Hyperoxic Exposure in Humans* Effects of 50 Percent Oxygen on Alveolar Macrophage Leukotriene 9 4 Synthesis David E. Griffith, M.D ., F.C.C.P.;Joe G. N. Garcia, M.D ., F.C.C .P.;t Harold L. James, Ph.D.; Karleen S. Callahan, Ph.D.; Sarah lriana, M.S.; and David Holiday, m.o. The pathogenesis of oxygen toxicity remains unknown but may involve leukocyte mediated injury. Tbe effects of byperoxia on several lower respiratory tract parameters were examined in broncboalveolar lavage 8uid of normal nonsmoking subjects who inhaled a fractional inspired oxygen concentration of 50 percent (mean exposure: 44 h). Evidence that 50 percent O. produced oxidative stress in the lung included recovery of Huorescent products of lipid peroxidation and partial oxidation of a1pha.-antitrypsin in BAL Huid obtained after O. exposure. To examine wbether alveolar macrophage-derived leukotriene B. may he generated in response to 50 percent 0., AM were isolated from O.-exposed subjects and compared with AM recovered from subjects breathing room air. Leukotriene B. levels were elevated in supernatants from both unstimulated and arachidonic acid-stimulated AM obtained from hyperoxia-
exposed subjects. In hyperoxia-exposed individuals, LTB. levels were also elevated in extracted BAL 8uid . The percentage of BAL neutrophils was also significantly increased after O. exposure (2.8±0.6 vs 1.2±0.4 percent, p 0.05). We conclude that an Flo. of 50 percent inhaled for 44 h is associated with enhanced oxidative stress, stimulation of AM to release LTB., and a small but significantly increased percentage of neutropbils recovered (Chest 1992; 101:392-97) in BAL 8uid.
toxicity is a function of both O concentraO xygen tion and length of O exposure. The threshold
stimulation is important in the pathogenesis of hyperoxia-induoed lung injury. Following hyperoxic exposure, AMs initiate procoagulant activitY·6 and perpetuate or amplify the inflammatory response by producing neutrophil chemoattractants, including the lipoxygenase product, LTB•. 7.8 Davis et al9 have also demonstrated that AMs from humans exposed to FIo2 near 100 percent for approximately 17 h release factors promoting lung Bbrosis," In this study, we report biochemical and inflammatory events in the lower respiratory tract of normal subjects before and after exposure to an FIo2 of 50 percent for a mean of 44 h . We evaluated two potential markers of hyperoxic stress; lipid peroxidation and AAT actiyity. Furthermore, lower respiratory tract inflammatory cell burden was evaluated by BAL and possible mechanisms of inflammatory cell recruitment to the lung were assessed by measurement of spontaneous and stimulated AM production of LTB•.
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2
concentration for pulmonary O 2 toxicity in humans, however, is unknown. Because patients exposed to FIo 2 of up to 50 percent for several days do not exhibit spirometric abnormalities, this level of oxygen inhalation has been considered to be clinically innocuous. 1 We have previously demonstrated, however, that an FIo2 of 50 percent inhaled for near 48 h in normal human volunteers is associated with reversible alterations in lung clearance of an inhaled radioactively labeled solute (99mTc-DTPA) and increased albumin concentration in BAL fluid. 2 These findings are compatible with increased vascular permeability caused by the low level hyperoxic exposure and suggests that this FIo2 may have adverse pulmonary effects. The cellular mechanisms ofhyperoxia-induced lung injury are not well understood. Animal models confirm an influx of inflammatory cells into the lung with hyperoxic exposure.s" although the precise pathophysiologic role of neutrophil influx with O2 toxicity remains unclear. It does appear, however, that AM *From the Departments of Medicine, Biochemistry, Epidemiology and Biomathematics. The University of Texas Health Center at Tyler, Tyler; and the tDepartment of Medicine, Physiology and Biophysics, Indiana University School of Medicine. Indianapolis. This study was supported by a grant from the American Heart AssociationTexas Affiliate86G662. Manuscript received February 19; revision accepted May 29. Reprint requests: Dr. Griffith, PO Box 2003, University of 'Iexas Health Center, Tyler 75710 392
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AA arachidonic acid; AM = alveolar macrophage; EIC = elastase inhibitory capacity; LTB. = leukotriene B.; MDA = maIonaIdehyde; MPR = methionine sulfoxide peptide reductase; PeA = procoagu!ant activity; PPE = puri6ed porcine pancreatic e~; RIA = radioimmunoassay; RFI relative Huorescence mtenslty
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METHODS
Study Population and ExperimentalProtocol Twelve nonsmolting volunteers, 8 men , 4 women (age range 18 to 45) without apparent lung disease were enrolled in the study. All subjects had baseline FVC and FEV, values within normal limits. Informed consent followingthe guidelines of the University of Texas Health Center at Tyler Institutional Review Board was obtained from each subject . Subjects breathed an Flo. of 50 percent (15 Umin oxygen Bow) via an air entrainment mask for a mean of 44±O.6hours. A two period crossover design'
exposed subjects. In hyperoxia-exposed individuals, LTB. levels were also elevated in extracted BAL 8uid . The percentage of BAL neutrophils was also significantly increased after O. exposure (2.8±0.6 vs 1.2±0.4 percent, p 0.05). We conclude that an Flo. of 50 percent inhaled for 44 h is associated with enhanced oxidative stress, stimulation of AM to release LTB., and a small but significantly increased percentage of neutropbils recovered (Chest 1992; 101:392-97) in BAL 8uid.
