Antibody to intercellular adhesion molecule 1 (CD54) decreases survival and not lung injury in baboons with sepsis.

Neutrophil influx into the lung is an important event in the pathogenesis of acute lung injury in gram-negative sepsis. We hypothesized that administration of a monoclonal antibody to intercellular adhesion molecule 1 (ICAM-1, CD54), a molecule mediating neutrophil adhesion to endothelial cells, would decrease neutrophil sequestration and transmigration in the lung and attenuate lung injury in Escherichia coli sepsis. Sepsis was induced in 12 baboons primed with heat-killed E. coli (1 x 10(9) CFU/kg) 12 h before infusion of live bacteria (1 x 10(10) CFU/kg). Six animals received monoclonal antibody to CD54 (1 mg/kg) intravenously at the time of live E. coli infusion. After 48 h or when blood pressure could not be maintained, tissues were harvested and bronchoalveolar lavage (BAL) samples were obtained. Median survival time was decreased in anti-CD54-treated animals. This group also had decreased mean arterial pressure, increased metabolic acidosis, and decreased urine output. Measures of lung injury including gas exchange, lung lavage protein and lactate dehydrogenase (LDH), lung thiobarbituric acid-reactive species, and lung histology, including alveolar neutrophil volumes, were unaffected by treatment. The effect of anti-CD54 on neutrophil influx into tissues as measured by myeloperoxidase was organ specific. These data show that monoclonal antibody to CD54 does not ameliorate acute lung injury in E. coli sepsis, and septic primates given anti-CD54 have worsened metabolic parameters and decreased survival.

The biological lifetime of nitric oxide: implications for the perivascular dynamics of NO and O2.

Endothelial nitric oxide (nitrogen monoxide) is synthesized at the intravascular/extravascular interface. We previously have reported the intravascular half-life of NO, as a result of consumption by erythrocytes, as approximately 2 ms. We report here studies designed to estimate the lifetime of NO in the parenchymal (extravascular) tissue and describe the implications of these results for the distribution of NO and oxygen concentration gradients away from the blood vessel. The rate of consumption of NO by parenchymal cells (hepatocytes) linearly depends on both NO and O(2) concentration. We estimate that the extravascular half-life of NO will range from 0.09 to > 2 s, depending on O2 concentration and thus distance from the vessel. Computer modeling reveals that this phenomenon, coupled with reversible NO inhibition of cellular mitochondrial oxygen consumption, substantially extends the zone of adequate tissue cellular oxygenation away from the blood vessel, with an especially dramatic effect during conditions of increased tissue work (oxygen consumption). This represents a second action of NO, in addition to vasodilation, in enhancing tissue cellular respiration and provides a possible physiological function for the known reversible inhibition of mitochondrial respiration by low concentrations of NO.

Clarithromycin for safe and cost-effective reduction of cyclosporine doses in lung allograft recipients.

BACKGROUND: This study was done to analyze the economic effect of clarithromycin on the daily dosing of cyclosporine in lung transplantation. METHODS: Nine consecutive patients (mean age +/- SEM, 34.6 +/- 5.2 years) had transplantation from June 1995 to June 1996. Median follow-up time was 649 days (range, 431 to 799 days). Preoperative diagnoses were cystic fibrosis (n = 4), idiopathic pulmonary fibrosis (n = 2), emphysema, bronchiectasis, and obliterative bronchiolitis. Median time from transplantation to addition of clarithromycin to a standard immunosuppressive regimen was 86 days (range, 14 to 181 days). RESULTS: Baseline cyclosporine dose (9.9 +/- 2.2 mg/kg/day) was reduced to 5.8 +/- 1.0 mg/kg/day and 4.1 +/- 0.8 mg/kg/day at 1 month and 1 year, respectively, after initiation of clarithromycin therapy. Estimated annual savings were $3,400 per patient. There was no increase in infection or rejection episodes. CONCLUSIONS: Clarithromycin safely reduced the dose and cost of cyclosporine in this series.

Regulation of tumor necrosis factor cytotoxicity by calcineurin.

