Fas and fas ligand are up-regulated in pulmonary edema fluid and lung tissue of patients with acute lung injury and the acute respiratory distress syndrome

Apoptosis mediated by Fas/Fas ligand (FasL) interaction has been implicated in human disease processes, including pulmonary disorders. However, the role of the Fas/FasL system in acute lung injury (ALI) and in the acute respiratory distress syndrome (ARDS) is poorly defined. Accordingly, we investigated both the soluble and cellular expression of the Fas/FasL system in patients with ALI or ARDS. The major findings are summarized as follows. First, the soluble expression of the Fas/FasL system was assessed in undiluted pulmonary edema fluid and simultaneous plasma. Pulmonary edema fluid obtained from patients with ALI or ARDS (n = 51) had significantly higher concentrations of both soluble Fas (27 ng/ml; median; P < 0.05) and soluble FasL (0.125 ng/ml; P < 0.05) compared to control patients with hydrostatic pulmonary edema (n = 40; soluble Fas, 12 ng/ml; soluble FasL, 0.080 ng/ml). In addition, the concentrations of both soluble Fas and soluble FasL were significantly higher in the pulmonary edema fluid of the patients with ALI or ARDS compared to simultaneous plasma samples (soluble Fas, 16 ng/ml; soluble FasL, 0.058 ng/ml; P < 0.05), indicating local release in the lung. Higher soluble Fas concentrations were associated with worse clinical outcomes. Second, cellular expression of the Fas/FasL system was assessed by semiquantitative immunofluorescence microscopy in lung tissue obtained at autopsy from a different set of patients. Both Fas and FasL were immunolocalized to a greater extent in the patients who died with ALI or ARDS (n = 10) than in the patients who died without pulmonary disease (n = 10). Both proteins were co-expressed by epithelial cells that lined the alveolar walls, as well as by inflammatory cells and sloughed epithelial cells that were located in the air spaces. Semiquantitative immunohistochemistry showed that markers of apoptosis (terminal dUTP nick-end labeling, caspase-3, Bax, and p53) were more prevalent in alveolar wall cells from the patients who died with ALI or ARDS compared to the patients who died without pulmonary disease. These findings indicate that alveolar epithelial injury in humans with ALI or ARDS is in part associated with local up-regulation of the Fas/FasL system and activation of the apoptotic cascade in the epithelial cells that line the alveolar air spaces.     

Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis

Apoptosis and inflammation play an important role in the pathogenesis of direct/pulmonary acute lung injury (ALI). However, the role of the Fas receptor-driven apoptotic pathway in indirect/nonpulmonary ALI is virtually unstudied. We hypothesized that if Fas or caspase-8 plays a role in the induction of indirect ALI, their local silencing using small interfering RNA (siRNA) should be protective in hemorrhage-induced septic ALI. Initially, as a proof of principle, green fluorescent protein-siRNA was administered intratracheally into transgenic mice overexpressing green fluorescent protein. Twenty-four hours after siRNA delivery, lung sections revealed a significant decrease in green fluorescence. Intratracheally administered Cy-5-labeled Fas-siRNA localized primarily in pulmonary epithelial cells. Intratracheal instillation of siRNA did not induce lung inflammation via toll-like receptor or protein kinase PKR pathways as assessed by lung tissue interferon-alpha, tumor necrosis factor-alpha, and interleukin (IL)-6 levels. Mice subjected to hemorrhagic shock and sepsis received either Fas-, caspase-8-, or control-siRNA intratracheally 4 hours after hemorrhage. Fas- or caspase-8-siRNA significantly reduced lung tissue Fas or caspase-8 mRNA, respectively. Only Fas-siRNA markedly diminished lung tissue tumor necrosis factor-alpha, IL-6, IL-10, interferon-gamma, IL-12, and caspase-3 activity. Fas-siRNA also preserved alveolar architecture and reduced lung neutrophil infiltration and pulmonary epithelial apoptosis. These data indicate the pathophysiological significance of Fas activation in nonpulmonary/shock-induced ALI and the feasibility of intrapulmonary administration of anti-apoptotic siRNA in vivo.     

