S100A9 is indispensable for survival of pneumococcal pneumonia in mice.

S100A8/A9 has important immunomodulatory roles in antibacterial defense, but its relevance in focal pneumonia caused by Streptococcus pneumoniae (S. pneumoniae) is understudied. We show that S100A9 was significantly increased in BAL fluids of patients with bacterial but not viral pneumonia and correlated with procalcitonin and sequential organ failure assessment scores. Mice deficient in S100A9 exhibited drastically elevated Zn2+ levels in lungs, which led to bacterial outgrowth and significantly reduced survival. In addition, reduced survival of S100A9 KO mice was characterized by excessive release of neutrophil elastase, which resulted in degradation of opsonophagocytically important collectins surfactant proteins A and D. All of these features were attenuated in S. pneumoniae-challenged chimeric WT→S100A9 KO mice. Similarly, therapy of S. pneumoniae-infected S100A9 KO mice with a mutant S100A8/A9 protein showing increased half-life significantly decreased lung bacterial loads and lung injury. Collectively, S100A9 controls central antibacterial immune mechanisms of the lung with essential relevance to survival of pneumococcal pneumonia. Moreover, S100A9 appears to be a promising biomarker to distinguish patients with bacterial from those with viral pneumonia. Trial registration: Clinical Trials register (DRKS00000620).

Cathepsin G and neutrophil elastase contribute to lung-protective immunity against mycobacterial infections in mice.

The neutrophil serine proteases cathepsin G (CG) and neutrophil elastase (NE) are involved in immune-regulatory processes and exert antibacterial activity against various pathogens. To date, their role and their therapeutic potential in pulmonary host defense against mycobacterial infections are poorly defined. In this work, we studied the roles of CG and NE in the pulmonary resistance against Mycobacterium bovis bacillus Calmette-Guérin (BCG). CG-deficient mice and even more pronounced CG/NE-deficient mice showed significantly impaired pathogen elimination to infection with M. bovis BCG in comparison to wild-type mice. Moreover, granuloma formation was more pronounced in M. bovis BCG-infected CG/NE-deficient mice in comparison to CG-deficient and wild-type mice. A close examination of professional phagocyte subsets revealed that exclusively neutrophils shuttled CG and NE into the bronchoalveolar space of M. bovis BCG-infected mice. Accordingly, chimeric wild-type mice with a CG/NE-deficient hematopoietic system displayed significantly increased lung bacterial loads in response to M. bovis BCG infection. Therapeutically applied human CG/NE encapsulated in liposomes colocalized with mycobacteria in alveolar macrophages, as assessed by laser scanning and electron microscopy. Importantly, therapy with CG/NE-loaded liposomes significantly reduced mycobacterial loads in the lungs of mice. Together, neutrophil-derived CG and NE critically contribute to deceleration of pathogen replication during the early phase of antimycobacterial responses. In addition, to our knowledge, we show for the first time that liposomal encapsulated CG/NE exhibit therapeutic potential against pulmonary mycobacterial infections. These findings may be relevant for novel adjuvant approaches in the treatment of tuberculosis in humans.

Oral zinc aspartate treats experimental autoimmune encephalomyelitis.

The essential trace element zinc plays a critical role in the regulation of immune homeostasis. Zinc deficiency or excess can cause severe impairment of the immune response, which points to the importance of the physiological and dietary control of zinc levels for a functioning immune system. We previously reported that injection of zinc aspartate suppresses experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), as well as effector T cell functions in vitro. Among the preferred characteristics of novel therapeutics for the treatment of autoimmune diseases such as MS are oral availability and a tolerable effective dose to minimize side effects. In this study, we investigated whether oral administration of zinc aspartate, an approved drug to treat zinc deficiency in humans, is effective in controlling EAE at clinically approved doses. We show that oral administration of 6 µg/day [0.3 mg/kg body weight (BW)] or 12 µg/day [0.6 mg/kg BW] of zinc aspartate reduces clinical and histopathological signs during the relapsing remitting phase of the disease in SJL mice. The clinical effect in mice was accompanied by suppression of IFN-γ, TNF-α, GM-CSF and IL-5 production in stimulated human T cells and mouse splenocytes in a dose-dependent manner. Furthermore, a large array of proinflammatory cytokines was modulated by zinc aspartate exposure in vitro. These data suggest that administration of oral zinc aspartate may have beneficial effects on autoimmune diseases like MS.

