Lung Leak Research Articles

The Role of Cyclooxygenase-2 in Mechanical Ventilation–Induced Lung Injury

Mechanical ventilation is necessary for patients with acute respiratory failure, but can cause or propagate lung injury. We previously identified cyclooxygenase-2 as a candidate gene in mechanical ventilation-induced lung injury. Our objective was to determine the role of cyclooxygenase-2 in mechanical ventilation-induced lung injury and the effects of cyclooxygenase-2 inhibition on lung inflammation and barrier disruption. Mice were mechanically ventilated at low and high tidal volumes, in the presence or absence of pharmacologic cyclooxygenase-2-specific inhibition with 3-(4-methylsulphonylphenyl)-4-phenyl-5-trifluoromethylisoxazole (CAY10404). Lung injury was assessed using markers of alveolar-capillary leakage and lung inflammation. Cyclooxygenase-2 expression and activity were measured by Western blotting, real-time PCR, and lung/plasma prostanoid analysis, and tissue sections were analyzed for cyclooxygenase-2 staining by immunohistochemistry. High tidal volume ventilation induced lung injury, significantly increasing both lung leakage and lung inflammation relative to control and low tidal volume ventilation. High tidal volume mechanical ventilation significantly induced cyclooxygenase-2 expression and activity, both in the lungs and systemically, compared with control mice and low tidal volume mice. The immunohistochemical analysis of lung sections localized cyclooxygenase-2 expression to monocytes and macrophages in the alveoli. The pharmacologic inhibition of cyclooxygenase-2 with CAY10404 significantly decreased cyclooxygenase activity and attenuated lung injury in mice ventilated at high tidal volume, attenuating barrier disruption, tissue inflammation, and inflammatory cell signaling. This study demonstrates the induction of cyclooxygenase-2 by mechanical ventilation, and suggests that the therapeutic inhibition of cyclooxygenase-2 may attenuate ventilator-induced acute lung injury.

Role of microRNA in 3,3-diindolylmethane (DIM)-mediated amelioration of SEB-induced acute lung injury (173.6)

Abstract Staphylococcal enterotoxin B (SEB) is a CDC select agent of bioterrorism and a public health threat during nosocomial infections. A deleterious effect of SEB exposure is acute lung injury (ALI) and acute respiratory distress syndrome characterized by pulmonary infiltration. 3,3-diindolylmethane (DIM) found in cruciferous vegetables, has been shown to have anti-inflammatory properties. We studied the mechanism underlying the ameliorative effects of DIM on SEB-induced ALI. Mice were exposed to SEB [50 mg; intranasal] and administered DIM [100 mg/kg] via oral gavage. Forty-eight hours after SEB exposure, serum, lung tissue and lung infiltrating cells were collected. DIM treatment led to a decrease in lymphocytic infiltration, specifically CD4+, CD8+ and Vβ8+ T cells as well as NKT cells. DIM also led to a reduction of fluid leak in lungs. DIM [10, 25, 50 µM] induced apoptosis in splenocytes and CD3+ T cells treated in vitro with SEB in a dose-dependent manner. DIM treatment led to downregulation in microRNA, miR-706 and miR-494 that target pro-apoptotic genes, and upregulation of miR-22 and miR-34a targeting anti-apoptotic genes in lung-infiltrating cells. Thus, studies are underway to examine whether in T cells, DIM treatment leads to modulation in the expression of genes involved in apoptosis. Together, these studies demonstrate that DIM treatment may suppress SEB-mediated ALI by induction of apoptosis in T cells via alteration in expression of miRNA.

NFkB signaling and inducible nitric oxide synthase activity during pulmonary ischemia‐reperfusion increase co‐localization of fibrinogen/fibrin and platelets at sites of vascular leakage in rabbit lung

Platelet adhesion and fibrinogen/fibrin (Fg/Fb) deposition cause endothelial dysfunction during acute lung injury. We examined the hypothesis that during pulmonary IR, platelet activation, recruitment of Fg/Fb to the vascular wall and subsequent leakage are associated with NFκB and inducible nitric oxide synthase (iNOS) activation pathways. Subpleural microvessels and alveoli were examined by video microscopy of the right lung in anesthetized rabbits with open chest and ventilated lungs. IR was caused by occluding the right pulmonary artery for 2 h. Injection of fluorescently labeled albumin and a polyclonal antibody to Fg/Fb into the pulmonary circulation via the right atrium during early reperfusion revealed areas of microvascular leakage and interstitial edema associated with Fg/Fb deposition. Platelets were detected by immunohistochemistry of tissue samples at sites of vascular leakage in conjunction with Fg/Fb. On average, IR (n=3) caused a 6–7 fold increase in albumin leakage, platelet adhesion, and albumin‐Fg/Fb‐platelet co‐localization compared to control. Additionally, IR increased NF‐κB activation in lung lysates as detected by phosphorylation of IκB. These effects were attenuated by treatment with an iNOS activity inhibitor (1400W) before IR. Our data suggest that NFκB and iNOS activation may play a critical role in the regulation of platelet Fg/Fb–induced microvascular leakage during pulmonary IR. Supported in part by American Lung Association‐Kentucky to AMR and NIH grant #HL080394 to DL.

