Introduction of the interleukin-10 gene into mice inhibited bleomycin-induced lung injury in vivo

Interleukin (IL)-10 has been shown to reduce many inflammatory reactions. We investigated the in vivo effects of IL-10 on a bleomycin-induced lung injury model. Hemagglutinating virus of Japan (HVJ)-liposomes containing a human IL-10 expression vector (hIL10-HVJ) or a balanced salt solution as a control (Cont-HVJ) was intraperitoneally injected into mice on day -3. This was followed by intratracheal instillation of bleomycin (0.8 mg/kg) on day 0. Myeloperoxidase activity of bronchoalveolar lavage fluid and tumor necrosis factor-alpha mRNA expression in bronchoalveolar lavage fluid cells on day 7 and hydroxyproline content of the whole lung on day 21 were inhibited significantly by hIL10-HVJ treatment. However, Cont-HVJ treatment could not suppress any of these parameters. We also examined the in vitro effects of IL-10 on the human lung fibroblast cell line WI-38. IL-10 significantly reduced constitutive and transforming growth factor-beta-stimulated type I collagen mRNA expression. However, IL-10 did not affect the proliferation of WI-38 cells induced by platelet-derived growth factor. These data suggested that exogenous IL-10 may be useful in the treatment of pulmonary fibrosis.     

Protective role of reactive oxygen species in endotoxin-induced lung inflammation through modulation of IL-10 expression

Reactive oxygen species (ROS) generated by NADPH oxidase are generally known to be proinflammatory, and it seems to be counterintuitive that ROS play a critical role in regulating the resolution of the inflammatory response. However, we observed that deficiency of the p47(phox) component of NADPH oxidase in macrophages was associated with a paradoxical accentuation of inflammation in a whole animal model of noninfectious sepsis induced by endotoxin. We have confirmed this observation by interrogating four separate in vivo models that use complementary methodology including the use of p47(phox-/-) mice, p47(phox-/-) bone marrow chimera mice, adoptive transfer of macrophages from p47(phox-/-) mice, and an isolated perfused lung edema model that all point to a relationship between excessive acute inflammation and p47(phox) deficiency in macrophages. Mechanistic data indicate that ROS deficiency in both cells and mice results in decreased production of IL-10 in response to treatment with LPS, at least in part, through attenuation of the Akt-GSK3-β signal pathway and that it can be reversed by the administration of rIL-10. Our data support the innovative concept that generation of ROS is essential for counterregulation of acute lung inflammation.     

Maternal glucocorticoids increase endotoxin-induced lung inflammation in preterm lambs

Antenatal betamethasone (Beta) is widely used in women with asymptomatic chorioamnionitis at risk for preterm delivery, but its effects on fetal inflammation are unstudied. Groups of ewes at 109 +/- 1 days of gestation received the following treatments: intra-amniotic (IA) saline (control), 0.5 mg/kg intramuscular Beta, 10 mg IA endotoxin (Endo), and Beta + 2 h later Endo (Beta + Endo). Beta suppressed Endo-induced lung inflammation at 1 day. However, compared with Endo 5 days after treatment, Beta + Endo lambs had increased alveolar neutrophils, proinflammatory cytokine mRNA expression, and serum amyloid A3 (SAA3) mRNA expression. IL-1beta mRNA expression was localized to the inflammatory cells, whereas SAA3 mRNA expression was induced in the bronchial epithelium and the inflammatory cells. Compared with Endo, Beta + Endo lambs had increased lung inflammation but equivalent lung volumes 15 days after treatment. The late increase in inflammation in the Beta + Endo animals suggests that glucocorticoids impair the ability of the preterm lung to downregulate Endo-induced inflammation after fetal clearance of the glucocorticoids. These results have implications for lung inflammation and bronchopulmonary dysplasia in preterm infants exposed to chorioamnionitis and maternal glucocorticoids.     

Impact of interleukin-6 on hypoxia-induced pulmonary hypertension and lung inflammation in mice

Background

Inflammation may contribute to the pathogenesis of various forms of pulmonary hypertension (PH). Recent studies in patients with idiopathic PH or PH associated with underlying diseases suggest a role for interleukin-6 (IL-6).

Methods

To determine whether endogenous IL-6 contributes to mediate hypoxic PH and lung inflammation, we studied IL-6-deficient (IL-6^-/-) and wild-type (IL-6^+/+) mice exposed to hypoxia for 2 weeks.

