedding of epithelial cells are key determinants of AHR. However, the cellular and molecular mechanism underlying the pathogenesis of epithelial damage and AHR has not been thoroughly investigated. In this part, a successive ozone-stressed rat model was constructed by exposing animals to ozone (2 ppm,30 min) for 1,2, or 4 days. The RL results showed that AHR was induced after ozone stress. Besides, AHR caused by ozone stress presented as increased RL to inhaled histamine but not baseline RL. The degree of AHR increased when ozone stress was increased. After ozone stress, we observed structural disruption, epithelial shedding, and significant down-regulation of the structural adhesion molecule integrinβ4. Furthermore, mRNA expression of integrinβ4 was negatively correlated with RL by inhaled histamine (at 0.32 mg/ml) after ozone stress, which indicated the possible relationship between integrinβ4 expression and AHR parthenogenesis after ozone stress.To explore the possible mechanism by which decreased integrinβ4 expression and induction of AHR after ozone stress, we silenced integrinβ4 on 16HBE14o- using siRNA. When integrinβ4 expression was down-regulated, we observed disrupted anti-oxidation ability and increased apoptosis of epithelial cells.Thus, these results indicates that down-regulation of integrinβ4 expression is an important element of AHR pathogeneses after ozone stress, presumably because it causes increased oxidative damage and apoptosis of airway epithelial cells.4) The role of integrinβ4 to the antigen presentation ability of airway epithelial cells and the derivation of T cellsThe airway epithelial cells can act as a kind of accessory antigen presentation cells (APC) to take antigen and present signals to T cell which can induce the activation and proliferation of T cells. It is an important way to regulate the local inflammation of the airway mucus. Asthma is charactered with typical Th2 airway inflammation. The second part of this study demonstrated that integrinβ4 down expressed on the airway mucus of asthma patients which have a close relationship to the parthenogenesis of asthma. Then, we suspect that down regulation of integrinβ4 may influence the antigen presentation processes of airway epithelial cells, which will have a close connection to the local Th2 inflammation bias in the asthma airway.To verify these hypotheses, we detected the expression of costimulus molecules on the human airway mucus and 16HBE14o-. Then integrinβ4 was silenced by specific siRNA vector. We found that integrinβ4 silenced cells showed a weakened ability to traffic antigen. After T cells was cocultured with the integrinβ4 silenced 16HBE14o- cells, the results showed that the proliferation of T cells was recede and the expression of IFN-gamma was decreased compared with the control group. Thus, these results indicated that down regulation of integrinβ4 not only weaken the antigen presentation ability but also inhibit the derivation of Th1 derivation, which may have a close connection to the local Th2 inflammation bias on asthma airway.Conclusion:This present study demonstrated that integrinβ4 was constitutively expressed on airway epithelial cell. Expression of integrinβ4 on airway epithelial cells was regulated by lots of outer stimulation. Besides adhesion, integrinβ4 involved in regulating wound repair and anti-oxidation ability on airway epithelial cells. On the airway mucus of asthma patients, integrinβ4 was down regulated. As the decreased expression of integrinβ4 led to the disruption of structural integrity, loss of functional homeostasis, occurrence of AHR and derivation of Th2 inflammation bias, it has a close relationship to the pathogenesis of asthma and other respiratory diseases.
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