Coagulation-mediated hypoxia and neutrophil-dependent hepatic injury in rats given lipopolysaccharide and ranitidine

Idiosyncrasy-like liver injury occurs in rats cotreated with nonhepatotoxic doses of ranitidine (RAN) and bacterial lipopolysaccharide (LPS). Hepatocellular oncotic necrosis is accompanied by neutrophil (PMN) accumulation and fibrin deposition in LPS/RAN-treated rats, but the contribution of PMNs to injury has not been shown. We tested the hypothesis that PMNs are critical mediators of LPS/RAN-induced liver injury and explored the potential for interaction between PMNs and hemostasis-induced hypoxia. Rats were given either LPS (44.4 x 10(6) endotoxin units/kg) or its vehicle and then RAN (30 mg/kg) or its vehicle 2 h later. They were killed 3 or 6 h after RAN treatment, and hepatocellular injury was estimated from serum alanine aminotransferase activity and liver histopathology. Plasma PMN chemokine concentration and the number of PMNs in liver increased after LPS treatment at 3 h and were not markedly altered by RAN cotreatment. Depletion of circulating PMNs attenuated hepatic PMN accumulation and liver injury and had no effect on coagulation system activation. Anticoagulation with heparin attenuated liver fibrin deposition and injury in LPS/RAN-treated rats; however, heparin had little effect on liver PMN accumulation or plasma chemokine concentration. Liver hypoxia occurred in LPS/RAN-cotreated rats and was significantly reduced by heparin. In vitro, hypoxia enhanced the killing of rat hepatocytes by PMN elastase and shortened its onset, indicating a synergistic interaction between PMNs and hypoxia. The results suggest that PMNs are involved in the hepatocellular injury caused by LPS/RAN-cotreatment and that hemostasis increases sensitivity to PMN-induced hepatocellular injury by causing liver hypoxia.     

Coagulation-dependent gene expression and liver injury in rats given lipopolysaccharide with ranitidine but not with famotidine

In an animal model of drug idiosyncrasy, rats cotreated with nonhepatotoxic doses of lipopolysaccharide (LPS) and ranitidine (RAN) develop hepatocellular injury, whereas rats treated with LPS and famotidine (FAM) do not. The coagulation system and neutrophils (PMNs) are requisite mediators of LPS/RAN-induced liver injury. We tested the hypothesis that unique gene expression in LPS/RAN-treated rats requires coagulation system activation and that these changes are absent in rats given LPS and FAM. Rats were treated with a nonhepatotoxic dose of LPS (44.4 x 10(6) endotoxin units/kg i.v.) or its vehicle, and then 1 h later, they were treated with heparin (3000 U/kg) or its vehicle. One hour thereafter, they were given RAN (30 mg/kg), FAM (6 mg/kg, a pharmacologically equiefficacious dose, or 28.8 mg/kg, an equimolar dose), or vehicle (i.v.). They were killed 2 or 6 h after drug treatment for evaluation of hepatotoxicity, coagulation system activation, and liver gene expression (2 h only). Statistical filtering of gene array results and real-time polymerase chain reaction identified groups of genes expressed in LPS/RAN-treated rats but not LPS/FAM-treated rats that were either changed or unchanged by heparin administration. For example, LPS/RAN-induced mRNA expression of the inflammatory mediators interleukin-6, cyclooxygenase-2, and macrophage inflammatory protein-2 (MIP-2) was reduced by anticoagulation. Enhancement of serum MIP-2 and plasminogen activator inhibitor-1 concentrations in LPS/RAN-treated rats was prevented by anticoagulation. The results suggest cross-talk between hemostasis-induced gene expression and inflammation (e.g., PMN function) in the genesis of hepatocellular injury in LPS/RAN-treated rats. In contrast, neither the expression of such genes nor hepatocellular necrosis occurred in rats treated with LPS/FAM.     