toxicity is a function of both O concentraO xygen tion and length of O exposure. The threshold
stimulation is important in the pathogenesis of hyperoxia-induoed lung injury. Following hyperoxic exposure, AMs initiate procoagulant activitY·6 and perpetuate or amplify the inflammatory response by producing neutrophil chemoattractants, including the lipoxygenase product, LTB•. 7.8 Davis et al9 have also demonstrated that AMs from humans exposed to FIo2 near 100 percent for approximately 17 h release factors promoting lung Bbrosis," In this study, we report biochemical and inflammatory events in the lower respiratory tract of normal subjects before and after exposure to an FIo2 of 50 percent for a mean of 44 h . We evaluated two potential markers of hyperoxic stress; lipid peroxidation and AAT actiyity. Furthermore, lower respiratory tract inflammatory cell burden was evaluated by BAL and possible mechanisms of inflammatory cell recruitment to the lung were assessed by measurement of spontaneous and stimulated AM production of LTB•.
2
2
concentration for pulmonary O 2 toxicity in humans, however, is unknown. Because patients exposed to FIo 2 of up to 50 percent for several days do not exhibit spirometric abnormalities, this level of oxygen inhalation has been considered to be clinically innocuous. 1 We have previously demonstrated, however, that an FIo2 of 50 percent inhaled for near 48 h in normal human volunteers is associated with reversible alterations in lung clearance of an inhaled radioactively labeled solute (99mTc-DTPA) and increased albumin concentration in BAL fluid. 2 These findings are compatible with increased vascular permeability caused by the low level hyperoxic exposure and suggests that this FIo2 may have adverse pulmonary effects. The cellular mechanisms ofhyperoxia-induced lung injury are not well understood. Animal models confirm an influx of inflammatory cells into the lung with hyperoxic exposure.s" although the precise pathophysiologic role of neutrophil influx with O2 toxicity remains unclear. It does appear, however, that AM *From the Departments of Medicine, Biochemistry, Epidemiology and Biomathematics. The University of Texas Health Center at Tyler, Tyler; and the tDepartment of Medicine, Physiology and Biophysics, Indiana University School of Medicine. Indianapolis. This study was supported by a grant from the American Heart AssociationTexas Affiliate86G662. Manuscript received February 19; revision accepted May 29. Reprint requests: Dr. Griffith, PO Box 2003, University of 'Iexas Health Center, Tyler 75710 392
=
=
AA arachidonic acid; AM = alveolar macrophage; EIC = elastase inhibitory capacity; LTB. = leukotriene B.; MDA = maIonaIdehyde; MPR = methionine sulfoxide peptide reductase; PeA = procoagu!ant activity; PPE = puri6ed porcine pancreatic e~; RIA = radioimmunoassay; RFI relative Huorescence mtenslty
=
METHODS
Study Population and ExperimentalProtocol Twelve nonsmolting volunteers, 8 men , 4 women (age range 18 to 45) without apparent lung disease were enrolled in the study. All subjects had baseline FVC and FEV, values within normal limits. Informed consent followingthe guidelines of the University of Texas Health Center at Tyler Institutional Review Board was obtained from each subject . Subjects breathed an Flo. of 50 percent (15 Umin oxygen Bow) via an air entrainment mask for a mean of 44±O.6hours. A two period crossover design'
