SUMMARY. Therapy directed against the toxic effects of reactive oxygen species may reduce the final extent of ischemic injury in otherwise viable tissue irreversibly injured by the abrupt reoxygenation of reperfusion. In four groups of dogs, superoxide dismutase plus catalase (groups I-III) or saline (controls) (group IV) was infused into the left atrium. Group I received the infusion for 2 hours, beginning 15 minutes before occlusion of the left circumflex coronary artery (90 minutes) and ending 15 minutes after reperfusion. Group II received the infusion for 1 hour starring 15 minutes before reperfusion. Group III received the infusion for 1 hour beginning 40 minutes after reperfusion. Dogs were killed the next day, and infarct size was determined by dissection and weighing, and confirmed histologically. Infarct size expressed as percent of the anatomic area at risk was: group I, 19.4 ± 5.0; group II, 21.8 ± 3.3; group III, 47.6 ± 10.3; group IV, 43.6 ± 3.5 (mean ± SEM). Analysis of variance followed by Duncan's multiple range test showed that ultimate infarct size as assessed in groups I and II differed significantly (P < 0.05) from that observed in the control animals in group IV, whereas infarct size between groups III and IV did not differ significantly (P > 0.05). The percent of left ventricle at risk did not differ between the four groups. The beneficial effects of superoxide dismutase plus catalase could not be explained by hemodynamic differences. Similar protection of jeopardized myocardium in groups I and II suggest that potentially viable tissue is salvaged by scavenging free radicals during early reperfusion. Lack of protection in group III suggests that injury has occurred within the first 40 minutes of reperfusion. The results of this investigation demonstrate that the '"primary' myqcardial cellular damage due to ischemia is additive to the cardiac cell damage during the phase of reperfusion, and that the "secondary" effects are mediated by toxic metabolites of oxygen. (Che Res 54: 277-285, 1984)

Although overproduction of proinflammatory 5-lipoxygenase (5-LO)-derived leukotrienes (LTs) has been demonstrated in the lungs of patients with pulmonary fibrosis, their causal involvement in this condition has not been established. Bleomycin-induced pulmonary fibrosis was studied in mice rendered LT deficient by knockout of the 5-LO gene (KO) and in wild-type (WT) control mice. Following administration of bleomycin, lung lavage fluid of WT mice demonstrated an approximately 5-fold increase in levels of cysteinyl-LTs over baseline levels at Day 1, with persistent elevation up to Day 21. As compared with WT mice, 5-LO KO mice demonstrated reduced amounts of histologically evident collagen as well as an approximately 60% reduction in lung hydroxyproline levels postbleomycin. Unlike WT mice, KO mice showed no increases in the numbers of lung inflammatory cells postbleomycin. Furthermore, in situ expression and stimulated production by mixed lung leukocytes of the antifibrotic cytokine interferon-gamma were significantly greater in cells from the 5-LO KO mice. Finally, lavage levels of the antiinflammatory and antifibrotic molecule, prostaglandin E(2), were significantly greater in the KO animals. These results provide strong evidence that LTs may participate in the pathogenesis of pulmonary fibrosis, and they may do so by direct effects as well as indirect effects occurring via their modulation of the synthesis of other inflammatory mediators.

Idiopathic pulmonary fibrosis (IPF) is a progressive disorder characterized by inflammation, fibroblast proliferation, and accumulation of extracellular matrix proteins. Leukotrienes (LTs) are pro-inflammatory and pro-fibrogenic mediators derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism. They are thought to play a role in a number of disease processes, but have received relatively little attention in investigations into the pathogenesis of IPF. In this study, we measured the levels of immunoreactive LTs B 4 and C 4 in homogenates of lung tissue obtained from patients with newly diagnosed, untreated IPF, as compared to levels measured in homogenates of uninvolved nonfibrotic lung tissue from patients undergoing resectional surgery for bronchogenic carcinoma. Compared to homogenates of nonfibrotic control lung, homogenates from IPF patients contained 15-fold more LTB 4 and 5-fold more LTC 4 . IPF homogenate levels of LTB 4 were significantly correlated with histologic indices of both inflammation ( r ϭ 0.861) and fibrosis ( r ϭ 0.926). Activation of 5-LO is known from in vitro studies to be associated with localization of the enzyme at the nuclear membrane. Immunohistochemical staining for 5-LO protein in alveolar macrophages (AMs) demonstrated that such an "activated" localization pattern was significantly more frequent in IPF lung (19.2 Ϯ 3.3% of cells) than in control lung (9.3 Ϯ 0.9%); this localization pattern was rarely seen (3.2%) in sections from a truly normal transplant donor lung. Consistent with these data, AMs obtained from IPF patients by bronchoalveolar lavage, purified by adherence, and cultured in the absence of a stimulus for 16 h elaborated significantly greater amounts of LTB 4 and LTC 4 than did control AMs obtained from normal volunteers. These data indicate that the 5-LO pathway is constitutively activated in the lungs of patients with IPF, and the AM represents at least one cellular source of LT overproduction in this disorder. We speculate that LTs participate in the pathogenesis of IPF, and their overproduction in this disorder may be amenable to specific pharmacotherapy. (

5-Lipoxygenase catalyzes the synthesis of leukotrienes from arachidonic acid. The subcellular distribution of 5-lipoxygenase is known to be cell type-dependent and is cytosolic in blood neutrophils. In this study, we asked whether neutrophil recruitment into sites of inflammation can alter the subcellular compartmentation of 5-lipoxygenase. In peripheral blood neutrophils from rats, 5-lipoxygenase was exclusively cytosolic, as expected. However, in glycogen-elicited peritoneal neutrophils, abundant soluble 5-lipoxygenase was in the nucleus. Upon activation with calcium ionophore A23187, intranuclear 5-lipoxygenase translocated to the nuclear envelope. Elicited neutrophils required a greater concentration of A23187 for activation than did blood neutrophils (half-maximal response, 160 versus 52 nM, respectively) but generated greater amounts of leukotriene B 4 upon maximal stimulation (26.6 versus 7.68 ng/10 6 cells, respectively). Intranuclear 5-lipoxygenase was also evident in human blood neutrophils after adherence to a variety of surfaces, suggesting that adherence alone is sufficient to drive 5-lipoxygenase redistribution. These results demonstrate a physiologically relevant circumstance in which the subcellular distribution of 5-lipoxygenase can be rapidly altered in resting cells, independent of 5-lipoxygenase activation. Nuclear import of 5-lipoxygenase may be a universal accompaniment of neutrophil recruitment into sites of inflammation, and this may be associated with alterations in enzymatic function.