Cyclosporin (CsA) inhibits mitochondrial death signaling and opposes tumor necrosis factor (TNF)-induced apoptosis in vitro. However, CsA is also a potent inhibitor of calcineurin, a phosphatase that may participate in cell death. Therefore, we tested the hypothesis that calcineurin regulates TNF cytotoxicity in rat hepatoma cells (FTO2B). TNF-treated FTO2B cells appeared apoptotic by DNA fragmentation, nuclear condensation, annexin V binding, and caspase activation. We studied two calcineurin inhibitors, CsA and FK506, and found that each potently inhibited TNF cytotoxicity. Western blot demonstrated calcineurin in FTO2B homogenates. In a model of mitochondrial permeability transition (MPT), we found that CsA prevented MPT and cytochrome c release, while FK506 inhibited neither. In summary, we present evidence that calcineurin participates in an apoptotic death pathway activated by TNF. CsA may oppose programmed cell death by inhibiting calcineurin activity and/or inhibiting mitochondrial signaling.

Proinflammatory cytokines increase in sepsis after anti-adhesion molecule therapy.

Cytokine mediators and leukocyte-endothelial cell adhesion molecules are critical and interdependent components of the acute inflammatory response in sepsis. We hypothesized that the administration of monoclonal antibodies to intercellular adhesion molecule-1 (CD54) or E- and L-selectin (CD62E/L) would decrease serum levels of the proinflammatory cytokines interleukin-1beta (IL-1), IL-6, and IL-8 and tumor necrosis factor receptor (TNFR-1) in baboons during sepsis. Adult male baboons received infusions of 1 x 10(9) colony forming units (CFU)/kg heat-killed Escherichia coli (E. coli) followed 12 h later by live E. coli (1 x 10(10) CFU/kg). At the time of live bacterial infusion, six septic animals were treated with a monoclonal antibody to CD54 and six with an antibody to CD62E and L (1 mg/kg). Eight untreated septic animals served as controls. Sequentially drawn serum samples were assayed for IL-1, IL-6, IL-8, and TNFR-1 using enzyme-linked immunoassay (ELISA). Data were compared using Mann-Whitney U tests and Chi-square analyses. Median survival was decreased in both treatment groups compared to controls (P < 0.05). Peak IL-1 level was higher than controls in septic animals treated with anti-CD54 but not anti-CD62E/L (P < 0.05, P = NS, respectively). Elevations in IL-6, IL-8, and TNFR-1 were increased and prolonged in both antibody treated groups compared to controls (P < 0.05). These results provide the first in vivo evidence that leukocyte-endothelial adhesion molecules CD54 and CD62E/L regulate cytokine production in sepsis.

Oxidative stress and adenine nucleotide control of mitochondrial permeability transition.

Mitochondria can initiate apoptosis by releasing cytochrome c after undergoing a calcium-dependent permeability transition (MPT). Although the MPT is enhanced by oxidative stress and prevented by adenine nucleotides such as adenosine 5'-diphosphate (ADP), the hypothesis has not been tested that oxidants regulate the effects of exogenous adenine nucleotides on the MPT and cytochrome c release. We found that cytochrome c release from intact rat liver mitochondria depended strictly on pore opening and not on membrane potential, and that MPT-enhancing oxidative stress also augmented cytochrome c release. At low oxidative stress, micromolar (ADP) and low adenosine 5'-triphosphate (ATP)/ADP ratio inhibited the MPT and cytochrome c release, whereas ATP or high ATP/ADP had only a slight effect. In freshly isolated mitochondria, the time to half-maximal MPT was related to the log of the ATP/ADP ratio. This function was shifted to shorter times by oxidative stress which decreased ADP protection and caused ATP to accelerate the calcium-dependent MPT. By comparison, mitochondria treated with reducing agents and those isolated from septic rats were protected from the MPT by both nucleotides. These results indicate that oxidation-sensitive site(s) in the membrane regulate the effects of adenine nucleotides on the MPT. The oxidant-based differences in the effects of ADP and ATP on the pore support the novel hypothesis that failure of the cell to consume ATP and provide adequate ADP at the adenine nucleotide transporter during oxidative stress predisposes to cytochrome c release and initiation of apoptosis.

