Activated pulmonary vascular neutrophils as early mediators of endotoxin-induced lung inflammation

Pulmonary vascular sequestration of leukocytes has been reported to increase in some models of lung injury, including that induced by gram-negative bacterial lipopolysaccharide (LPS). Neutrophils recruited to the lung likely participate in LPS-induced lung inflammation and associated injury, but the functional activities of these pulmonary vascular neutrophils have not been directly assessed. In the current study, cells were recovered by pulmonary vascular lavage (PVL) of isolated rat lungs, harvested 2 h after intravenous infusion of LPS (3 mg/kg) or saline in intact rats, at which time LPS-induced neutrophil recruitment to the lung could be appreciated histologically but not by airway lavage. Relative concentrations of leukocytes recovered from the pulmonary vasculature by PVL were compared with those present in circulating blood, normalizing for lavage dilution on the basis of erythrocyte counts. Excess neutrophils, lymphocytes, monocytes, and eosinophils were recovered from the pulmonary vasculature of controls, and LPS infusion increased recovery of neutrophils (most prominently), lymphocytes, and monocytes. Compared with cells recovered from controls, PVL neutrophils from LPS-infused animals were primed for increased zymosan-stimulated superoxide generation, determined by ferricytochrome C reduction, and were more adherent to nylon wool columns. Northern blots of extracted RNA demonstrated that LPS infusion also upregulated interleukin-1 beta (IL-1 beta) mRNA expression in PVL leukocyte samples, but not BAL or circulating blood samples. Ficoll-hypaque separation demonstrated that the LPS-induced IL-1 beta signal in PVL leukocytes was derived primarily from polymorphonuclear rather than mononuclear leukocytes. In conclusion, all circulating leukocyte populations are sequestered in rat lungs, and LPS increases pulmonary vascular sequestration of leukocytes, recruiting most prominently an activated pool of neutrophils that are more adherent, primed for increased oxygen radical production, and expressing increased IL-1 beta message. These findings suggest a more prominent role than previously appreciated for sequestered neutrophils in sepsis-induced lung inflammation.