Allergic airway inflammation in mice deficient for the antigen-processing protease cathepsin E.

BACKGROUND: Allergic asthma is a Th2-type chronic inflammatory disease of the lung. It is characterized by infiltration of eosinophils, neutrophils, mast cells and T lymphocytes into the airways. Th2 cytokines like interleukin (IL)-4, IL-5 and chemokines like eotaxin are increased in the asthmatic response. The processing and presentation of exogenous antigens is important in the sensitization to an allergen. Cathepsin E (Ctse) is an intracellular aspartic endoprotease which is expressed in immune cells like dendritic cells (DCs). It was found to play an essential role in the processing and presentation of ovalbumin (OVA). The aim of the present study was to investigate the inhibition of Ctse in two different experimental models of allergic airway inflammation. METHODS: Ctse wild-type (Ctse(+/+)) and Ctse-deficient (Ctse(-/-)) bone marrow-derived DCs (BMDCs) were pulsed with OVA/OVA peptide and cocultured with OVA transgenic T II (OT II) cells whose proliferation was subsequently analyzed. Two different in vivo asthma models with Ctse(+/+) and Ctse(-/-) mice were performed: an acute OVA-induced and a subchronic Phleum pratense-induced airway inflammation. RESULTS: Proliferation of OT II cells was decreased when cocultured with BMDCs of Ctse(-/-) mice as compared to cells cocultured with BMDCs of Ctse(+/+) mice. In vivo, Ctse deficiency led to reduced lymphocyte influx after allergen sensitization and challenge in both investigated airway inflammation models, compared to their control groups. CONCLUSION: Ctse deficiency leads to a reduced antigen presentation in vitro. This is followed by a distinct effect on lymphocyte influx in states of allergic airway inflammation in vivo.

Interleukin 6, lipopolysaccharide-binding protein and interleukin 10 in the prediction of risk and etiologic patterns in patients with community-acquired pneumonia: results from the German competence network CAPNETZ.

BACKGROUND: The aim of our study was to investigate the predictive value of the biomarkers interleukin 6 (IL-6), interleukin 10 (IL-10) and lipopolysaccharide-binding protein (LBP) compared with clinical CRB and CRB-65 severity scores in patients with community-acquired pneumonia (CAP). METHODS: Samples and data were obtained from patients enrolled into the German CAPNETZ study group. Samples (blood, sputum and urine) were collected within 24 h of first presentation and inclusion in the CAPNETZ study, and CRB and CRB-65 scores were determined for all patients at the time of enrollment. The combined end point representative of a severe course of CAP was defined as mechanical ventilation, intensive care unit treatment and/or death within 30 days. Overall, a total of 1,000 patients were enrolled in the study. A severe course of CAP was observed in 105 (10.5%) patients. RESULTS: The highest IL-6, IL-10 and LBP concentrations were found in patients with CRB-65 scores of 3-4 or CRB scores of 2-3. IL-6 and LBP levels on enrollment in the study were significantly higher for patients with a severe course of CAP than for those who did not have severe CAP. In receiver operating characteristic analyses, the area under the curve values for of IL-6 (0.689), IL-10 (0.665) and LPB (0.624) in a severe course of CAP were lower than that of CRB-65 (0.764) and similar to that of CRB (0.69). The accuracy of both CRB and CRB-65 was increased significantly by including IL-6 measurements. In addition, higher cytokine concentrations were found in patients with typical bacterial infections compared with patients with atypical or viral infections and those with infection of unknown etiology. LBP showed the highest discriminatory power with respect to the etiology of infection. CONCLUSIONS: IL-6, IL-10 and LBP concentrations were increased in patients with a CRB-65 score of 3-4 and a severe course of CAP. The concentrations of IL-6 and IL-10 reflected the severity of disease in patients with CAP. The predictive power of IL-6, IL-10 and LBP for a severe course of pneumonia was lower than that of CRB-65. Typical bacterial pathogens induced the highest LBP, IL-6 and IL-10 concentrations.

ApoE-deficient mice on cholate-containing high-fat diet reveal a pathology similar to lung sarcoidosis.