Induction of arginase isoforms in the lung during hyperoxia.

L-Arginine can be metabolized by nitric oxide (NO) synthase (NOS) to produce NO or by arginase to produce urea and L-ornithine. In the liver, arginase (the AI isoform) is a key enzyme in the urea cycle. In extrahepatic organs including the lung, the function of arginase (the AII isoform) is less clear. Because we found that lung AII was upregulated during 100% O2 exposure in preliminary experiments, we sought to characterize expression of the arginase isoforms and inducible NOS and to assess the functions of arginase in hyperoxic lung injury. Male Sprague-Dawley rats were exposed to 100% O2 for 60 h. Protein expression of AI and AII and their cellular distribution were determined. The activities of arginase and NOS were also measured. Expression of arginase was correlated with that of ornithine decarboxylase, a biochemical marker for tissue repair, in a separate group of rats allowed to recover in room air for 48 h. We found by Western blot analyses that both AI and AII proteins were upregulated after 60 h of hyperoxic exposure (403 and 88% increases by densitometry, respectively) and, like ornithine decarboxylase, remained elevated during the recovery phase. Arginase activity increased by 37%. Immunostaining showed that increases in AI and AII were mainly in the peribronchial and perivascular connective tissues. NOS activity was unchanged and inducible NOS was not induced, but the level of nitrogen oxides in the lung decreased by 67%. Our study showed in vivo induction of arginase isoforms during hyperoxia. The strong expression of arginase in the connective tissues suggests that the function of pulmonary arginase may be linked to connective tissue elements, e.g., fibroblasts, during lung injury and recovery.

Mitochondrial respiration after sepsis and prolonged hypoxia.

Recently, marked oxygen dependence of respiration by isolated mitochondria after exposure to prolonged hypoxia has been described. Because mitochondrial oxygen-dependent respiration could significantly influence oxygen consumption during critical illness, we sought to confirm the oxygen-dependent behavior of mitochondria. We hypothesized that mitochondria isolated during sepsis would exhibit increased oxygen dependence. We isolated rat liver mitochondria 16 h after cecal ligation and puncture and found a 30-40% greater oxygen uptake compared with control rats under state 3 conditions. Mitochondria incubated in deoxygenated buffer were studied for oxygen dependence at 10-min intervals for 90 min. Mitochondrial respiration after reoxygenation was stable over a 60-min period of hypoxia for control rats and decreased slightly for septic rats (10-15%). State 3 respiration was 10% lower when mitochondria were reoxygenated at low (15-25 Torr) versus high (90-100 Torr) and low (10-15 Torr) versus intermediate (40-45 Torr) oxygen tension. Oxygen consumption with ascorbate+N, N, N', N'-tetramethyl-p-phenylenediamine was 20% lower at low versus high oxygen tension. No increase in oxygen dependence was observed during 1 h of hypoxic incubation. Our data indicate only a modest oxygen dependence of respiration between 10 and 100 Torr, which is similar for septic and control mitochondria. Additionally, oxygen dependence did not increase significantly during a 1-h hypoxic exposure for well-coupled mitochondrial preparations.

Bacterial priming increases lung injury in gram-negative sepsis.

Sepsis syndrome is a leading cause of acute respiratory distress syndrome (ARDS), but the development of acute lung injury is highly variable for reasons that are poorly understood. We hypothesized that nonlethal systemic exposure to gram-negative bacteria, with its consequent activation of inflammatory processes, would increase functional and structural lung injury on a second exposure to live organisms, as compared with exposure of naive animals. Sixteen adult baboons received 1 to 2 x 10(10) colony-forming-units (cfu)/kg Escherichia coli by intravenous infusion. Eight animals received live bacteria as a single infusion, whereas the other eight received 10% of the total dose as heat-killed organisms (priming dose) 12 h before the live infusion. Pulmonary gas exchange and hemodynamics were monitored for 48 h or until blood pressure could not be maintained. The animals were killed and one lung was processed for electron microscopy and morphometry. Group data were compared through analysis of variance (ANOVA). The systemic circulatory responses to the bacterial challenge were similar, although less severe shock occurred in primed animals. In contrast, primed animals had increased structural damage involving lung epithelium and endothelium, and showed increased cellularity of the interstitium. The morphologic evidence of increased lung injury in septic animals with prior exposure to heat-killed bacteria suggests that prior activation of systemic inflammatory responses is a contributing factor in the pathogenesis of ARDS.