An antibody to neutrophils attenuates alpha-naphthylisothiocyanate-induced liver injury.

alpha-Naphthylisothiocyanate (ANIT) causes cholestasis and injury to bile duct epithelium and hepatic parenchymal cells in rats. The mechanism of toxicity is unknown. Neutrophils (PMNs) infiltrate periportal regions of the liver after ANIT intoxication. Because PMNs play a causal role in other extrahepatic models of tissue injury, we determined whether PMNs might be involved in ANIT-induced liver injury in rats by reducing circulating PMN numbers with a polyclonal antibody (antineutrophil serum). ANIT treatment caused cholestasis and elevations in serum of total bilirubin concentration, total bile acid concentration, aspartate amino-transferase activity, gamma-glutamyltransferase activity and histologic lesions consistent with acute, neutrophilic cholangiohepatitis. Cotreatment of rats with antineutrophil serum reduced circulating PMN numbers, prevented ANIT-induced cholestasis and attenuated other markers of liver injury elevated by ANIT. In addition, antineutrophil serum treatment attenuated the severity of histologic lesions within the liver and reduced the number of PMNs in periportal regions. Numbers of PMNs in liver sections correlated positively with markers of liver injury, histologic evidence of cholangiohepatitis and numbers of circulating PMNs in peripheral blood. The protection afforded by antineutrophil serum appeared to result from a specific reduction of PMNs and not lymphocytes, because administration of an antilymphocyte serum reduced circulating lymphocyte numbers without offering protection. Inasmuch as ANIT stimulates PMNs in vitro to release O2- and since PMN-derived oxygen species may cause tissue injury, we determined whether administration of agents which degrade oxygen radicals afforded protection against the liver injury caused by ANIT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombin and protease-activated receptor-1 agonists promote lipopolysaccharide-induced hepatocellular injury in perfused livers

Bacterial lipopolysaccharide (LPS) is a potent inflammatory agent capable of producing liver injury, the pathogenesis of which depends on numerous mediators, including thrombin. Previous studies showed that thrombin promotes LPS-induced liver injury independent of its ability to form fibrin clots. In isolated, buffer-perfused livers from LPS-treated rats, thrombin added to the perfusion buffer caused dose-dependent liver injury with an EC(50) value of 0.4 nM, consistent with activation by thrombin of a protease-activated receptor (PAR). Actions of thrombin at PARs can be mimicked by thrombin receptor-activating peptides (TRAPs). TRAPs for PAR-1 reproduced the injury caused by thrombin in isolated livers, suggesting that one mechanism by which thrombin promotes LPS-induced liver injury is by activating PAR-1. Immunocytochemistry demonstrated the presence of PAR-1 on sinusoidal endothelial cells and Kupffer cells but not on parenchymal cells or neutrophils. Previous studies showed that thrombin interacts with neutrophils in the genesis of liver injury after LPS treatment. To explore this interaction further, the influence of thrombin on mediators that modulate neutrophil function were evaluated. Inhibition of thrombin in LPS-treated rats prevented liver injury but did not prevent up-regulation of cytokine-induced neutrophil chemoattractant-1, macrophage inflammatory protein-2, or intercellular adhesion molecule-1. Thrombin inhibition did, however, prevent neutrophil (PMN) degranulation in vivo as measured by plasma elastase levels. In addition, elastase concentration was increased in the perfusion medium of livers isolated from LPS-treated rats and perfused with TRAPs. These results suggest that activation of PAR-1 after LPS exposure promotes PMN activation and hepatic parenchymal cell injury.     

Pulmonary leukostasis and the inhibition of airway neutrophil recruitment are early events in the endotoxemic rat

