KLF2 And L-selectin、CXCR1/2 Affecting Adhesion And Migration Of Neutrophil In Patients With Asthma

Bronchial asthma is a chronic inflammatory airway disease involved in a variety of cells and cellular components. During the past years it was thought that asthma is an eosinophilic inflammation dominated allergic disease. However, recent years, more and more studies have found that some types of asthma are neutrophils dominated, especially in severe asthma, refractory asthma. By far the mechanism of airway neutrophils increasing in bronchial asthma patients has not been elucidated. On the basis of our laboratory’s previous studies on cells and animals, We aimed to explore the effects of KLF2, L-selectin, CXCR1, CXCR2, CD11 a, CD11 b and CD18 on the adhesion and migration of neutrophils in patients with bronchial asthma.Method:29 asthma Patients with acute exacerbation were collected, then patients were separated to mild asthma group(13 cases), moderate asthma group(11 cases) and severe asthma group(5 cases), according to their disease state. Another 12 healthy volunteers were recruited as healthy control group(group C). Neutrophils were isolated by gradient centrifugation and appraisaled by Diff-Quik staining. The m RNA expressions of KLF2, L-selectin, CXCR1, CXCR2, CD11 a, CD11 b,CD18 were examined by Real-time PCR. The protein expressions of KLF2, L-selectin, CXCR1, CXCR2 were examined by Western-blot. Mean fluorescence intensity of L-selectin,CD11a, CD11b, CD18 is measured by Flow cytometry. The concentration of IL-8 in serum were examined by enzyme linked immunosorbent assay(ELISA). The migration and adhesion ability of neutrophils were detected by using cell adhesion assay and IL-8 migration assay in vivo.Results:1 The counts of leukocyte in Peripheral bloodThe percentage of neutrophils isolated from peripheral blood were significantly higher in mild, moderate and severe asthma group than in healthy control group(P<0.05). The percentage of eosnophils isolated from peripheral blood were significantly higher in mild group than in moderate, severe asthma group and healthy control group(P<0.05).2 Neutrophils isolation and identificationSuccessfully isolated neutrophils from peripheral blood, neutrophils were appraisaled by Diff-Quik staining,the neutrophils purity was more than 95%.3 The m RNA expressions of KLF2, L-selectin, CXCR1, CXCR2, CD11 a, CD11 b, CD18 in neutrophils isolated from peripheral blood in each groupThe relative m RNA expressions of KLF2 was more lower in moderate and severe asthma group than in healthy control group(p<0.01),and more lower in severe asthma group than in mild asthma group(P<0.01). But the m RNA expressions of L-selectin, CXCR1, CXCR2, CD11 a, CD11 b and CD18 were more higher in moderate, severe asthma group than in healthy control group(p<0.01), and more higher in severe asthma group than in mild asthma group(P<0.01).4 The protein expressions of KLF2, L-selectin, CXCR1, CXCR2 in neutrophils isolated from peripheral blood in each groupThe protein expressions of KLF2 was more lower in moderate and severe asthma group than in healthy control group(p<0.05), and more lower in severe asthma group than in mild asthma group(P<0.01). But the protein expressions of L-selectin was more higher in mild, moderate and severe asthma group than in healthy control group(p<0.05). The protein expressions of CXCR1, CXCR2 were more higher in moderate, severe asthma group than in healthy control group(p<0.01).5 Mean fluorescence intensity of L-selectin, CD11 a, CD11 b, CD18 in neutrophils isolated from peripheral blood in each groupMean fluorescence intensity of L-selectin, CD11 a, CD11 b, CD18 were more higher in moderate, severe asthma group than in healthy control group(p<0.01), and more higher in severe asthma group than in mild asthma group(P<0.01).6 The consentration of IL-8 in peripheral blood serum in each groupThe expressions of serum IL-8 more higher in moderate, severe asthma group than in healthy control group(p<0.01), and more higher in severe asthma group than in mild asthma group(P<0.01).7 Migration of neutrophils isolated from peripheral blood in each groupAccording to the results of preliminary experiments,we selected 100ng/ml IL-8 to finish transwell experiment, results showed: The rate of migration of neutrophils were higher in mild, moderate and severe asthma group than in healthy control group(p<0.05), and higher in severe asthma group than in mild asthma group(P<0.05).8 Adhesion ability of neutrophils isolated from peripheral blood in each groupThe rate of adhesion of neutrophils were higher in moderate and severe asthma group than in healthy control group(p<0.01), and higher in severe asthma group than in mild asthma group(P<0.01).9 Pearson analysis on KLF2 and L-selectin, CXCR1, CXCR2 m RNA and protein. The result shows that there is a negative correlation between them.Conclusion:(1) The expression of KLF2 decreased in asthma groups, and decline with the severity. But it made the neutrophils have more activity to adhension and migration.(2) KLF2 may affect neutrophils, adhesion and migration of patients asthma by regulating the expressions of related Adhesion molecules(L-selectin, CD11 a, CD11 b and CD18) and chemokine receptors(CXCR1, CXCR2).