Antibody to E- and L-selectin does not prevent lung injury or mortality in septic baboons.

Recruitment of polymorphonuclear leukocytes (PMN) through upregulation of cellular adhesion molecules is a proposed mechanism of injury in sepsis and acute respiratory distress syndrome (ARDS). We hypothesized that pretreatment of baboons with a monoclonal antibody to human E- and L-selectin (EL-246) during sepsis would decrease PMN influx into tissues and result in less organ injury during gram-negative sepsis. We studied 14 anesthetized, ventilated adult baboons; six animals received 1 mg/kg of EL-246 before infusion of an LD100 of live Escherichia coli and six received the E. coli infusion without antibody therapy. Two other animals received 1 mg/kg of EL-246 intravenously without an infusion of bacteria. Intermittent measurements were made of circulatory pressures, cardiac output, urine output, arterial blood gases, ventilation:perfusion ratio (VA/Q), and hematologic status. The experiments were ended at 48 h or at the time of death. Tissues were harvested for pathology and biochemical measurements. The E. coli infusions were associated with a hyperdynamic state, pulmonary hypertension, systemic hypotension, decreased urine output (UOP), and metabolic acidosis. The antibody partly blocked PMN migration, but there were few significant physiologic or biochemical differences between the EL-246-treated and untreated animals. In the antibody-treated animals, UOP was decreased, metabolic acidosis was worsened, and median survival time was decreased significantly. We conclude that treatment with an antibody to E- and L-selectin in gram-negative sepsis does not improve gas exchange or protect against lung injury, and is associated with decreased survival time in primates.

Oxidative metabolism in rat hepatocytes and mitochondria during sepsis.

We hypothesized that cellular oxygen consumption is abnormal during sepsis as a result of increased oxidative stress and selective mitochondrial damage. In a rat model of sepsis (cecal ligation and puncture), we studied the respiratory characteristics of isolated hepatocytes and liver mitochondria 16 h after onset of septic injury. Endogenous respiration by isolated cells was decreased during sepsis, while cyanide-resistant (nonmitochondrial) respiration was unaffected. Maximal oxygen consumption in ADP-supplemented, permeabilized hepatocytes was decreased with succinate as the substrate, but not with malate + glutamate or TMPD + ascorbate. In contrast, maximum oxygen consumption (State 3) by isolated liver mitochondria increased up to 35% during sepsis using either succinate or malate + glutamate as substrate. The electrophoretic features and mobility of nondenatured mitochondrial respiratory complexes were similar in control and septic hepatocytes, with the exception of decreased Complex V protein in sepsis. Structural evaluation of mitochondria in fixed liver slices by electron microscopy showed mitochondrial swelling in most of the septic animals. Measurements of oxidative stress during sepsis suggested an increase in hydroxylation of salicylate by isolated hepatocytes, and mitochondrial protein carbonyl content was increased significantly. Induction of iNOS in hepatocytes after 16 h of sepsis was variable, and little release of the oxidation products of NO. was detected. These findings are interpreted to mean that hepatocytes contain a mixed population of injured and hyperfunctional mitochondria during sepsis.

Aerosolized manganese SOD decreases hyperoxic pulmonary injury in primates. I. Physiology and biochemistry.

