Exposure to particulate hexavalent chromium exacerbates allergic asthma pathology

Abstract

Airborne hexavalent chromate, Cr(VI), has been identified by the Environmental Protection Agency as a possible health threat in urban areas, due to the carcinogenic potential of some of its forms. Particulate chromates are produced in many different industrial settings, with high levels of aerosolized forms historically documented. Along with an increased risk of lung cancer, a high incidence of allergic asthma has been reported in workers exposed to certain inhaled particulate Cr(VI) compounds. However, a direct causal association between Cr(VI) and allergic asthma has not been established. We recently showed that inhaled particulate Cr(VI) induces an innate neutrophilic inflammatory response in BALB/c mice. In the current studies we investigated how the inflammation induced by inhaled particulate Cr(VI) might alter the pathology of an allergic asthmatic response. We used a well-established mouse model of allergic asthma. Groups of ovalbumin protein (OVA)-primed mice were challenged either with OVA alone, or with a combination of OVA and particulate zinc chromate, and various parameters associated with asthmatic responses were measured. Co-exposure to particulate Cr(VI) and OVA mediated a mixed form of asthma in which both eosinophils and neutrophils are present in airways, tissue pathology is markedly exacerbated, and airway hyperresponsiveness is significantly increased. Taken together these findings suggest that inhalation of particulate forms of Cr(VI) may augment the severity of ongoing allergic asthma, as well as alter its phenotype. Such findings may have implications for asthmatics in settings in which airborne particulate Cr(VI) compounds are present at high levels.

Figure 1

Regimen of acute allergic lung inflammation and intranasal delivery of particulate chromium. On day 0, female BALB/c mice were primed i.p. with 50 μg OVA mixed 1:1 with Imject alum adjuvant. On days 7–10, mice were challenged intranasally (i.n.) with 50 μl of the indicated agent: (A) Saline-treated mice (control) received saline challenges. (B) Cr-treated mice were challenged with 24 μg Cr on days 7 & 9 and saline on days 8 & 10. (C) OVA-treated mice were administered 50 μg OVA for all challenges. (D) OVA+Cr-treated mice were challenged with 50 μg OVA + 24 μg Cr on days 7 & 9 and 50 μg OVA on days 8 & 10. All mice were sacrificed on day 12 and analyses were conducted.

Figure 2

Cellular infiltrate in BAL fluid. Mice were primed and challenged as indicated in Figure 1 and sacrificed on day 12. BAL was performed on each mouse, and airway cells were isolated, counted, and leukocyte subtypes were identified by flow cytometric analysis using forward light scatter/side light scatter distribution. Bar graphs show: (A) The total number of living cells in the airways, (B) Neutrophil cell numbers in the airways, and (C) Eosinophil cell numbers in the airways. Data are the mean ± SE from two independent experiments, with a total of 8–12 animals per group. Statistically significant differences among treatment groups were determined using a 1-Way ANOVA, * p < 0.05.

Figure 3

Histology of lung tissue. Mice were primed and challenged as indicated in Figure 1 and sacrificed on day 12. Whole lungs were isolated, embedded in parafin, cut into 6-μm sections, and stained. H&E staining was performed on lung sections representative of 4–6 animals of each treatment group from 2 independent experiments. All images, captured at 10X magnification, show tissue areas surrounding bronchioles. Scale bar: 10 μm.

Figure 4

PAS staining of lung tissue. (A) PAS staining was performed on lung sections representative of 4–6 animals of each treatment group from 1 independent experiment. Bar graphs show: (B) The percentage of PAS-positive cells in the airways, (C) Area of PAS-positive staining in the airways. Data are the mean ± SE from 4–6 animals per group. Statistically significant differences among treatment groups were determined using a 1-Way ANOVA, * p < 0.05.

Figure 5

Airway hyperresponsiveness to methylcholine. Mice were primed and challenged as indicated in Figure 1. On day 12, individual mice were anesthetized i.p. with ketamine/xylazine, a tracheostomy tube was inserted and then attached to a respirator. The animals were challenged with aerosolized PBS (baseline) followed by increasing doses of methylcholine ranging from 0–50 mg/ml. Maximum resistance (R_L, cm H₂O/m/s) was recorded during a 3-minute period following each challenge. Data are the mean ± SE from two independent experiments, with a total of 8–12 animals per group. Statistically significant differences among treatment groups was determined using a 1-Way ANOVA, * p < 0.05.