Rationale: Acute lung injury (ALI) causes high mortality, but its molecular mechanisms and therapeutic options remain ill-defined. Gram-negative bacterial infections are the main cause of ALI, leading to lung neutrophil infiltration, permeability increases, deterioration of gas exchange, and lung damage. Platelets are activated during ALI, but insights into their mechanistic contribution to neutrophil accumulation in the lung are elusive. Objectives: To determine mechanisms of platelet-mediated neutrophil recruitment in ALI. Methods: Interference with platelet-neutrophil interactions using antagonists to P-selectin and glycoprotein IIb/IIIa or a small peptide antagonist disrupting platelet chemokine heteromer formation in mouse models of ALI. Measurements and Main Results: In a murine model of LPS-induced ALI, we uncover important roles for neutrophils and platelets in permeability changes and subsequent lung damage. Furthermore, platelet depletion abrogated lung neutrophil infiltration, suggesting a sequential participation of platelets and neutrophils. Whereas antagonists to Pselectin and glycoprotein IIb/IIIa had no effects on LPS-mediated ALI, antibodies to the platelet-derived chemokines CCL5 and CXCL4 strongly diminished neutrophil eflux and permeability changes. The two chemokines were found to form heteromers in human and murine ALI samples, positively correlating with leukocyte influx into the lung. Disruption of CCL5-CXCL4 heteromers in LPS-, acid-, and sepsis-induced ALI abolished lung edema, neutrophil infiltration, and tissue damage, thereby revealing a causal contribution.Conclusions: Taken together, our data identify a novel function of platelet-derived chemokine heteromers during ALI and demonstrate means for therapeutic interference.Keywords: neutrophil; platelet; chemokine; recruitment; acute lung injury Acute lung injury (ALI) is a life-threatening disease with an ageadjusted incidence of 86.2 per 100,000 person-years (1). Despite innovations in intensive care medicine, the mortality of ALI remains approximately 40%. ALI is characterized by an increased permeability of the alveolar-capillary barrier, resulting in lung edema with protein-rich fluid and consequently in impaired arterial oxygenation. A major cause for development of ALI is sepsis, wherein gram-negative bacteria are the dominating factor. LPS inhalation mimics human gram-negative ALI, leading to recruitment of neutrophils, pulmonary edema, and finally impairment of gas exchange (2).Recruitment of neutrophils is a key event in development of ALI (3) resulting in plasma leakage and deterioration of oxygenation. The importance of neutrophils in ALI is supported by studies, where lung injury was abolished or reversed by depletion of neutrophils (4,5). Much of the neutrophil-dependent ALI is thought to be mediated by granule proteins released from activated neutrophils. For example, azurocidin and a-defensins have been found to directly affect permeability changes (6, 7), whereas proteases of neutrophilic origin, such as neutrophil ela...

In acute and chronic inflammation, neutrophils and platelets, both of which promote monocyte recruitment, are often activated simultaneously. We investigated how secretory products of neutrophils and platelets synergize to enhance the recruitment of monocytes. We found that neutrophil-borne human neutrophil peptide 1 (HNP1, α-defensin) and platelet-derived CCL5 form heteromers. These heteromers stimulate monocyte adhesion through CCR5 ligation. We further determined structural features of HNP1-CCL5 heteromers and designed a stable peptide that could disturb proinflammatory HNP1-CCL5 interactions. This peptide attenuated monocyte and macrophage recruitment in a mouse model of myocardial infarction. These results establish the in vivo relevance of heteromers formed between proteins released from neutrophils and platelets and show the potential of targeting heteromer formation to resolve acute or chronic inflammation.

Rationale The leukocyte response in acute inflammation is characterized by an initial recruitment of neutrophils preceding a second wave of monocytes. Neutrophil-derived granule proteins were suggested to hold an important role in this cellular switch. The exact mechanisms by which neutrophils mediate these processes are only partially understood. Objective To investigate the role of neutrophils and their granule contents in the adhesion of monocyte subpopulations in acute inflammation. Methods and Results Here, we show that neutrophil-derived cathelicidins (human: LL37, mouse: CRAMP) induce adhesion of classical monocytes but not of non-classical monocytes in the mouse cremaster muscle and in in vitro flow chamber assays. CRAMP is released from emigrated neutrophils and then transported across the endothelium where it is presented to rolling leukocytes. Endothelial-bound cathelicidin activates FPR2 on classical monocytes, resulting in monocytic β1- and β2-integrin conformational change towards an extended, active conformation that allows for adhesion to their respective ligands VCAM-1 and ICAM-1. Conclusions These data elucidate a novel mechanism of neutrophil-mediated monocyte recruitment, which could be targeted in conditions where recruitment of classical monocytes plays an unfavorable role.