牛磺酸对大鼠肢体缺血再灌注后肺损伤时细胞凋亡的影响

目的:从细胞凋亡的角度探讨肢体缺血再灌注(LIR)后急性肺损伤(ALI)的发病机制及牛磺酸的影响。方法:复制大鼠肢体缺血再灌注(LIR)损伤动物模型,采用TUNEL法、电泳法、半定量逆转录聚合酶链反应(SqRT-PCR)及免疫组织化学等技术观察LIR后肺损伤发生过程中,肺泡上皮及血管内皮细胞凋亡变化以及Fas/FasL系统蛋白质和mRNA表达的改变。结果:大鼠LIR后,肺泡上皮细胞和肺血管内皮细胞凋亡明显增加;肺组织Fas/FasLmRNA和蛋白质表达明显上调,DNA断链率、组织钙含量和活性氧(ROS)升高,且与肺泡上皮及血管内皮细胞凋亡的增加相一致。结论:肺泡上皮及血管内皮细胞凋亡以及Fas/FasL系统表达明显上调可能参与LIR后ALI的发生;牛磺酸可减少肺组织细胞凋亡,但并非通过影响Fas/FasL基因表达而实现其保护效应。     

Opposing effect by cytokines on Fas-mediated apoptosis in A549 lung epithelial cells.

Tissue repair is determined by many signals provided in the local environment. Central to this process is the commitment of the parenchymal cell to undergo apoptosis, survive, or proliferate following inflammation. We hypothesize that lung epithelial cell apoptosis is influenced by exposure to cytokines released into the alveolar microenvironment during the inflammatory process. In this investigation we demonstrate that interferon (IFN)-gamma and interleukin (IL)-1beta have opposing effects on Fas-mediated apoptosis in A549 cells, a human lung epithelial cell line. Exposure to IFN-gamma before Fas activation significantly increased caspase activity, caspase processing of CK-18, a key cytoskeletal protein in epithelial cells, and increased the appearance of apoptotic nuclei. Induction of Fas-mediated death by IFN-gamma was 3-fold higher than with Fas activation alone. In contrast, pretreatment with IL-1beta before Fas activation completely inhibited apoptosis. Furthermore, our results demonstrate that IFN-gamma and IL-1beta induce opposite effects at multiple checkpoints during Fas-mediated apoptosis. Most striking, IL-1beta prevented the activation of caspases involved in Fas-mediated death by inducing an anti-apoptotic effect proximal to or at the point of caspase-8 activation. Finally, our investigation demonstrates that the differential impact of IL-1beta and IFN-gamma on Fas-mediated apoptosis are in part dependent on modulation of the PI 3-K/Akt survival pathway.     

Induction of interleukin-8 secretion and apoptosis in bronchiolar epithelial cells by Fas ligation.

Epithelial cell injury is the common manifestation of lung injury. Contributing to such injury of epithelial cells is apoptosis. Although apoptosis is part of the normal process of epithelial renewal, in excess it is pathologic. We previously demonstrated the excessive apoptosis of lung epithelial cells and the upregulation of Fas and Fas ligand (FasL) in fibrosing lung diseases. We also showed that inhalation of anti-Fas antibody induced lung injury and fibrosis in mice. Interleukin (IL)-8 is one of the most important cytokines in the pathophysiology of acute lung injury and pulmonary fibrosis. In this study we investigated whether Fas ligation induces IL-8 secretion in addition to apoptosis in bronchiolar epithelial cells in vitro. Bronchiolar epithelial cells underwent apoptosis and also secreted IL-8 in response to tumor necrosis factor (TNF)-alpha or Fas ligation. New gene expression and protein synthesis were not necessary for Fas ligation- and TNF-alpha- mediated apoptosis, but were necessary for IL-8 secretion. We further found that Fas ligation induced activation of nuclear factor-kappa B. We conclude that the Fas/FasL pathway not only mediates apoptosis but also plays a proinflammatory role, and that stimulation of the Fas/FasL pathway in bronchiolar epithelial cells leads to IL-8 production, which may amplify the inflammatory cascade in lung injury and pulmonary fibrosis.     