Sarcoidosis is a chronic disease of unknown etiology characterized by the formation of non-necrotizing epithelioid granulomas in various organs, especially in the lungs. The lack of an adequate animal model reflecting the pathogenesis of the human disease is one of the major impediments in studying sarcoidosis. In this report, we describe ApoE-/- mice on a cholate-containing high-fat diet that exhibit granulomatous lung inflammation similar to human sarcoidosis. Histological analysis revealed well-defined and non-necrotizing granulomas in about 40% of mice with the highest number of granulomas after 16 weeks on a cholate-containing high-fat diet. Granulomas contained CD4+ and CD8+ T cells, and the majority of the cells in granulomas showed immunoreactivity for the macrophage marker Mac-3. Cells with morphological features of epithelioid cells expressed angiotensin-converting enzyme, osteopontin, and cathepsin K, all characteristics of epithelioid and giant cells in granulomas of human sarcoidosis. Giant cells and nonspecific inclusions such as Schaumann's bodies and crystalline deposits were also detected in some lungs. Granulomatous inflammation resulted in progressive pulmonary fibrosis. Removal of cholate from the diet prevented the formation of lung granulomas. The observed similarities between the analyzed mouse lung granulomas and granulomas of human sarcoidosis, as well as the chronic disease character leading to fibrosis, suggest that this mouse model might be a useful tool to study sarcoidosis.

Modulation of cytokine production and silica-induced lung fibrosis by inhibitors of aminopeptidase N and of dipeptidyl peptidase-IV-related proteases.

AIMS: Dipeptidyl peptidase IV (DP IV)-related proteases and aminopeptidase N (APN) are drug targets in various diseases. Here we investigated for the first time the effects of DP-IV-related protease inhibitors and APN inhibitors on chronic inflammatory lung diseases. MAIN METHODS: A murine model of silica (SiO2)-induced lung fibrosis and in vitro cultures of human lung epithelial cells and monocytes have been used and the influence of silica-treatment and inhibitors on inflammation and fibrosis has been measured. KEY FINDINGS: We found increased inflammation and secretion of the chemokines IL-6, MCP-1 and MIP-alpha 2 weeks after SiO2 application, and increased lung fibrosis after 3 months. Treatment with the APN inhibitor actinonin reduced chemokine secretion in the lung and bronchoalveolar lavage fluid, and in cell culture, and decreased the level of fibrosis after 3 months. Treatment with inhibitors of DP-IV-related proteases, or a combination of DP IV inhibitors and APN inhibitors, had no significant effect. We found no obvious side effects of long-term treatment with inhibitors of APN and DP IV. SIGNIFICANCE: Overall, our findings show that actinonin, an inhibitor of aminopeptidase N, might modulate chemokine secretion in the lung and thus attenuate the development of lung fibrosis. Additional targeting of DP-IV-related proteases had no significant effect on these processes.

Radiation-induced matrix production of lung fibroblasts is regulated by interleukin-8.

PURPOSE: Lung fibrosis can be caused by radiation therapy during cancer treatment and therefore can be the limiting factor of the treatment. The factors that cause the actual fibrosis and the interaction between different cell types were investigated. MATERIALS AND METHODS: Epithelial lung cells and fibroblasts were irradiated and different cytokines were measured in the supernatant. Also effects of radiation on the matrix production of fibroblasts were investigated. RESULTS: Irradiation of isolated lung fibroblasts did not cause increased extracellular matrix production; however, the co-culturing of fibroblasts and irradiated lung epithelial cells or the treatment of fibroblasts with supernatants of irradiated epithelial cells did result in an increase. We were able to show that increased interleukin-8 (IL-8) levels led to increased matrix production. CONCLUSIONS: IL-8 is not only a proinflammatory cytokine but it also stimulates collagen synthesis and matrix production and therefore could be a possible drug target in preventing radiation damage during cancer therapy.

Overexpression of cathepsin K in mice decreases collagen deposition and lung resistance in response to bleomycin-induced pulmonary fibrosis.