흰쥐에서 내독소로 유도된 급성 폐손상에서 moxofloxacin의 개선효과

흰쥐에서 내독소로 유도된 급성 폐손상에서 moxifloxacin의 효과를 형태학적으로 호중구의 폐장 내 침윤 및 세포질형 포스포리파아제(cytosolic phospholipase A2) 발현과 관련하여 알아보았다. 내독소는 잘 알려진대로 흰쥐에서 급성 폐손상을 유발하였고 이때 폐장 내의 호중구의 침윤 및 폐장 및 호중구에서의 세포질형 포스포리파아제의 발현이 증가하였고, 또한 염증성 사이토카인의 일종인 종양괴사성인자(tumor necrosis factor)의 발현도 증가하였다. 전자현미경을 이용한 산소기 생성의 검사에서도 내독소에 의하여 산소기의 생성이 폐장 내에 증가한 사실을 확인하였고, 산화성 스트레스에 유발되는 제2형 폐포세포에서의 층상체의 변화도 관찰하였다. 면역변환효과가 있다고 알려진 moxifloxacin은 이러한 변화들을 억제하고 형태학적으로 내독소에 의한 급성 폐손상을 현저히 감소시켰다. The immunomodulating effects of moxifloxacin seem to be effective in downregulating inflammatory reactions. This presumed effect was tested in endotoxin (ETX)-induced acute lung injury (ALI) in rats. After moxifloxacin treatment (10 mg/kg) of ETX-given rats, lung myeloperoxidase (MPO) activity, bronchoalveolar-lavage (BAL) protein, and the number of neutrophils in the BAL cells were measured. Light and electron microscopic structures were also examined. Electron microscopic <TEX>$CeCl_3$</TEX> histochemistry for the detection of hydrogen peroxide in the lungs and immunohistochemistry of cytosolic phospholipase A2 (cPLA2) in the lung tissues and BAL cells were performed. To examine the expression of TNF<TEX>${\alpha}$</TEX> in the lungs, western blotting was carried out with the lung tissues. ETX had accumulated neutrophils in the lungs, which was followed by lung leak. Oxidative stress occurred, and increased expression of cPLA2 in the lung tissues and BAL cells was observed in the ETX-given rats. Simultaneously, the expression of TNF<TEX>${\alpha}$</TEX> was enhanced by ETX. Moxifloxacin, however, decreased all these parameters, indicating that ALI may have been ameliorated. Moxifloxacin appears to ameliorate ETX-induced ALI partially through the suppression of cPLA2 in the lungs of rats.

TIMAP protects endothelial barrier from LPS-induced vascular leakage and is down-regulated by LPS

TIMAP is a regulatory subunit of protein phosphatase 1, whose role remains largely unknown. Our recent data suggested that TIMAP is involved in the regulation of barrier function in cultured pulmonary endothelial monolayers [Csortos et al., 2008. Am. J. Physiol. Lung Cell. Mol. Physiol. 295, L440-L450]. Here we showed that TIMAP depletion exacerbates lipopolysaccharide (LPS)-induced vascular leakage in murine lung, suggesting that TIMAP has a barrier-protective role in vivo. Real-Time RT PCR analysis revealed that treatment with LPS significantly suppressed Timap mRNA level. This suppression was not achieved via the down-regulation of Timap promoter activity, suggesting that LPS decreased Timap mRNA stability. Pretreatment with protein kinase A (PKA) inhibitor H-89 reduced TIMAP mRNA level, whereas pretreatment with PKA activator, bnz-cAMP, increased this level and attenuated LPS-induced decrease in TIMAP mRNA. Altogether, these data confirmed the barrier-protective role of TIMAP and suggested that barrier-disruptive and barrier-protective agents may employ modulation of TIMAP expression as a mechanism affecting barrier permeability.

