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Regulation of hepatocyte growth factor in mice with pneumonia by peptidases and trans-alveolar flux.

Raymond WW, Xu X, Nimishakavi S, Le C, McDonald DM, Caughey GH - PLoS ONE (2015)

Bottom Line: These findings are consistent with trans-alveolar flux rather than local production as the source of increased HGF in lavage fluid.Consistent with the presence of active HGF, increased expression of activated receptor c-Met was observed in infected tissues.These data suggest that HGF entering alveoli from the bloodstream during pneumonia compensates for destruction by Dppi-activated inflammatory proteases to allow HGF to contribute to epithelial repair.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Institute, School of Medicine, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Hepatocyte growth factor (HGF) promotes lung epithelial repair after injury. Because prior studies established that human neutrophil proteases inactivate HGF in vitro, we predicted that HGF levels decrease in lungs infiltrated with neutrophils and that injury is less severe in lungs lacking HGF-inactivating proteases. After establishing that mouse neutrophil elastase cleaves mouse HGF in vitro, we tested our predictions in vivo by examining lung pathology and HGF in mice infected with Mycoplasma pulmonis, which causes neutrophilic tracheobronchitis and pneumonia. Unexpectedly, pneumonia severity was similar in wild type and dipeptidylpeptidase I-deficient (Dppi-/-) mice lacking neutrophil serine protease activity. To assess how this finding related to our prediction that Dppi-activated proteases regulate HGF levels, we measured HGF in serum, bronchoalveolar lavage fluid, and lung tissue from Dppi(+/+) and Dppi(-/-) mice. Contrary to prediction, HGF levels were higher in lavage fluid from infected mice. However, serum and tissue concentrations were not different in infected and uninfected mice, and HGF lung transcript levels did not change. Increased HGF correlated with increased albumin in lavage fluid from infected mice, and immunostaining failed to detect increased lung tissue expression of HGF in infected mice. These findings are consistent with trans-alveolar flux rather than local production as the source of increased HGF in lavage fluid. However, levels of intact HGF from infected mice, normalized for albumin concentration, were two-fold higher in Dppi(-/-) versus Dppi(+/+) lavage fluid, suggesting regulation by Dppi-activated proteases. Consistent with the presence of active HGF, increased expression of activated receptor c-Met was observed in infected tissues. These data suggest that HGF entering alveoli from the bloodstream during pneumonia compensates for destruction by Dppi-activated inflammatory proteases to allow HGF to contribute to epithelial repair.

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Localization of c-Met expression by confocal immunofluorescence microscopy in pathogen-free and mycoplasma-infected lungs.A: representative image of endothelial cell (green, PECAM-1) and c-MET (red) staining of alveoli in a pathogen-free mouse. Scale bar = 20 μm. B: Representative image of PECAM-1 (green) and c-Met (red) staining in lung section of a pathogen-free mouse. Airway epithelium is marked by an arrowhead. Scale bar = 50 μm. C: representative image of PECAM-1 (green) and c-Met (red) staining in lung section of a 7-day M. pulmonis-infected mouse. Scale bar = 50 μm. D, E, and F: c-Met staining only, corresponding to A, B, and C, respectively.
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pone.0125797.g006: Localization of c-Met expression by confocal immunofluorescence microscopy in pathogen-free and mycoplasma-infected lungs.A: representative image of endothelial cell (green, PECAM-1) and c-MET (red) staining of alveoli in a pathogen-free mouse. Scale bar = 20 μm. B: Representative image of PECAM-1 (green) and c-Met (red) staining in lung section of a pathogen-free mouse. Airway epithelium is marked by an arrowhead. Scale bar = 50 μm. C: representative image of PECAM-1 (green) and c-Met (red) staining in lung section of a 7-day M. pulmonis-infected mouse. Scale bar = 50 μm. D, E, and F: c-Met staining only, corresponding to A, B, and C, respectively.

Mentions: To identify and compare local sources of HGF and of its receptor c-Met in pathogen-free and mycoplasma-infected mice, lung tissue sections were immunostained with antibodies detecting HGF, c-Met and activated phospho-c-Met (Fig 5). HGF expression was diffuse but similar in airway and alveolar epithelial structures in pathogen-free and infected mice. No non-specific staining was seen in sections incubated with secondary antibody alone, omitting primary antibody. c-Met immunoreactivity was strong in airway epithelium but weak to absent in stromal tissues. Phospho-c-Met staining was lighter but with similar overall distribution to that of c-Met in epithelial structures from pathogen-free and infected mice. However, compared to tissues from pathogen-free mice, lungs from infected mice were infiltrated by inflammatory cells, some of which were recognized by antibodies to c-Met and phospho-c-Met. Therefore, overall expression of activated c-Met increased in infected lungs. To localize expression of c-Met in airway and alveolar structures in relation to vascular structures, lung tissues were subjected to confocal immunofluorescence microscopy using antibodies to c-Met and to endothelium-selective CD31. These results, shown in Fig 6, confirm strong expression of c-Met in airway epithelial cells and localize alveolar expression to a subset of epithelial cells and intra-alveolar mononuclear cells, in which expression increased in lungs from infected mice. c-Met was not detected in alveolar endothelium, as indicated by the lack of co-localization of the c-Met signal with CD31.