BACKGROUND: Lung fibrosis is a devastating pulmonary disorder characterized by alveolar epithelial injury, extracellular matrix deposition and scar tissue formation. Due to its potent collagenolytic activity, cathepsin K, a lysosomal cysteine protease is an interesting target molecule with therapeutic potential to attenuate bleomycin-induced pulmonary fibrosis in mice. We here tested the hypothesis that over-expression of cathepsin K in the lungs of mice is protective in bleomycin-induced pulmonary fibrosis. METHODS: Wild-type and cathepsin K overexpressing (cathepsin K transgenic; cath K tg) mice were challenged intratracheally with bleomycin and sacrificed at 1, 2, 3 and 4 weeks post-treatment followed by determination of lung fibrosis by estimating lung collagen content, lung histopathology, leukocytic infiltrates and lung function. In addition, changes in cathepsin K protein levels in the lung were determined by immunohistochemistry, real time RT-PCR and western blotting. RESULTS: Cathepsin K protein levels were strongly increased in alveolar macrophages and lung parenchymal tissue of mock-treated cathepsin K transgenic (cath K tg) mice relative to wild-type mice and further increased particularly in cath K tg but also wild-type mice in response to bleomycin. Moreover, cath K tg mice responded with a lower collagen deposition in their lungs, which was accompanied by a significantly lower lung resistance (RL) compared to bleomycin-treated wild-type mice. In addition, cath K tg mice responded with a lower degree of lung fibrosis than wild-type mice, a process that was found to be independent of inflammatory leukocyte mobilization in response to bleomycin challenge. CONCLUSION: Over-expression of cathepsin K reduced lung collagen deposition and improved lung function parameters in the lungs of transgenic mice, thereby providing at least partial protection against bleomycin-induced lung fibrosis.

Tiotropium suppresses acetylcholine-induced release of chemotactic mediators in vitro.

The driving force in the progression of COPD is the development of exacerbations which are mostly the result of excessive inflammation. Bronchodilatators play an important role in the treatment of COPD. The reported reduction in exacerbation rates in COPD is due to the inhibition of vagal-mediated bronchoconstriction and mucus secretion. However, recent studies have highlighted the existence of muscarinic receptors on inflammatory cells and we have explored the possibility that tiotropium bromide might also inhibit neutrophil migration. We analysed the influence of tiotropium on the release of neutrophil chemotactic activity in response to acetylcholine (ACh) and the expression of muscarinic receptors on human alveolar macrophages (AM), A549 cells, MonoMac6 cells, and human lung fibroblasts. We found significant levels of all muscarinic receptor subtypes on all analysed cells except the fibroblasts. Fibroblasts expressed predominantly M2, receptors and did not release chemotactic activity. AM, A549 cells, and MonoMac6 cells released chemotactic active mediators after incubation with ACh. The secretion could be suppressed by more than 70% after coincubation with tiotropium. Tiotropium alone did not influence the granulocyte migration. Most of the chemotactic activity could be attributed to leukotriene B4 (LTB4). The release of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) was not induced by ACh. From this, we suggest that the suppression of the Ach-mediated release of chemotactic substances like LTB4 modulates the inflammatory reaction. This may contribute to the decreased rate of exacerbations in COPD, which was observed in clinical trials.

Proteases and their role in chronic inflammatory lung diseases.

Proteases play an essential role in modulating the turnover of extracellular matrix. Furthermore, they are involved in the processing of various proteins thus regulating fundamental cellular functions such as apoptosis, cells growth and activation, protein secretion and phagocytosis. At the tissue and organ levels, proteases influence mechanisms including cell migration and invasion, cellular interactions and signal transduction as well as tissue formation and stabilization. Proteases are classified based on their catalytic mechanisms into serin, aspartic, metallo, threonin and cysteine proteases and are localized extracellularly, at the cellular surface, in the cytoplasm of cells or within specific subcellular structures such as lysosomes. The present review focuses on the specific functions of lysosomal cysteine proteases and the potential effects of modulators of cysteine protease activity.

Pronapsin A and B gene expression in normal and malignant human lung and mononuclear blood cells.