Neutrophil (polymorphonuclear leukocyte [PMN]) migration into pulmonary airspaces is a prerequisite for clearance of bacteria commonly found in nosocomial pneumonia. Patients at risk for nosocomial pneumonia often experience endotoxemia, and neutrophil dysfunction is associated with endotoxemia in both humans and animals. Using a rodent model of endotoxemia-associated pneumonia, we characterized the altered kinetics of pulmonary PMN trafficking and addressed the roles of platelets, tumor necrosis factor (TNF), and products of complement activation as potential mediators in the modulation of PMN migratory function. In male Sprague-Dawley rats made endotoxemic with intravenously (i.v.) administered endotoxin (lipopolysaccharide [LPS]), recruitment of PMNs into the lung airspaces in response to intratracheally (i.t.) instilled LPS was inhibited. In animals given IT LPS alone (0.5 mg/rat), numbers of airway PMNs were significantly elevated by 2 h, and immunohistochemical evaluation revealed PMNs in alveolar airspaces, alveolar walls, and in interstitium surrounding large airways. LPS (2 mg/kg i.v.) caused neutropenia and pulmonary PMN sequestration within 15 min of administration. Inhibition of airway PMN accumulation occurred by 30 min and lasted for at least 6 h after i.v. LPS. Factors present or activated after 30 min of endotoxemia were hypothesized to mediate the inhibitory effect of i.v. LPS. We found that pretreatment of rats with cobra venom factor to deplete complement (and C5a production) or immunodepletion of platelets or TNF did not affect the ability of i.v. LPS to inhibit pulmonary PMN recruitment or to cause pulmonary leukostasis. In summary, our results show that the inhibitory effects of i.v. LPS on PMN trafficking are rapid and persist for several hours and suggest that neither TNF, C5a, nor platelets are sufficient to mediate the inhibitory response.     

Metformin prevents endotoxin-induced liver injury after partial hepatectomy

Metformin [2-(N,N-dimethylcarbamimidoyl)guanidine] is a drug used in the treatment of type 2 diabetes. Recent studies have suggested that metformin may have effects in addition to lowering serum glucose concentrations (e.g., anti-inflammatory). The aim of the present study was to determine whether metformin prevents the inflammatory reaction and liver damage in a model of postsurgical sepsis. Accordingly, rats underwent 2/3 partial hepatectomy (PH; or sham surgery); 48 h after surgery, animals were administered endotoxin (LPS; 1.5 mg/kg i.v.). Both PH and LPS alone caused some minor liver damage. However, their combined effect (PH/LPS) was synergistic, leading to robust hepatic damage, as indicated by plasma enzymes and histological assessment. Although metformin treatment did not alter changes caused by PH alone, it almost completely blunted the effects of LPS in the PH/LPS group. Increases in biomarkers of inflammation (e.g., interleukin 6, interferon gamma, and neutrophil number) were also blunted by metformin treatment. Furthermore, PH/LPS caused a >200x increase in hepatic plasminogen activator inhibitor 1 (PAI-1) mRNA expression and plasma PAI-1 protein. These increases were associated with inhibition of hepatic urokinase plasminogen activator activity and an increase in fibrin deposition, indicative of local thrombosis. These effects were markedly reduced by metformin treatment. In conclusion, these data demonstrate that metformin prevents liver damage in a model of postsurgical sepsis in rats by decreasing proinflammatory and hemostatic responses.     

Modest inflammation enhances diclofenac hepatotoxicity in rats: role of neutrophils and bacterial translocation

Idiosyncratic adverse drug reactions (IADRs) represent an important human health problem, yet animal models for preclinical prediction of these reactions are lacking. Recent evidence in animals suggests that some IADRs arise from drug interaction with an inflammatory episode that renders the liver sensitive to injury. Diclofenac (DCLF) is one of those drugs for which the clinical use is limited by idiosyncratic liver injury. We tested the hypothesis that modest inflammation triggered in rats by a small dose of lipopolysaccharide (LPS) renders a nonhepatotoxic dose of DCLF injurious to liver. Cotreatment of rats with nonhepatotoxic doses of LPS and DCLF resulted in elevated serum alanine aminotransferase activity and liver histopathologic changes 6 h after DCLF administration. Neither LPS nor DCLF alone had such an effect. Gene array analysis of livers revealed a unique gene expression pattern in the LPS/DCLF-cotreated group compared with groups given either agent alone. Antiserum-induced neutrophil (PMN) depletion in LPS/DCLF-cotreated rats protected against liver injury, demonstrating a role for PMNs in the pathogenesis of this LPS/DCLF interaction. Gut sterilization of LPS/DCLF-treated rats did not protect against liver injury. In contrast, gut sterilization did attenuate liver injury caused by a large, hepatotoxic dose of DCLF, suggesting that hepatotoxicity induced by large doses of DCLF is caused in part by its ability to increase intestinal permeability to endotoxin or other bacterial products. These results demonstrate that inflammation-DCLF interaction precipitates hepatotoxicity in rats and raise the possibility of creating animal models that predict human IADRs.     

