PMN recruitment in inflammatory lung injury models follows classical transendothelial migration paradigms requiring PECAM-1 and CD99.

Abstract

Immune cells are recruited to sites of inflammation in a stepwise process involving a symphony of signals and receptors. In the systemic circulation, the step at which immune cells migrate out of the blood and across the endothelium, transendothelial migration, occurs via homophilic interactions between leukocyte PECAM-1 and CD99 and endothelial cell PECAM-1 and CD99. Previous work showed that rolling and adhesion of immune cells in the lung vasculature does not follow the classical paradigm of inflammatory recruitment; however, the transmigration step of this process has largely gone understudied. In this study, we demonstrate that polymorphonuclear cells (PMNs) use PECAM-1 and CD99 when transmigrating in response to murine chemical, bacterial, and ischemia/reperfusion lung injury (IRI). We demonstrate that recruitment of PMNs in response to both Gram-positive and Gram-negative bacteria is PECAM-1- and CD99-dependent. We implemented a method of intravital microscopy (IVM) of the pulmonary vasculature after IRI, with which we directly visualized and quantified transmigration. We demonstrate, in real time, that PMN enter the alveoli by crossing alveolar capillaries. Because PMNs are known to be independent mediators of both tissue damage and resolution of inflammation, we tested these effective blocking antibodies for survival effects in models of 50-60% mortality, but found none. In summary, our study shows that the classical transmigration protein interactions are necessary for the transmigration of PMNs into the airspace during response to four distinct inflammatory stimuli.NEW & NOTEWORTHY Previous studies have shown that neutrophil extravasation in the lung was selectin-independent and the requirement for leukocyte integrins was stimulus-dependent. This study demonstrates that PECAM-1 and CD99 are required for PMN transmigration during chemical, bacterial, and ischemia/reperfusion lung inflammation. We show directly in real time, using intravital microscopy, that neutrophils extravasate from alveolar capillaries. Blocking antibodies against PECAM-1 or CD99 prevented transmigration into the lung airspace, just as they prevent transmigration in the systemic circulation.