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Background: The activation of poly (adenosine diphosphate) ribose synthetase (PARS) is known to be important in the cellular response to oxidative stress. Previous studies have reported that PARS inhibition confers protection in models of endotoxic shock and ischemia-reperfusion. The purpose of this study was to determine the role of PARS inhibition in lung ischemia-reperfusion injury (LIRI)., Methods: Left lungs of Long-Evans rats were rendered ischemic for 90 minutes and reperfused for up to 4 hours. Treated animals received 3 mg/kg of INO-1001 (a PARS inhibitor) intravenously 30 minutes before ischemia. Injury was quantitated in terms of tissue myeloperoxidase (MPO) content, vascular permeability ((125)I radiolabeled bovine serum albumin extravasation) and bronchoalveolar lavage (BAL) leukocyte content. BAL fluid was assessed for cytokine and chemokine content by enzyme-linked immunoassay. Further samples were processed for nuclear protein analysis by electromobility shift assay (EMSA) and cellular death by terminal deoxyribonucleotidyl transferase-mediated d-UTP biotin nick-end labeling (TUNEL) assay and caspase-3 staining., Results: Lung vascular permeability was reduced in treated animals by 73% compared with positive controls (p < 0.009). The protective effects of PARS inhibition correlated with a 46% reduction in tissue MPO content (p < 0.008) and marked reductions in BAL leukocyte accumulation. This positively correlated with the diminished expression of pro-inflammatory mediators and nuclear transcription factors, as well as decreased levels of cellular death., Conclusions: The deleterious effects of LIRI are in part mediated by the formation of free radicals and superoxides, which lead to DNA single-strand breaks. This leads to activation of PARS, which causes rapid cellular energy depletion and cell death. PARS inhibition is protective against this and represents a potentially useful therapeutic tool in the prevention of LIRI.

Background: Alpha chemokines function predominantly to recruit and activate neutrophils, which are important effectors of acute lung injury. This study evaluated whether blockade of 2 potent alpha chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC), is protective against lung ischemia-reperfusion injury in a warm in situ hilar clamp model., Methods: Left lungs of Long-Evans rats underwent normothermic ischemia for 90 minutes and reperfusion for up to 4 hours. Treated animals received antibodies to MIP-2 or CINC immediately prior to reperfusion. Lung injury was quantitated by vascular permeability to (125)I-radiolabeled bovine serum albumin, lung tissue neutrophil sequestration (myeloperoxidase [MPO] content), and alveolar leukocyte content in bronchoalveolar lavage (BAL) fluid. CINC and MIP-2 mRNA expression were assessed by northern blot, while ribonuclease protection assays were performed to evaluate mRNA expression for a number of early response cytokines. MIP-2 and CINC protein expression in injured lungs was determined by immunoblotting., Results: Treatment with antibodies to CINC or MIP-2 was associated with significant protection against increases in vascular permeability, MPO content and alveolar leukocyte sequestration in injured lungs. Expression of CINC and MIP-2 mRNA peaked after 2 hours of reperfusion in injured lungs, and protein levels were evident on immunoblotting after 3 hours of reperfusion. Neither CINC nor MIP-2 blockade appeared to modulate cytokine mRNA expression., Conclusions: CINC and MIP-2 are important mediators involved in direct lung ischemia-reperfusion injury. They appear to function by modulating neutrophil recruitment, but not inflammatory cytokine release.