Results

Right ventricular systolic pressure, right ventricle hypertrophy, and the number and media thickness of muscular pulmonary vessels were decreased in IL-6^-/- mice compared to wild-type controls after 2 weeks'' hypoxia, although the pressure response to acute hypoxia was similar in IL-6^+/+ and IL-6^-/- mice. Hypoxia exposure of IL-6^+/+ mice led to marked increases in IL-6 mRNA and protein levels within the first week, with positive IL-6 immunostaining in the pulmonary vessel walls. Lung IL-6 receptor and gp 130 (the IL-6 signal transducer) mRNA levels increased after 1 and 2 weeks'' hypoxia. In vitro studies of cultured human pulmonary-artery smooth-muscle-cells (PA-SMCs) and microvascular endothelial cells revealed prominent synthesis of IL-6 by PA-SMCs, with further stimulation by hypoxia. IL-6 also markedly stimulated PA-SMC migration without affecting proliferation. Hypoxic IL-6^-/- mice showed less inflammatory cell recruitment in the lungs, compared to hypoxic wild-type mice, as assessed by lung protein levels and immunostaining for the specific macrophage marker F4/80, with no difference in lung expression of adhesion molecules or cytokines.

Conclusion

These data suggest that IL-6 may be actively involved in hypoxia-induced lung inflammation and pulmonary vascular remodeling in mice.     

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.     

Activated protein C protection from lung inflammation in endotoxin-induced injury

We studied the protection of recombinant human activated protein C (rhAPC) in endotoxin-induced lung inflammation and injury and whether this effect is correlated with modulation of lung matrix metalloproteinase (MMP) activity. We randomly assigned 12 Large White pigs to receive intravenous Escher-ichia coli lipopolysaccharide (LPS; 40 mu g/kg/hr), rhAPC (24 mu g/ kg/hr), or both. We monitored respiratory mechanics and function, cell counts, and cytokine concentrations in bron-choalveolar lavage fluid (BALF). Lung samples were collected for the zymography of MMP-2 and MMP-9 activities and for histology. In septic pigs, rhAPC decreased proMMP-9 release as well as MMP-9 activation, and increased proMMP-2 presence without any evident activation compared with specimens that were given LPS alone. In addition, lung injury in rhAPC-treated animals was significantly attenuated, as shown by higher respiratory compliance, delayed increase in tumor necrosis alfa and interleukin-1beta as well as neutrophil recruitment in the BALF, reduced lung edema, and histologic changes. In conclusion, rhAPC is beneficial in acute lung injury, and the protection may depend, at least in part, on modulation of MMP-2/9 activity.     

Resistance to bleomycin-induced lung fibrosis in MMP-8 deficient mice is mediated by interleukin-10

Background

Matrix metalloproteinases (MMPs) may have pro and antifibrotic roles within the lungs, due to its ability to modulate collagen turnover and immune mediators. MMP-8 is a collagenase that also cleaves a number of cytokines and chemokines.

Methodology and Principal Findings

To evaluate its relevance in lung fibrosis, wildtype and Mmp8^−/− mice were treated with either intratracheal bleomycin or saline, and lungs were harvested at different time points. Fibrosis, collagen, collagenases, gelatinases, TGFβ and IL-10 were measured in lung tissue. Mmp8^−/− mice developed less fibrosis than their wildtype counterparts. This was related to an increase in lung inflammatory cells, MMP-9 and IL-10 levels in these mutant animals. In vitro experiments showed that MMP-8 cleaves murine and human IL-10, and tissue from knockout animals showed decreased IL-10 processing. Additionally, lung fibroblasts from these mice were cultured in the presence of bleomycin and collagen, IL-10 and STAT3 activation (downstream signal in response to IL-10) measured by western blotting. In cell cultures, bleomycin increased collagen synthesis only in wildtype mice. Fibroblasts from knockout mice did not show increased collagen synthesis, but increased levels of unprocessed IL-10 and STAT3 phosphorylation. Blockade of IL-10 reverted this phenotype, increasing collagen in cultures.

Conclusions

According to these results, we conclude that the absence of MMP-8 has an antifibrotic effect by increasing IL-10 and propose that this metalloprotease could be a relevant modulator of IL-10 metabolism in vivo.     

Inhibition of tumor growth by interleukin 10 gene transfer in B16(F10) melanoma cells

Interleukin 10 (IL-10) is a potent immunosuppressive cytokine with an antitumor activity. The effect of IL-10 on tumor growth was tested in murine melanoma cells manipulated by gene transfer to secrete IL-10. In mice bearing B16(F10) tumors expressing IL-10 tumor growth was decreased depending on the amount of secreted IL-10.     

