Abrogation of bleomycin-induced epithelial apoptosis and lung fibrosis by captopril or by a caspase inhibitor

Angiotensin-converting enzyme is involved in apoptosis of alveolar epithelial cells (Wang R, Zagariya A, Ang E, Ibarra-Sunga O, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 277: L1245-L1250, 1999). This study tested the ability of the angiotensin-converting enzyme inhibitor captopril or the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD-fmk) to block alveolar epithelial cell apoptosis and lung fibrosis in vivo in response to bleomycin (Bleo). Male Wistar rats received 8 U/kg of Bleo (bleomycin sulfate) or vehicle intratracheally. Subgroups of Bleo-treated rats received captopril, ZVAD-fmk, or vehicle alone. Lung collagen was assessed by picrosirius red or hydroxyproline assay at 1, 7, and 14 days post-Bleo, and apoptosis was detected by in situ end labeling (ISEL). Bleo increased alveolar septal and peribronchial collagen by 100 and 133%, respectively (both P < 0.01), by day 14 but not earlier. In contrast, ISEL was increased in alveolar and airway cells at all time points. Captopril or ZVAD-fmk inhibited collagen accumulation by 91 and 85%, respectively (P < 0. 01). Both agents also inhibited ISEL in alveoli by 99 and 81% and in airways by 67 and 63%, respectively. These data suggest that the efficacy of captopril to inhibit experimental lung fibrogenesis is related to inhibition of apoptosis. They also demonstrate the antifibrotic potential of a caspase inhibitor.     

Neutralization of IL-18 by IL-18 binding protein ameliorates bleomycin-induced pulmonary fibrosis via inhibition of epithelial-mesenchymal transition

Idiopathic pulmonary fibrosis (IPF) is a fatal parenchymal lung disease with limited effective therapies. Interleukin (IL)-18 belongs to a rather large IL-1 gene family and is a proinflammatory cytokine, which acts in both acquired and innate immunity. We have previously reported that IL-18 play an important role in lipopolysaccharide-induced acute lung injury in mice. Persistent inflammation often drives fibrotic progression in the bleomycin (BLM) injury model. However, the role of IL-18 in pulmonary fibrosis (PF) is still unknown. IL-18 binding protein (IL-18BP) is able to neutralize IL-18 biological activity and has a protective effect against renal fibrosis. The aim of this study was to investigate the effects of IL-18BP on BLM-induced PF. In the present study, we found that IL-18 was upregulated in lungs of BLM-injured mice. Neutralization of IL-18 by IL-18BP improved the survival rate and ameliorated BLM-induced PF in mice, which was associated with attenuated pathological changes, reduced collagen deposition, and decreased content of transforming growth factor-β1 (TGF-β1). We further demonstrated that IL-18BP treatment suppressed the BLM-induced epithelial mesenchymal transition (EMT), characterized by decreased α-smooth muscle actin (α-SMA) and increased E-cadherin (E-cad) in vivo. In addition, we provided in vitro evidence demonstrating that IL-18 promoted EMT through upregulation of Snail-1 in A549 cells. In conclusion, our findings raise the possibility that the increase of IL-18 is involved in the development of BLM-induced PF through modulating EMT in a Snail-1-dependent manner. IL-18BP may be a worthwhile candidate option for PF therapy.     

Targeting the extracellular membrane-proximal domain of membrane-bound IgE by passive immunization blocks IgE synthesis in vivo

The classical allergic reaction starts seconds or minutes after Ag contact and is committed by Abs produced by a special subset of B lymphocytes. These Abs belong to the IgE subclass and are responsible for Type I hyperreactivity reactions. Treatment of allergic diseases with humanized anti-IgE Abs leads primarily to a decrease of serum IgE levels. As a consequence, the number of high-affinity IgE receptors on mast cells and basophils decreases, leading to a lower excitability of the effector cells. The biological mechanism behind anti-IgE therapy remains partly speculative; however, it is likely that these Abs also interact with membrane IgE (mIgE) on B cells and possibly interfere with IgE production. In the present work, we raised a mouse mAb directed exclusively against the extracellular membrane-proximal domain of mIgE. The interaction between the monoclonal anti-mIgE Ab and mIgE induces receptor-mediated apoptosis in vitro. Passive immunization experiments lead to a block of newly synthesized specific IgEs during a parallel application of recombinant Bet v1a, the major birch pollen allergen. The decrease of allergen-specific serum IgE might be related to tolerance-inducing mechanisms stopping mIgE-displaying B cells in their proliferation and differentiation.     

