Convergent sets of data from in vivo and in vitro methods point to an active role of Hsp60 in chronic obstructive pulmonary disease pathogenesis

Abstract

Background: It is increasingly clear that some heat shock proteins (Hsps) play a role in inflammation. Here, we report results showing participation of Hsp60 in the pathogenesis of chronic obstructive pulmonary diseases (COPD), as indicated by data from both in vivo and in vitro analyses.

Methods and results: Bronchial biopsies from patients with stable COPD, smoker controls with normal lung function, and non-smoker controls were studied. We quantified by immunohistochemistry levels of Hsp10, Hsp27, Hsp40, Hsp60, Hsp70, Hsp90, and HSF-1, along with levels of inflammatory markers. Hsp10, Hsp40, and Hsp60 were increased during progression of disease. We found also a positive correlation between the number of neutrophils and Hsp60 levels. Double-immunostaining showed that Hsp60-positive neutrophils were significantly increased in COPD patients. We then investigated in vitro the effect on Hsp60 expression in bronchial epithelial cells (16HBE) caused by oxidative stress, a hallmark of COPD mucosa, which we induced with H₂O₂. This stressor determined increased levels of Hsp60 through a gene up-regulation mechanism involving NFkB-p65. Release of Hsp60 in the extracellular medium by the bronchial epithelial cells was also increased after H₂O₂ treatment in the absence of cell death.

Conclusions: This is the first report clearly pointing to participation of Hsps, particularly Hsp60, in COPD pathogenesis. Hsp60 induction by NFkB-p65 and its release by epithelial cells after oxidative stress can have a role in maintaining inflammation, e.g., by stimulating neutrophils activity. The data open new scenarios that might help in designing efficacious anti-inflammatory therapies centered on Hsp60 and applicable to COPD.

Figure 1. Measurement of Hsps in the bronchial epithelium and lamina propria: Comparison between groups of patients.

Hsp10, Hsp40, and Hsp60 immunopositive cells in the epithelium (top panels A, C, E) and in the lamina propria (bottom panels B, D, F) of the four groups studied: Control non Smokers, Control Smokers, Mild/Moderate COPD, and Severe/Very Severe COPD. Results are expressed as the median and interquartile range (IQR) of scored (0–3, vertical axis) immunopositivity in the epithelium, or as number of immunopositive cells per square millimeter (vertical axis) in the lamina propria; “p” values are shown on top of the lines spanning the two groups being compared.

Figure 2. Hsps in the bronchial epithelium and lamina propria: Representative images.

Photomicrographs showing frozen sections of bronchial mucosa from a control non smoker (A, C, E) and from a patient with severe stable COPD (B, D, F) immunostained to identify Hsp10 (A, B), Hsp40 (C, D), and Hsp60 (E, F). Nuclei were counterstained with haematoxylin (blue). Cells positive for Hsps are in red. Inset in F shows a cell double stained for neutrophil elastase (red) and Hsp60 (brown). Bar  = 50 microns.

Figure 3. Correlations between neutrophils and Hsp60.

Regression analysis between number of Hsp60 positive cells (vertical axis) and number of neutrophils (horizontal axis), panels A and B, and between number of Hsp60 positive cells (vertical axis) and number of MPO positive neutrophils (horizontal axis), panels C and D, in the lamina propria of all smokers (panels A and C) and of patients with COPD alone, considered as a single group (panels B and D).

Figure 4. Impact of oxidative stress on the levels of Hsp60 in a human bronchial epithelial cell line.

Panel A: Western blotting showing significantly increased levels of Hsp60 in 16HBE after treatment with 50 or 100 μM of H₂O₂ compared to untreated (UT) cells. The difference between 100 and 50 μM was also significant. Histograms represent the mean (SD) of the Hsp60/actin ratio. Panel B: RT-PCR showing significantly increased levels of Hsp60 only after treatment with 100 μM of H₂O₂ compared to UT treated cells. Panel C: Hsp60 increased levels in the extracellular medium (measured by ELISA tests) after treatment with 50 or 100 μM of H₂O₂ compared to untreated (UT) cells. ^: p<0.005; *: p<0.001.

Figure 5. Quantification of mRNA in the bronchial biopsies and binding of NFkB-p65 to the human hsp60-gene promoter region.

Panel A: Levels of HSF-1 and NFKB-p65 mRNA did not change significantly after treatment (50 or 100 µM of H₂O₂). Panel B: the binding of NFkB-p65 to the hsp60 promoter was lower in untreated (UT) cells (lane 2) compared to the binding in cells treated with 50 or 100 µM of H₂O₂ (lanes 3 and 4, respectively). Lanes: 1, Marker; 5, DNA UT Positive control; 6, DNA UT Negative control; and 7, Input DNA.