Exudate macrophages attenuate lung injury by the release of IL-1 receptor antagonist in gram-negative pneumonia

Exudate macrophages are key players in host defense towards invading pathogens. Their anti-inflammatory and epithelial-protective potential in gram-negative pneumonia, however, remains elusive. Objective: We investigated whether exudate macrophages contributed to preservation of alveolar epithelial barrier integrity and analyzed the molecular pathways involved. Methods: We evaluated the anti-inflammatory and epithelial-protective effects of exudate macrophages in a model of LPS- and K. pneumoniae-induced lung injury comparing wild-type and CCR2 (CC-chemokine receptor 2)-deficient mice with defective lung macrophage recruitment and in in vitro studies using primary alveolar epithelial cells. CCR2–/– mice exhibited enhanced alveolar epithelial cell apoptosis and lung leakage upon intratracheal LPS treatment which could be attributed to lack of exudate macrophage recruitment from the circulating pool as demonstrated in a model of wild-type/CCR2–/– bone-marrow chimeric mice. Among various anti-inflammatory and proliferative mediators analysed, the endogenous counterpart of resident macrophage-expressed interleukin-1β (IL-1β), interleukin-1 receptor antagonist (IL-1ra), was highly upregulated in flow-sorted exudate macrophages in LPS-treated wild-type mice. LPS/IL-1β-induced impairment of alveolar epithelial cell integrity was antagonized by IL-1ra in vitro. Finally, intratracheal substitution of IL-1ra or intravenous adoptive transfer of IL-1ra+/+ but not IL-1ra–/– blood mononuclear cells attenuated alveolar inflammation, epithelial apoptosis, and loss of barrier function in LPS-challenged or K. pneumoniae-infected CCR2–/– mice and enhanced survival after K. pneumoniae infection. We conclude that recruited lung macrophages attenuate IL-1β-mediated acute lung injury in gram-negative pneumonia by release of IL-1ra.

Factor VIIa Bound to Endothelial Cell Protein C Receptor Activates Protease Activated Receptor 1-Mediated Cell Signaling and Barrier-Protective Response In Endothelial Cells

AbstractAbstract 346Endothelial cell protein C receptor (EPCR) is the cellular receptor for protein C and activated protein C (APC). In addition to controlling coagulation by modulating the protein C-mediated anticoagulant pathway, EPCR has been shown to play a critical role in supporting APC-induced cell signaling, which could be responsible for some of the non-hemostatic functions of EPCR and APC. Recent studies from our laboratory and others have shown that factor VIIa (FVIIa), a coagulation factor whose primary function is to initiate tissue factor (TF)-dependent coagulation, also binds to EPCR on endothelium. At present, the physiological significance of this interaction is unclear. APC binding to EPCR has been shown to provide cytoprotective effects via protease activated receptor (PAR) 1-mediated cell signaling. In earlier studies using exogenously expressed PAR1 and PAR2 reporter constructs in a heterologus cell model system, we were unable to find measurable n-terminal cleavage (activation) of PARs by FVIIa bound to EPCR. It is possible that transfected PAR constructs may segregate differently on the cell surface membrane than that of endogenous PARs, and thus may have decreased susceptibility for cleavage by FVIIa-EPCR. In the present study, we have investigated whether FVIIa, upon binding to EPCR on endothelial cells, activates endogenous PAR1 and induces PAR1-mediated cell signaling. To determine whether FVIIa cleaves endogenously expressed PAR1 on endothelial cells, unperturbed cultures of human umbilical vein endothelial cells (HUVEC) were exposed to varying concentrations of FVIIa (0-40 nM) and the cleavage of PAR1 at the cell surface was measured quantitatively in a cell-surface ELISA using a cleavage-specific PAR1 monoclonal antibody. The data show that FVIIa, in a dose- and time-dependent manner, cleaves PAR1 on endothelial cells. FVIIa cleavage of PAR1 on endothelial cells is dependent on FVIIa binding to EPCR, as prevention of FVIIa binding to EPCR by pretreating HUVEC with EPCR polyclonal antibody completely abolished FVIIa cleavage of PAR1. Similarly, silencing EPCR with EPCR-specific siRNA fully attenuated FVIIa cleavage of PAR1. FVIIa cleavage of PAR1 on endothelial cells is independent of TF as pretreatment of HUVEC with anti-TF antibodies or transduction of HUVEC with adenovirus encoding TF had no significant effect on FVIIa cleavage of PAR1. The efficiency of PAR1 cleavage by FVIIa appears to be comparable to that of APC, as both at 10 nM cleave PAR1 to a similar extent. FVIIa (10 nM) cleaves only a fraction of PAR1 (∼25 to 30%) on endothelial cell surface; increasing either FVIIa concentration or duration of treatment has not resulted in additional cleavage of remaining PAR1. Low expression of PAR2 in endothelial cells and lack of cleavage specific antibodies to PAR2 prevented us from determining whether FVII bound to EPCR also cleaves PAR2. FVIIa (10 nM) induced p44/42 MAPK activation in HUVEC and this activation was dependent on EPCR and PAR1 but not PAR2, as silencing EPCR or PAR1 but not PAR2 attenuated FVIIa-induced p44/42 MAPK phosphorylation. In additional studies, FVIIa (10 nM) was found to elicit protection against thrombin-induced barrier disruption in endothelial cells as analyzed in a dual-chamber system using Evans blue-labeled BSA or measurements of transendothelial electrical resistance. FVIIa-induced barrier-protective effect is EPCR-dependent. F-actin staining of HUVEC exposed to thrombin showed formation of transcellular actin stress fibers, cellular contractions and paracellular gap formation. Pretreatment of HUVEC with FVIIa maintained actin at the cell periphery, and reduced formation of central stress fibers and paracellular gaps. FVIIa-induced p44/42 MAPK activation and barrier protective effect are mediated via Rac1, as specific inhibitors against Rac1 or transduction of Rac1 dominant negative mutant abolished these FVIIa-induced effects. Consistent with in vitro findings, in vivo studies in mice showed that administration of FVIIa prior to LPS attenuated the LPS-induced vascular leakage in lung and kidney. Overall, our present data provide strong and convincing evidence that FVIIa bound to EPCR on endothelial cells activates PAR1-mediated cell signaling and provides a barrier protective effect. These findings are novel and assume a great clinical significance as FVIIa is used prophylactically for prevention of bleeding in hemophiliacs. Disclosures:No relevant conflicts of interest to declare.