Regulation of found in inflammatory zone 1 expression in bleomycin-induced lung fibrosis: role of IL-4/IL-13 and mediation via STAT-6

Found in inflammatory zone (FIZZ)1, also known as resistin-like molecule alpha, belongs to a novel class of cysteine-rich secreted protein family, named FIZZ/resistin-like molecule, with unique tissue expression patterns. FIZZ1 is induced in alveolar type II epithelial cells (AECs) in bleomycin (BLM)-induced lung fibrosis, and found to induce myofibroblast differentiation in vitro. The objective of this study was to elucidate the regulation of AEC FIZZ1 expression in pulmonary fibrosis. AECs were isolated from rat lungs and the effects of a number of cytokines on FIZZ1 expression were evaluated by RT-PCR. Of all cytokines examined, only IL-4 and IL-13 were effective in stimulating FIZZ1 expression in AECs. Stimulation by IL-4/IL-13 was accompanied by increases in phosphorylated STAT6 and JAK1. FIZZ1 expression was also stimulated by transfection with a STAT6 expression plasmid, but was inhibited by antisense oligonucleotides directed against STAT6. In vivo studies showed that compared with wild-type controls, both IL-4- and IL-13-deficient mice showed reduced BLM-induced lung FIZZ1 expression and fibrosis, which were essentially abolished in IL-4 and IL-13 doubly deficient mice. Furthermore, STAT6-deficient mice showed marked reduction in BLM-induced lung FIZZ1 expression. Thus, IL-4 and IL-13 are potent inducers of AEC FIZZ1 expression via STAT6 and play key roles in BLM-induced lung FIZZ1 expression and fibrosis. This represents a potential mechanism by which IL-4/IL-13 could play a role in the pathogenesis of lung fibrosis.     

Inactivation of IL-6 and soluble IL-6 receptor by neutrophil derived serine proteases in cystic fibrosis

The ability of IL-6 to signal via both membrane bound and soluble receptors is thought to explain the capacity of this cytokine to act in both the initiation and resolution of acute inflammatory responses. In cystic fibrosis (CF), poorly resolved neutrophillic inflammation of the lungs is a primary cause of morbidity and mortality. Expression of IL-6 has been reported to be low in CF lung secretions, despite ongoing inflammation, but the status of soluble IL-6 receptor (sIL-6R) in these patients is unknown. We hypothesised that sIL-6R may be an important potentiator of IL-6 activity in CF associated lung disease. IL-6, sIL-6R and sgp130 (a natural antagonist of responses mediated by the sIL-6R) were analysed by ELISA and Western blot in bronchoalveolar lavage fluid (BALF) from 28 paediatric CF patients and nine non-CF controls. Total cell counts in CF were four fold higher compared to controls (median: 1.4 × 10⁶ cells/ml v. 0.35 × 10⁶ cells/ml in controls) (p < 0.001) and the infiltrate was dominated by neutrophils which were elevated by 89 fold (0.62 × 10⁶ cells/ml v. 0.007 × 10⁶ cells/ml in controls) (p < 0.001). Other markers of inflammation such as IL-8 and MCP-1 were elevated 17.5 and 3.8 fold respectively (IL-8; median: 1122 pg/ml v. 64 pg/ml in controls, p < 0.01 and MCP-1; median: 692 pg/ml v. 182 pg/ml in controls, p < 0.05). IL-6, although present in 23/32 CF BALF specimens compared to 1/9 controls (p < 0.01), was weakly expressed (median: 50 pg/ml). Expression of sIL-6R and sgp130 in CF was no different to control patients. We tested whether weak expression of all three molecules was due to degradation by CF BALF. Degradative activity was observed in association with BALF elastase activity and could be specifically blocked by serine protease inhibitors. Degradation of sIL-6R by purified serine proteases (elastase, cathepsin G and proteinase 3) was also observed leading to a loss of trans-signalling activity. Interestingly, sIL-6R was protected from proteolysis by interaction with IL-6. Our data identify and define a novel protease mediated deficiency of IL-6 signalling in the CF lung.     

