Figure 3

HIF-1α and CCL2 are both increased in BALF and lung tissue after challenge in asthmatic patients. A. Representative photomicrographs of HIF-1α (a and b) and CCL2 (c and d) immunostaining observed at magnification of 100X in epithelial cells from bronchial lavage before (a and c) and after (b and d) challenge. In the right panel, we quantified HIF-1α and CCL2 positive cells, before and after the challenge and found a significant increase in the expression of both genes. Student t-test *p < 0.05 pre vs. post challenge HIF-1α and CCL2 expression. B. The colocalization of HIF-1α and CCL2 in BAL cells from asthmatic patients was performed using double-immunofluorescence. The cells were incubated with anti-HIF-1α antibody and then with FITC-conjugated anti-rabbit antibody (a). For CCL2 detection, the cells were treated with goat anti-CCL2 antibody and then treated with a secondary antibody with red fluorescence (b). The merging of fluorescence resulted in significant yellow fluorescence that indicated co-expression of HIF-1α and CCL2 (c). C. Representative photomicrographs of HIF-1α (a and b) and CCL2 expression (c and d) before (a and c) and after (b and d) challenge, observed at magnification of 40X in lung tissue (left panel). Both HIF-1α and CCL2 expression was enhanced after allergen challenge in the lung tissue. In the right panel, we quantified the density of the expression of HIF-1α and CCL2 before and after challenge. This analysis was performed from data collected from 9 patients. Student t-test *p < 0.05 Pre vs. post challenge HIF-1α and CCL2 expression. D. Correlation analysis between HIF-1α and CCL2 positively immunostained cells after challenge. Pearson’s analysis (p = 0.001, r = 0.878) post challenge HIF-1α vs. CCL2.