Impact of TREM-2 gene silencing on inflammatory response of endotoxin-induced acute lung injury in mice

Acute lung injury (ALI) is one of the critical clinical respiratory diseases, of which infection is the main cause and the first risk factor. This study investigated the impact of triggering receptor of myeloid cells expression (TREM)-2 gene silencing on inflammatory response of endotoxin-induced ALI in mice. Lentivirus-mediated TREM-2-shRNA was transfected into healthy male C57BL/6 mice, and the lipopolysaccharide-induced ALI model was established. The immunohistochemistry, immunofluorescence, fluorescence quantitative PCR, western blot, and ELISA were applied to detect the pathological changes of lung tissue and expressions of TREM-2, tumor necrosis factor-α (TNF-α), and interleukin 10 (IL-10) in bronchoalveolar lavage fluid. The lentivirus group, saline control group, ALI model group, blank control group, and negative control group were set up at the same time. Results found that, in lentivirus group, the pathological change of lung tissue was significantly lighter than ALI model group (P < 0.05), and the expression of TREM-2 was significantly reduced compared with all control groups (P < 0.05). The levels of TNF-α and IL-10 were significantly increased than all control groups (P < 0.05), while above indexes in negative control group and blank control group showed no significant difference with ALI group (P > 0.05). This study indicates that TREM-2 has a protective effect on inflammatory response of endotoxin-induced ALI in mice, which has provided new potential targets for prevention and treatment of ALI.     

Regulation of chemokine expression by IL-10 in lung inflammation

We have been interested in understanding the mechanisms regulating the inflammatory process underlying acute lung injury. The current studies have employed a model of acute lung inflammation in mice triggered by bacterial lipopolysaccharide. The development of this injury was associated with increased expression of the chemokines, MIP-1alpha and MIP-2, that coordinate recruitment of neutrophils to the lung. IL-10 is a potent, endogenous anti-inflammatory molecule that has been shown to decrease lung inflammation partly on the basis of TNF-alpha and IL-1beta inhibition. In these studies we tested the hypothesis that endogenous IL-10 modulates chemokine expression using the IL-10 knock-out mouse, and then explored the molecular mechanisms by which IL-10 might do so. The results demonstrate that significant elevations in both chemokines were observed in the absence of IL-10 and that these findings were associated with significant increases in lung neutrophil accumulation. In vitro studies defined two, gene-specific, mechanisms by which IL-10 regulated chemokine expression: mRNA destabilization and NF-kappaB inhibition. These results suggested that IL-10 is an important, endogenous regulator of chemokine expression in acute lung inflammation.     

Inhibition of tobacco smoke-induced lung inflammation by a catalytic antioxidant

Cigarette smokers experience airway inflammation and epithelial damage, the mechanisms of which are unknown. One potential cause may be free radicals either in tobacco smoke or produced during persistent inflammation. Inflammation may also be a driving force to cause airway epithelium to undergo changes leading to squamous cell metaplasia. To test whether tobacco smoke-induced inflammation could be reduced by a catalytic antioxidant, manganese(III)meso-tetrakis(N,N'-diethyl-1,3-imidazolium-2-yl) porphyrin (AEOL 10150) was given by intratracheal instillation to rats exposed to filtered air or tobacco smoke. Exposure to tobacco smoke for 2 d or 8 weeks (6 h/d, 3 d/week) significantly increased the number of cells recovered by bronchoalveolar lavage (BAL). AEOL 10150 significantly decreased BAL cell number in tobacco smoke-treated rats. Significant reductions in neutrophils were noted at 2 d and macrophages at 8 weeks. Lymphocytes were significantly reduced by AEOL 10150 at both time points. Squamous cell metaplasia following 8 weeks of tobacco smoke exposure was 12% of the total airway epithelial area in animals exposed to tobacco smoke without AEOL 10150, compared with 2% in animals exposed to tobacco smoke, but treated with AEOL 10150 (p <.05). We conclude that a synthetic catalytic antioxidant decreased the adverse effects of exposure to tobacco smoke.     

Induction of neutrophil apoptosis and secondary necrosis during endotoxin-induced pulmonary inflammation in mice

The present study investigated the relationship between apoptotic and necrotic cell death and their role in pulmonary inflammatory response to endotoxin. Pulmonary administration of lipopolysaccharide (LPS) caused a rapid increase in the levels of pro-inflammatory cytokine TNF-alpha and inflammatory cell influx in the bronchoalveolar lavage (BAL) fluids. Control mice showed only resident alveolar macrophages with no apoptosis, whereas LPS-treated mice showed clear apoptosis of BAL cells. Microscopic studies confirmed the presence of apoptotic neutrophils and macrophages ingesting apoptotic bodies. The number of apoptotic neutrophils increased concomitantly with the increase in neutrophil influx which peaked 1 day after the treatment. However, necrosis was not detected at this early time, but increased subsequently and peaked at day 3. The levels of necrosis and apoptosis were both elevated and prolonged at high LPS doses. Treatment of mice with phosphatidylserine (PS)-containing liposome, known to inhibit macrophage phagocytosis of apoptotic cells, increased the level of apoptosis and necrosis caused by LPS, whereas control non-PS liposome or saline treatment had no effects. We conclude that necrosis occurs secondary to apoptosis in LPS-treated lung model and that this development is not the result of direct insult by LPS. Instead, our results and previous studies suggest that inefficient clearance of apoptotic cells by macrophages contributes, at least in part, to the levels of apoptosis and necrosis induced by LPS. Because necrosis is associated with cell damage and release of histotoxic contents, this development is likely to play a role in determining the severity and duration of lung toxicity induced by endotoxin.     

