Vasoactive intestinal peptide re-balances TREM-1/TREM-2 ratio in acute lung injury

Vasoactive intestinal peptide (VIP) is one of the most plentiful neuropeptides in the lung and it has anti-inflammatory effects in the respiratory system. Triggering receptors expressed on myeloid cells-1 (TREM-1) and triggering receptors expressed on myeloid cells-2 (TREM-2) regulate immune responses to lipopolysaccharide (LPS). In the present study, we tested the expressions of TREM-1 and TREM-2 in various pulmonary cell lines and/or tissue using an animal model of LPS-induced acute lung injury (ALI), and determined the effects of VIP on expression of the TREM-1 and TREM-2 in lung tissues and cells from ALI mice. We found 1) expression of the TREM-1 mRNA from lung tissues of ALI was significantly increased, whereas the expression of TREM-2 mRNA was decreased in these tissues; 2) TREM-1 mRNA was only expressed in macrophages, while TREM-2 mRNA was detected in HBECs, lung fibroblasts, lung adenocarcinoma cells and macrophages; 3) the ratio of TREM-1 mRNA to TREM-2 mRNA was increased in LPS-induced lung tissues and macrophages; 4) VIP inhibited expression of the TREM-1 mRNA in a time- and dose-dependent manner in lung cells from LPS-induced ALI mice; however, it increased expression of the TREM-2 mRNA. As a result of these effects, VIP normalized the ratio of TREM-1 to TREM-2 mRNA in these cells. Our results suggest that VIP might exert its anti-inflammatory effect through a mechanism involved in regulation of expression of the TREM-1 and TREM-2 in LPS-induced ALI.     

Protective role of vascular endothelial growth factor in endotoxin-induced acute lung injury in mice

Background

Vascular endothelial growth factor (VEGF), a substance that stimulates new blood vessel formation, is an important survival factor for endothelial cells. Although overexpressed VEGF in the lung induces pulmonary edema with increased lung vascular permeability, the role of VEGF in the development of acute lung injury remains to be determined.

Methods

To evaluate the role of VEGF in the pathogenesis of acute lung injury, we first evaluated the effects of exogenous VEGF and VEGF blockade using monoclonal antibody on LPS-induced lung injury in mice. Using the lung specimens, we performed TUNEL staining to detect apoptotic cells and immunostaining to evaluate the expression of apoptosis-associated molecules, including caspase-3, Bax, apoptosis inducing factor (AIF), and cytochrome C. As a parameter of endothelial permeability, we measured the albumin transferred across human pulmonary artery endothelial cell (HPAEC) monolayers cultured on porous filters with various concentrations of VEGF. The effect of VEGF on apoptosis HPAECs was also examined by TUNEL staining and active caspase-3 immunoassay.

Results

Exogenous VEGF significantly decreased LPS-induced extravascular albumin leakage and edema formation. Treatment with anti-VEGF antibody significantly enhanced lung edema formation and neutrophil emigration after intratracheal LPS administration, whereas extravascular albumin leakage was not significantly changed by VEGF blockade. In lung pathology, pretreatment with VEGF significantly decreased the numbers of TUNEL positive cells and those with positive immunostaining of the pro-apoptotic molecules examined. VEGF attenuated the increases in the permeability of the HPAEC monolayer and the apoptosis of HPAECs induced by TNF-α and LPS. In addition, VEGF significantly reduced the levels of TNF-α- and LPS-induced active caspase-3 in HPAEC lysates.

Conclusion

These results suggest that VEGF suppresses the apoptosis induced by inflammatory stimuli and functions as a protective factor against acute lung injury.     

MLCK210 gene knockout or kinase inhibition preserves lung function following endotoxin-induced lung injury in mice

Barrier dysfunction, involving the endothelium or epithelium, is implicated in the pathophysiology of many disease states, including acute and ventilator-associated lung injury. Evidence from cell culture, in vivo and clinical studies, has identified myosin light chain kinase as a drug discovery target for such diseases. Here, we measured disease-relevant end points to test the hypothesis that inhibition of myosin light chain kinase is a potential therapeutic target for treatment of barrier dysfunction resulting from acute lung injury. We used a combined gene knockout and chemical biology approach with an in vivo intact lung injury model. We showed that inhibition of myosin light chain kinase protects lung function, preserves oxygenation, prevents acidosis, and enhances survival after endotoxin exposure with subsequent mechanical ventilation. This protective effect provided by the small molecule inhibitor of myosin light chain kinase is present when the inhibitor is administered during a clinically relevant injury paradigm after endotoxin exposure. Treatment with inhibitor confers additional protection against acute lung injury to that provided by a standard protective mode of ventilation. These results support the hypothesis that myosin light chain kinase is a potential therapeutic target for acute lung injury and provide clinical end points of arterial blood gases and pulmonary compliance that facilitate the direct extrapolation of these studies to measures used in critical care medicine.     