Prolonged hyperoxia causes lung injury and respiratory failure secondary to oxidative tissue damage mediated, in part, by the superoxide anion. We hypothesized that aerosol treatment with recombinant human manganese superoxide dismutase (rhMnSOD) would attenuate hyperoxic lung damage in primates. Adult baboons were anesthetized and ventilated with 100% oxygen for 96 h or until death. Six animals were treated with aerosolized rhMnSOD (3 mg . kg-1 . day-1 in divided doses), and six control animals did not receive enzyme therapy. Physiological variables were recorded every 12 h, and ventilation-perfusion ratio relationships were evaluated by using the multiple inert-gas elimination technique. After the experiments, surfactant composition and lung edema were measured. We found that rhMnSOD significantly decreased pulmonary shunt fraction (P < 0.01) and preserved arterial oxygenation (P < 0.01) during hyperoxia. The rhMnSOD increased lung phospholipids, phosphatidylcholine and disaturated phosphatidylcholine, and decreased lung edema in this model. Testing of higher and lower doses of MnSOD (1 and 10 mg . kg-1 . day-1) in two other groups of baboons produced variable physiological protection, suggesting a "window" of effective dosage. We conclude that aerosolized MnSOD (3 mg . kg-1 . day-1) affords significant preservation of pulmonary gas exchange during hyperoxic lung injury.

Aerosolized manganese SOD decreases hyperoxic pulmonary injury in primates. II. Morphometric analysis.

Hyperoxia damages lung parenchyma via increased cellular production of reactive oxygen species that exceeds antioxidant defenses. We hypothesized that aerosolized human recombinant manganese superoxide dismutase (rhMnSOD) would augment extracellular antioxidant defenses and attenuate epithelial injury in the lung during hyperoxia in primates. Twenty-four adult male baboons were anesthetized and mechanically ventilated with 100% oxygen for 96 h. The baboons were divided equally into four groups. Oxygen alone and oxygen plus rhMnSOD given at 3 mg . kg-1 . day-1 were compared to assess efficacy of the drug. Subsequently, aerosolized rhMnSOD was given at 1 or 10 mg . kg-1 . day-1 to study dose effects and toxicity. Quantitative morphometry showed protection of alveolar epithelium from hyperoxia by 3 mg . kg-1 . day-1 rhMnSOD (P < 0.05). In addition, interstitial fibroblast volumes were increased in the treatment group (P = 0.06). This effect appeared greater at the two higher doses of the rhMnSOD. The aerosolized drug was localized to the surface of airways and air spaces and macrophages by immunolabeling studies, suggesting efficacy via physicochemical properties that localize it to cell surfaces or by effects on alveolar macrophage function.

Hypoxia inhibits nitric oxide synthesis in isolated rabbit lung.

Nitric oxide (NO.) has been proposed to modulate hypoxic vasoconstriction in the lung. The activity of nitric oxide synthase (NOS) can be inhibited by hypoxia because molecular oxygen is a necessary substrate for the enzyme. On the basis of this mechanism, we hypothesized that NOS activity has a key role in regulation of pulmonary vascular tone during hypoxia. We measured oxidation products of NO. released into the vasculature of isolated buffer-perfused rabbit lung ventilated with normoxic (21% O2), moderately hypoxic (5% O2), or anoxic (0% O2) gas using two methods. Mean PO2 in perfusate exiting the lung was 25 Torr during anoxic ventilation and 47 Torr during moderately hypoxic ventilation. We found that the amount of the NO. oxidation product nitrite released into the perfusate was suppressed significantly during ventilation with anoxic but not moderately hypoxic gas. During normoxic ventilation, nitrite release was inhibited by pretreatment with NG-monomethyl-L-arginine, a competitive inhibitor of NOS. To confirm that changes in nitrite concentration reflected changes in NO. release into the perfusate, major oxidation products of NO. (NOx) were assayed using a method for reduction of these products to NO. by vanadium(III) Cl. Release of NOx into the perfusate was suppressed by severe hypoxia (anoxic ventilation), and this effect was reversed by normoxia. Pulmonary vasoconstriction was observed during severe but not moderate hypoxia and was related inversely to the rate of nitrite release. These observations provide evidence that decreased NO. production contributes to the pulmonary vasoconstrictor response during severe hypoxia.

Idiopathic pneumonia syndrome: changing spectrum of lung injury after marrow transplantation.