Tumor necrosis factor (TNF) is a proinflammatory cytokine which has recently been shown to have beneficial effects in the setting of acquired host immunity. However, the role of TNF in innate immune responses, as in the setting of bacterial pneumonia, has been incompletely characterized. To determine the role of TNF in gram-negative bacterial pneumonia, CBA/J mice were challenged with 10 2 CFU of Klebsiella pneumoniae intratracheally, resulting in the time-dependent expression of TNF MRNA and protein within the lung. Passive immunization of animals with a soluble TNF receptor-immunoglobulin (Ig) construct (sTNFR:Fc) intraperitoneally 2 h prior to K. pneumoniae inoculation resulted in a significant reduction in bronchoalveolar lavage neutrophils, but not macrophages, at 48 h, as compared with animals receiving control IgG1. Furthermore, treatment with sTNFR:Fc resulted in 19.6-and 13.5-fold increases in K. pneumoniae CFU in lung homogenates and plasma, respectively, as compared with animals receiving control IgG1. Finally, treatment of Klebsiellainfected mice with sTNFR:Fc markedly decreased both short-and long-term survival of these animals. In conclusion, our studies indicate that endogenous TNF is a critical component of antibacterial host defense in murine Klebsiella pneumonia.

A B S T R A C T Angiotensin II was determined by radioimmunoassay in systemic arterial, pulmonary arterial, and renal venous plasma and in renal hilar lymph in dogs. Levels of the peptide were determined prior to and during progressive graded hemorrhage or reduction in renal perfusion pressure. Levels of angiotensin II in plasma consistently rose during transit through the lung indicating pulmonary conversion of angiotensin I to angiotensin II. On the other hand, angiotensin II in the renal vein plasma was less than that in arterial plasma indicating renal extraction of the peptide from plasma. When renal hilar lymph was sampled under similar conditions, angiotensin II in lymph was consistently higher than that in arterial or renal venous plasma. Furthermore, in some experiments angiotensin II in lymph increased at a time when the concentration in plasma was undetectable. No evidence was found to indicate that angiotensin II in plasma entered renal lymph. It was concluded that angiotensin II levels in lymph reflected the concentration of angiotensin II in renal tissue. The data further suggested that angiotensin II is partially removed from arterial plasma by hydrolysis during transit through the -kidney. INTRODUCTIONSeveral investigators have proposed models in which angiotensin II (AII) is a critical mediator for the control of glomerular filtration rate (1), renal autoregulation (2), and tubular sodium reabsorption (3,4). It is not clear whether the AII postulated to mediate these control mechanisms is produced within the kidney or reaches the kidney from the systemic circulation.

The purpose of this investigation was to correlate the development of the various enzyme activities associated with the renin-angiotensin system with age-related differences in the steady-state concentrations of angiotensin I (AI) and II (AII). Angiotensin was quantified by radioimmunoassay. Plasma renin activity and concentration increased between birth and 3 wk of age, and declined thereafter to adult values. Renal renin content, on the other hand, increased throughout the first 6 wk of postnatal life. The concentration of AII in plasma also increased following birth; however, maximum concentrations were not attained until 5 wk of age. In contrast, plasma AI did not increase between 3 and 6 wk of age. These data suggest that the steady-state concentration of AII in neonatal rat plasma may be partially limited by the low plasma renin substrate concentration. The increase in AII between 3 and 6 wk of age may reflect the increasing converting enzyme activity.

Fibrosis of the lung constitutes a major clinical challenge and novel therapies are required to alleviate the associated morbidity and mortality. Investigating the antifibrotic efficacy of drugs that are already in clinical practice offers an efficient strategy to identify new therapies. The phosphodiesterase 4 (PDE4) inhibitors, approved for the treatment of chronic obstructive pulmonary disease, harbor therapeutic potential for pulmonary fibrosis by augmenting the activity of endogenous antifibrotic mediators that signal through cyclic AMP. In this study, we tested the efficacy of several PDE4 inhibitors including a novel compound (Compound 1) in a murine model of lung fibrosis that results from a targeted type II alveolar epithelial cell injury. We also compared the antifibrotic activity of PDE4 inhibition to the two therapies that are FDA‐approved for idiopathic pulmonary fibrosis (pirfenidone and nintedanib). We found that both preventative (day 0–21) and therapeutic (day 11–21) dosing regimens of the PDE4 inhibitors significantly ameliorated the weight loss and lung collagen accumulation that are the sequelae of targeted epithelial cell damage. In a therapeutic protocol, the reduction in lung fibrosis with PDE4 inhibitor administration was equivalent to pirfenidone and nintedanib. Treatment with this class of drugs also resulted in a decrease in plasma surfactant protein D concentration, a reduction in the plasma levels of several chemokines implicated in lung fibrosis, and an in vitro inhibition of fibroblast profibrotic gene expression. These results motivate further investigation of PDE4 inhibition as a treatment for patients with fibrotic lung disease.