Figure 4

Fibrotic human lungs are enriched for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and CD45⁺Pro-collagen (Col)-Iα1⁺ cells. (a,b) Assessment of cleaved caspase 3 (brown cytoplasmic stain) on lung biopsy sample from (a) non-fibrotic and (b) idiopathic pulmonary fibrosis (IPF) lung sample. Nuclei are counterstained with hematoxylin (60 × original magnification). An increase in caspase cleavage is seen in the IPF lung. (c,d) TUNEL staining (black nuclear stain) performed on (c) non-fibrotic and (d) IPF lung shows an increase in TUNEL+ nuclei in the IPF lung. Nuclei are counterstained with nuclear fast red, 40 × original magnification. (e) Compared to non-fibrotic samples (left) TUNEL staining is increased in lungs obtained from subjects with IPF (middle) or connective tissue disease interstitial lung disease (CTD-ILD) (right). (f) CD45⁺Pro-Col-Iα1⁺ cells are increased in fibrotic lungs. Top left: intracellular isotype (x axis) versus CD45-PE (y axis) on cells obtained from a non-fibrotic lung. The diagonal morphology of the cells reflects the high inherent autofluorescence of lung inflammatory cells. Top right: Pro-Col-Iα1- fluorescein isothiocyanate (FITC) (x axis) versus CD45-PE (y axis) on non-fibrotic lung. There is only minimal shift past the isotype control. Bottom left: intracellular isotype (x axis) versus CD45-PE (y axis) in cells obtained from an IPF lung. Bottom right: Pro-Col-Iα1-FITC (x axis) versus D45-PE (y axis) on IPF lung. (g) Compared to non-fibrotic subjects (n = 6) (left), detection of CD45⁺Pro-Col-Iα1⁺ cells is increased in the lungs of subjects with either IPF (n = 4, middle) or CTD-ILD (n = 4, right). *P < 0.05 **P < 0.01.