Biliverdin administration protects against endotoxin-induced acute lung injury in rats

Given the high morbidity and mortality rates associated with pulmonary inflammation in sepsis, there is a pressing need for new therapeutic modalities to prevent acute respiratory distress. The enzyme heme oxygenase-1 (HO-1) provides potent cytoprotection against lung injury; however, the mechanism by which it does so is unclear. HO-1 catabolizes heme into biliverdin (BV), which is rapidly converted to bilirubin by BV reductase. We tested the hypothesis that BV administration could substitute for the effects observed with HO-1. Using the well-described rat model of LPS-induced shock, we demonstrate that exposure to BV imparts a potent defense against lethal endotoxemia systemically, as well as in the lungs, and effectively abrogates the inflammatory response. BV administration before a lethal dose of LPS leads to a significant improvement in long-term survival: 87% vs. 20% in sham-treated controls. BV treatment suppressed LPS-induced increases in lung permeability and lung alveolitis and significantly reduced serum levels of the LPS-induced proinflammatory cytokine IL-6. Moreover, bilirubin administered just after LPS also abrogated lung inflammation. BV treatment also augmented expression of the anti-inflammatory cytokine IL-10. Similar effects on production were observed with BV treatment in vitro in mouse lung endothelial cells and RAW 264.7 macrophages treated with LPS. In conclusion, these data demonstrate that BV can modulate the inflammatory response and suppress pathophysiological changes in the lung and may therefore have therapeutic application in inflammatory disease states of the lung.     

Intratracheal synthetic CpG oligodeoxynucleotide causes acute lung injury with systemic inflammatory response

Bacterial genome is characterized by frequent unmethylated cytosine-phosphate-guanine (CpG) motifs. Deleterious effects can occur when synthetic oligodeoxynucleotides (ODN) with unmethylated CpG dinucleotides (CpG-ODN) are administered in a systemic fashion. We aimed to evaluate the effect of intratracheal CpG-ODN on lung inflammation and systemic inflammatory response. C57BL/6J mice received intratracheal administration of CpG-ODN (0.01, 0.1, 1.0, 10, or 100 μM) or control ODN without CpG motif. Bronchoalveolar lavage (BAL) fluid was obtained 3 or 6 h or 1, 2, 7, or 14 days after the instillation and subjected to a differential cell count and cytokine measurement. Lung permeability was evaluated as the BAL fluid-to-plasma ratio of the concentration of human serum albumin that was injected 1 h before euthanasia. Nuclear factor (NF)-κB DNA binding activity was also evaluated in lung homogenates. Intratracheal administration of 10 μM or higher concentration of CpG-ODN induced significant inflammatory cell accumulation into the airspace. The peak accumulation of neutrophils and lymphocytes occurred 1 and 2 days after the CpG-ODN administration, respectively. Lung permeability was increased 1 day after the 10 μM CpG-ODN challenge. CpG-ODN also induced nuclear translocation of NF-κB and upregulation of various inflammatory cytokines in BAL fluid and plasma. Histopathology of the lungs and liver revealed acute lung injury and liver damage with necrosis, respectively. Control ODN without CpG motif did not induce any inflammatory change. Since intratracheal CpG-ODN induced acute lung injury as well as systemic inflammatory response, therapeutic strategies to neutralize bacterial DNA that is released after administration of bactericidal agents should be considered.     

Glycosaminoglycan synthesis in endotoxin-induced lung injury

Endotoxin-induced lung injury has previously been shown to produce lesions that resemble emphysema morphologically and biochemically as demonstrated by the reduction in the content of lung elastin. The purpose of this study was to define the changes in one other connective tissue component, glycosaminoglycans, during the acute phase of the lung injury. Intravenous administration of a single dose of endotoxin in rats resulted in an increase in the total synthesis of glycosaminoglycans by the pulmonary parenchyma. There was a significant increase in the proportion of dermatan sulfate synthesized during the first 48 hr and a concomitant decrease in heparin/heparan sulfate synthesis. At 48 hr the increased synthesis of dermatan sulfate had reached 7.3 times control values and began to decline, whereas the synthesis of chondroitin-4-sulfate rose from 4.1 to 10.7 times control values between 48 and 72 hr. Analysis of the rates of synthesis revealed that the total amount of heparin/heparan sulfate remained constant while the synthesis of chondroitin-6-sulfate increased proportionally to the overall synthesis of glycosaminoglycans. These findings indicate that dramatic changes in glycosaminoglycan synthesis are an integral part of endotoxin lung injury.     