Interleukin-1으로 유도된 급성폐손상에서 rutin의 효과

흰쥐에서 Interleukin-1 (IL-1)으로 유도된 급성폐손상에서의 group II phospholipase <TEX>$A_2$</TEX> (<TEX>$PLA_2$</TEX>) 억제제인 rutin의 효과를 알아보기 위하여 본 연구를 시행하였다. Rutin은 IL-1에 의해 증가한 폐장내의 myeloperoxidase의 활성도를 감소시키지는 못하였으나 폐포세척액 내의 호중구의 수 및 모세혈관의 손상지표로 알려져 있는 폐장 모세혈관에서의 단백질 누출양을 감소시켰다. 동시에 rutin은 IL-1에 의하여 증가한 폐장의 염증조절효소인 <TEX>$PLA_2$</TEX>의 활성도를 감소시키고 결과적으로 호중구에서의 산소기의 생성을 감소시켰다. Rutin 뿐만 아니라 manoalide, scalaradial 같은 group II <TEX>$PLA_2$</TEX>의 억제제도 호중구의 respiratory burst를 감소시킴을 확인하였다. IL-1에 의하여 증가한 폐포세척액 내에서의 cytokine induced neutrophil chemoattractant의 농도는 rutin에 의해 영향을 받지 않았다. 형태학적으로는 IL-1에 의한 폐장조직에서의 산소기의 형성이 관찰되었고 rutin은 이러한 산소기의 생성을 현저히 감소시켰다. 이러한 결과로 미루어 group II <TEX>$PLA_2$</TEX> 억제제인 rutin은 호중구에서의 활성 산소기의 생성을 효과적으로 억제함으로써 IL-1에 의한 급성폐손상의 감소를 가져 오는 것으로 결론지을 수 있다. Rutin, a group II phospholipase <TEX>$A_2$</TEX> (<TEX>$PLA_2$</TEX>) inhibitor, was tested on interleukin-1 (IL-1) induced acute lung injury (ALI) in male Sprague-Dawley rats. Rutin did not alter the increased lung myeloperoxidase activity by IL-1. However, the number of neutrophils in bronchoalveolar lavage fluid (BALF) and IL-1 induced lung leak were decreased by rutin (p<0.001). Simultaneously, rutin decreased lung <TEX>$PLA_2$</TEX> activity, which was increased by IL-1 (p<0.001). The reduction of neutrophilic respiratory burst by the inhibition of <TEX>$PLA_2$</TEX> was confirmed by group II <TEX>$PLA_2$</TEX> inhibitors such as rutin, manoalide and scalaradial. The increased level of cytokine-induced neutrophilic chemoattractant (CINC) in BALF by IL-1 was not affected by rutin. Ultrastructural changes of ALI and increased generation of free radicals in the lung by IL-1 were found, and rutin ameliorated these pathological findings. Taken together, rutin seems to be effective in decreasing IL-1 induced ALI through inhibition of group II <TEX>$PLA_2$</TEX>.

Protective role of PI3-kinase/Akt/eNOS signaling in mechanical stress through inhibition of p38 mitogen-activated protein kinase in mouse lung