Objective: Tumor necrosis factor-alpha is a proinflammatory mediator required for the development of experimental lung ischemia-reperfusion injury. The alveolar macrophage is a rich source of tumor necrosis factor-alpha in multiple models of acute lung injury. The present study was undertaken to determine whether the alveolar macrophage is an important source of tumor necrosis factor-alpha in lung ischemia-reperfusion injury and whether suppression of its function protects against injury., Methods: Left lungs of Long-Evans rats underwent normothermic ischemia for 90 minutes and reperfusion for up to 4 hours. Treated animals received gadolinium chloride, a rare earth metal that inhibits macrophage function. Injury was quantitated via lung tissue neutrophil accumulation (myeloperoxidase content), lung vascular permeability, and bronchoalveolar lavage fluid leukocyte, cytokine, and chemokine content. Separate samples were generated for immunohistochemistry., Results: Tumor necrosis factor-alpha secretion occurred at 15 minutes of reperfusion and was localized to the alveolar macrophage by immunohistochemistry. In gadolinium-treated animals, lung vascular permeability was reduced by 66% at 15 minutes (P <.03) of reperfusion and by 34% at 4 hours (P <.02) of reperfusion. Suppression of macrophage function resulted in a 35% reduction in lung myeloperoxidase content (P <.03) and similar reductions in bronchoalveolar lavage leukocyte accumulation. Tumor necrosis factor-alpha and microphage inflammatory protein-1alpha protein levels were markedly reduced in the bronchoalveolar lavage of gadolinium-treated animals by enzyme-linked immunosorbent assay., Conclusions: The alveolar macrophage secretes tumor necrosis factor-alpha protein by 15 minutes of reperfusion, which orchestrates the early events that eventually result in lung ischemia-reperfusion injury at 4 hours. Gadolinium pretreatment markedly reduces tumor necrosis factor-alpha elaboration, resulting in significant protection against lung ischemia-reperfusion injury.

Background: Although chemokines are functionally important in models of ischemia-reperfusion injury, little is known about their role in lung ischemia-reperfusion injury (LIRI). This study examined the role of the beta-chemokines, macrophage inflammatory protein (MIP)-1alpha, monocyte chemoattractant protein (MCP)-1, and regulated upon activation normal T cells expressed and secreted (RANTES) in LIRI., Methods: Left lungs of Long-Evans rats underwent normothermic ischemia for 90 minutes and reperfusion for up to 4 hours. Treated animals received anti-MIP-1alpha, anti-MCP-1, or anti-RANTES antibodies before reperfusion. Changes in lung vascular permeability were measured with iodine 125-labeled bovine serum albumin. Neutrophil accumulation in the lung parenchyma was determined by myeloperoxidase activity, and bronchoalveolar lavage was performed to measure leukocyte cell counts. Western blots, Northern blots, and ribonuclease protection assays assessed beta-chemokine messenger RNA and protein levels., Results: Animals receiving anti-MIP-1alpha demonstrated reduced vascular permeability compared with controls (p < 0.001). Attenuation of permeability was less dramatic in animals treated with anti-MCP-1 and anti-RANTES antibody, which demonstrated permeability decreases of 15% and 16%, respectively (p < 0.02). Lung neutrophil accumulation was reduced in animals receiving anti-MIP-1alpha antibody (p < 0.005) but was unchanged in animals receiving either anti-MCP-1 or anti-RANTES. Bronchoalveolar lavage leukocyte content was also reduced by treatment with anti-MIP-1alpha (p < 0.003) and was unchanged in anti-MCP-1-treated and anti-RANTES-treated animals. MIP-1alpha treatment decreased tumor necrosis factor-alpha messenger RNA in injured left lungs., Conclusions: MIP-1alpha is functionally significant in the development of LIRI. It likely exerts its effects in part by mediating the expression of proinflammatory and antiinflammatory cytokines and influencing tissue neutrophil recruitment. MCP-1 and RANTES seem to play relatively minor roles in the development of direct LIRI.

Objectives: We previously demonstrated that calcineurin inhibitors given intravenously ameliorate experimental lung ischemia-reperfusion injury. This study evaluates whether these effects can be achieved when these agents are delivered endotracheally., Methods: Left lungs of Long Evans rats were rendered ischemic for 90 minutes and reperfused for up to 4 hours. Treated animals received tacrolimus endotracheally at doses of 0.2, 0.1, or 0.025 mg/kg 60 minutes before ischemia. Injury was quantitated in terms of vascular permeability. Additional animals treated at a dose of 0.1 mg/kg were assessed for lung tissue myeloperoxidase content and bronchoalveolar lavage leukocyte content. Bronchoalveolar lavage fluid was assessed for cytokine and chemokine content by enzyme-linked immunosorbent assay. Tissue samples were processed for nuclear factor-kappaB activation, and blood levels of tacrolimus were measured in treated animals., Results: Left lung vascular permeability was reduced in treated animals in a dose-dependent fashion compared with controls. The protective effects correlated with a 47% (0.50% +/- 0.06% vs 0.27% +/- 0.08%, respectively) reduction in tissue myeloperoxidase content (P <.004) and marked reductions in bronchoalveolar lavage leukocyte accumulation. This protection was also associated with decreased nuclear factor-kappaB activation and diminished expression of proinflammatory mediators. Blood tacrolimus levels in treated animals at 4 hours of reperfusion were undetectable., Conclusions: Tacrolimus administered endotracheally is protective against lung ischemia-reperfusion injury in our model. This protection is associated with a decrease in nuclear factor-kappaB activation. This route of tacrolimus administration broadens its potential clinical use and decreases concerns about systemic and renal toxicity. It may be a useful therapy in lung donors to protect against lung ischemia-reperfusion injury.

