Adrenergic and glucocorticoid receptor antagonists reduce ozone-induced lung injury and inflammation

Abstract

Recent studies showed that the circulating stress hormones, epinephrine and corticosterone/cortisol, are involved in mediating ozone-induced pulmonary effects through the activation of the sympathetic-adrenal-medullary (SAM) and hypothalamus-pituitary-adrenal (HPA) axes. Hence, we examined the role of adrenergic and glucocorticoid receptor inhibition in ozone-induced pulmonary injury and inflammation. Male 12-week old Wistar-Kyoto rats were pretreated daily for 7days with propranolol (PROP; a non-selective β adrenergic receptor [AR] antagonist, 10mg/kg, i.p.), mifepristone (MIFE; a glucocorticoid receptor [GR] antagonist, 30mg/kg, s.c.), both drugs (PROP+MIFE), or respective vehicles, and then exposed to air or ozone (0.8ppm), 4h/d for 1 or 2 consecutive days while continuing drug treatment. Ozone exposure alone led to increased peak expiratory flow rates and enhanced pause (Penh); with greater increases by day 2. Receptors blockade minimally affected ventilation in either air- or ozone-exposed rats. Ozone exposure alone was also associated with marked increases in pulmonary vascular leakage, macrophage activation, neutrophilic inflammation and lymphopenia. Notably, PROP, MIFE and PROP+MIFE pretreatments significantly reduced ozone-induced pulmonary vascular leakage; whereas PROP or PROP+MIFE reduced neutrophilic inflammation. PROP also reduced ozone-induced increases in bronchoalveolar lavage fluid (BALF) IL-6 and TNF-α proteins and/or lung Il6 and Tnfα mRNA. MIFE and PROP+MIFE pretreatments reduced ozone-induced increases in BALF N-acetyl glucosaminidase activity, and lymphopenia. We conclude that stress hormones released after ozone exposure modulate pulmonary injury and inflammatory effects through AR and GR in a receptor-specific manner. Individuals with pulmonary diseases receiving AR and GR-related therapy might experience changed sensitivity to air pollution.

Keywords: Adrenergic receptor antagonist; Glucocorticoid receptor antagonist; Lung inflammation; Lung injury; Ozone; Stress response.

Figure 1:. Schema of the experimental design.

For all three studies, the timing for drug pretreatments and the information on air or ozone exposure, plethysmography and necropsies are indicated by corresponding arrows. Animals assigned to 1 day (4 hr) air or ozone exposure are referred as group D+1 and those assigned to 2 consecutive days of exposure are referred as group D+2. Animals assigned to group D+2 were subjected to plethysmography prior to the start of drug pretreatment, after 3 days of drug pretreatment (D-4) and immediately after each day of air/ozone exposure. Necropsy and tissue collection were performed immediately after exposure (D+1) or after exposure and plethysmography (D+2) (within 1–2 hours of exposure). Vehicles: SAL, saline; CO, corn oil; drugs: PROP, propranolol; MIFE, mifepristone.

Figure 2:. Ventilatory parameters in vehicle- or drug-pretreated rats after each day of air or ozone.

Ventilatory parameters determined after the first (D+1) and second (D+2) day of air or 0.8 ppm ozone exposure are shown (pre-air or -ozone exposure data are not shown). The breathing parameters indicate mean ± standard error of mean (SEM) of n=6–8 animals/group. Significant differences between groups (P ≤ 0.05) are indicated by * for ozone effect and by † for drug pretreatment effect. A) Breathing frequency, B) minute volume (MV), C) peak expiratory flow (PEF), D) enhanced pause (PenH).

Figure 3:. The influence of drug pretreatments on ozone-induced BALF protein leakage and N-acetyl glucosaminidase (NAG) activity.

Protein (A), albumin (B), and NAG activity (C) were determined in BALF collected immediately following exposure to air or 0.8 ppm ozone, 4 hr/day for 1 day (D+1) or 2 days (D+2). Values indicate mean ± SE of n=6–8 animals/group. Significant differences between groups (P ≤ 0.05) are indicated by * for ozone effect in matching pretreatment groups, and by † for drug effect in matching exposure groups.

Figure 4:. The influence of drug pretreatments on ozone-induced changes in lung inflammation as determined by BALF cell count.

Total cells (A), neutrophils (B) and lymphocytes (C) were determined in BALF collected immediately following exposure to air or ozone (0.8 ppm), 4 hr/day for 1 day (D+1) or 2 consecutive days (D+2). Values indicate mean ± SE of n=6–8 animals/group. Significant differences between groups (P ≤ 0.05) are indicated by * for ozone effect in matching pretreatment groups, and by † for drug effect in matching exposure groups.

Figure 5:. The effects of drug pretreatments on ozone-induced changes in circulating white blood cells (WBC) in rats.

Circulating total white blood cells (A), neutrophils (B) and lymphocytes (C) were determined in rats immediately after exposure to air or 0.8 ppm ozone, 4 hr/day for 1 day (D+1). Values indicate mean ± SE of n=6–8 animals/group. Significant differences between groups (P ≤ 0.05) are indicated by * for ozone effect in matching pretreatment groups, and by † for drug effect in matching exposure groups.

Figure 6:. Ozone-induced changes in Tnfα and Il6 lung mRNA and BALF proteins in rats pretreated with βAR and GR antagonists.

Lung mRNA expression of Il6 (A) and Tnfα (B) were determined in D+1 groups while BALF protein levels of IL-6 (C) and TNF-α (D) were assessed in D+2 groups. Values indicate mean ± SE of n=6–8 animals/group. Significant differences between groups (P ≤ 0.05) are indicated by * for ozone effect in matching pretreatment groups, and by † for drug effect in matching exposure groups.

Figure 7:. The effect of drug pretreatments on ozone-induced increases in pulmonary Cxcl2, Mt2a and Tsc22d3 mRNA expression.

Relative lung mRNA expression was determined in D+1 groups for Cxcl2 (also known as Mip2; A), Mt2a (B) and Tsc22d3 (C). Values indicate mean ± SE of n=6–8 animals/group. Significant differences between groups (P ≤ 0.05) are indicated by * for ozone effect in matching pretreatment groups, and by † for drug effect in matching exposure groups.

Figure 8:. Proposed mechanism by which βAR and GR antagonists reduce ozone-induced pulmonary protein leakage, cytokine expression, neutrophilic inflammation and lymphopenia.

This schematic is based on our earlier studies that ozone-induced lung injury and inflammation are mediated through neuroendocrine activation of stress response and involves the release of adrenal-derived stress hormones, epinephrine and corticosterone (Kodavanti, 2016). Ozone-induced effects are inhibited by pretreatment of rats with PROP and/or MIFE.