Interleukin-15 increases neutrophil adhesion onto human respiratory epithelial A549 cells and attracts neutrophils in vivo

Abstract

Interleukin-15 (IL-15) is a neutrophil agonist that plays a role in inflammatory disorders, including a variety of pulmonary diseases. Adhesion of neutrophils onto pulmonary cells is a major event leading to development of inflammation. Recently, elevated levels of IL-15 have been associated with different pulmonary diseases. There is no clear evidence that IL-15 modulates cell surface expression of adhesion molecules in neutrophils, or that IL-15 is involved in neutrophil adhesion onto pulmonary cells. Also, it is not clear if IL-15 induces a neutrophilic inflammation in vivo. This study was aimed at elucidation of these issues. Neutrophils were treated with IL-15 and cell surface expression of CD11a, CD11b, CD11c and CD18 was monitored by flow cytometry. The human respiratory epithelial A549 cell line was used as a substrate for the neutrophil adhesion assay and cell surface expression of CD50, CD54 and CD106 was monitored in IL-15-induced A549 cells. The murine air pouch model was used for investigating potential neutrophilic inflammation induced by IL-15 in vivo. IL-15 significantly increased neutrophil cell surface expression of CD11b and CD18 and up-regulated A549 cell surface expression of CD54. Moreover, A549 cells were found to express IL-15R components and adhesion of neutrophils onto A549 cells was increased when neutrophils or A549 cells were treated with IL-15. Finally, IL-15 induced neutrophilic inflammation in vivo and concentrations of IL-6 and CXCL2/MIP-2 were increased in IL-15-induced pouches. IL-15 might participate in inflammatory pulmonary diseases by attracting neutrophils, modulating cell surface expression molecules and increasing neutrophil adhesion onto pulmonary cells.

Fig. 1

IL-15 up-regulates cell surface expression of CD11b and CD18 in human neutrophils. Neutrophils were treated with buffer (Ctrl), LPS (1 µg/ml), IL-15, IL-21 or IL-15 + IL-21 and cell surface expression of (a) CD11a, (b) CD11b, (c) CD11c and (d) CD18 was monitored by flow cytometry as described in Methods. Results are means ± SEM (n = 5). *P < 0·05 versus control by anova.

Fig. 2

IL-15 up-regulates cell surface expression of CD54 in human respiratory epithelial A549 cells. Cells were incubated for 24 h with buffer (Ctrl), 100 ng/ml TNF-α (A), 1000 ng/ml TNF-α (C), or IL-15. Cell surface expression of (a) CD50, (b) CD54 and (c) CD106 was monitored by flow cytometry as described in Methods. Results are means ± SEM (n = 3). *P < 0·05 versus control (Ctrl) by anova.

Fig. 3

A549 cells express IL-15R components. Cells were cultivated, harvested and cell surface expression of (a) IL-15Rα, (b) CD122, (c) CD132 and (d) CD25 was monitored by flow cytometry as described in Methods. Results are from one experiment out of three. Dashed lines represent appropriate isotypic control.

Fig. 4

IL-15 enhances adhesion of neutrophils onto A549 cells. (a) neutrophils were stimulated with buffer (Ctrl), LPS, or IL-15 for 2 h, labelled with calcein AM, incubated on confluent A549 cells for 30 min and adhesion was measured as described in Methods. Results are means ± SEM (n = 4). (b) The adhesion assay was performed as in A, except that A549 cells were stimulated with buffer (Ctrl), TNF-α or IL-15 for 24 h. Results are means ± SEM (n = 3). Pictures on the right part of the figure illustrated typical data that were plotted for the graph. *P < 0·05 versus Ctrl by anova.

Fig. 5

IL-15 induces a leucocyte influx in vivo. (a) air pouches were raised before injection of PBS or IL-15, exudates were harvested 6 h later and the number of leucocytes was calculated (means ± SEM (n ≥ 8)). C, representative preparations of cells harvested from concentrated PBS-injected mice and from non concentrated IL-15-injected mice. *P < 0·05 versus control by anova.

Fig. 6

IL-15 induces a neutrophilic inflammation in vivo. (a) the percentage of each cell type attracted into PBS- or IL-15-induced pouch was evaluated (means ± SEM; n ≥ 8). (b) cells were stained with 7/4 or F4/80 antibody. Open histogram, appropriate isotypic controls. Exudates from PBS-treated mice were concentrated to obtain a good number of cells for identification. *P < 0·05 versus control by anova.

Fig. 7

IL-15 increases CXCL2/MIP-2 and IL-6 production. Air pouches were created and buffer PBS (Ctrl), or IL-15 (10–500 ng/ml) were administered into pouch as described in Methods. Exudates were harvested after 6 h and CXCL2 (a) and IL-6 (b) production were measured using specific ELISA kits. Results are means ± SEM (n = 10).