Regulation of hepatocyte growth factor in mice with pneumonia by peptidases and trans-alveolar flux.

Raymond WW, Xu X, Nimishakavi S, Le C, McDonald DM, Caughey GH - PLoS ONE (2015)

Localization of c-Met expression by confocal immunofluorescence microscopy in pathogen-free and mycoplasma-infected lungs.A: representative image of endothelial cell (green, PECAM-1) and c-MET (red) staining of alveoli in a pathogen-free mouse. Scale bar = 20 μm. B: Representative image of PECAM-1 (green) and c-Met (red) staining in lung section of a pathogen-free mouse. Airway epithelium is marked by an arrowhead. Scale bar = 50 μm. C: representative image of PECAM-1 (green) and c-Met (red) staining in lung section of a 7-day M. pulmonis-infected mouse. Scale bar = 50 μm. D, E, and F: c-Met staining only, corresponding to A, B, and C, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418689&req=5

pone.0125797.g006: Localization of c-Met expression by confocal immunofluorescence microscopy in pathogen-free and mycoplasma-infected lungs.A: representative image of endothelial cell (green, PECAM-1) and c-MET (red) staining of alveoli in a pathogen-free mouse. Scale bar = 20 μm. B: Representative image of PECAM-1 (green) and c-Met (red) staining in lung section of a pathogen-free mouse. Airway epithelium is marked by an arrowhead. Scale bar = 50 μm. C: representative image of PECAM-1 (green) and c-Met (red) staining in lung section of a 7-day M. pulmonis-infected mouse. Scale bar = 50 μm. D, E, and F: c-Met staining only, corresponding to A, B, and C, respectively.
Mentions: To identify and compare local sources of HGF and of its receptor c-Met in pathogen-free and mycoplasma-infected mice, lung tissue sections were immunostained with antibodies detecting HGF, c-Met and activated phospho-c-Met (Fig 5). HGF expression was diffuse but similar in airway and alveolar epithelial structures in pathogen-free and infected mice. No non-specific staining was seen in sections incubated with secondary antibody alone, omitting primary antibody. c-Met immunoreactivity was strong in airway epithelium but weak to absent in stromal tissues. Phospho-c-Met staining was lighter but with similar overall distribution to that of c-Met in epithelial structures from pathogen-free and infected mice. However, compared to tissues from pathogen-free mice, lungs from infected mice were infiltrated by inflammatory cells, some of which were recognized by antibodies to c-Met and phospho-c-Met. Therefore, overall expression of activated c-Met increased in infected lungs. To localize expression of c-Met in airway and alveolar structures in relation to vascular structures, lung tissues were subjected to confocal immunofluorescence microscopy using antibodies to c-Met and to endothelium-selective CD31. These results, shown in Fig 6, confirm strong expression of c-Met in airway epithelial cells and localize alveolar expression to a subset of epithelial cells and intra-alveolar mononuclear cells, in which expression increased in lungs from infected mice. c-Met was not detected in alveolar endothelium, as indicated by the lack of co-localization of the c-Met signal with CD31.

Bottom Line: These findings are consistent with trans-alveolar flux rather than local production as the source of increased HGF in lavage fluid.Consistent with the presence of active HGF, increased expression of activated receptor c-Met was observed in infected tissues.These data suggest that HGF entering alveoli from the bloodstream during pneumonia compensates for destruction by Dppi-activated inflammatory proteases to allow HGF to contribute to epithelial repair.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Institute, School of Medicine, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Hepatocyte growth factor (HGF) promotes lung epithelial repair after injury. Because prior studies established that human neutrophil proteases inactivate HGF in vitro, we predicted that HGF levels decrease in lungs infiltrated with neutrophils and that injury is less severe in lungs lacking HGF-inactivating proteases. After establishing that mouse neutrophil elastase cleaves mouse HGF in vitro, we tested our predictions in vivo by examining lung pathology and HGF in mice infected with Mycoplasma pulmonis, which causes neutrophilic tracheobronchitis and pneumonia. Unexpectedly, pneumonia severity was similar in wild type and dipeptidylpeptidase I-deficient (Dppi-/-) mice lacking neutrophil serine protease activity. To assess how this finding related to our prediction that Dppi-activated proteases regulate HGF levels, we measured HGF in serum, bronchoalveolar lavage fluid, and lung tissue from Dppi(+/+) and Dppi(-/-) mice. Contrary to prediction, HGF levels were higher in lavage fluid from infected mice. However, serum and tissue concentrations were not different in infected and uninfected mice, and HGF lung transcript levels did not change. Increased HGF correlated with increased albumin in lavage fluid from infected mice, and immunostaining failed to detect increased lung tissue expression of HGF in infected mice. These findings are consistent with trans-alveolar flux rather than local production as the source of increased HGF in lavage fluid. However, levels of intact HGF from infected mice, normalized for albumin concentration, were two-fold higher in Dppi(-/-) versus Dppi(+/+) lavage fluid, suggesting regulation by Dppi-activated proteases. Consistent with the presence of active HGF, increased expression of activated receptor c-Met was observed in infected tissues. These data suggest that HGF entering alveoli from the bloodstream during pneumonia compensates for destruction by Dppi-activated inflammatory proteases to allow HGF to contribute to epithelial repair.

Show MeSH
Related in: MedlinePlus