CXCR4+ Cytokeratin5+ CD45+ Progenitor Epithelial Cells Are Present in Bone Marrow and Are Recruited during Airway Epithelial Injury.

Abstract Background: Human lung transplants have demonstrated recipient epithelial cells in donor organs, supporting the notion of a circulating epithelial progenitor cell. In the proximal airway, the submucosal gland ducts and basal epithelium are the classically described locations of cytokeratin 5/14 progenitor airway epithelial cells. This would be the preferred site of circulating epithelial progenitor cell recruitment during airway injury. Ischemia/reperfusion injury associated with transplantation leads to increased CXCL12 expression, creating a chemotactic gradient for CXCR4+ progenitor cells. Regeneration of the pseudostratified columnar pulmonary epithelium occurs within 7 days after tracheal transplantation in the mouse model. Purpose of study: To determine the existence of circulating progenitor epithelial cells, and to identify the mechanism by which these cells traffic and contribute to regeneration of the epithelium during airway injury. Methods used: Flow cytometric analysis of cytokeratin 5 (CK5), CXCR4 and CD45 expressing cells in the buffy-coat and bone marrow of naive mice and mice undergoing tracheal transplantation. Subcutaneous implantation of dissected female tracheas into the flanks of male recipient mice. Real-time PCR for quantitation of male DNA in implanted female tracheas. In situ hybridization identification of the cell types expressing the Y chromosome in female tracheal implants. Time course of immunohistochemical analysis of CXCL12, CK5 and CXCR4 in tracheal implants. Summary of results: A population of cells was identified which express CD45, CK5 (i.e., a marker of progenitor epithelial cells) and CXCR4 in the bone marrow (mean of 6% of cells) and buffy-coat (mean of 12% of cells) in naive mice (n=6 mice). Twelve hours after tracheal implantation, CD45+, CK5+, CXCR4+ cells are undetectable in the bone marrow, but present in the tracheal implants. Real-time PCR detected a mean of 5ng of male DNA in the female tracheal implants by 5 days post-tracheal implantation, when compared to a standard quantitative curve of male DNA. In situ hybridization localized the Y chromosome expressing cells to cells of the regenerating pulmonary epithelium and to ducts of submucosal glands, the previously described location of progenitor pulmonary epithelial cells and the site of CK5+ cells. Immunohistochemistry with primary antibody to CXCL12 demonstrated staining in the ducts of submucosal glands of the regenerating pulmonary epithelium at 3 days post tracheal implantation. At day 7, CXCL12 expression was predominantly found in the basal cell population of the airway epithelium, and by day 14, CXCL12 expression was found to be predominantly in the apical airway epithelial cells. CXCR4+ cells were identified at 3 days post-transplant in the regenerating pulmonary epithelium, but were almost absent by 7 days post-transplant. Conclusions: There is a population of CD45+CK5+CXCR4+ cells in the bone marrow and circulation of naive mice. Tracheal implantation results in significant proximal pulmonary epithelial cell ischemia-reperfusion injury, which is associated with recruitment of male recipient circulating cells that are CD45+CK5+CXCR4+ to the ducts of submucosal glands where CXCL12 is expressed. The CXCR4/CXCL12 biological axis may provide a mechanism for recruitment of progenitor epithelial cells from the bone marrow during airway epithelial cell injury.