NOX1 is responsible for cell death through STAT3 activation in hyperoxia and is associated with the pathogenesis of Acute Respiratory Distress Syndrome

Reactive oxygen species (ROS) contribute to alveolar cell death in Acute Respiratory Distress Syndrome (ARDS) and we previously demonstrated that NOX1-derived ROS contributed to hyperoxia-induced alveolar cell death in mice. The study investigates whether NOX1 expression is modulated in epithelial cells concomitantly to cell death and associated to STAT3 signaling in the exudative phase of ARDS. In addition, the role of STAT3 activation in NOX1-dependent epithelial cell death was confirmed by using a lung epithelial cell line and in mice exposed to hyperoxia. NOX1 expression, cell death and STAT3 staining were evaluated in the lungs of control and ARDS patients by immunohistochemistry. In parallel, a stable NOX1-silenced murine epithelial cell line (MLE12) and NOX1-deficient mice were used to characterize signalling pathways. In the present study, we show that NOX1 is detected in alveolar epithelial cells of ARDS patients in the exudative stage. In addition, increased alveolar epithelial cell death and phosphorylated STAT3 are observed in ARDS patients and associated with NOX1 expression. Phosphorylated STAT3 is also correlated with TUNEL staining. We also confirmed that NOX1-dependent STAT3 activation participates to alveolar epithelial cell death. Silencing and acute inhibition of NOX1 in MLE12 led to decreased cell death and cleaved-caspase 3 induced by hyperoxia. Additionally, hyperoxia-induced STAT3 phosphorylation is dependent on NOX1 expression and associated with cell death in MLE12 and mice. This study demonstrates that NOX1 is involved in human ARDS pathophysiology and is responsible for the damage occurring in alveolar epithelial cells at least in part via STAT3 signalling pathways.     

Regulation of inflammation and apoptosis by GPR43 via JNK/ELK1 in acute lung injury

Acute lung injury (ALI) is mostly relevant to acute and severe lung inflammation. We first utilized lipopolysaccharide (LPS) to induce mice for establishing a mouse model of ALI and detected decreased expression of GPR43 in the lung tissue in mice with ALI. Mice expressing increased GPR43 showed improvement in lung injury compared to LPS-treated mice. Additionally, ALI mice transfected with lenti-GPR43 significantly decreased the mRNA levels of TNF-α, IL-1β, IL-6, MPO, COX2 and iNOS, and apoptosis levels in the lungs, as well as the phosphorylation levels of JNK and ELK1 compared to LPS-treated mice with lenti-vector infection. Subsequently, we employed LPS to induce alveolar type ii epithelial cells and observed that Ov-GPR43 infection markedly reduced the expression levels of inflammatory factors and apoptosis levels, while exposure of cells to anisomycin was also effective in blunting the effects of Ov-GPR43 on these processes. Taken together, these results demonstrate a role of GPR43 in mediating lung injury through JNK/ELK1 and imply the therapeutic potential of targeting GPR43 against ALI.

     

Fas (CD95) induces alveolar epithelial cell apoptosis in vivo: implications for acute pulmonary inflammation