To test the hypothesis that PI3K/Akt/eNOS signaling has a protective role in a murine model of ventilation associated lung injury (VALI) through down-regulation of p38 MAPK signaling. Male C57BL/J6 (wild-type, WT) or eNOS knockout mice (eNOS(-/-)) were exposed to mechanical ventilation (MV) with low (LV(T), 7 mL/kg) and high tidal volume (HV(T), 20 mL/kg) for 0-4 h. A subset of WT mice was administered the specific inhibitors of PI3K (100 nmol/L Wortmannin [Wort], ip) or of p38 MAPK (SB203580, 2 mg/kg, ip) 1 h before MV. Cultured type II alveolar epithelial cells C10 were exposed to 18% cyclic stretch for 2 h with or without 20 nmol/L Wort pretreatment. At the end of the experiment, the capillary leakage in vivo was assessed by extravasation of Evans blue dye (EBD), wet/dry weight ratio and lung lavage protein concentration. The lung tissue and cell lysate were also collected for protein and histological review. MV decreased PI3K/Akt phosphorylation and eNOS expression but increased phospho-p38 MAPK expression along with a lung leakage of EBD. Inhibitions of phospho-Akt by Wort worsen the lung edema, whereas inhibition of p38 MAPK kinase restored activation of Akt together with alleviated capillary leakage. eNOS(-/-) mice showed an exacerbated lung edema and injury. The stretched C10 cells demonstrated that Wort diminished the activation of Akt, but potentiated phosphorylation of MAPK p38. Our results indicate that PI-3K/Akt/eNOS pathway has significant protective effects in VALI by preventing capillary leakage, and that there is a cross-talk between PI3K/Akt and p38 MAPK pathways in vascular barrier dysfunction resulting from VALI.

Evaluation of the effects of ozone therapy in the treatment of intra-abdominal infection in rats

INTRODUCTIONThe antibacterial effect of ozone (O3) has been described in the extant literature, but the role of O3 therapy in the treatment of certain types of infection remains controversial.OBJECTIVESTo evaluate the effect of intraperitoneal (i.p.) O3 application in a cecal ligation/puncture rat model on interleukins (IL-6, IL-10) and cytokine-induced neutrophil chemoattractant (CINC)-1 serum levels, acute lung injury and survival rates.METHODSFour animal groups were used for the study: a) the SHAM group underwent laparotomy; b) the cecal ligation/puncture group underwent cecal ligation/puncture procedures; and c) the CLP+O2 and CLP+O3 groups underwent CLP+ corresponding gas mixture infusions (i.p.) throughout the observation period. IL-6, CINC-1 and IL-10 concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Acute lung injury was evaluated with the Evans blue dye lung leakage method and by lung histology. P<0.05 was considered significant.RESULTSCINC-1 was at the lowest level in the SHAM group and was lower for the CLP+O3 group vs. the CLP+O2 group and the cecal ligation/puncture group. IL-10 was lower for the SHAM group vs. the other three groups, which were similar compared to each other. IL-6 was lower for the SHAM group vs. all other groups, was lower for the CLP+O3 or CLP+O2 group vs. the cecal ligation/puncture group, and was similar for the CLP+O3 group vs. the CLP+O2 group. The lung histology score was lower for the SHAM group vs. the other groups. The Evans blue dye result was lower for the CLP+O3 group vs. the CLP+O2 group and the cecal ligation/puncture group but similar to that of the SHAM group. The survival rate for the CLP+O3 group was lower than for the SHAM group and similar to that for the other 2 groups (CLP and CLP+O2).CONCLUSIONOzone therapy modulated the inflammatory response and acute lung injury in the cecal ligation/puncture infection model in rats, although there was no improvement on survival rates.

Mechanisms of nitric oxide synthase uncoupling in endotoxin-induced acute lung injury: Role of asymmetric dimethylarginine

Acute lung injury (ALI) is associated with severe alterations in lung structure and function and is characterized by hypoxemia, pulmonary edema, low lung compliance and widespread capillary leakage. Asymmetric dimethylarginine (ADMA), a known cardiovascular risk factor, has been linked to endothelial dysfunction and the pathogenesis of a number of cardiovascular diseases. However, the role of ADMA in the pathogenesis of ALI is less clear. ADMA is metabolized via hydrolytic degradation to l-citrulline and dimethylamine by the enzyme, dimethylarginine dimethylaminohydrolase (DDAH). Recent studies suggest that lipopolysaccharide (LPS) markedly increases the level of ADMA and decreases DDAH activity in endothelial cells. Thus, the purpose of this study was to determine if alterations in the ADMA/DDAH pathway contribute to the development of ALI initiated by LPS-exposure in mice. Our data demonstrate that LPS exposure significantly increases ADMA levels and this correlates with a decrease in DDAH activity but not protein levels of either DDAH I or DDAH II isoforms. Further, we found that the increase in ADMA levels cause an early decrease in nitric oxide (NO x ) and a significant increase in both NO synthase (NOS)-derived superoxide and total nitrated lung proteins. Finally, we found that decreasing peroxynitrite levels with either uric acid or Manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTymPyp) significantly attenuated the lung leak associated with LPS-exposure in mice suggesting a key role for protein nitration in the progression of ALI. In conclusion, this is the first study that suggests a role of the ADMA/DDAH pathway during the development of ALI in mice and that ADMA may be a novel therapeutic biomarker to ascertain the risk for development of ALI.

