Transfusion-Related Acute Lung Injury (TRALI) Requires Heterotypic Interactions of Platelets with Specific Neutrophil Microdomains

Neutrophil and platelet recruitment and activation into inflamed organs play key roles in the acute inflammatory response. Leukocyte recruitment is initiated by interactions with endothelial selectins, followed by activation of integrins which interact with their counter-receptors on the inflamed endothelium. Before migrating into the surrounding tissue, neutrophils actively crawl on the inflamed endothelium displaying a characteristic polarization with distinct microdomains at the leading (LE) and trailing edges (TE). During this period, they establish frequent interactions with circulating erythrocytes through the LE, which in the context of sickle cell disease can lead to vascular occlusion (Turhan et al., P.N.A.S. 2002 and unpublished data). During high-speed multichannel fluorescence intravital microscopy (MFIM) experiments, we have noted that neutrophils (identified by CD45+ Gr-1+ F4/80- expression) crawling on the inflamed endothelium actively interact with circulating platelets (identified in vivo by CD41 expression). In conditions in which inflammation was induced by surgical trauma only, platelet interactions were relatively rare and mediated by both the TE (identified in vivo by L-selectin clustering) and LE (opposite to L-selectin clusters). By contrast, in venules inflamed by the administration of TNFα, platelet interactions with the LE of crawling neutrophils were markedly increased. We have previously reported (Blood110: 145; Nov. 2007) that sickle erythrocyte interactions in inflamed venules occurred at the LE of neutrophils through E-selectin-induced activation of the integrin Mac-1. We analyzed the role of E-selectin and Mac-1 in platelet capture and found a marked reduction in captures at the LE in mice lacking E-selectin, whereas these were virtually absent in mice deficient in Mac-1. These findings indicate that the increase in platelet capture during inflammation is the result of Mac-1 activation by endothelial E-selectin. Transfusion-related acute lung injury (TRALI), the leading cause of transfusion-related morbidity and mortality, is an acute inflammatory process mediated by intravascular neutrophils. Using a murine model of TRALI in which Balb/c mice are infused with high doses of anti-MHC-I antibodies (anti-H2d), we find that the induction of lung injury (assessed by accumulation of plasma proteins in the broncho-alveolar lavage (BAL)) correlates with a moderate thrombocytopenia and that depletion of platelets prior to anti-H2d infusion results in complete prevention of lung injury. We thus reasoned that neutrophil-platelet interactions might be required for TRALI. To assess this possibility, we analyzed intravascular events by MFIM of inflamed cremasteric venules before and after injection of anti-H2d. Platelet captures by crawling neutrophils were increased by 2-fold after anti-H2d antibody infusion, and this increase was prevented by blocking E-selectin. Infusion of anti-H2d markedly increased (9-fold) vascular permeability (measured by FITC-dextran leakage) that could be prevented by blocking E-selectin or Mac-1, or by depleting platelets. We next assessed in vivo activation of crawling neutrophils after anti-H2d infusion by analyzing the presence of reactive oxygen species (ROS) using the fluorescent probe dihydroxyrhodamine-123. We found that ROS were rapidly induced in crawling leukocytes following anti-H2d infusion, and that this induction could be blunted by platelet depletion, indicating a requirement for heterotypic interactions during neutrophil activation. To determine whether observations made in the cremasteric circulation correlated with the pulmonary injury caused by anti- H2d infusion, we measured protein accumulation in BAL fluids of mice pretreated with antibodies against E-selectin or Mac-1. Inhibition of either receptor strongly prevented the development of lung injury. In addition, n-acetyl-cysteine, a ROS scavenging molecule, completely prevented lung injury induced by anti-H2d. These results suggest that E-selectin-induced, Mac-1-mediated platelet capture by the LE of crawling neutrophils result in ROS production, which are responsible for the vascular and organ injuries observed in this model of TRALI. The heterotypic interactions described here share mechanisms with those triggering vaso-occlusive episodes in a humanized murine model of sickle cell disease, suggesting that they play important and previously unappreciated contributions to thrombo-inflammatory complications.