纤维蛋白止血敷料的止血效果

目的评价纤维蛋白止血敷料对股动脉切口和肝脏损伤出血模型的止血效果。方法制造大鼠股动脉切口出血模型和兔肝脏损伤出血模型,分别用纤维蛋白止血敷料和医用胶原蛋白海绵进行止血,记录止血时间和出血量。结果对大鼠股动脉切口模型,纤维蛋白止血敷料止血时间为(90.60±33.12)s,出血量为(0.51±0.26)ml,医用胶原蛋白海绵止血时间为(164.20±53.70)s,出血量为(1.04±0.50)ml,二者差异有统计学意义(P<0.05);对兔肝损伤模型,纤维蛋白止血敷料止血时间为(48.67±8.14)s,出血量为(0.82±0.09)ml,医用胶原蛋白海绵止血时间为(107.67±6.66)s,出血量为(1.07±0.13)ml,二者差异有统计学意义(P<0.01)。结论纤维蛋白止血敷料的止血效果优于医用胶原蛋白海绵,是一种良好的止血材料。     

大鼠内毒素血症时外周血及肺泡内中性粒细胞死亡方式及呼吸爆发

本研究观察大鼠内毒素血症时肺组织中及外周血多形核中性粒细胞（PMN）凋亡，坏死及功能改变的差异。采用Wistar大鼠20只。腹腔注射LPS（O55B5，5mg/kg)造成内毒素血症，给予LPS后2，4，8，12小时（每组5只动物）取血及支气管肺泡灌洗，密度梯度法分离PMN，用流式细胞仪测定凋亡和坏死比例以及呼吸爆发功能的改变，同时采用5只大鼠作为正常对照。结果显示，内毒素血症时外周血和支气管肺泡灌洗液中PMN凋亡细胞比例相似。但与对照相比，外周血PMN坏死比例明显增加，呼吸爆发能力明显受抑，而支气管肺泡灌洗液PMN坏死比例显减少，呼吸爆发能力显增强。结论：在内毒素血症时，扣押于肺组织中的PMN在凋亡和坏死上表现出与比例显减少，呼吸爆发能力显增强，结论：在内毒素血症时，扣押于肺组织中的PMN在凋亡和坏死上表现出与外周血PMN不同的改变，其结果是组织中PMN存活增加，并持续处于活化状态，这与PMN造成组织损伤有关。     

Ciprofloxacin transport by chemoattractant-activated polymorphonuclear leukocytes: regulation by priming and protein kinase C

At infection sites, polymorphonuclear leukocyte (PMN) function is enhanced ("primed") by granulocyte-macrophage colony-stimulating factor (GM-CSF) or lipopolysaccharide (LPS) and activated by formyl peptides. In this study, GM-CSF or LPS alone had no significant effects on PMN ciprofloxacin transport. Through a mechanism involving protein kinase C, activation by formyl-Met-Leu-Phe (fMLP) significantly decreased the K(m) of ciprofloxacin transport and enhanced ciprofloxacin accumulation. This effect was dramatically enhanced when PMNs were primed with GM-CSF or LPS prior to activation by fMLP.     