Background: Functional roles for chemokines have been demonstrated in several models of ischemia-reperfusion injury. The redundancy inherent to chemokine pathways makes administration of neutralizing antibodies to any single chemokine ineffective in ameliorating injury. This study was undertaken to define the pattern of chemokine expression in lung ischemia-reperfusion injury (LIRI), and to determine whether a broad-spectrum chemokine inhibitor, NR58-3.14.3, could confer significant protection against LIRI., Methods: Left lungs of rats were rendered ischemic for 90 minutes and then reperfused for 4 hours. Chemokine secretion into the alveolar space was quantified by enzyme-linked immunoassay. Treated animals received NR58-3.14.3 prior to reperfusion. Vascular injury was measured by lung permeability index, neutrophil accumulation in lung parenchyma was determined by myeloperoxidase (MPO) activity, and alveolar leukocyte counts were quantified in bronchoalveolar lavage (BAL) effluent. A ribonuclease protection assay evaluated mRNA expression of various chemokines., Results: Lavage effluent in untreated animals demonstrated significant increases in the secretion of cytokine-induced neutrophil chemoattractant (CINC), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2), MIP-1alpha and monocyte chemoattractant protein-1 (MCP-1). Animals receiving NR58-3.14.3 demonstrated a 37% (p < 0.001) reduction in vascular injury and a marked reduction in lung MPO activity (p < 0.001) and alveolar cell counts (p = 0.005). Chemokine inhibition decreased mRNA expression of a number of early response cytokines when compared with positive control animals, and caused a significant decrease (p < 0.04) in the secretion of TNF-alpha., Conclusions: These studies demonstrate that chemokines are expressed after lung ischemia and reperfusion, and that broad-spectrum chemokine inhibition ameliorates reperfusion injury. mRNA expression of early response cytokines was modulated, and the secretion of TNF-alpha was decreased.

Objectives: Statins are lipid-lowering drugs with anti-inflammatory and antioxidant properties. This study explores the potential of these commonly prescribed agents to ameliorate lung ischemia-reperfusion injury., Methods: Left lungs of Long-Evans rats were rendered ischemic for 90 minutes and reperfused for up to 4 hours. Treated animals received simvastatin orally (0.5 mg/kg) for 5 days before the experiment. Injury was quantitated in terms of tissue myeloperoxidase content, vascular permeability ((125)I bovine serum albumin extravasation), and bronchoalveolar lavage leukocyte and cytokine content. Changes in nuclear translocation of transcription factors were evaluated by electromobility shift assay. Additional animals received N(G)-nitro-L-arginine methyl ester before ischemia-reperfusion to assess whether inhibition of nitric oxide synthase could reverse simvastatin's protective effects. The presence of nicotinamide adenine dinucleotide phosphate oxidase was also evaluated using enzyme staining both histologically and in native electrophoresis., Results: Lung vascular permeability was reduced in treated animals by 71% compared with positive controls (P <.001). Administration of N(G)-nitro-L-arginine methyl ester reversed this protection. The protective effects of statin pretreatment correlated with a 68% reduction in tissue myeloperoxidase content (P <.01), marked reductions in bronchoalveolar lavage leukocyte accumulation, and decreased expression of proinflammatory cytokines. Nicotinamide adenine dinucleotide phosphate oxidase expression also decreased with statin treatment., Conclusion: In addition to its antioxidant properties, the protective effects of simvastatin are likely mediated by modulation of endothelial nitric oxide synthase. The potential to pretreat recipients of lung transplantation with statins to ameliorate reperfusion injury is promising.