The two human pronapsin genes which are located immediately adjacent to one another on chromosome 19 were shown, using quantitative RT-PCR, to have distinctly different expression patterns. Transcription of the pronapsin A gene in normal human lung tissue was not appreciably altered in lung carcinomas and comparable pronapsin A mRNA levels were also quantified in lung epithelial cell lines and in tumour cell lines originating from human lung epithelium. Pronapsin A may thus have considerable diagnostic value as a marker for primary lung cancer. In contrast, the pronapsin B gene, which lacks an in-frame stop codon and so may be a transcribed pseudogene, is expressed at comparable levels in normal human spleen, thymus, cytotoxic and helper T-lymphocytes, natural killer (NK) cells and B-lymphocytes. The mRNA levels quantified in B-cells from adults with chronic lymphoblastic leukaemia were not significantly different from those measured in B-cells from healthy individuals. Similarly, comparable levels of expression were quantified in monocytes from healthy individuals and from a patient with acute myeloid leukaemia, whereas in alveolar macrophages, which are terminally differentiated myeloid cells, transcription of the pronapsin B gene was down-regulated by approximately one order of magnitude. Reciprocally, an approximately 20-fold up-regulation in expression of the procathepsin D gene in the macrophages relative to the circulating monocytes was revealed by quantitative measurements made in parallel throughout all of this study for the respective mRNAs encoding the intracellular aspartic proteinases, procathepsin D and procathepsin E. Thus, while there appears to be little difference in expression of the pronapsin A and B genes between healthy and malignant human cells and tissues, the reciprocal alterations in the levels of transcription of the pronapsin B and procathepsin D genes may have significance in the developmental processes associated with myeloid cell differentiation into macrophages.

Altered expression of membrane-bound and soluble CD95/Fas contributes to the resistance of fibrotic lung fibroblasts to FasL induced apoptosis.

BACKGROUND: An altered susceptibility of lung fibroblasts to Fas-induced apoptosis has been implicated in the pathogenesis of pulmonary fibrosis; however, the underlying mechanism is not completely understood. Here, we studied the susceptibility of lung fibroblasts, obtained from patients with (f-fibs) and without pulmonary fibrosis (n-fibs), to FasL- (CD95L/APO-1) induced apoptosis in relation to the expression and the amounts of membrane-bound and soluble Fas. We also analysed the effects of tumor necrosis factor-beta on FasL-induced cell death. METHODS: Apoptosis was induced with recombinant human FasL, with and without prior stimulation of the fibroblasts with tumor necrosis factor-alpha and measured by a histone fragmentation assay and flow cytometry. The expression of Fas mRNA was determined by quantitative PCR. The expression of cell surface Fas was determined by flow cytometry, and that of soluble Fas (sFas) was determined by enzyme-linked immunosorbent assay. RESULTS: When compared to n-fibs, f-fibs were resistant to FasL-induced apoptosis, despite significantly higher levels of Fas mRNA. F-fibs showed lower expression of surface-bound Fas but higher levels of sFas. While TNF-alpha increased the susceptibility to FasL-induced apoptosis in n-fibs, it had no pro-apoptotic effect in f-fibs. CONCLUSIONS: The data suggest that lower expression of surface Fas, but higher levels of apoptosis-inhibiting sFas, contribute to the resistance of fibroblasts in lung fibrosis against apoptosis, to increased cellularity and also to increased formation and deposition of extracellular matrix.

Overexpression of cathepsin K during silica-induced lung fibrosis and control by TGF-beta.

BACKGROUND: Lung fibrosis is characterized by tissue remodeling resulting from an imbalance between synthesis and degradation of extracellular organic matrices. To examine whether cathepsin(s) (Cat) are important in the development of pulmonary fibrosis, we assessed the expression of four Cat known for their collagenolytic activity in a model of silica-induced lung fibrosis. METHODS: Different strains of mice were transorally instilled with 2.5 mg crystalline silica or other particles. Cat expression (Cat K, S, L and B) was quantified in lung tissue and isolated pulmonary cells by quantitative RT-PCR. In vitro, we assessed the effect of different cytokines, involved in lung inflammatory and fibrotic responses, on the expression of Cat K by alveolar macrophages and fibroblasts. RESULTS: In lung tissue, Cat K transcript was the most strongly upregulated in response to silica, and this upregulation was intimately related to the fibrotic process. In mouse strains known for their differential response to silica, we showed that the level of Cat K expression following silica treatment was inversely related to the level of TGF-beta expression and the susceptibility of these strains to develop fibrosis. Pulmonary macrophages and fibroblasts were identified as Cat K overproducing cells in the lung of silicotic mice. In vitro, Cat K was downregulated in mouse and human lung fibroblasts by the profibrotic growth factor TGF-beta1. CONCLUSION: Altogether, these data suggest that while Cat K may contribute to control lung fibrosis, TGF-beta appears to limit its overexpression in response to silica particles.