IL-13 induces skin fibrosis in atopic dermatitis by thymic stromal lymphopoietin

Skin fibrotic remodeling is a major feature in human atopic dermatitis (AD). Inflammation and tissue fibrosis are common consequences of Th2 responses. Elevated IL-13 and thymic stromal lymphopoietin (TSLP) have been found in the AD skin lesions. Fibrocytes can be recruited to inflamed tissues to promote wound healing and fibrosis. Dermal transgenic expression of IL-13 causes an AD-like phenotype with fibrosis and increased TSLP. However, the role of TSLP in fibrotic remodeling is unknown. In this study, we investigated the role of TSLP and fibrocytes in the generation of IL-13-induced skin fibrosis. In AD lesion, cessation of IL-13 transgene expression resulted in reduced skin inflammation but with no effect on further progression of fibrosis. This was accompanied by markedly increased CD34(+)/procollagen 1(+) fibrocytes. Furthermore, fibrocytes express TSLP receptor (TSLPR), and TSLP directly promotes PBMC-derived fibrocytes to produce collagen. Neutralization of TSLP or genetic deletion of TSLPR in IL-13 transgenic mice resulted in a significant reduction in fibrocytes and in skin fibrosis. Furthermore, reduction of fibrosis by depletion of TSLP was independent of IL-13. Interestingly, the number of fibrocytes was highly increased in the skin samples of AD patients. These data indicate that the progression of skin fibrosis in IL-13-induced AD occurs via TSLP/TSLPR-dependent but IL-13-independent novel mechanisms by promoting fibrocyte functions.     

TLR2-mediated production of IL-27 and chemokines by respiratory epithelial cells promotes bleomycin-induced pulmonary fibrosis in mice

Idiopathic pulmonary fibrosis is a fatal disease characterized by progressive destruction of the lung. Although TLR2 bridges innate and adaptive immunity by sensing tissue damage, its role in pulmonary fibrosis remains unclear. To address this issue, TLR2(-/-) and WT mice were examined for bleomycin-induced pulmonary fibrosis (BIPF). Flow cytometric and immunohistochemical analysis revealed that TLR2 expression in bronchial epithelial and immune cells of the lungs was upregulated in WT mice during BIPF. Levels of IL-27, TGF-β, chemokines, and hydroxyproline were lower in lungs of TLR2(-/-) mice than in those of WT mice, but IL-17 levels were higher in TLR2(-/-) mice. In in vivo experiments using bone marrow-chimeric mice, TLR2 expression on respiratory epithelial cells, rather than immune cells, induced IL-27 and chemokine production in the lungs, further stimulating BIPF. This effect of TLR2 depended on IRF complexes and MyD88. BIPF was more severe in IL-17A(-/-) mice and in TLR2(-/-) mice treated with anti-IL-17 mAb than in TLR2(-/-) and WT mice. Furthermore, IL-27 blockade in WT mice reduced hydroxyproline levels by enhancing IL-17 production, whereas the treatment of TLR2(-/-) mice with a chemokine mixture increased hydroxyproline levels by recruiting inflammatory cells into the lungs. TLR2 signaling promotes BIPF by inducing IL-27 and chemokine production by respiratory epithelial cells, thereby inhibiting IL-17 production and recruiting inflammatory cells into the lungs.     

Intracellular pH topography of Penicillium cyclopium protoplasts. Maintenance of delta pH by both passive and active mechanisms

The intracellular pH distribution in protoplasts of Penicillium cyclopium has been studied using the recently developed fluorescent probe microscopic technique. The technique gives detailed pH maps of the interior of the protoplasts with the exception of vacuoles (no fluorescence signal from vacuoles was observed). In the cytoplasm two separate layers were distinguished: a thinner outer layer with acidic pH (around 5) and the larger core region with near neutral pH. The pH of the core region is decreased by the addition of uncouplers, inhibitors of respiration and during the uptake of L-phenylalanine. These compounds do not change the pH of the surface layer, which is, however, acidified by addition of vanadate, an inhibitor of the proton pump of the plasmalemma. We suggest that the pH of the surface layer is maintained by the combined effects of a Donnan distribution of protons (bound to postulated anion binding proteins) and the proton extrusion via the plasmalemma proton pump. This mechanism explains the protection of the cytoplasmic core of acidophilic eukaryotes from the influence of the usually acidic environment.     

Betaine or taurine administration prevents fibrosis and lipid peroxidation induced by rat liver by ethanol plus carbon tetrachloride intoxication

Summary. The aim of this study was to investigate the effect of betaine or taurine on liver fibrogenesis and lipid peroxidation in rats. Fibrosis was induced by treatment of rats with drinking water containing 5% ethanol and CCl₄ (2×weekly, 0.2ml/kg, i.p.) for 4 weeks. Ethanol plus CCl₄ treatment caused increased lipid peroxidation and disturbed antioxidant system in the liver. Histopathological findings suggested that the development of liver fibrosis was prevented in rats treated with betaine or taurine (1% v/v in drinking water) together with ethanol plus CCl₄ for 4 weeks. When hepatic taurine content was depleted with -alanine (3% v/v in drinking water), portal-central fibrosis induced by ethanol+CCl₄ treatment was observed to proceed cirrhotic structure. Betaine or taurine was also found to decrease serum transaminase activities and hepatic lipid peroxidation without any change in hepatic antioxidant system in rats with hepatic fibrosis. In conclusion, the administration of betaine or taurine prevented the development of liver fibrosis probably associated with decreased oxidative stress.     

