Divergent effects of neutrophils on Fas-induced pulmonary inflammation, apoptosis, and lung damage

Abstract

Pulmonary Fas activation is a hallmark of the acute respiratory distress syndrome (ARDS). Fas-signalling induces chemokine release by lung epithelial cells (LEC) with subsequent immigration of activated neutrophils (PMN), LEC apoptosis, and lung damage. So far it remained unclear whether lung damage after Fas activation is the consequence of LEC apoptosis, PMN influx, or both. Therefore, mice (C57Bl/6) were injected with PMN-depleting antibody (Gr-1) or isotype 48 h prior to intratracheal (i.t.) instillation of Fas-activating antibody (Jo2) or isotype. 6 or 18 h later lung tissue, plasma, and bronchoalveolar lavage fluid (BALF) were harvested. Lung cytokines were quantified by multiplex or enzyme-linked immunosorbant assay (ELISA), lung active caspase-3 (C-3) by western blotting. Terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) and hematoxylin-eosin (H&E) staining were performed in lung sections. Lung myeloperoxidase activity (MPO) was evaluated. Total protein concentration in BALF was assessed. PMN depletion markedly reduced the increase of lung MPO activity and PMN influx in BALF observed after Fas activation in non-depleted mice. In the presence of PMN, keratinocyte-derived chemokine (KC) (lung, plasma), interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) (lung, plasma, BALF) were markedly increased 18 h after Jo2; in the absence of PMN no such increase was observed for lung KC or IL-6, whereas MCP-1 was even further enhanced in lungs and BALF but not in plasma. KC was also further enhanced in BALF and plasma, whereas IL-6 remained similar to mice treated with Fas in the presence of PMN. 6 h after Jo2 BALF protein was markedly increased in the presence but not in the absence of PMN. Following Fas activation apoptosis was increased (TUNEL, C-3) irrespectively whether PMNs were present or absent. Taken together, in the absence of PMN, Fas induced lung inflammation was divergently affected, whereas pulmonary apoptosis remained unaffected. The degree of lung injury, however, was blunted after PMN depletion. This indicates, that secondary lung damage via recruitment of activated PMN into the lungs in response to Fas mediated inflammation is an important mechanism in the development of structural lung damage and needs to be differentiated from the direct effects of Fas induced lung epithelial cell death.