Clinical proteomics in lung diseases.

Proteomics is a relatively new approach for understanding the pathology and pathogenesis of various diseases. It has also been used for characterizing the modifications in protein expression during the development of interstitial lung diseases, in lung tumors, or following exposure to exogenous stress signals. We compared the protein composition of primary human lung fibroblasts derived from patients with lung fibrosis and control fibroblasts of unaffected lung tissues. We found a predominant modulation in proteins related to the cytoskeleton, including decreased expression of vimentin and lamin A/C, and increased expression of moesin. Furthermore, we observed lower levels of components of the antioxidative system, such as omega class glutathione S-transferase and an up-regulation of an intracellular chloride channel. In fibroblasts obtained from fibrotic lungs, the expression of a major histocompatibility complex class I C was decreased, and so was the expression of tripeptidyl-peptidase-I-precursor, a collagen-degrading exopeptidase. Our results and the studies reviewed in this paper represent just the beginning of detailed studies that should unravel the relevance and the functional consequences of differential protein expressions in the diseased lung.

Gene targeting of the cysteine peptidase cathepsin H impairs lung surfactant in mice.

BACKGROUND: The 11 human cysteine cathepsins are proteases mainly located in the endolysosomal compartment of all cells and within the exocytosis pathways of some secretory cell types. Cathepsin H (Ctsh) has amino- and endopeptidase activities. In vitro studies have demonstrated Ctsh involvement in the processing and secretion of the pulmonary surfactant protein B (SP-B). Furthermore, Ctsh is highly expressed in the secretory organelles of alveolar type II pneumocytes where the surfactant proteins are processed. METHODOLOGY/PRINCIPAL FINDINGS: Hence, we generated Ctsh null mice by gene targeting in embryonic stem cells to investigate the role of this protease in surfactant processing in vivo. The targeting construct contains a ß-galactosidase (lacZ) reporter enabling the visualisation of Ctsh expression sites. Ctsh-deficiency was verified by northern blot, western blot, and measurement of the Ctsh aminopeptidase activity. Ctsh(-/-) mice show no gross phenotype and their development is normal without growth retardation. Broncho-alveolar lavage (BAL) from Ctsh(-/-) mice contained lower levels of SP-B indicating reduced SP-B secretion. The BAL phospholipid concentration was not different in Ctsh(+/+) and Ctsh(-/-) mice, but measurement of surface tension by pulsating bubble surfactometry revealed an impairment of the tension reducing function of lung surfactant of Ctsh(-/-) mice. CONCLUSIONS/SIGNIFICANCE: We conclude that cathepsin H is involved in the SP-B production and reduced SP-B levels impair the physical properties of the lung surfactant. However, Ctsh defiency does not reproduce the severe phenotype of SP-B deficient mice. Hence, other proteases of the secretory pathway of type II pneumocytes, i.e. cathepsins C or E, are still able to produce surfactant of sufficient quality in absence of Ctsh.

Increased carcinogenic potential of myeloid tumor cells induced by aberrant TGF-beta1-signaling and upregulation of cathepsin B.

The TGF-beta signaling pathways are implicated in cancer. Cysteine cathepsins can contribute to the carcinogenic potential of tumor cells. The aim of this study was to investigate the regulation of cysteine cathepsin expression by TGF-beta1 and the functional implications in tumor cells. We found an upregulation of cathepsin B (CathB, 2- to 5-fold) in different myeloid tumor cells (THP-1, MonoMac-1, MonoMac-6) after incubation with TGF-beta1. No upregulation was found in monocytes, and there was suppression of CathB expression in epithelial tumor cells (A549). Increased cathepsin B activity led to enhanced carcinogenic potential, which was reflected by increased migration and invasion of the cells and resistance to inhibitor-induced apoptosis. Analysis of the TGF-beta signaling pathways showed no alterations in TGF-beta/BMP receptor expression or SMAD2/3 phosphorylation, and no influence of MAP kinase pathways. However, a reduction in SMAD1 expression was detected. The lack of BMP action on cysteine cathepsin expression in myeloid tumor cells, but not in epithelial tumor cells, suggests a defect in the Smad1/Smad5 pathway. We located a related TGF-beta1-responsive element within the first intron of the CathB gene. In conclusion, alterations in the TGF-beta1 signaling pathway lead to upregulation of CathB, which contributes to the carcinogenic potential of tumor cells.