BACKGROUND: The aim of our study was to describe the incidence, clinical course, and risk factors for the idiopathic pneumonia syndrome (IPS), compared with those previously described for "idiopathic pneumonia," after bone marrow transplantation (BMT). METHODS: Our study design was a case-series review with determination of risk by comparison with unaffected controls by log-rank or Fisher's exact (two-tailed) test and logistic regression analyses. The study group comprised 1165 consecutive marrow recipients at a single center from 1988 to 1991. RESULTS: IPS was documented in 85 BMT recipients (7.3%) by bronchoalveolar lavage (n=68), open lung biopsy (n=3), or autopsy (n=14). The calculated actuarial incidence for IPS within 120 days after BMT was 7.7%. Median time to onset was 21 days (mean 34+/-30). Hospital mortality was 74%, and 53 BMT recipients (62%) died with progressive respiratory failure. IPS resolved in 22 patients (26%); 18 patients (21%) survived to discharge. Mechanical ventilation was required by 59 BMT recipients (69%), within a median of 2 days of onset of infiltrates. Two of these 59 recipients (3%) survived to discharge. Pulmonary infection (predominantly fungal) was noted in 7 of 25 (28%) BMT recipients who had an autopsy. Potential risk factors for IPS were assessed in univariate and multivariate logistic regression analyses. Although the incidence was not significantly different between autologous (5.7%) and allogeneic marrow recipients (7.6%), risks were identified only for the latter: malignancy other than leukemia (odds ratio=6.5 compared with aplastic anemia), and grade 4 graft-versus-host disease (odds ratio=5.4 compared with lower grades). No factors were associated with recovery. CONCLUSIONS: The incidence of idiopathic lung injury seems lower, the onset earlier, and the risk factors different from those previously reported. The major risks seem to be regimen-related toxicity and multi-organ dysfunction associated with alloreactive processes.

Release of cytochrome c from liver mitochondria during permeability transition.

The mitochondrial permeability transition (PT) follows opening of megachannels in the inner membrane and may be part of a programmed death pathway. Recently a role for cytochrome c in programmed cell death has been proposed, although its relationship to PT has not been been determined. We studied the release of cytochrome c from liver mitochondria undergoing PT. Well-coupled mitochondria treated with 5 mM atractyloside (ATR) or 100 microM calcium chloride were found to undergo PT and release cytochrome c into the incubation buffer within 5 minutes. Control mitochondria and mitochondria treated with the uncoupler FCCP did not undergo PT or release cytochrome c at 5 or 15 minutes. PT induced by ATR could be prevented by pretreatment with 10 microM cyclosporin A. Mitochondria incubated with ATR or calcium caused a 20-30% decrease in electron transfer rate via cytochrome c and cytochrome c oxidase. We conclude that cytochrome c release is an early event during mitochondrial PT, and is sufficient to decrease electron transfer through the terminal electron transport components of the mitochondrion.

The CD4/CD8 ratio in BAL fluid is highly variable in sarcoidosis.

Patients with pulmonary sarcoidosis frequently have increased numbers of lymphocytes and a high ratio of CD4+ to CD8+ T-lymphocytes (CD4/CD8 ratio) in bronchoalveolar lavage (BAL) fluid. Some investigators have suggested that these parameters can be used to distinguish sarcoidosis from other types of interstitial lung disease with a high degree of reliability. However, we hypothesized that the BAL CD4/CD8 ratio measured during the initial diagnostic evaluation of patients with biopsy-proven sarcoidosis is highly variable. BAL lymphocytes were analysed via flow cytometry to determine the CD4/CD8 ratio in a population of 86 patients with histological and clinical evidence of sarcoidosis, who underwent BAL as part of their initial diagnostic evaluation. In these patients, the CD4/CD8 ratio ranged 0.5-37.3, with a median value of 3.35 (mean 6.49). The CD4/CD8 ratio was greater than 4 in only 36 (42%) subjects. Ten patients (12%) had a CD4/CD8 ratio less than 1. The distribution of CD4/CD8 ratios was similar in the presence or absence of BAL lymphocytosis. In conclusion, the CD4/CD8 ratio in bronchoalveolar lavage fluid is highly variable in biopsy-proven sarcoidosis. Bronchoalveolar lavage lymphocyte subset determination is a diagnostic test with low sensitivity for this disease.