Cellular inflammatory infiltrate in pneumonitis induced by a single moderate dose of thoracic x radiation in rats

Abstract

The goal of these studies was to characterize the infiltrating inflammatory cells during pneumonitis caused by moderate doses of radiation. Two groups of male rats (WAG/RijCmcr, 8 weeks old) were treated with single 10- or 15-Gy doses of thoracic X radiation; a third group of age-matched animals served as controls. Only 25% rats survived the 15-Gy dose. Bronchoalveolar lavage fluid and whole lung mounts were subjected to cytological and histological evaluation after 8 weeks for distribution of resident macrophages, neutrophils, lymphocytes and mast cells. There was a modest increase in airway and airspace-associated neutrophils in lungs from rats receiving 15 Gy. Mast cells (detected by immunohistochemistry for tryptase) increased over 70% with 10 Gy and over 13-fold after 15 Gy, with considerable leakage of tryptase into blood vessels and airways. Circulating levels of eight inflammatory cytokines were not altered after 10 Gy but appeared to decrease after 15 Gy. In summary, there were only modest increases in cellular inflammatory infiltrate during pneumonitis after a non-lethal dose of 10 Gy, but there was a dramatic rise in mast cell infiltration after 15 Gy, suggesting that circulating levels of mast cell products may be useful markers of severe pneumonitis.

FIG. 1

Actuarial survival curves for control rats and rats receiving 10 and 15 Gy thoracic radiation. Numbers of animals: 10 Gy: 8, 15 Gy: 12. Numbers in parentheses show the numbers of animals still at risk at various times.

FIG. 2

Differential counts of cells recovered in bronchoalveolar lavage fluid. Bars are means ± SD for differential counts of neutrophils (open bars), lymphocytes (hatched bars) and macrophages (shaded bars). The number of rats for each group is shown within each bar. There were no significant differences between groups.

FIG. 3

Lung sections stained with hematoxylin and eosin. Panel A: Typical section from a control rat showing normal alveoli. Panel B: Rat lung after 10 Gy demonstrating focal minor reactive alveolar change (solid arrow). Panel C: Rat lung after 15 Gy showing focal mild increase in perivascular cellularity with inflammation (between arrows) and reactive alveolar changes.

FIG. 4

Rat lung stained with anti-tryptase antibody to show mast cells in brown. Panel A: Control lung showing only rare perivascular mast cells, with no notable peripheral parenchymal mast cells. Panel B: Lung irradiated with 10 Gy showing distinct perivascular mast cell infiltration, with very few peripheral parenchymal mast cells. Panel C: Lung irradiated with 15 Gy showing diffuse infiltration of the parenchyma (including alveolar walls) by mast cell. Panel D: Cell counts from sections stained with anti-tryptase antibody. Cells were counted in five randomly selected fields from each lung. Bars are means ± SD; the number of rats is shown within each bar.

FIG. 5

Rat lungs stained with anti-tryptase antibody for mast cells. Panel A: 10 Gy, interstitial mast cell. There is no tryptase leakage onto the alveolar lining (dotted arrow). Panel B: 15 Gy, interstitial mast cell in alveolar walls, with tryptase (post degranulation) adsorbed onto the alveolar lining (solid arrow). Panel C: 10 Gy, endothelial lining free of stain from leaked tryptase (dotted arrows). Panel D: 15 Gy, membranous endothelial stain (solid arrow) and red cell stain (double-lined arrow) secondary to mast cell degranulation and tryptase leakage.

FIG. 6

Rat lung stained with anti-CD 45 antibody to show lymphocytes and larger macrophages that stain with light membranous positivity. Panel A: Control rat lung demonstrating scattered small lymphocytes (dotted arrow) and larger macrophages (solid arrow) in the interstitium as well as within the alveoli (red arrows). Panel B: Rat lung irradiated with 10 Gy demonstrating perivascular accentuation of lymphocytes (dotted arrow). The solid arrow points to a macrophage. Panel C: Rat lung irradiated with 15 Gy showing lymphocytic vasculitis (example of most severe lesion), with numerous lymphocytes infiltrating through vascular wall and a few into the subendothelial space (dotted arrows), and macrophages (solid arrows) showing membranous stain in perivascular parenchyma. The grouped arrows encompass a subendothelial fibrinoid cushion in the artery. Panel D: Counts of anti-CD45-positive cells. Bars are mean numbers of cells ± SD for five randomly selected fields. The number of rats is shown within each bar.

FIG. 7

Rat lung stained with anti-CD 45 antibody demonstrating vasculitis and endotheliitis at 15 Gy. Panel A: Vascular wall of unirradiated lung with lymphocytes rolling on the endothelial surface, but not infiltrating. Solid arrow points to intact endothelial cell. Panel B: Vascular wall of lung after 10 Gy showing early endotheliitis/subendotheliitis in the endothelial layer. Solid arrow points to an endothelial cell. Panel C: Rat lung irradiated with 15 Gy showing marked lymphocytic subendotheliitis. Numerous lymphocytes (dotted arrows) traversing and underlying the endothelium (solid arrows), which is partially lifted.

FIG. 8

Rat lungs stained with anti-myeloperoxidase antibody to show neutrophils in brown. Panel A: Control rat. Panel B: Rat irradiated with 10 Gy. Panel C: Rat irradiated with 15 Gy. Panel D: Cell counts from sections stained with anti-myeloperoxidase showing an increase in neutrophils after 15 Gy. Bars show number of cells for five randomly selected from each lung. The number of rats is given within each bar.

FIG. 9

Rat lung sections treated with trichrome stain to demonstrate fibrosis (blue) at 8 weeks after irradiation. Panel A: Control rat. Panel B: Rat irradiated with 10 Gy. Panel C: Rat irradiated with 15 Gy. Panel D: Scores for relative areas of blue stain (marked by arrows) compared to counterstain (red). Bars are the shows mean scores ± SD for each group. The number of rats/group is indicated within each bar.

FIG. 10

Levels of eight rat cytokines measured in serum from rats at 8 weeks after irradiation of the whole thorax with 10 Gy (solid bars) or 15 Gy of X rays (hatched bars) and age-matched controls (open bars). Values are the percentage of the control ± SD for each cytokine.