Efficacy Profiles of Daptomycin for Treatment of Invasive and Noninvasive Pulmonary Infections with Streptococcus pneumoniae

Daptomycin is a novel lipopeptide antibiotic with excellent activity against Gram-positive bacterial pathogens, but its therapeutic value for the treatment of invasive pneumococcal disease compared to that for the treatment of pneumococcal pneumonia is incompletely defined. We investigated the efficacy of daptomycin in two models of Streptococcus pneumoniae-induced lung infection, i.e., pneumococcal pneumonia and septic pneumococcal disease. Mice were infected with a bioluminescent, invasive serotype 2 S. pneumoniae strain or a less virulent serotype 19 S. pneumoniae strain and were then given semitherapeutic or therapeutic daptomycin or ceftriaxone. Readouts included survival; bacterial loads; and septic disease progression, as determined by biophotonic imaging. Semitherapeutic daptomycin treatment fully protected the mice against the progression of septic disease induced by serotype 2 S. pneumoniae, while therapeutic treatment of the mice with daptomycin or ceftriaxone led to approximately 70% or approximately 60% survival, respectively. In contrast, mice infected with serotype 19 S. pneumoniae developed severe pneumonia and lung leakage even in the presence of increased intra-alveolar daptomycin levels, resulting in only 40% survival, whereas the ceftriaxone-treated mice had 100% survival. Together, although daptomycin demonstrates little efficacy in the treatment of pneumococcal pneumonia, daptomycin is highly effective in preventing S. pneumoniae-induced septic death, thus possibly offering a therapeutic option for patients with life-threatening septic pneumococcal disease.

High mobility group box 1 as a mediator of endotoxin administration after hemorrhagic shock-primed lung injury

High mobility group box 1 (HMGB1) was discovered as a novel late-acting cytokine that contributes to acute lung injury (ALI). However, the contribution of HMGB1 to two-hit-induced ALI has not been investigated. To examine the participation of HMGB1 in the pathogenesis of ALI caused by the two-hit hypothesis, endotoxin was injected intratracheally in a hemorrhagic shock-primed ALI mouse model. Concentrations of HMGB1 in the lung of the shock group were markedly increased at 16 h (1.63 +/- 0.05, compared to the control group: 1.02 +/- 0.03; P < 0.05), with the highest concentration being observed at 24 h. In the sham/lipopolysaccharide group, lung HMGB1 concentrations were found to be markedly increased at 24 h (1.98 +/- 0.08, compared to the control group: 1.07 +/- 0.03; P < 0.05). Administration of lipopolysaccharide to the hemorrhagic shock group resulted in a notable HMGB1 increase by 4 h, with a further increase by 16 h. Intratracheal lipopolysaccharide injection after hemorrhagic shock resulted in the highest lung leak at 16 h (2.68 +/- 0.08, compared to the control group: 1.05 +/- 0.04; P < 0.05). Compared to the hemorrhagic shock/lipopolysaccharide mice, blockade of HMGB1 at the same time as lipopolysaccharide injection prevented significantly pulmonary tumor necrosis factor-alpha, interleukin-1beta and myeloperoxidase. Lung leak was also markedly reduced at 16 h; blockade of HMGB1 24 h after lipopolysaccharide injection failed to alter lung leak or myeloperoxidase at 48 h. Our observations suggest that HMGB1 plays a key role as a late mediator when lipopolysaccharide is injected after hemorrhagic shock-primed ALI and the kinetics of its release differs from that of one-hit ALI. The therapeutic window to suppress HMGB1 activity should not be delayed to 24 h after the disease onset.

Kidney ischemia-reperfusion injury induces caspase-dependent pulmonary apoptosis

Distant organ effects of acute kidney injury (AKI) are a leading cause of morbidity and mortality. While little is known about the underlying mechanisms, limited data suggest a role for inflammation and apoptosis. Utilizing a lung candidate gene discovery approach in a mouse model of ischemic AKI-induced lung dysfunction, we identified prominent lung activation of 66 apoptosis-related genes at 6 and/or 36 h following ischemia, of which 6 genes represent the tumor necrosis factor receptor (TNFR) superfamily, and another 23 genes are associated with the TNFR pathway. Given that pulmonary apoptosis is an important pathogenic mechanism of acute lung injury (ALI), we hypothesized that AKI leads to pulmonary proapoptotic pathways that facilitate lung injury and inflammation. Functional correlation with 1) terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and 2) active caspase-3 (aC3) activity, immunoblotting, and immunohistochemistry (IHC) identified kidney IRI-induced pulmonary apoptosis at 24 h, and colocalization studies with CD34 identified predominantly endothelial apoptosis. Mice were treated with the caspase inhibitor Z-VAD-FMK (0.25 mg ip) or vehicle 1 h before and 8 h after sham or kidney IRI, and bronchoalveolar lavage fluid protein was measured at 36 h as a surrogate for lung leak. Caspase inhibition reduced lung microvascular changes after kidney IRI. The pulmonary apoptosis seen in wild-type control mice during AKI was absent in TNFR(-/-) mice. Using an initial genomic approach to discovery followed by a mechanistic approach to disease targeting, we demonstrate that pulmonary endothelial apoptosis is a direct mediator of the distant organ dysfunction during experimental AKI.