Inhibition of MAPK signaling by eNOS gene transfer improves ventricular remodeling after myocardial infarction through reduction of inflammation

Endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) may play an important role in attenuating cardiac remodeling and apoptosis after myocardial infarction. However, the anti-inflammation effects of eNOS in infarcted myocardium and the role of MAPK signaling in eNOS/NO mediated cardiac remodeling have not yet been elucidated. Adenovirus carrying Human eNOS gene was delivered locally into heart 4 days prior to induction of myocardial infarction (MI) by left anterior descending coronary artery ligation. Monocyte/macrophage infiltration was detected by ED-1 immunohistochemistry. Western blot was employed to examine the activation of MAPK. eNOS gene transfer significantly reduced myocardial infarct size and improved cardiac contractility as well as left ventricle (LV) diastolic function at 7 days after MI. In addition, eNOS gene transfer decreased monocyte/macrophage infiltration in the infarct region of the heart. Phosphorylation of MAPK after MI were also dramatically reduced by eNOS gene transfer. All the protective effects of eNOS were blocked by N(ω)-nitro-l-arginine methyl ester (L-NAME) administration, indicating a NO-mediated event. These results demonstrate that the eNOS/NO system provides cardiac protection after MI injury through inhibition of inflammation and suppression of MAPK signaling.     

Mutual modulation between interleukin-10 and interleukin-6 induced by Rhodococcus aurantiacus infection in mice

The interaction between interleukin-10 (IL-10) and interleukin-6 (IL-6) was investigated in the inflammatory response to Rhodococcus aurantiacus (R. aurantiacus) infection, in which both cytokines act as anti-inflammatory cytokines. Compared with wild-type (WT) counterparts, IL-6 gene-deficient (IL-6(-)/(-)) mice mounted a more robust production of IL-10 and tumor necrosis factor-alpha (TNF-alpha) during the initial phase of infection. Administration of anti-IL-10 antibody resulted in all the mice dying within 3 days post-infection as well as a further elevated TNF-alpha release. In vitro challenge of the macrophages from IL-6(-)/(-) and WT mice with heat-killed R. aurantiacus also showed similar results. Addition of exogenous IL-6 depressed IL-10 and TNF-alpha production by either IL-6(-)/(-) mice or IL-6(-)/(-) mouse macrophages. Likewise, WT mouse macrophages pretreated with anti-IL-10 or anti-IL-6 antibody exhibited increased production of TNF-alpha and IL-6 or IL-10 respectively. Moreover, neutralization of both IL-10 and IL-6 induced a further increase in TNF-alpha production by WT mouse cells. Overall, we conclude that IL-10 is a key element in protecting mice against mortality, and that IL-10 and IL-6 production are negatively regulated by each other although they are additive in suppressing TNF-alpha release in R. aurantiacus-infected mouse model.     

Role of metallothionein in lung inflammation induced by ozone exposure in mice

Metallothionein (MT) is a free radical scavenger induced by inflammatory stimuli; however, its roles in inflammation have not been fully investigated. In the present study, we genetically determined the role of MT in ozone (O₃)-induced lung inflammation using MT-I/II null (–/–) mice. Subacute (65 h) exposure to O₃ (0.3 ppm) induced lung inflammation and enhanced vascular permeability, which was significantly greater in MT(–/–) than in corresponding wild-type mice. Electron microscopically, O₃ exposure induced vacuolar degeneration of pulmonary endothelial and epithelial cells, and interstitial edema with focal loss of the basement membrane, which was more prominent in MT(–/–) than in wild-type mice. O₃ -induced lung expression of interleukin-6 was significantly greater in MT(–/–) than in wild-type mice; however, lung expression of the chemokines examined was comparable in both genotypes of mice in the presence of O₃. Following O₃ exposure, the formation of oxidative stress-related molecules/adducts, such as heme oxidase-1, inducible nitric oxide synthase, 8-hydroxy-2′-deoxyguanosine, and nitrotyrosine, in the lung was significantly greater in MT(–/–) than in wild-type mice. Collectively, MT protects against O₃-induced lung inflammation, at least partly, via the regulation of pulmonary endothelial and epithelial integrity and its antioxidative property.