Tranexamic acid-loaded starch hemostatic microspheres

Efficacious hemostatics have significant potential for use in rapid exsanguinating hemorrhage control by emergency medical technicians or military medics nowadays. Current hemostatics focus primarily on speeding up the formation of blood clots, but inhibiting fibrinolysis is also critical for promoting coagulation and improving survival rates. Here we report a drug-loaded cross-linked microporous starch (TACMS) fabricated by loading tranexamic acid (TA) with antifibrinolytic properties into cross-linked microporous starch (CMS). The results showed that the cross-linking modification improved the mechanical properties and the particle density. The introduction of TA had no influence on water absorption of CMS. TACMS retained good physical hemostatic capacity and excellent biocompatibility. The prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) of TACMS with 20 mg g⁻¹ of TA were shortened greatly, indicating the chemical hemostasis of TACMS. TACMS demonstrated a 70% reduction in clotting time in vitro compared to CMS, which effectively inhibited the dissolution of fibrin and increased the strength of blood clots. Importantly, TACMS presented excellent hemostatic performance in rabbit ear artery injury and rabbit liver injury and even better hemostatic ability than Arista®. In conclusion, cross-linking, enzyme hydrolysis and modification of starch greatly improved absorption speed, blood uptake capacity and mechanical strength, and the introduction of TA simultaneously amplified the physical hemostasis and inhibited the dissolution of fibrin. The potent hemostatic ability of TACMS resulted from the synergistic role of physical hemostasis and drug hemostasis. The results of the present study put forward TACMS as a safe and effective hemostatic system and present a platform for further optimization studies of materials with enhanced hemostatic capabilities for specific injury types.

Efficacious hemostatics have significant potential for use in rapid exsanguinating hemorrhage control by emergency medical technicians or military medics nowadays.     

TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADPH oxidase

Interactions of polymorphonuclear neutrophils (PMNs) with endothelial cells may contribute to the activation of endothelial cell responses involved in innate immunity. We explored a novel function of PMN NADPH oxidase in the mechanism of Toll-like receptor-2 (TLR2) upregulation induced by LPS-TLR4 signaling in endothelial cells. We showed that LPS induced TLR2 up-regulation through TLR4- and MyD88-dependent signaling. In neutropenic mice, the LPS-induced NF-kB activation and TLR2 expression were significantly reduced, and both responses were restored upon repletion by PMN obtained from WT mice but not by PMNs from NADPH oxidase gp91pho(-/-) mice. These findings were recapitulated in mouse lung vascular endothelial cells cocultured with PMNs, indicating that the augmented NF-kB activation and the resultant TLR2 upregulation in endothelial cells were secondary to oxidant signaling generated by PMN NADPH oxidase. The functional relevance of NADPH oxidase in mediating TLR4-induced TLR2 expression in endothelial cells was evident by markedly elevated and stable ICAM-1 expression as well as augmented PMN migration in response to sequential challenge with LPS and peptidoglycan. Thus, PMN NADPH oxidase-derived oxidant signaling is an important determinant of the cross talk between TLR4 and TLR2 and the control of endothelial cell activation.     

Microarray analysis of lipopolysaccharide potentiation of trovafloxacin-induced liver injury in rats suggests a role for proinflammatory chemokines and neutrophils

Idiosyncratic drug toxicity refers to toxic reactions occurring in a small subset of patients and usually cannot be predicted during preclinical or early phases of clinical trials. One hypothesis for the pathogenesis of hepatic idiosyncratic drug reactions is that, in certain individuals, underlying inflammation results in sensitization of the liver, such that injury occurs from an agent that typically would not cause hepatotoxicity at a therapeutic dose. We explored this possibility by cotreating rats with nonhepatotoxic doses of bacterial lipopolysaccharide (LPS) and trovafloxacin (TVX), a drug that caused idiosyncratic hepatotoxicity in humans. The combination of LPS and TVX resulted in hepatotoxicity in rats, as determined by increases in serum alanine aminotransferase activity and hepatocellular necrosis, which were not observed with either agent alone. In contrast, treatment with LPS and levofloxacin, a fluoroquinolone without human idiosyncratic liability, did not result in these changes. Liver gene expression analysis identified unique changes induced by the combination of TVX and LPS, including enhanced expression of chemokines, suggestive of liver neutrophil (PMN) accumulation and activation. Consistent with a role for PMN in the hepatotoxicity induced by LPS/TVX, prior depletion of PMN attenuated the liver injury. The results suggest that gene expression profiles predictive of idiosyncratic liability can be generated in rats cotreated with LPS and drug. Furthermore, they identify gene expression changes that could be explored as biomarkers for idiosyncratic toxicity and lead to enhanced understanding of the mechanism(s) underlying hepatotoxicity induced by TVX.     