Activation of the Fas/FasL system induces apoptosis of susceptible cells, but may also lead to nuclear factor kappaB activation. Our goal was to determine whether local Fas activation produces acute lung injury by inducing alveolar epithelial cell apoptosis and by generating local inflammatory responses. Normal mice (C57BL/6) and mice deficient in Fas (lpr) were treated by intranasal instillation of the Fas-activating monoclonal antibody (mAb) Jo2 or an irrelevant control mAb, and studied 6 or 24 hours later using bronchoalveolar lavage (BAL), histopathology, DNA nick-end-labeling assays, and electron microscopy. Normal mice treated with mAb Jo2 had significant increases in BAL protein at 6 hours, and BAL neutrophils at 24 hours, as compared to lpr mice and to mice treated with the irrelevant mAb. Neutrophil recruitment was preceded by increased mRNA expression for tumor necrosis factor-alpha, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-2, macrophage chemotactic protein-1, and interleukin-6, but not interferon-gamma, transforming growth factor-ss, RANTES, eotaxin, or IP-10. Lung sections from Jo2-treated normal mice showed neutrophilic infiltrates, alveolar septal thickening, hemorrhage, and terminal dUTP nick-end-labeling-positive cells in the alveolar septae and airspaces. Type II pneumocyte apoptosis was confirmed by electron microscopy. Fas activation in vivo results in acute alveolar epithelial injury and lung inflammation, and may be important in the pathogenesis of acute lung injury.     

Human epidermal growth factor receptor signaling in acute lung injury

Acute lung injury (ALI) is a syndrome marked by increased permeability across the pulmonary epithelium resulting in pulmonary edema. Recent evidence suggests that members of the human epidermal growth factor receptor (HER) family are activated in alveolar epithelial cells during ALI and regulate alveolar epithelial barrier function. These tyrosine kinase receptors, which also participate in the pathophysiology of pulmonary epithelial malignancies, regulate cell growth, differentiation, and migration as well as cell-cell adhesion, all processes that influence epithelial injury and repair. In this review we outline mechanisms of epithelial injury and repair in ALI, activation patterns of this receptor family in pulmonary epithelial cells as a consequence injury, how receptor activation alters alveolar permeability, and the possible intracellular signaling pathways involved. Finally, we propose a theoretical model for how HER-mediated modulation of alveolar permeability might affect lung injury and repair. Understanding how these receptors signal has direct therapeutic implications in lung injury and other diseases characterized by altered epithelial barrier function.     

Association of endogenous G-CSF with anti-inflammatory mediators in patients with acute respiratory distress syndrome.

Upregulation of the anti-inflammatory mediators, soluble tumor necrosis factor-alpha receptors I and II (sTNFRI/RII) and interleukin-1 receptor antagonist (IL-1RA), by granulocyte colony-stimulating factor (G-CSF) may contribute to the pathophysiology of lung injury. We determined the relation of endogenous G-CSF to proinflammatory and anti-inflammatory mediators in bronchoalveolar lavage fluid (BALF) and serum of patients with acute respiratory distress syndrome (ARDS) and acute lung injury (ALI). Nineteen patients with ARDS and 10 with ALI were included in this prospective investigation. BAL was performed within 12 h and 24 h after onset of lung injury. Concentrations of G-CSF, TNF-alpha, IL-6, sTNFRI and sTNFRII, IL-1RA and IL-10 in BALF as well as in serum were determined by ELISA. G-CSF was associated with alveolar neutrophilia. Results in patients with ARDS and ALI exhibited significant positive correlations in BALF of G-CSF levels with that of IL-6, sTNFRII, and IL-1RA and of G-CSF levels in serum with that of serum IL-6, IL-1RA, and IL-10. Given the potential of G-CSF to directly induce anti-inflammatory cytokines in vitro, significant associations of endogenous G-CSF levels with these mediators early in the development of severe lung injury suggest an endogenous anti-inflammatory role of G-CSF in vivo.     

