Morphometric analysis of inflammation in bronchial biopsies following exposure to inhaled diesel exhaust and allergen challenge in atopic subjects

Abstract

Background: Allergen exposure and air pollution are two risk factors for asthma development and airway inflammation that have been examined extensively in isolation. The impact of combined allergen and diesel exhaust exposure has received considerably less attention. Diesel exhaust (DE) is a major contributor to ambient particulate matter (PM) air pollution, which can act as an adjuvant to immune responses and augment allergic inflammation. We aimed to clarify whether DE increases allergen-induced inflammation and cellular immune response in the airways of atopic human subjects.

Methods: Twelve atopic subjects were exposed to DE 300 μg.m(-3) or filtered air for 2 h in a blinded crossover study design with a four-week washout period between arms. One hour following either filtered air or DE exposure, subjects were exposed to allergen or saline (vehicle control) via segmental challenge. Forty-eight hours post-allergen or control exposure, bronchial biopsies were collected. The study design generated 4 different conditions: filtered air + saline (FAS), DE + saline (DES), filtered air + allergen (FAA) and DE + allergen (DEA). Biopsies sections were immunostained for tryptase, eosinophil cationic protein (ECP), neutrophil elastase (NE), CD138, CD4 and interleukin (IL)-4. The percent positivity of positive cells were quantified in the bronchial submucosa.

Results: The percent positivity for tryptase expression and ECP expression remained unchanged in the bronchial submucosa in all conditions. CD4 % positive staining in DEA (0.311 ± 0.060) was elevated relative to FAS (0.087 ± 0.018; p = 0.035). IL-4% positive staining in DEA (0.548 ± 0.143) was elevated relative to FAS (0.127 ± 0.062; p = 0.034). CD138 % positive staining in DEA (0.120 ± 0.031) was elevated relative to FAS (0.017 ± 0.006; p = 0.015), DES (0.044 ± 0.024; p = 0.040), and FAA (0.044 ± 0.008; p = 0.037). CD138% positive staining in FAA (0.044 ± 0.008) was elevated relative to FAS (0.017 ± 0.006; p = 0.049). NE percent positive staining in DEA (0.224 ± 0.047) was elevated relative to FAS (0.045 ± 0.014; p = 0.031).

Conclusions: In vivo allergen and DE co-exposure results in elevated CD4, IL-4, CD138 and NE in the respiratory submucosa of atopic subjects, while eosinophils and mast cells are not changed.

Trial registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01792232.

Figures

Fig. 1

Immunohistochemical staining of tryptase-positive mast cells in human bronchial submucosa tissue. a Representative 20X image of positive staining using mAb AA1 for tryptase with positive staining in red with Mayer’s hematoxylin counterstain in blue from a subject exposed to FAA. b Zoom region (40X) highlighted in panel a black box. c Positive pixel count quantification of submucosa region for tryptase staining. Data are expressed as mean ± SEM. n = 10 for each experimental condition

Fig. 2

Immunohistochemical staining of ECP-positive eosinophils in human bronchial submucosa tissue. a Representative 20X image of positive staining using mAb EG2 for ECP with positive staining in red with Mayer’s hematoxylin counterstain in blue from a subject exposed to FAA. b Zoom region (40X) highlighted in panel a black box. c Positive pixel count quantification of submucosa region for ECP staining. Data are expressed as mean ± SEM. n =10 for each experimental condition

Fig. 3

Immunohistochemical staining of elastase-positive neutrophils in human bronchial submucosa tissue. a Representative 20X image of positive staining using mAb NP57 for NE with positive staining in red with Mayer’s hematoxylin counterstain in blue from a subject exposed to FAA. b Zoom region (40X) as highlighted in black box of panel a. c Positive pixel count quantification of submucosa region for NE staining. Data are expressed as mean ± SEM. n = 10 for each experimental condition. *p < 0.05

Fig. 4

Immunohistochemical staining of CD138-positive plasma cells in human bronchial submucosa tissue. a Representative 20X image of positive staining using mAb B-A38 for CD138 with positive staining in red with Mayer’s hematoxylin counterstain in blue from a subject exposed to FAA. b Zoom region (40X) highlighted in black box of panel a. c Positive pixel count quantification of submucosa region for CD138 staining. Data are expressed as mean ± SEM. n = 9 for each experimental condition. *p < 0.05; **p < 0.01

Fig. 5

Immunohistochemical staining of CD4-positive T cells in human bronchial submucosa tissue. a Representative 20X image of positive staining using mAb 4B12 for CD4 with positive staining in red with Mayer’s hematoxylin counterstain in blue from a subject exposed to FAA. b Zoom region (40X) highlighted in black box of panel a. c Positive pixel count quantification of submucosa region for CD4 staining. Data are expressed as mean ± SEM. n = 10 for each experimental condition. *p < 0.05

Fig. 6

Immunohistochemical staining of IL-4-positive cells in human bronchial submucosa tissue. a Representative 20X image of positive staining using mAb 4D9 for IL-4 with positive staining in red with Mayer’s hematoxylin counterstain in blue from a subject exposed to FAA. b Zoom region (40X) highlighted in black box of panel a. c Positive pixel count quantification of submucosa region for IL-4 staining. Data are expressed as mean ± SEM. n = 12 for each experimental condition. *p < 0.05

Fig. 7

Schematic of exposure protocol. Study subjects were exposed to DE (300 μg.m−3 of PM2.5) or FA (filtered air) for 2 h. One hour post-exposure, a segmental allergen challenge was performed with allergen or saline vehicle in the right upper and middle lobe or left lingular lobe. Forty-eight hours post-exposure endobronchial biopsies were obtained via bronchoscopy. The process was repeated following a washout period of 1 month with exposure conditions reversed compared to the first visit

Fig. 8

Demonstration of image analysis using Aperio® ImageScope™ software. A positive pixel count algorithm was used to quantify positive staining in the submucosa (blue region) of bronchial biopsies for tryptase, ECP, NE, CD138, CD4, and IL-4. The airway epithelium was not examined and positive staining in the epithelium was excluded from analysis. a Representative image of tryptase positive staining from a subject exposed to FAA, Black arrows denote positive staining in submucosa area that are selected by the positive pixel count. b Image from a with submucosa region selected by manual trace followed by positive pixel count recognition of tryptase stain (red colour) within submucosa region (blue colour)