Background: Regulatory cytokines play functional roles in experimental heart, hindlimb, and liver ischemia reperfusion injury. However, little is known about their involvement in direct lung ischemia reperfusion injury (LIRI). These studies were undertaken to investigate the role of two regulatory cytokines, interleukin-4 (IL-4) and IL-10, in an in vivo model of LIRI., Methods: Left lungs of Long-Evans rats underwent normothermic ischemia for 90 minutes and reperfusion for up to 4 hours. Treated animals received either recombinant IL-4 or recombinant IL-10, or antibodies to IL-4 or IL-10 immediately before reperfusion. Lung injury was quantitated by permeability indices, lung parenchymal neutrophil sequestration (myeloperoxidase [MPO] content), and alveolar leukocyte content in bronchoalveolar lavage (BAL) effluent. Expression of IL-4 and IL-10 was determined by immunoblotting, and mRNA expression for early response cytokines was evaluated by ribonuclease protection assays., Results: IL-4 and IL-10 protein expression was significant after 2 hours of reperfusion. Animals receiving anti-IL-4 (p = 0.05) and anti-IL-10 (p = 0.01) antibodies demonstrated increased permeabilities compared with positive controls. Lung tissue neutrophil accumulation (p < 0.004) and BAL leukocyte content (p < 0.04) were also significantly increased in animals receiving anti-IL-10 antibodies. Conversely, animals receiving recombinant IL-4 and recombinant IL-10 demonstrated reduced permeabilities and lung MPO content. Both anti-IL-4 and anti-IL-10 treatment increased mRNA expression for a number of early response cytokines, including TNF-alpha and IL-1beta., Conclusions: IL-4 and IL-10 are expressed in response to LIRI and function to decrease injury severity. These effects are partly due to modulated expression of early proinflammatory cytokines.

Background: Peroxynitrite is a potent cytotoxic free radical produced by the reaction of nitric oxide with the superoxide ion produced in conditions of oxidative stress. The purpose of the study was to examine the role of this reactive nitrogen species in lung ischemia-reperfusion injury., Methods: Left lungs of male Long-Evans rats were rendered ischemic for 90 minutes and reperfused for up to 4 hours. Treated animals received FP-15 (a water-soluble iron containing metalloporphyrin that acts as a peroxynitrite decomposition catalyst). Injury was quantitated in terms of tissue neutrophil accumulation (myeloperoxidase content) and vascular permeability ((125)I bovine serum albumin [BSA] extravasation) and bronchoalveolar lavage cytokine, transcriptional factor and leukocyte content. Separate tissue samples were processed for immunohistology and nuclear protein analysis., Results: Lung vascular permeability was reduced in treated animals by 61% compared with control animals (p < 0.005). The protective effects of enhanced peroxynitrite decomposition correlated with a 72% reduction in tissue myeloperoxidase content (p < 0.001) and marked reductions in brochoalveolar lavage leukocyte accumulation. This correlated positively with the diminished expression of pro-inflammatory chemokines and nuclear transcription factors., Conclusions: The deleterious effects of lung ischemia-reperfusion injury are in part mediated by the formation of peroxynitrite, as enhanced decomposition of this species is protective in this model. The development of potent water-soluble decomposition catalysts represents a potentially useful therapeutic tool in the prevention of lung ischemia-reperfusion injury after lung transplantation.

Objectives: Activation of the alveolar macrophage is critical to the development of nonischemic inflammatory lung injury. The present studies were undertaken to determine whether the alveolar macrophage plays a similarly important role in lung ischemia-reperfusion injury., Methods: The left lungs of male rats were rendered ischemic for 90 minutes and reperfused for up to 4 hours. Treated animals received liposome-encapsulated clodronate, which depletes alveolar macrophages. Injury was quantitated in terms of vascular permeability, tissue neutrophil accumulation, and bronchoalveolar lavage fluid leukocyte, chemokine, and cytokine content. Lung homogenates were also analyzed for nuclear translocation of the transcription factors nuclear factor kappaB and activator of protein 1., Results: Depletion of alveolar macrophages reduced lung vascular permeability by 53% compared with that seen in control animals (permeability indices: 0.88 +/- 0.07 to 0.46 +/- 0.04, P <.001). The protective effects of alveolar macrophage depletion correlated with a 50% reduction in tissue myeloperoxidase content (0.62 +/- 0.07 to 0.33 +/- 0.03, P <.006) and marked reductions in bronchoalveolar lavage fluid leukocyte accumulation. Alveolar macrophage-depleted animals also demonstrated marked reductions of the elaboration of multiple proinflammatory chemokines and cytokines in the lavage effluent and nuclear transcription factors in lung homogenates., Conclusion: It is likely that the alveolar macrophage is the key early source of multiple proinflammatory mediators that orchestrate lung ischemia-reperfusion injury. Depleting alveolar macrophages is protective against injury, supporting its central role in oxidant stress-induced cytokine and chemokine release and the subsequent development of lung injury.