In vivo neutralization of human IL-6 (hIL-6) achieved by immunization of hIL-6-transgenic mice with a hIL-6 receptor antagonist

Neutralization of IL-6 represents an attractive therapeutic option in several diseases, including B cell neoplasia, osteoporosis, and autoimmunity. Therapeutic attempts in humans have shown that administration of injectable doses of a mAb to IL-6 does not provide efficient neutralization of the cytokine in vivo. Therefore, alternative approaches are needed. In this study, we evaluated whether the Ab response to human IL-6 (hIL-6) elicited by vaccination with Sant1 (a hIL-6 variant with seven amino acid substitutions) was able to fully correct in vivo the clinical and biological effects of a chronic endogenous overproduction of hIL-6 in the hIL-6-transgenic NSE/hIL-6 mice. Because of the overexpression of hIL-6, occurring since birth, with circulating levels in the nanogram per milliliter range, NSE/hIL-6 mice have a marked decrease in growth rate, associated with decrease in insulin-like growth factor I levels, and represent an animal model of the growth impairment associated with human chronic inflammatory diseases. Following immunization with Sant1, but not with hIL-6, NSE/hIL-6 mice developed high titers of polyclonal Abs to hIL-6. The Abs, acquired by transplacental transfer, effectively neutralized IL-6 activities in vivo as shown by the complete correction of the growth defect and normalization of insulin-like growth factor levels in the hIL-6-transgenic offspring. Immunization with Sant1 could therefore represent a novel and simple therapeutic approach for the specific neutralization of IL-6 in humans.     

Dynamics of the contamination of mice during the treatment of burn sepsis due to Pseudomonas aeruginosa using tobramycin alone or combined with active and passive immunization

An experimental study was made with a view to finding out the possible bacteriological advantages of the combined use of tobramycin (Tb) and P. aeruginosa corpuscular polyvalent vaccine (PaCPV) or P. aeruginosa hyperimmune plasma (PaHIP) in burn sepsis caused by P. aeruginosa. The use of the median therapeutic dose of Tb (2.5 mg/kg body weight per day), alone or in combination with immunopreparations, ensured the survival rate of the animals equal to 100%. The contamination of the body with P. aeruginosa after treatment with Tb and PaHIP or PaCPV was lower than after the administration of Tb alone, this phenomenon becoming manifest starting from day 5 of observation in the first case and from day 10 in the second case. The combined use of Tb and immunopreparations (PaCPV or PaHIP) in acute P. aeruginosa infection proved to be more effective than treatment with Tb alone.     

Reduced protection of RIPK3-deficient mice against influenza by matrix protein 2 ectodomain targeted active and passive vaccination strategies

RIPK3 partially protects against disease caused by influenza A virus (IAV) infection in the mouse model. Here, we compared the immune protection of active vaccination with a universal influenza A vaccine candidate based on the matrix protein 2 ectodomain (M2e) and of passive immunization with anti-M2e IgG antibodies in wild type and Ripk3^−/− mice. We observed that the protection against IAV after active vaccination with M2e viral antigen is lost in Ripk3^−/− mice. Interestingly, M2e-specific serum IgG levels induced by M2e vaccination were not significantly different between wild type and Ripk3^−/− vaccinated mice demonstrating that the at least the humoral immune response was not affected by the absence of RIPK3 during active vaccination. Moreover, following IAV challenge, lungs of M2e vaccinated Ripk3^−/− mice revealed a decreased number of immune cell infiltrates and an increased accumulation of dead cells, suggesting that phagocytosis could be reduced in Ripk3^−/− mice. However, neither efferocytosis nor antibody-dependent phagocytosis were affected in macrophages isolated from Ripk3^−/− mice. Likewise following IAV infection of Ripk3^−/− mice, active vaccination and infection resulted in decreased presence of CD8+ T-cells in the lung. However, it is unclear whether this reflects a deficiency in vaccination or an inability following infection. Finally, passively transferred anti-M2e monoclonal antibodies at higher dose than littermate wild type mice completely protected Ripk3^−/− mice against an otherwise lethal IAV infection, demonstrating that the increased sensitivity of Ripk3^−/− mice could be overcome by increased antibodies. Therefore we conclude that passive immunization strategies with monoclonal antibody could be useful for individuals with reduced IAV vaccine efficacy or increased IAV sensitivity, such as may be expected in patients treated with future anti-inflammatory therapeutics for chronic inflammatory diseases such as RIPK inhibitors.Subject terms: Infection, Viral infection