TLR4 is Essential in Acute Lung Injury Induced by Unresuscitated Hemorrhagic Shock

Acute lung injury (ALI) and acute respiratory distress syndrome in patients with hemorrhagic shock (HS) or resuscitation is associated with the expression of TLR4. However, the role of TLR4 in ALI induced by unresuscitated HS remains obscure. The lung pathologic change was observed by hematoxylin and eosin staining. Interleukin-1beta and tumor necrosis factor-alpha were analyzed by enzyme-linked immunosorbent assay. Polymorphonuclear leukocyte sequestration and lung leak were analyzed by pulmonary myeloperoxidase activity and Evans blue dye. The expressions of TLR4 mRNA and protein were analyzed by reverse transcription-polymerase chain reaction and Western blot, respectively. TLR4 distribution was analyzed by immunohistochemistry. Lung neutrophil accumulation and microvascular permeability were significantly increased after unresuscitated HS, meanwhile, lung interleukin-1beta and tumor necrosis factor-alpha were gradually augmented. TLR4 mRNA, TLR4 distribution and TLR4 protein were also significantly increased in TLR4 wt mice, however, no above-mentioned changes appeared in TLR4 mutant mice. TLR4 is strongly associated with the pathogenesis of ALI induced by unresuscitated HS, which may serve as a useful therapeutic target.

Lung epithelial apoptosis in influenza virus pneumonia: the role of macrophage-expressed TNF-related apoptosis-inducing ligand

Mononuclear phagocytes have been attributed a crucial role in the host defense toward influenza virus (IV), but their contribution to influenza-induced lung failure is incompletely understood. We demonstrate for the first time that lung-recruited “exudate” macrophages significantly contribute to alveolar epithelial cell (AEC) apoptosis by the release of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in a murine model of influenza-induced pneumonia. Using CC-chemokine receptor 2–deficient (CCR2−/−) mice characterized by defective inflammatory macrophage recruitment, and blocking anti-CCR2 antibodies, we show that exudate macrophage accumulation in the lungs of influenza-infected mice is associated with pronounced AEC apoptosis and increased lung leakage and mortality. Among several proapoptotic mediators analyzed, TRAIL messenger RNA was found to be markedly up-regulated in alveolar exudate macrophages as compared with peripheral blood monocytes. Moreover, among the different alveolar-recruited leukocyte subsets, TRAIL protein was predominantly expressed on macrophages. Finally, abrogation of TRAIL signaling in exudate macrophages resulted in significantly reduced AEC apoptosis, attenuated lung leakage, and increased survival upon IV infection. Collectively, these findings demonstrate a key role for exudate macrophages in the induction of alveolar leakage and mortality in IV pneumonia. Epithelial cell apoptosis induced by TRAIL-expressing macrophages is identified as a major underlying mechanism.

Role of Oxidative Stress and NFkB in Hypoxia-Induced Pulmonary Edema

Hypoxia is well known to increase the free radical generation in the body, leading to oxidative stress. In the present study, we have determined whether the increased oxidative stress further upregulates the nuclear transcription factor (NFkB) in the development of pulmonary edema. The rats were exposed to hypobaric hypoxia at 7620 m (280 mm Hg) for different durations, that is, 3 hrs, 6 hrs, 12 hrs, and 24 hrs at 25+/-1 degrees C. The results revealed that exposure of animals to hypobaric hypoxia led to a significant increase in vascular leakage, with time up to 6 hrs (256.38+/-61 rfu/g) as compared with control (143.63+/-60.1 rfu/g). There was a significant increase in reactive oxygen species, lipid peroxidation, and superoxide dismutase levels, with a concurrent decrease in lung glutathione peroxidase activity. There was 13-fold increase in the expression of NFkB level in nuclear fraction of lung homogenates of hypoxic animals over control rats. The DNA binding activity of NFkB was found to be increased significantly (P<0.001) in the lungs of rats exposed to hypoxia as compared with control. Further, we observed a significant increase in proinflammatory cytokines such as IL-1, IL-6, and TNF-alpha with concomitant upregulation of cell adhesion molecules such as ICAM-I, VCAM-I, and P-selectin in the lung of rats exposed to hypoxia as compared with control. Interestingly, pretreatment of animals with curcumin (NFkB blocker) attenuated hypoxia-induced vascular leakage in lungs with concomitant reduction of NFkB levels. The present study therefore reveals the possible involvement of NFkB in the development of pulmonary edema.