Objective: Proinflammatory cytokines are known to play roles in ischemia-reperfusion injury of the heart, kidney, small bowel, skin, and liver. Little is known about their roles in ischemia-reperfusion injury of the lung. This study was undertaken to define the role of 2 proinflammatory cytokines, tumor necrosis factor alpha and interleukin 1beta, in ischemia-reperfusion injury of the lung., Methods: Left lungs of male rats were rendered ischemic for 90 minutes and reperfused for up to 4 hours. Treated animals received anti-tumor necrosis factor alpha or anti-interleukin 1beta antibody before reperfusion. Increased vascular permeability in the lung was measured by using iodine 125-labeled bovine serum albumin. Neutrophil sequestration in the lung parenchyma was determined on the basis of activity. Bronchoalveolar lavage was performed to measure cell counts. Separate tissue samples were processed for histology, cytokine protein, and messenger RNA content by using Western blotting and the ribonuclease protection assay., Results: Animals receiving anti-tumor necrosis factor alpha and anti-interleukin 1beta demonstrated reduced injury compared with that seen in positive control animals (vascular permeability of 48.7% and 29.4% lower, respectively; P <.001). Vascular injury was reduced by 71% when antibodies to tumor necrosis factor alpha and interleukin 1beta were administered together. Lung neutrophil accumulation was markedly reduced among animals receiving anti-tumor necrosis factor alpha and anti-interleukin 1beta (myeloperoxidase content of 30.9% and 38.5% lower, respectively; P <.04) and combination blockade afforded even greater protection (52.4% decrease, P <.01). Bronchoalveolar lavage leukocyte content was also reduced by treatment with anti-tumor necrosis factor alpha, anti-interleukin 1beta, and combination treatment. Reductions in permeability, myeloperoxidase, and bronchoalveolar lavage leukocyte content also resulted in a decrease in a histologic injury. Finally, anti-tumor necrosis factor alpha and anti-interleukin 1beta treatment resulted in decreased messenger RNA expression for a number of early response and regulatory cytokines., Conclusion: Tumor necrosis factor alpha and interleukin 1beta help regulate the development of lung ischemia-reperfusion injury. They appear to promote injury by altering expression of proinflammatory and anti-inflammatory cytokines and influencing tissue neutrophil recruitment.

Background: We have recently demonstrated a role for selected chemokines in a rat model of lung ischemia reperfusion injury (LIRI). We have further shown that pretreatment with cyclosporine A (CSA) is protective. The precise cellular events regulating this model are unknown. The alveolar macrophage (AM) is a key effector cell in multiple models of acute lung injury, and it likely plays a central role in LIRI as well. The present studies were undertaken to determine whether CSA functions in part by modifying the chemokine response of AMs to hypoxia and reoxygenation in vitro., Methods: Alveola macrophages were rendered hypoxic (0.5%) for 2 hours and reoxygenated for 6 hours. The secreted chemokine content in the media was quantified by enzyme-linked immunosorbent assay, and nuclear protein was analyzed after electro-mobility shift assay. When employed, CSA was administered 30 minutes before hypoxia., Results: Alveolar macrophages demonstrated a marked increase in the secretion of the chemokines, MIP-2, MIP-1alpha, CINC, and MCP-1, in response to hypoxia and reoxygenation. This increase was dependent on mRNA transcription and de novo protein synthesis. It was also blocked by a specific inhibitor of the nuclear translocation factor, NF-kappaB. Pretreatment with CSA (500 ng/mL) significantly reduced expression of chemokines and activation of NF-kappaB., Conclusions: Cyclosporine A attenuates the chemokine response of AMs in vitro to hypoxia and reoxygenation at the pretranscriptional level through modulation of NF-kappaB. These findings suggest the potential mechanism of action of CSA's protective effects in lung ischemia reperfusion injury.