Dual role of TGF-β1 on Fas-induced apoptosis in lung epithelial cells

Recent evidence suggests that TGF-β1 has a dual role in regulating cell response to Fas/Fas ligand (FasL)-induced apoptosis. TGF-β1 may play a positive or negative role on cell sensitivity to apoptosis via Fas/FasL system, depending on cell types and their specific environment. TGF-β1 and the Fas/FasL system are also involved in pathological processes of acute lung injury (ALI) and interstitial lung diseases including early lung injury and subsequent tissue repair. However, it is not well understood how TGF-β1 regulates Fas/FasL mediated apoptotic signaling in lung epithelium. In this study, we found that TGF-β1 could affect the sensitivity of lung epithelial A549 cells to Fas/FasL mediated apoptosis in a time-dependent manner. Apoptosis of A549 cells could be enhanced significantly by co-treatment with TGF-β1 and FasL, or pretreatment with TGF-β1 followed by FasL exposure, as evidenced by markedly increased caspase-8 and JNK activities. However, prolonged exposure to TGF-β1 could result in an obvious inhibition of the Fas/FasL-induced apoptosis, accompanied by down-regulation of Fas and up-regulation of c-Flip. Our results also showed that the effect of TGF-β1 on cell sensitivity to Fas-mediated apoptosis was independent of Akt pathway activation. These findings suggest that timely interplay of TGF-β1 and the Fas/FasL system could determine the final outcomes of cell survival/death signaling, for example, switching cell death signaling to survival signaling during early injury and later repair process of lung epithelium.     

Key mechanisms governing resolution of lung inflammation

Innate immunity normally provides excellent defence against invading microorganisms. Acute inflammation is a form of innate immune defence and represents one of the primary responses to injury, infection and irritation, largely mediated by granulocyte effector cells such as neutrophils and eosinophils. Failure to remove an inflammatory stimulus (often resulting in failed resolution of inflammation) can lead to chronic inflammation resulting in tissue injury caused by high numbers of infiltrating activated granulocytes. Successful resolution of inflammation is dependent upon the removal of these cells. Under normal physiological conditions, apoptosis (programmed cell death) precedes phagocytic recognition and clearance of these cells by, for example, macrophages, dendritic and epithelial cells (a process known as efferocytosis). Inflammation contributes to immune defence within the respiratory mucosa (responsible for gas exchange) because lung epithelia are continuously exposed to a multiplicity of airborne pathogens, allergens and foreign particles. Failure to resolve inflammation within the respiratory mucosa is a major contributor of numerous lung diseases. This review will summarise the major mechanisms regulating lung inflammation, including key cellular interplays such as apoptotic cell clearance by alveolar macrophages and macrophage/neutrophil/epithelial cell interactions. The different acute and chronic inflammatory disease states caused by dysregulated/impaired resolution of lung inflammation will be discussed. Furthermore, the resolution of lung inflammation during neutrophil/eosinophil-dominant lung injury or enhanced resolution driven via pharmacological manipulation will also be considered.     

Acute lung injury: apoptosis in effector and target cells of the upper and lower airway compartment

Apoptotic cell death has been considered an underlying mechanism in acute lung injury. To evaluate the evidence of this process, apoptosis rate was determined in effector cells (alveolar macrophages, neutrophils) and target cells (tracheobronchial and alveolar epithelial cells) of the respiratory compartment upon exposure to hypoxia and endotoxin stimulation in vitro. Cells were exposed to 5% oxygen or incubated with lipopolysaccharide (LPS) for 4, 8 and 24 h, and activity of caspase‐3, ‐8 and ‐9 was determined. Caspase‐3 of alveolar macrophages was increased at all three time‐points upon LPS stimulation, while hypoxia did not affect apoptosis rate at early time‐points. In neutrophils, apoptosis was decreased in an early phase of hypoxia at 4 h. However, enhanced expression of caspase‐3 activity was seen at 8 and 24 h. In the presence of LPS a decreased apoptosis rate was observed at 8 h compared to controls, while it was increased at 24 h. Tracheobronchial as well as alveolar epithelial cells experienced an enhanced caspase‐3 activity upon LPS stimulation with no change of apoptosis rate under hypoxia. While increased apoptosis rate is triggered through an intrinsic and extrinsic pathway in alveolar macrophages, intrinsic signalling is activated in tracheobronchial epithelial cells. The exact pathway pattern in neutrophils and alveolar epithelial cells could not be determined. These data clearly demonstrate that upon injury each cell type experiences its own apoptosis pattern. Further experiments need to be performed to determine the functional role of these apoptotic processes in acute lung injury.     