Caspase-1-deficient mice have delayed neutrophil apoptosis and a prolonged inflammatory response to lipopolysaccharide-induced acute lung injury

Caspase-1, the prototypic caspase, is known to process the cytokines IL-1beta and IL-18 to mature forms but it is unclear whether, like other caspases, it can induce apoptosis by activation of downstream protease cascades. Neutrophils are known to express caspase-1, to release IL-1beta and to undergo rapid, caspase-dependent apoptosis. We examined apoptosis and IL-1beta production in peripheral blood neutrophils of caspase-1-deficient and wild-type mice. Constitutive apoptosis of caspase-1-deficient neutrophils was delayed compared with wild-type neutrophils and LPS-mediated inhibition of apoptosis was absent, but caspase-1-deficient neutrophils were susceptible to Fas-mediated apoptosis. LPS-stimulated IL-1beta production was absent from caspase-1-deficient neutrophils. To ascertain whether these differences in apoptosis and IL-1beta production would alter the response to acute lung injury, we studied pulmonary neutrophil accumulation following intratracheal administration of LPS. Caspase-1-deficient mice showed increased, predominantly neutrophilic pulmonary inflammation, but inflammation had resolved in both wild-type and deficient animals by 72 h after LPS instillation. IL-1beta production was increased in wild-type lungs but was also detected in caspase-1-deficient mice. We conclude that caspase-1 modulates apoptosis of both peripheral blood and inflammatory neutrophils, but is not essential for IL-1beta production in the lung.     

Regulatory effects of eotaxin on acute lung inflammatory injury

Eotaxin, which is a major mediator for eosinophil recruitment into lung, has regulatory effects on neutrophil-dependent acute inflammatory injury triggered by intrapulmonary deposition of IgG immune complexes in rats. In this model, eotaxin mRNA and protein were up-regulated during the inflammatory response, resulting in eotaxin protein expression in alveolar macrophages and in alveolar epithelial cells. Ab-induced blockade of eotaxin in vivo caused enhanced NF-kappaB activation in lung, substantial increases in bronchoalveolar lavage levels of macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC), and increased MIP-2 and CINC mRNA expression in alveolar macrophages. In contrast, TNF-alpha levels were unaffected, and IL-10 levels fell. Under these experimental conditions, lung neutrophil accumulation was significantly increased, and vascular injury, as reflected by extravascular leak of (125)I-albumin, was enhanced. Conversely, when recombinant eotaxin was administered in the same inflammatory model of lung injury, bronchoalveolar lavage levels of MIP-2 were reduced, as was neutrophil accumulation and the intensity of lung injury. In vitro stimulation of rat alveolar macrophages with IgG immune complexes greatly increased expression of mRNA and protein for MIP-2, CINC, MIP-1alpha, MIP-1beta, TNF-alpha, and IL-1beta. In the copresence of eotaxin, the increased levels of MIP-2 and CINC mRNAs were markedly diminished, whereas MIP-1alpha, MIP-1beta, TNF-alpha, and IL-1beta expression of mRNA and protein was not affected. These data suggest that endogenous eotaxin, which is expressed during the acute lung inflammatory response, plays a regulatory role in neutrophil recruitment into lung and the ensuing inflammatory damage.     

Intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxin-induced acute lung injury in mice

Recent in vivo and in vitro work suggests that mesenchymal stem cells (MSC) have anti-inflammatory properties. In this study, we tested the effect of administering MSC directly into the airspaces of the lung 4 h after the intrapulmonary administration of Escherichia coli endotoxin (5 mg/kg). MSC increased survival compared with PBS-treated control mice at 48 h (80 vs 42%; p < 0.01). There was also a significant decrease in excess lung water, a measure of pulmonary edema (145 +/- 50 vs 87 +/- 20 microl; p < 0.01), and bronchoalveolar lavage protein, a measure of endothelial and alveolar epithelial permeability (3.1 +/- 0.4 vs 2.2 +/- 0.8 mg/ml; p < 0.01), in the MSC-treated mice. These protective effects were not replicated by the use of further controls including fibroblasts and apoptotic MSC. The beneficial effect of MSC was independent of the ability of the cells to engraft in the lung and was not related to clearance of the endotoxin by the MSC. MSC administration mediated a down-regulation of proinflammatory responses to endotoxin (reducing TNF-alpha and MIP-2 in the bronchoalveolar lavage and plasma) while increasing the anti-inflammatory cytokine IL-10. In vitro coculture studies of MSC with alveolar macrophages provided evidence that the anti-inflammatory effect was paracrine and was not cell contact dependent. In conclusion, treatment with intrapulmonary MSC markedly decreases the severity of endotoxin-induced acute lung injury and improves survival in mice.     

Unscheduled apoptosis during acute inflammatory lung injury

Apoptosis is a mode of cell death currently thought to occur in the absence of inflammation. In contrast, inflammation follows unscheduled events such as acute tissue injury which results in necrosis, not apoptosis. We examined the relevance of this paradigm in three distinct models of acute lung injury; hyperoxia, oleic acid, and bacterial pneumonia. In every case, it was found that apoptosis is actually a prominent component of the acute and inflammatory phase of injury. Moreover, using strains of mice that are differentially sensitive to hyperoxic lung injury we observed that the percent of apoptotic cells was well correlated with the severity of lung injury. These observations suggest that apoptosis may be one of the biological consequences during acute injury and the failure to remove these apoptotic cells may also contribute to the inflammatory response.     

Genomic insights into acute inflammatory lung injury

Acute lung injury (ALI) is a devastating syndrome (usually associated with sepsis) that represents a major healthcare burden in the United States. We have focused our studies on unraveling the genetic underpinnings of this syndrome utilizing a candidate gene approach to identify novel genes for ALI susceptibility. Two novel genes identified by this approach include pre-B cell colony-enhancing factor (PBEF) and the gene for myosin light chain kinase (MLCK). PBEF protein levels were elevated in human bronchoalveolar lavage and serum samples from patients with ALI, and DNA sequencing identified two single nucleotide polymorphisms in the PBEF promoter (T-1001G, C-1543T) that were overrepresented in patients with sepsis-induced ALI. More recently, we found MLCK single polymorphisms and haplotypes to be associated with human ALI with unique variants observed in African-Americans with ALI. Thus genomic and genetic approaches represent powerful strategies in the identification of novel candidate genes and potential targets for ALI therapies.     

The bacterial metabolite 2,3-butanediol ameliorates endotoxin-induced acute lung injury in rats

Widely identified in bacteria, yeasts and human beings, 2,3-butanediol has been studied for decades.This chemical reportedly functions as a neutralization agent to counteract lethal acidification by bacterial growth and as a signaling molecule involved in interactions among insects, and between bacteria and the plant host. While 2,3-butanediol is produced by many pathogenic bacterial species, its significance and effect on mammals remains basically uncharacterized. Herein, we show that gastric intubation of 2,3-butanediol in rats significantly ameliorates acute lung injury (ALI) and the inflammatory responses induced by the bacterial endotoxin lipopolysaccharide (LPS), with an efficacy comparable to that of the polyphenol compound resveratrol. Such effect was further demonstrated to occur via modulation of the NF-kappaB signaling pathway. These results indicate that bacterial metabolite, 2,3-butanediol has a negative regulatory effect on host innate immunity response, suggesting bacteria may use some metabolites for host immune evasion.     

Inhibition of endotoxin-induced lung inflammation by interleukin-10 gene transfer in mice

Interleukin (IL)-10 is an anti-inflammatory cytokine that has great potential for use in the treatment of inflammatory and immune illnesses. In this study, gene transfer was used to induce IL-10 transgene expression in murine lungs for treatment of endotoxin-induced lung inflammation. Gene transfer was performed with a cytomegalovirus (CMV)-IL-10 plasmid with the aid of the liposomal agents LipofectAMINE and N-[1-(2,3-dioleoyl)propyl]-N,N, N-trimethylammonium methylsulfate (DOTAP). Administration of the endotoxin caused a marked increase in lung inflammation as indicated by increased tumor necrosis factor (TNF)-alpha release and neutrophil count. Pretreatment of the mice with IL-10 plasmid with and without LipofectAMINE had no inhibitory effect on lung inflammation and IL-10 transgene expression. LipofectAMINE by itself induced lung inflammation, an effect that was not observed with DOTAP. IL-10 plasmid when codelivered with DOTAP expressed biologically active IL-10 protein and caused a reduction in endotoxin-induced inflammation. Transgene expression was observed as early as 3 h after administration, peaked at 12 h, and declined thereafter. We conclude that IL-10 gene transfer is a feasible approach for the treatment of lung inflammation.     

