Journal of Leukocyte Biology 46:41-50 (1989) © 1989 Alan R. Liss, Inc. Characterization of Arachidonic Acid Metabolism, Superoxide Production, and Bacterial Killing in Bovine Circulating Neutrophils and Elicited Alveolar Neutrophils Jerry R. Heidel, Stephen M. Taylor, William W. Laegreid, Ron M. Silfiow, H. Denny Liggitt, and R. Wes Leid Laboratory of Molecular and Cellular Inflammation, Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington (J.R.H., S.M.T., W.W.L., R.M.S., R.W.L.), and Genentech, Inc., South San Francisco, California (H.D.L.) The In vitro generation and release of 5-Iipoxygenase metabolites of arachidonic acid by bovine peripheral blood neutrophlls and alveolar neutrophlls elicited with either a heat- killed bacterium, Haemophllus somnus, or platelet-activating factor, were compared. Af- ter stimulation with calcium lonophore A23187 for 2.5-60 mm, up to 4.5 ± 0.7 (mean ± SEM) ng of LTB4 per 106 cells was released into the media by circulating neutrophils. LTB4 release by alveolar neutrophils was significantly less (P#{176}z.05) than that of peripheral blood neutrophils from the same animal; 5-HETE release by circulating neutrophils was maximal after 5 mm stimulation by ionophore (1.2 ± 0.1 ng/106 cells) but was not Iden- tified in cell culture media after 20 mm. Alveolar neutrophils released similar amounts of 5-HETE when compared to circulating neutrophils, and release of 5-HETE by alveolar neutrophils was maximal after 5 mm of stimulation. However, the 5-HETE released into the culture media persisted throughout the 60 mm time period at levels which were maxImal (1.5 ± 0.2 to 1.8 ± 0.3 ng/1 0 cells). Bacterial killing and the release of superoxide anion were not different between the two cell populations. Key words: eicosanolds, leukotrienes, inflammation INTRODUCTION Neutrophils enter the lung alveolus in response to a variety of noxious and chemotactic stimuli. In an acti- vated state, neutrophils are directly responsible for dam- age to the lung parenchyma [7,11,17,28]. Neutrophil- mediated lung disease is of sufficient significance in mammalian species as to warrant studies aimed at char- acterizing the biology of this cell as it moves from the vasculature to the pulmonary microenvironment. The peripheral blood neutrophil of numerous species is a potent source of 5-lipoxygenase metabolites of arachidonic acid. Neutrophils isolated from man and other animals characteristically produce relatively large quantities of LTB4 and 5-HETE when stimulated either by soluble agonists such as calcium ionophore A23 187 [9,12,22,25,27] and arachidonic acid [14] or by partic- ulate stimuli during phagocytosis [6]. As LTB4 and 5- HETE are proinflammatory mediators [13], the release of these metabolites by alveolar neutrophils could mark- edly influence the development of the inflammatory re- sponse within the lung. Therefore, lipid mediator release would be important in the pathogenesis of neutrophil- mediated pulmonary disease. We have used the bovine lung as a model system for eliciting and harvesting al- veolar neutrophils. We have characterized the metabo- lism of arachidonic acid, as well as the capacity to gen- erate superoxide anion and to kill Staphylococcus epidermidis, by neutrophils obtained from the pulmonary environment and compared these results to those ob- tained from circulating neutrophils isolated concurrently from the same animal. MATERIALS AND METHODS Animals Conventionally reared Holstein steers weighing ap- proximately 350 kg with no clinical signs or history of respiratory disease were housed indoors in cement- floored stalls, bedded with wood shavings, and fed al- falfa cubes and water ad libitum. Received August 23, 1988; accepted February 16, 1989. Reprint requests: R. Wes Leid, Department of Veterinary Microbiol- ogy and Pathology. Washington State University, Pullman, WA 99164-7040.