Seawater induces apoptosis in alveolar epithelial cells via the Fas/FasL-mediated pathway

Our previous study showed that seawater can cause lung tissue cell apoptosis; in the present study, the immunohistochemistry and Western blot analysis results demonstrated that Fas, FasL, and cleaved caspase-8 and caspase-3 were up-regulated in the rat lungs exposed to seawater. We found that seawater-induced human lung alveolar epithelial A549 cell apoptosis was concentration and time dependent. Moreover, seawater increased the expression of Fas, FasL, and cleaved caspase-8 and caspase-3 in A549 cells. The incubation of A549 cells in the presence of FasL-neutralising antibody (NOK-2) or caspase-8 inhibitor (Z-IETD-FMK) resulted in a decrease of seawater-induced cell apoptosis. NOK-2 inhibited Fas/FasL interaction and reduced the cleavage of caspase-8 and caspase-3, and Z-IETD-FMK blocked caspase-8 and caspase-3 activation. Seawater similarly produced a significant increase in rat alveolar type II cell apoptosis and expression of Fas and cleaved caspase-8. In summary, the Fas/FasL pathway involved in alveolar epithelial cell (AEC) apoptosis could be important in the pathogenesis of seawater-induced acute lung injury (SW-ALI).     

Concise review: Mesenchymal stem cells for acute lung injury: role of paracrine soluble factors

Morbidity and mortality have declined only modestly in patients with clinical acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), despite extensive research into the pathophysiology. Current treatment remains primarily supportive with lung-protective ventilation and a fluid conservative strategy. Pharmacologic therapies that reduce the severity of lung injury in preclinical models have not yet been translated to effective clinical treatment options. Consequently, further research in translational therapies is needed. Cell-based therapy with mesenchymal stem cells (MSCs) is one attractive new therapeutic approach. MSCs have the capacity to secrete multiple paracrine factors that can regulate endothelial and epithelial permeability, decrease inflammation, enhance tissue repair, and inhibit bacterial growth. This review will focus on recent studies, which support the potential therapeutic use of MSCs in ALI/ARDS, with an emphasis on the role of paracrine soluble factors.     

IL-27 is Elevated in Acute Lung Injury and Mediates Inflammation

Cytokines play a critical role in the development of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Here we investigated whether IL-27 was elevated in patients with ALI/ARDS and its potential clinical significance. Bronchoalveolar lavage (BAL) and serum samples were obtained from 58 ALI/ARDS patients, and 25 control healthy volunteers. IL-27 and other inflammatory mediators were measured in BAL and serum by ELISA. Besides, a mouse model of cecal ligation and puncture (CLP)-induced lung inflammation/injury was established, and serum, BAL fluid and tissues were collected for analyses in the presence or absence of IL-27 neutralizing antibodies. BAL IL-27 was found to be significantly higher in patients with ALI/ARDS than that in controls, particularly of pulmonary origin; serum IL-27 was also significantly higher. Increased IL-27 was associated with markers of inflammation, and correlated with disease severity of patients in ALI/ARDS. In a mouse model of CLP-induced lung inflammation/injury, elevated IL-27 levels were observed in the lung, serum, and BAL fluids. IL-27 neutralizing antibody treatment reduced pulmonary inflammation and lung injury and improved mouse survival in response to CLP. Therefore, IL-27 is a critical cytokine in ALI/ARDS and inhibition of IL-27 may open a promising approach for ALI/ARDS patients.     

Inhibition of PKR ameliorates lipopolysaccharide-induced acute lung injury by suppressing NF-κB pathway in mice

