Cysteinyl leukotriene 1 receptor expression associated with bronchial inflammation in severe exacerbations of COPD

Abstract

Background: Cysteinyl leukotriene 1 (CysLT1) receptor expression is known to be increased in the airway mucosa of patients with asthma, especially during exacerbations; however, nothing is known of its expression in COPD.

Methods: We applied immunohistochemistry and in situ hybridization to endobronchial biopsies to determine inflammatory cell CysLT1 receptor protein and mRNA expression in the following: (1) 15 nonsmoker control subjects (NSC), (2) 16 smokers with moderate to severe COPD in its stable phase (S-COPD), and (3) 15 smokers with COPD hospitalized for a severe exacerbation (SE-COPD).

Results: The total number of bronchial mucosal inflammatory cells (CD45+) and those expressing CysLT1 receptor protein were significantly greater in SE-COPD (CysLT1 receptor protein: median [range] = 139 [31-634]) as compared with S-COPD (32 [6-114]) or NSC (16 [4-66]) (P < .001 for both). CysLT1 receptor gene expression showed similar differences. A greater proportion of CD451 cells expressed CysLT1 receptor protein in SE-COPD (median [range] = 22% [8-81]) compared with S-COPD (10% [4-32]) (P < .03) or NSC (7% [1-19]) (P < .002). In SE-COPD, the relative frequencies of CysLT1 receptor-expressing cells were as follows: tryptase1 mast cells > CD681 monocytes/macrophage > neutrophils > CD201 B lymphocytes = EG21 eosinophils. Moreover, there were positive correlations between the numbers of cells expressing CysLT1 receptor protein and the numbers of CD451 cells (r = 0.78; P < .003) and tryptase1 mast cells (r = 0.62; P < .02).

Conclusions: Bronchial mucosal CysLT1 receptor-positive inflammatory cells are present in the bronchial mucosa in COPD in greatest number in those experiencing a severe exacerbation.

Figure 1.

Nonisotopic in situ hybridization (ISH) of a bronchial biopsy tissue section illustrating cysteinyl leukotriene 1 (CysLT₁) receptor mRNA expression. The positive signal is visualized as blue/black with the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) technique. Cells are counterstained with nuclear fast red. A, A healthy nonsmoker control subject (NSC) showing relatively few positive cells in the subepithelial zone weakly expressing CysLT₁ receptor mRNA. B, A subject with COPD experiencing a severe exacerbation showing many strongly stained CysLT₁ receptor mRNA positive inflammatory cells infiltrating the subepithelial zone. C, The sense control probe shows an absence of signal in a biopsy specimen of a COPD subject experiencing an exacerbation (internal scale bar = 20 μm for A-C).

Figure 2.

Immunohistochemistry (IHC) localizing CysLT₁ receptor protein in bronchial biopsy specimens. The protein is seen as red fuchsin positivity with cell nuclei counterstained blue by hematoxylin. A, A healthy NSC showing expression of CysLT₁ receptor protein in only a few of the subepithelial inflammatory cells. B, A subject with COPD with severe exacerbation (SE-COPD) demonstrating more CysLT₁ receptor protein positive inflammatory cells in both subepithelium and epithelium (arrows). C, Method control: using normal goat serum instead of anti-CysLT₁ receptor antibody, there is no positive stain for protein in the biopsy specimen of a COPD subject experiencing an exacerbation (internal scale bar = 20 μm for A-C). See Figure 1 legend for expansion of other abbreviations.

Figure 3.

A, B, Counts for CysLT₁ receptor. mRNA (A) and protein-positive cells (B) in bronchial biopsy specimens of patients with NSC, S-COPD, and SE-COPD. The data are expressed as the number of positive cells per mm² of subepithelium. ● = Individual counts; horizontal bars = median values (Mann-Whitney U test). S-COPD = COPD in its stable phase. See Figure 1 and 2 legends for expansion of other abbreviations.

Figure 4.

A, Counts for CD45⁺ inflammatory cells. B, A percentage of CD45⁺ inflammatory cells expressing CysLT₁ receptor protein in subepithelium of bronchial biopsy specimens of patients with NSC, S-COPD, and SE-COPD. The data are expressed as (A) the number of positive cells per mm² of subepithelium and (B) percentage of CD45⁺ cells expressing CysLT₁ receptor protein. ● = Individual counts; horizontal bars = median values (Mann-Whitney U test). See Figure 1-3 legends for expansion of abbreviations.

Figure 5.

Graphs of counts for inflammatory cells in subepithelium of bronchial biopsy specimens of patients with NSC, S-COPD, and SE-COPD. A, CD68⁺. B, CD20⁺. C, EG2⁺ eosinophils. D, Tryptase⁺ mast cells. E, CD4⁺. F, CD8⁺. The data are expressed as the number of positive cells per mm² of subepithelium. ● = Individual counts; horizontal bars = median values (Mann-Whitney U test). See Figure 1-3 legends for expansion of abbreviations.

Figure 6.

Double immunofluorescence staining to demonstrate colocalization of CysLT₁ receptor to inflammatory cells of distinct phenotype in a bronchial biopsy specimen from a patient with SE-COPD. A, D, G, and J, CysLT₁ receptor protein immunopositivity is illustrated with Texas Red fluorescence. B, E, H, K, Neutrophil elastase⁺ neutrophils (B), CD68⁺ monocytes/macrophages (E), tryptase⁺ mast cells (H), and CD20⁺ B lymphocytes (K) shown by the green fluorescence of fluorescein isothiocyanate (FITC). C, F, I, L, Coexpression of CysLT₁ receptor seen as yellow fluorescence in each case in neutrophils (C), monocytes/macrophages (F), mast cells (I) and CD20⁺ cells (L) (internal scale bars = 10 μm for A-L). Nuclei are counterstained blue with 4′,6-diamidino-2-phenylindole (DAPI). See Figure 1 and 2 legends for expansion of other abbreviations.

Figure 7.

A, B, Double immunofluorescence staining for CysLT₁ receptor is illustrated with Texas Red fluorescence and CD4⁺ (A) and CD8⁺ T lymphocytes (B) are shown by green FITC fluorescence. In this case, there are no yellow fluorescent double-labeled cells. Nuclei are counterstained blue with DAPI (internal scale bar = 10 μm for A and B). See Figure 1 and 6 legends for expansion of abbreviations.

Figure 8.

A-C, Correlations between the numbers of subepithelial. CD45⁺ inflammatory cells (A, B) or (C) tryptase⁺ mast cells and CysLT₁ receptor mRNA⁺ or CysLT₁ receptor protein⁺ cells in the SE-COPD group, expressed as the number of cells/mm² of subepithelium (Spearman rank correlation; n = 15). See Figure 1 and 2 legends for expansion of abbreviations.