We performed bronchoalveolar lavage (BAL) 0.5 to 24 h after thrombin-induced pulmonary microembolization in spontaneously breathing sheep to examine the inflammatory events that occur after pulmonary intravascular coagulation. Neutrophil alveolitis was evident as early as 0.5 h after microembolization and was maximal at 4 h (4.9 ± 1.5% neutrophils of total BAL cells at baseline versus 26.2 ± 2.8% at 4 h post-thrombin). Neutrophils obtained both at baseline (isolated from peripheral blood) and at 0.5 to 24 h after thrombin (isolated from BAL) did not demonstrate significant basal production of superoxide anion (O2̄) and produced similar amounts of O2̄ upon challenge with phorbol myristate acetate (PMA) 200 μg/ml. The basal O2̄ production by alveolar macrophages was also not increased. However, alveolar macrophages recovered after fibrin microembolization produced greater amounts of O2̄ (29.1 ± 6.3 nm O2̄/106 cells at 0.5 h) after challenge with PMA compared with alveolar macrophages recovered prior to embolization (10.6 ± 1.6 nm O2̄/106 cells baseline), suggesting that thrombin-induced microembolization primes alveolar macrophages and enhances their O2̄ generation. Neutrophil chemotactic activity was detected in BAL fluid at 0.5 h post-microembolization and reached a peak level at 2 h. Alveolar macrophages were a source of the chemotactic activity since conditioned medium obtained from 2-h post-thrombin macrophages induced neutrophil chemotaxis, whereas baseline cells did not. The addition of the thrombin to macrophages did not result in the generation of chemotactic activity from baseline macrophages, indicating that macrophages were activated during the process of intravascular coagulation rather than by thrombin per se. Post-thrombin BAL fluid also stimulated O2̄ generation from sheep neutrophils. Therefore, BAL fluid contained factor(s) capable of activating neutrophils and also inducing neutrophil chemotaxis. Characterization of the neutrophil chemoattractant and activating (i.e., O2̄ generation) factor demonstrated that both activities resided in the BAL ether-extracted phase. Analysis of BAL fluid by reverse-phase high pressure liquid chromatography revealed a time-dependent increase in the 5-lipoxygenase product, LTB4, a potent neutrophil chemotaxin. The role of LTB4 as the putative neutrophil chemotaxin was demonstrated by the finding that chemotactic activity of the post-thrombin BAL fluid resided in the fraction with elution time the same as LTB45 standard. In summary, this study indicates that lipoxygenase products generated by alveolar macrophages may be involved in the time-dependent migration of neutrophils into the air space after fibrin microembolization and that priming of alveolar macrophages may be an important pathogenic mechanism in acute lung injury.
ASJC Scopus subject areas
- Pulmonary and Respiratory Medicine