Acute lung injury (ALI) is characterized by dramatic lung inflammation and alveolar epithelial cell death. Although protein kinase R (PKR) (double-stranded RNA-activated serine/threonine kinase) has been implicated in inflammatory response to bacterial cell wall components, whether it plays roles in lipopolysaccharide (LPS)-induced ALI remains unclear. This study was aimed to reveal whether and how PKR was involved in LPS-induced ALI pathology and the potential effects of its specific inhibitor, C16 (C₁₃H₈N₄OS). During the experiment, mice received C16 (100 or 500 ug/kg) intraperitoneally 1?h before intratracheal LPS instillation. Then, whole lung lavage was collected for analysis of total protein levels and proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6. The lungs were tested for Western blot, transferase-mediated dUTP nick-end labeling (TUNEL) stain and immunohistochemistry. Results showed that PKR phosphorylation increased significantly after LPS instillation. Furthermore, PKR specific inhibition attenuated LPS-induced lung injury (hematoxylin and eosin stain), reduced lung protein permeability (total protein levels in whole lung lavage) and suppressed proinflammatory cytokines (TNF-α, IL-1β and IL-6) and lung apoptosis (TUNEL stain and caspase3 activation). Moreover, mechanism-study showed that C16 significantly suppressed I kappa B kinase (IKK)/I kappa B alpha (IκBα)/NF-κB signaling pathway after LPS challenge. These findings suggested that PKR inhibition ameliorated LPS-induced lung inflammation and apoptosis in mice by suppressing NF-κB signaling pathway.     

FK506 (tacrolimus) improves lung injury through inhibition of Fas-mediated inflammation

Objective To investigate whether FK506 (tacrolimus) can inhibit Fas- or A23187-induced interleukin (IL)-8 expression and cell death in A549 human alveolar epithelial cells, plus Fas-mediated acute lung injury in vivo. Methods Assays for IL-8, cell death, and caspase-3 activity were performed. A549 cells were treated with 25 μmol A23187 or 0.2 μg/ml agonistic anti-Fas antibody plus 5 ng/ml interferon-gamma (IFN-γ). Tacrolimus was treated at 0.1–10 ng/ml. For in vivo experiment, agonistic anti-Fas antibody (Jo2) at 2.5 μg/g was intratracheally instilled into C57BL/6 mice. Neutrophils and protein contents in bronchoalveolar lavage (BAL) fluid were measured within 24 h of instillation. Mice were orally treated with 32 mg/kg of tacrolimus 24 h and 1 h prior to instillation. Results Both Fas and A23187 caused significant IL-8 expression and cell death in A549 cells. Tacrolimus inhibited A23187-induced IL-8 expression alone while it protected all Fas-mediated responses. Mice instilled intratracheally with Jo2 at 2.5 μg/g had significant increases in neutrophils, protein contents in BAL fluid and in expression of chemoattractants for neutrophils. These increases were reversed by tacrolimus. Conclusions Tacrolimus serves as a therapeutic option for improving lung injury through inhibition of Fas-mediated inflammation. Received 7 November 2005; returned for revision 28 December 2005; accepted by G. Wallace 2 February 2006     

Effect of inducible nitric oxide synthase on apoptosis in Candida-induced acute lung injury

Excessive nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) aggravates acute lung injury (ALI) by producing peroxinitrite. We previously showed that the expression of iNOS and lung injury were suppressed by inhalation of a novel iNOS inhibitor, ONO-1714, in mice with Candida-induced ALI, and that nitric oxide produced by iNOS and apoptosis of epithelial cells were found to have a crucial role in Candida-induced ALI. In the present study, we investigated the effect of NO on the apoptosis of alveolar epithelial cells in Candida-induced ALI. Mice were pretreated by inhalation of ONO-1714 or saline (vehicle control of ONO-1714), and were given an intravenous injection of Candida albicans to induce ALI. After 24 h from injection of Candida albicans, we performed bronchoalveolar lavage and removed lung tissues. We assessed apoptosis on the basis of TUNEL staining and caspase 3 activity. Our results showed that apoptosis was suppressed by inhibition of iNOS-derived NO production by ONO-1714 inhalation. The augmented production of NO increased FasL, TNF-alpha, and mRNA production of Bax of lung that induced apoptosis of alveolar epithelial cells. Inhibition of iNOS-derived NO production by ONO-1714 inhalation ameliorated Candida-induced ALI and improved survival by suppressing apoptosis of alveolar epithelial cells.