The specialized role of mouse Gr-1high monocytes in local inflammatory reactions has been well documented, but the trafficking and responsiveness of this subset during systemic inflammation and their contribution to sepsis-related organ injury has not been investigated. Using flow cytometry, we studied monocyte subset margination to the pulmonary microcirculation during sub-clinical endotoxemia in mice, and investigated if marginated monocytes contribute to lung injury in response to further septic stimuli. Sub-clinical low-dose i.v. LPS induced a rapid (within 2h), large scale mobilization of bone marrow Gr-1high monocytes and their prolonged margination to the lungs. With secondary LPS challenge, membrane TNF expression on these pre-marginated monocytes substantially increased, indicating their functional priming in vivo. Zymosan challenge produced small increases in pulmonary vascular permeability, which were markedly enhanced by the pre-administration of low-dose LPS. The LPS-zymosan induced permeability increases were effectively abrogated by pre-treatment (30 min before zymosan challenge) with the platelet-activating factor (PAF) antagonist WEB 2086, in combination with the phosphatidylcholine-phospholipase C inhibitor D609, suggesting the involvement of PAF/ceramide mediated pathways in this model. Depletion of monocytes (at 18h post clodronate-liposome treatment) significantly attenuated the LPS-zymosan induced permeability increase. However, restoration of normal LPS-induced Gr-1high monocyte margination to the lungs (at 48h post clodronate-liposome treatment) resulted in the loss of this protective effect. These results demonstrate that mobilization and margination of Gr-1high monocytes during sub-clinical endotoxemia primes the lungs toward further septic stimuli, and suggest a central role for this monocyte subset in the development of sepsis-related acute lung injury.

Background:The mechanisms responsible for up-regulation of TNF␣-converting enzyme (TACE) catalytic activity in primary human monocytes have not been elucidated. Results: TACE activation by lipopolysaccharide was dependent on reactive oxygen species (ROS) and the p38 MAPK pathway. Conclusion: ROS mediate TACE catalytic activation indirectly through the p38 pathway. Significance: This redefines the mechanisms of TACE activation in primary cells with a physiological stimulus.

Margination and activation of monocytes within the pulmonary microcirculation contribute substantially to the development of acute lung injury in mice. The enhanced LPS-induced TNF expression exhibited by Gr-1high compared with Gr-1low monocytes within the lung microvasculature suggests differential roles for these subsets. We investigated the mechanisms responsible for such heterogeneity of lung-marginated monocyte proinflammatory response using a combined in vitro and in vivo approach. The monocyte subset inflammatory response was studied in vitro in mouse peripheral blood mononuclear cell-lung endothelial cell coculture and in vivo in a two-hit model of intravenous LPS-induced monocyte margination and lung inflammation in mice, by flow cytometry-based quantification of proinflammatory genes and intracellular phospho-kinases. With LPS stimulation in vitro, TNF expression was consistently higher in Gr-1high than Gr-1low monocytes, markedly enhanced by coculture with endothelial cells, and abrogated by p38 MAPK inhibitors. Expression of IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) was only detectable under coculture conditions, was substantially higher in Gr-1high monocytes, and was attenuated by p38 inhibition. Consistent with these differential responses, phosphorylation of p38 and its substrate MAPK-activated protein kinase 2 (MK2) was significantly higher in the Gr-1high subset. In vivo, p38 inhibitor treatment significantly attenuated LPS-induced TNF expression in “lung-marginated” Gr-1high monocytes. LPS-induced p38/MK2 phosphorylation was higher in lung-marginated Gr-1high than Gr-1low monocytes and neutrophils, mirroring TNF expression. These results indicate that the p38/MK2 pathway is a critical determinant of elevated Gr-1high subset responsiveness within the lung microvasculature, producing a coordinated proinflammatory response that places Gr-1high monocytes as key orchestrators of pulmonary microvascular inflammation and injury.