Ethyl pyruvate reduces mortality in an endotoxin-induced severe acute lung injury mouse model

Background

Desmosine and Isodesmosine (D/I) are cross-linking amino acids which are present only in mature elastin. Changes in their concentration in body fluids indicate changes in elastin degradation and can be a reflection of tissue elastase activity. This study was undertaken to determine whether continuous therapy with the long-acting bronchodilator Tiotropium bromide (TTP) could result in reductions in D/I as measured by mass spectrometry in plasma, urine and sputum.

Methods

Twelve not currently smoking patients with chronic obstructive pulmonary disease (COPD), never on TTP, were selected for study. Levels of D/I, along with measurements of FVC, FEV₁ and FEV₁/FVC. were determined before starting TTP daily, and then one and two months after.

Results

D/I decreased in plasma (10 of 12 patients), in sputum all (12 of 12), and in the percentage of free D/I in urine (10 of 12). Most patients showed slight increases in FVC and FEV₁ percent predicted over two months.

Conclusion

The results are consistent with an effect of prolonged bronchodilitation by anti-cholinergic blockade to also result in reduced lung elastin degradation.     

The immunomodulation of endotoxin-induced acute lung injury by hesperidin in vivo and in vitro

To investigate the modulation of lung local immune responses of hesperidin (HES) on the acute lung inflammation induced by LPS in vivo. Mice were challenged with intratracheal lipopolysaccharide (100 μg) 30 min before with treatment hesperidin (200 mg/kg oral administration) or vehicle. After 4 and 24 h, bronchoalveolar lavage fluid was obtained to measure proinflammatory (TNF-α, IL-1β, IL-6), anti-inflammatory (IL-10, IL-4, IL-12) cytokines, chemokines (KC, MCP-1 and MIP-2), total cell counts, nitric oxide production, and proteins. Lung histology was performed in inflated-fixed lungs. Hesperidin downregulate the LPS-induced expression of TNF-α, IL-1β, IL-6, KC, MIP-2, MCP-1, and IL-12. It also enhanced the production of IL-4, IL-10. Total leukocyte counts; nitric oxide production, iNOS expression, and proteins were significantly decreased by hesperidin. In vitro, HES suppressed the expression of IL-8 on A549 cells and THP-1 cells, the expression of TNF-α, IL-1β, and IL-6 on THP-1 cells, the expression of ICAM-1 and VCAM-1 on A549 cells which effect cell adhesion function. The suppression of those molecules is controlled by NF-κB and AP-1, which are activated by IκB and MAPK pathways. HES inhibits those pathways, thereby suppressing the expression of IL-8, TNFα, IL-1β, IL-6, IL-12, ICAM-1 and VCAM-1. This study indicates that HES had a markedly immunomodulatory effect in a clinically relevant model of ARDS. Nevertheless, further investigations are required to determine the potential clinical usefulness of HES in the adjunctive therapy of ARDS.     

Preprotachykinin-A gene deletion protects mice against acute pancreatitis and associated lung injury

Impaired lung function in severe acute pancreatitis is the primary cause of morbidity and mortality in this condition. Preprotachykinin-A (PPT-A) gene products substance P and neurokinin (NK)-A have been shown to play important roles in neurogenic inflammation. Substance P acts primarily (but not exclusively) via the NK1 receptor. NKA acts primarily via the NK2 receptor. Earlier work has shown that knockout mice deficient in NK1 receptors are protected against acute pancreatitis and associated lung injury. NK1 receptors, however, bind other peptides in addition to substance P, not all of which are derived from the PPT-A gene. To examine the role of PPT-A gene products in acute pancreatitis, the effect of PPT-A gene deletion on the severity of acute pancreatitis and the associated lung injury was investigated. Deletion of PPT-A almost completely protected against acute pancreatitis-associated lung injury, with a partial protection against local pancreatic damage. These results show that PPT-A gene products are critical proinflammatory mediators in acute pancreatitis and the associated lung injury.