Differentiated transplant derived airway epithelial cell cytokine secretion is not regulated by cyclosporine

Abstract

Background: While lung transplantation is an increasingly utilized therapy for advanced lung diseases, chronic rejection in the form of bronchiolitis obliterans syndrome (BOS) continues to result in significant allograft dysfunction and patient mortality. Despite correlation of clinical events with eventual development of BOS, the causative pathophysiology remains unknown. Airway epithelial cells within the region of inflammation and fibrosis associated with BOS may have a participatory role.

Methods: Transplant derived airway epithelial cells differentiated in air liquid interface culture were treated with IL-1β and/or cyclosporine, after which secretion of cytokines and growth factor and gene expression for markers of epithelial to mesenchymal transition were analyzed.

Results: Secretion of IL-6, IL-8, and TNF-α, but not TGF-β1, was increased by IL-1β stimulation. In contrast to previous studies using epithelial cells grown in submersion culture, treatment of differentiated cells in ALI culture with cyclosporine did not elicit cytokine or growth factor secretion, and did not alter IL-6, IL-8, or TNF-α production in response to IL-1β treatment. Neither IL-1β nor cyclosporine elicited expression of markers of the epithelial to mesenchymal transition E-cadherin, EDN-fibronectin, and α-smooth muscle actin.

Conclusion: Transplant derived differentiated airway epithelial cell IL-6, IL-8, and TNF-α secretion is not regulated by cyclosporine in vitro; these cells thus may participate in local inflammatory responses in the setting of immunosuppression. Further, treatment with IL-1β did not elicit gene expression of markers of epithelial to mesenchymal transition. These data present a model of differentiated airway epithelial cells that may be useful in understanding epithelial participation in airway inflammation and allograft rejection in lung transplantation.

Figure 1

Pulmonary allograft epithelial cells in culture. A. Phase-contrast image of cells in submersion culture. B. Phase-contrast image of cells in air liquid interface culture for 3 weeks. C and D. Confocal microscopy of air liquid interface cells at 3 weeks. Cells were labeled with antibodies directed against ciliated cells (blue), goblet cells (red), or basal cells (green). White represents the overlap of all three colors and denotes an indeterminate cell. Original magnification of for A and B, 40 ×, and C and D, 400 ×.

Figure 2

Secretion of IL-8 by transplant-derived airway epithelial cells after stimulation with IL-1β and cyclosporine. A. IL-8 secretion in basal medium after basal stimulation. *, P = 0.03 for IL-1β vs. control; †, P = 0.04 for IL-1β and cyclosporine vs. cyclosporine alone. B. IL-8 secretion in basal medium after apical stimulation. *, P = 0.006 for IL-1β vs. control; † P = 0.03 for IL-1β and cyclosporine vs. cyclosporine alone. N = 5 unique patient samples.

Figure 3

Secretion of IL-6 by transplant-derived airway epithelial cells after stimulation with IL-1β and cyclosporine. A. IL-6 secretion in basal medium after basal stimulation. *, P < 0.0001 for IL-1β vs. control and for IL-1β and cyclosporine vs. cyclosporine alone. B. IL-6 secretion in basal medium after apical stimulation. *, P = 0.002 for IL-1β vs. control; †, P = 0.02 for IL-1β and cyclosporine vs. cyclosporine alone. N = 5 unique patient samples.

Figure 4

Secretion of TNF-α by transplant-derived airway epithelial cells after stimulation with IL-1β and cyclosporine. A. TNF-α secretion in basal medium after basal stimulation. *, P < 0.0001 for IL-1β vs. control and for IL-1β and cyclosporine vs. cyclosporine alone. B. TNF-α secretion in basal medium after apical stimulation. *, P = 0.0001 for IL-1β vs. control and for IL-1β and cyclosporine vs. cyclosporine alone. N = 5 unique patient samples except for apical stimulation with both IL-1β and cyclosporine, for which N = 3.

Figure 5

Expression of TGF-β1 in transplant-derived differentiated airway epithelial cells. Expression after either basal (Figure 5A) or apical (Figure 5B) addition of mediators is shown. N = 5 unique patient samples for each. *, P < 0.05 versus vehicle control. CSA, cyclosporine.

Figure 6

Expression of markers of epithelial-mesenchymal transformation in transplant-derived differentiated airway epithelial cells. Expression after either basal or apical addition of mediators is shown. A. Expression of α-smooth muscle actin (SMA). B. Expression of EDN-fibronectin. C. Expression of E-cadherin. N = 5 unique patient samples. *, P < 0.05 versus vehicle control. CSA, cyclosporine, SMA, smooth muscle actin, FN, fibronectin.