     

Montelukast reduces eosinophilic inflammation by inhibiting both epithelial cell cytokine secretion (GM-CSF, IL-6, IL-8) and eosinophil survival

Leukotriene receptor antagonists, such as montelukast (MK), are currently used to treat rhinitis and asthma, but their anti-inflammatory role in eosinophil inflammation is not well understood. The aim of this study is to investigate the effect of MK on an in vitro model of upper-airway eosinophil inflammation by reducing pro-inflammatory cytokines from both nasal mucosa (NM) and polyp (NP) epithelial cells and reducing eosinophil survival primed by epithelial cell secretions. Epithelial cells were stimulated with fetal bovine serum (FBS) with or without MK for 24 hours, and cytokine concentrations in epithelial secretions were measured by ELISA. After incubating peripheral blood eosinophils with epithelial cell-conditioned media (ECM) with or without MK up to 3 days, eosinophil survival was assessed by Trypan blue dye exclusion. Results are expressed as mean±SEM of cytokine concentration (percent of control) or eosinophil survival (percent). Epithelial cell stimulation increased GM-CSF, IL-6, IL-8, and sICAM-1 secretion in both NM and NP. MK had a significant inhibitory effect on FBS-induced GM-CSF, IL-6, and IL-8 secretion, but not sICAM-1, in both NM and NP. MK also showed an inhibitory effect (p<0.05) on ECM-induced eosinophil survival from both NM (from 10(-5)M to 10(-7)M, n=7) and NP (at 10(-5)M, n=7), after 3 days of incubation. These anti-inflammatory effects on epithelial cell cytokine secretion and on eosinophil survival suggest that montelukast may contribute to the reduction of eosinophilic inflammation in upper-airway inflammatory diseases such as rhinitis and nasal polyposis.     

PKC-dependent IL-6 production and inhibition of IL-8 production by PKC activation in normal human skin fibroblasts under extremely high hydrostatic pressure

Normal human dermal fibroblasts were found to survive and to be active in producing interleukin (IL)-6 and IL-8 under extremely high hydrostatic pressure, up to 40 MPa (1 atm=0.101325 MPa=1.03323 kgf/cm(2)), for 20 min. An inhibitor of protein kinase C (PKC) reduced the amount of IL-6 production, whereas IL-8 production was increased following pressure application. The activation of PKC in response to exposure to the pressure stress was detected by using the PKC-specific probe Rim-1. These findings indicate that IL-6 production induced by hydrostatic pressure stresses was dependent on the PKC signaling pathway. In contrast, pressure-induced IL-8 production was inhibited by PKC activity.     

Elevated plasma IL-6 associates with increased risk of advanced fibrosis and cholangiocarcinoma in individuals infected by Opisthorchis viverrini

Opisthorchis viverrini is considered among the most important of the food-borne trematodes due to its strong association with advanced periductal fibrosis and bile duct cancer (cholangiocarcinoma). We investigated the relationship between plasma levels of Interleukin (IL)-6 and the risk of developing advanced fibrosis and bile duct cancer from chronic Opisthorchis infection. We show that IL-6 circulates in plasma at concentrations 58 times higher in individuals with advanced fibrosis than age, sex, and nearest-neighbor matched controls and 221 times higher in individuals with bile duct cancer than controls. We also observed a dose-response relationship between increasing levels of plasma IL-6 and increasing risk of advanced fibrosis and bile duct cancer; for example, in age and sex adjusted analyses, individuals with the highest quartiles of plasma IL-6 had a 19 times greater risk of developing advanced periductal fibrosis and a 150 times greater risk of developing of bile duct cancer than individuals with no detectable level of plasma IL-6. Finally, we show that a single plasma IL-6 measurement has excellent positive predictive value for the detection of both advanced bile duct fibrosis and bile duct cancer in regions with high O. viverrini transmission. These data support our hypothesis that common mechanisms drive bile duct fibrosis and bile duct tumorogenesis from chronic O. viverrini infection. Our study also adds a unique aspect to the literature on circulating levels of IL-6 as an immune marker of hepatobiliary pathology by showing that high levels of circulating IL-6 in plasma are not related to infection with O. viverrini, but to the development of the advanced and often lethal pathologies resulting from chronic O. viverrini infection.