Gut Ischemia/Reperfusion Induced Acute Lung Injury is an Alveolar Macrophage Dependent Event

Although the role of the lung alveolar macrophage (AM) as a mediator of acute lung injury (ALI) after lung ischemia/reperfusion (I/R) has been suggested by animal experiments, it has not been determined whether AMs mediate ALI after intestinal I/R. The objective of this study was to determine the effect of AM elimination on ALI after intestinal I/R in rats. Male Wistar rats (n = 90) were randomly divided into three groups: the clodronate-liposomes (CLOD-LIP) group received intratracheal treatment with CLOD-LIP; the liposomes (LIP) group received intratracheal treatment with LIP; and the nontreated (UNTREAT) group received no treatment. Twenty-four hours later each group was randomly divided into three subgroups: the intestinal I/R subgroup was subjected to 45-minute intestinal ischemia and 2-hour reperfusion; the laparotomy (LAP) subgroup was subjected to LAP and sham procedures; the control (CTR) subgroup received no treatment. At the end of reperfusion, ALI was quantitated in all the animals by the Evans blue dye (EBD) method. ALI values are expressed as EBD lung leakage (microg EBD/g dry lung weight). EBD lung leakage values in the CLOD-LIP group were 32.59 +/- 12.74 for I/R, 27.74 +/- 7.99 for LAP, and 33.52 +/- 10.17 for CTR. In the LIP group, lung leakage values were 58.02 +/- 18.04 for I/R, 31.90 +/- 8.72 for LAP, and 27.17 +/- 11.48 for CTR. In the UNTREAT group, lung leakage values were 55.60 +/- 10.96 for I/R, 35.99 +/- 6.89 for LAP, and 30.83 +/- 8.41 for CTR. Within each group, LAP values did not differ from CTR values. However, in the LIP and UNTREAT groups, values for both the LAP and CTR subgroups were lower than values for the I/R subgroup (p < 0.001). The CLOD-LIP I/R subgroup value was less (p < 0.001) than the I/R subgroup values in the LIP and UNTREAT groups. These results indicated that I/R provokes ALI that can be prevented by CLOD-LIP treatment, and further suggested that AMs are essential for ALI occurrence induced by intestinal I/R in rats.

Low Level Laser Therapy (LLLT) Decreases Pulmonary Microvascular Leakage, Neutrophil Influx and IL-1β Levels in Airway and Lung from Rat Subjected to LPS-Induced Inflammation

Low level laser therapy (LLLT) is a known anti-inflammatory therapy. Herein we studied the effect of LLLT on lung permeability and the IL-1beta level in LPS-induced pulmonary inflammation. STUDY DESIGN/METHODOLOGY: Rats were divided into 12 groups (n = 7 for each group). Lung permeability was measured by quantifying extravasated albumin concentration in lung homogenate, inflammatory cells influx was determined by myeloperoxidase activity, IL-1beta in BAL was determined by ELISA and IL-1beta mRNA expression in trachea was evaluated by RT-PCR. The rats were irradiated on the skin over the upper bronchus at the site of tracheotomy after LPS. LLLT attenuated lung permeability. In addition, there was reduced neutrophil influx, myeloperoxidase activity and both IL-1beta in BAL and IL-1beta mRNA expression in trachea obtained from animals subjected to LPS-induced inflammation. LLLT reduced the lung permeability by a mechanism in which the IL-1beta seems to have an important role.

Cytosolic Phospholipase A2 Activity in Neutrophilic Oxidative Stress of Platelet-activating Factor-induced Acute Lung Injury

Background: The present investigation was performed in rats and isolated human neutrophils in order to confirm the presumptive role of the positive feedback loop of cytosolic phospholipase () activation by plateletactivating factor (PAF). Methods: The possible formation of the positive feedback loop of the activation and neutrophilic respiratory burst was investigated in vivo and in vitro by measurement of the parameters denoting acute lung injury. In addition, morphological examinations and electron microscopic cytochemistry were performed for the detection of free radicals in the lung. Results: Five hours after intratracheal instillation of PAF (), the lung leak index, lung myeloperoxidase (MPO) activity, the number of neutrophils and the concentration of cytokine-induced neutrophil chemoattractant (CINC) in bronchoalveolar lavage fluid were increased by PAF as compared with those of control rats. The NBT assay and cytochrome-c reduction assay revealed an increased neutrophilic respiratory burst in isolated human neutrophils following exposure to PAF. Lung and neutrophilic activity were increased following PAF exposure and exposure to hydrogen peroxide increased activity in the lung. Histologically, inflammatory findings of the lung were observed after PAF treatment. Remarkably, as determined by cytochemical electron microscopy, increased production of hydrogen peroxide was identified in the lung after PAF treatment. Conclusion: PAF mediates acute oxidative lung injury by the activation of , which may provoke the generation of free radicals in neutrophils.