Proinflammatory effects of bacterial lipoprotein on human neutrophil activation status, function and cytotoxic potential in vitro

Bacterial lipoprotein (BLP) is the most abundant protein in gram-negative bacterial cell walls, heavily outweighing lipopolysaccharide (LPS). Herein we present findings demonstrating the potent in vitro effects of BLP on neutrophil (PMN) activation status, function, and capacity to transmigrate an endothelial monolayer. PMNs are the principal effectors of the initial host response to injury or infection and constitute a significant threat to invading bacterial pathogens. The systemic inflammatory response syndrome (SIRS) is characterised by significant host tissue injury mediated, in part, by uncontrolled regulation of PMN cytotoxic activity. We found that BLP-activated human PMN as evidenced by increased CD11b/CD18 (Mac-1) expression. Up-regulation of PMN Mac-1 in response to BLP occurred independently of membrane-bound CD14 (mCD14). A similar up-regulation of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells was observed whilst E-Selectin expression was unaffected. PMN transmigration across a human umbilical vein endothelial cell (HUVEC) monolayer was markedly increased after treating either PMN's or HUVEC independently with BLP. This increased transmigration did not occur as a result of any direct effect of BLP on HUVEC monolayer permeability, assessed objectively using the passage of FITC-labeled Dextran-70. BLP primed PMN for enhanced respiratory burst and superoxide anion production in response to PMA, but did not influence phagocytosis of opsonized Escherichia coli. BLP far exceeds LPS as a gram-negative bacterial wall component, these findings therefore implicate BLP as an additional putative mediator of SIRS arising from gram-negative infection.     

Potential role of recombinant factor VIIa as a hemostatic agent

Recombinant factor VIIa (rFVIIa) has been shown to induce hemostasis in hemophilia patients with inhibitors against factor VIII or factor IX independent of factor VIII/factor IX. Factor VIIa binds to tissue factor (TF) exposed at the site of injury and generates, through factor X activation on the TF-bearing cells, enough thrombin to activate factors VIII, V, and XI, as well as platelets. The thrombin-activated platelets provided a perfect template for binding of activated factors VIII, IX, and V, further activation of factor X, and thrombin generation. Factor VIIa in high concentrations binds to thrombin-activated platelets and is capable of activating factor X, thereby generating thrombin independent of the presence of factor VIII or factor IX. Accordingly, rFVIIa has been shown to initiate hemostasis in severe hemophilila patients with inhibitors subjected to major surgery and suffering from serious limb- and life-threatening bleeding. Since rFVIIa enhances thrombin generation-thereby providing the formation of tight, stable fibrin hemostatic plugs resistance to premature lysis-it should be hemostatic in other situations characterized by impaired thrombin generation. A hemostatic effect has been reported in patients with various platelet disorders and factor XI deficiency. Further, a hemostatic effect of rFVIIa has been reported in patients subjected to trauma and extensive surgery who have developed profuse, excessive bleeding resulting in hemodilution and changes in coagulation patterns. rFVIIa was developed to treat bleeding in hemophilia patients with inhibitors against factor VIII or factor IX and has been shown to induce effective hemostasis in most such patients and also in life- and limb-threatening bleeding. It has also been used successfully to stop bleeding in patients who do not have hemophilia but who do have acquired antibodies against factor VIII (acquired hemophilia). rFVIIa initiates hemostasis by forming a complex with TF exposed as a result of vessel wall injury. Pharmacologic doses of rFVIIa can enhance thrombin generation on platelets that are already thrombin-activated, resulting in the formation of full thrombin burst. By enhancing thrombin generation, rFVIIa helps to form tight, stable, fibrin plugs resistant to premature fibrinolysis. This also maintains hemostasis in the absence of factor VIII or factor IX. Pharmacologic doses of rFVIIa may accordingly be of benefit in producing hemostasis in situations other than hemophilia characterized by profuse bleeding and impaired thrombin generation. There is now clinical experience indicating a hemostatic effect in patients with thrombocytopenia and functional platelet defects. rFVIIa has also been successfully used in acute trauma patients with profuse bleedings and in other bleeding situations.     

Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice

Mice deleted for the plasminogen activator inhibitor-1 (PAI-1) gene are relatively protected from developing pulmonary fibrosis induced by bleomycin. We hypothesized that PAI-1 deficiency reduces fibrosis by promoting plasminogen activation and accelerating the clearance of fibrin matrices that accumulate within the damaged lung. In support of this hypothesis, we found that the lungs of PAI-1(-/-) mice accumulated less fibrin after injury than wild-type mice, due in part to enhanced fibrinolytic activity. To further substantiate the importance of fibrin removal as the mechanism by which PAI-1 deficiency limited bleomycin-induced fibrosis, bleomycin was administered to mice deficient in the gene for the Aalpha-chain of fibrinogen (fib). Contrary to our expectation, fib(-/-) mice developed pulmonary fibrosis to a degree similar to fib(+/-) littermate controls, which have a plasma fibrinogen level that is 70% of that of wild-type mice. Although elimination of fibrin from the lung was not in itself protective, the beneficial effect of PAI-1 deficiency was still associated with proteolytic activity of the plasminogen activation system. In particular, inhibition of plasmin activation and/or activity by tranexamic acid reversed both the accelerated fibrin clearance and the protective effect of PAI-1 deficiency. We conclude that protection from fibrosis by PAI-1 deficiency is dependent upon increased proteolytic activity of the plasminogen activation system; however, complete removal of fibrin is not sufficient to protect the lung.     

Cyclooxygenase inhibition in lungs or in neutrophils attenuates neutrophil-dependent edema in rat lungs perfused with phorbol myristate acetate

Results from previous studies indicate that injury in isolated rat lungs perfused with buffer containing phorbol myristate acetate (PMA) and rat neutrophils (PMNs) is dependent on the production of reactive oxygen species and thromboxane (Tx) A2. The purpose of this study was to determine whether the lung or the PMN was the source of TxA2 required to produce lung injury in this model. Prostanoid synthesis by rat lungs or PMNs was inhibited selectively by pretreatment of either rats or isolated PMNs with aspirin (100 mg/kg p.o. or 100 microM, respectively). Unbound aspirin was removed from the lungs and PMNs before use in experiments. Lungs from vehicle-pretreated rats that were perfused with PMA and untreated PMNs exhibited increases in weight, lavage fluid albumin content and TxB2 production with respect to lungs perfused with PMA but no PMNs. Increases in these markers were prevented when cyclooxygenase from either the lungs or the PMNs was inhibited. These results indicate that TxA2 is produced by both PMNs and by lung cells in this preparation, and that TxA2 production by both of these sources is required for the manifestation of edema.     

Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung

In models of acute lung injury, CXC chemokine receptor 2 (CXCR2) mediates migration of polymorphonuclear leukocytes (PMNs) into the lung. Since CXCR2 ligands, including CXCL1 and CXCL2/3, are chemotactic for PMNs, CXCR2 is thought to recruit PMNs by inducing chemotactic migration. In a model of PMN recruitment to the lung, aerosolized bacterial LPS inhalation induced PMN recruitment to the lung in wild-type mice, but not in littermate CXCR2-/- mice. Surprisingly, lethally irradiated wild-type mice reconstituted with CXCR2-/- BM still showed about 50% PMN recruitment into bronchoalveolar lavage fluid and into lung interstitium, but CXCR2-/- mice reconstituted with CXCR2-/- BM showed no PMN recruitment. Conversely, CXCR2-/- mice reconstituted with wild-type BM showed a surprisingly large defect in PMN recruitment, inconsistent with a role of CXCR2 on PMNs alone. Cell culture, immunohistochemistry, flow cytometry, and real-time RT-PCR were used to show expression of CXCR2 on pulmonary endothelial and bronchial epithelial cells. The LPS-induced increase in lung microvascular permeability as measured by Evans blue extravasation required CXCR2 on nonhematopoietic cells. Our data revealed what we believe to be a previously unrecognized role of endothelial and epithelial CXCR2 in LPS-induced PMN recruitment and lung injury.