Mediator responses of alveolar macrophages and kinetics of mononuclear phagocyte subset recruitment during acute primary and secondary mycobacterial infections in the lungs of mice.

Little is known about the activation programme induced in alveolar macrophages upon phagocytosis of mycobacteria and the concomitant mononuclear phagocyte migratory responses that shape the acute phase of mycobacterial infection. Using high-speed cell sorting in conjunction with real-time RT-PCR analysis, we show that sorted alveolar macrophages of transgenic CX3CR1+/GFP mice infected with red fluorescent-labelled Mycobacterium bovis BCG exhibited weak transcriptional changes of lysosomal cathepsins B, L, D, K and S, whereas antimicrobial cathepsin G was strongly induced upon infection. Moreover, mRNA levels of pattern recognition receptors TLR2, TLR4 and NOD2 were downregulated as were neutrophil chemoattractants KC, MIP-2 and IP-10, whereas highly upregulated mRNA levels of the monocyte chemoattractant CCL2 were observed. M. bovis BCG infection of the mice elicited the alveolar accumulation of highly CX3CR1-positive alveolar dendritic cells but not neutrophils within the alveolar compartment, whereas no increased accumulation of CX3CR1high lung parenchymal dendritic cells (lung DC) or CX3CR1neg lung macrophages compared with controls was noted. In contrast, CX3CR1+/GFP mice previously immunized with M. bovis BCG rapidly responded with increased accumulations of both CX3CR1high alveolar and lung parenchymal DC and CX3CR1neg lung macrophages upon intratracheal M. bovis BCG challenge. Moreover, alveolar and lung macrophages and lung DC were found to contribute to early uptake of M. bovis BCG. Together, acute mycobacterial infection triggers both rapid changes in gene expression profiles in infected alveolar macrophages and a compartment-specific accumulation of mononuclear phagocyte subsets contributing to M. bovis BCG uptake in vivo.

Protease-activated receptor-1 in human lung fibroblasts mediates a negative feedback downregulation via prostaglandin E2.

Among the four protease-activated receptors (PARs), PAR-1 plays an important role in normal lung functioning and in the development of lung diseases, including fibrosis. We compared the expression and functional activity of PARs in normal and fibrotic human lung fibroblasts. Both normal and fibrotic cells express PAR-1, -2, and -3, with PAR-2 showing the lowest level. There was no significant difference between normal and fibrotic fibroblasts in expression levels of PAR-1 and PAR-3, whereas a fourfold higher expression level of PAR-2 was observed in fibrotic cells compared with normal cells. Ca(2+) imaging studies revealed apparently only PAR-1-induced Ca(2+) signaling in lung fibroblasts. PAR-1 agonists, thrombin and synthetic activating peptide, induced concentration-dependent Ca(2+) mobilization with EC(50) values of 5 nM and 1 microM, respectively. The neutrophil protease cathepsin G produced a transient Ca(2+) response followed by disabling PAR-1, whereas elastase did not affect Ca(2+) level. PAR-1 activation by thrombin or receptor-activating peptide downregulated expression of all three PARs in lung fibroblasts, with maximal effect at 3-6 h, whereas expression returned toward basal level after 24 h. Furthermore, PAR-1 agonists dose dependently increased PGE(2) secretion from lung fibroblasts and induction of cyclooxygenase-2 expression. We then found that PGE(2) downregulated expression of all three PARs. The effect of PGE(2) was continuously growing with time. Furthermore, PGE(2) exerts its effect through the EP2 receptor that was confirmed using the selective EP2 agonist butaprost. This novel autocrine feedback mechanism of PGE(2) in lung fibroblasts seems to be an important regulator in lung physiology and pathology.