Postnatal exposure history and airways: oxidant stress responses in airway explants

Abstract

Postnatally, the lung continues to grow and differentiate while interacting with the environment. Exposure to ozone (O(3)) and allergens during postnatal lung development alters structural elements of conducting airways, including innervation and neurokinin abundance. These changes have been linked with development of asthma in a rhesus monkey model. We hypothesized that O(3) exposure resets the ability of the airways to respond to oxidant stress and that this is mediated by changes in the neurokinin-1 receptor (NK-1R). Infant rhesus monkeys received episodic exposure to O(3) biweekly with or without house dust mite antigen (HDMA) from 6 to 12 months of age. Age-matched monkeys were exposed to filtered air (FA). Microdissected airway explants from midlevel airways (intrapulmonary generations 5-8) for four to six animals in each of four groups (FA, O(3), HDMA, and HDMA+O(3)) were tested for NK-1R gene responses to acute oxidant stress using exposure to hydrogen peroxide (1.2 mM), a lipid ozonide (10 μM), or sham treatment for 4 hours in vitro. Airway responses were measured using real-time quantitative RT-PCR of NK-1R and IL-8 gene expression. Basal NK-1R gene expression levels were not different between the exposure groups. Treatment with ozonide or hydrogen peroxide did not change NK-1R gene expression in animals exposed to FA, HDMA, or HDMA+O(3). However, treatment in vitro with lipid ozonide significantly increased NK-1R gene expression in explants from O(3)-exposed animals. We conclude that a history of prior O(3) exposure resets the steady state of the airways to increase the NK-1R response to subsequent acute oxidant stresses.

Figure 1.

Experimental design and exposure timeline. Sensitized animals were randomly assigned to one of four exposure groups and subjected to 11 2-week cycles of filtered air (FA), ozone (0.5 ppm 8 h/d, 5 d on and 9 d off), house dust mite antigen (HDMA) (2 h/d, 3 d on and 11 d off), or ozone (0.5 ppm+HDMA, allergen exposed last 3 days of ozone sequentially).

Figure 2.

Neurokinin-1 receptor (NK-1R) gene expression in midlevel airway explants from monkeys exposed to 11 cycles of filtered air (FA), 0.5 ppm ozone (O₃), HDMA, or 0.5 ppm O₃+HDMA in vivo and subjected to oxidant exposure (1.2 mM H₂O₂ or 10 μM lipid ozonide) in vitro. Some airways were also cultured in vitro with NK-1R agonist (Substance P [Sub P]) or agonist and antagonist (Substance P+L119). All values were analyzed relative to exposure sham control (e.g., FA animals relative to FA sham control). Baseline NK-1R expression in FA-exposed (A) and O₃–exposed (B) animals. NK-1R expression in airway explants from FA- (C), O₃– (D), HDMA- (E), or O₃+HDMA- (F) exposed animals treated with vehicle control, agonist, or agonist+antagonist and with sham, H₂O₂, or ozonide. n = 4 to 6 animals. Significance was considered at P < 0.05. ^aP ≤ 0.001. *Different from matched control group. ^†Different from matched SP+L119 group. ^‡Different from matched sham group. ^§Different from matched H₂O₂ group.

Figure 3.

Neurokinin-1/Nur77 receptor protein expression. Immunohistochemistry for NK-1R or Nur77 orphan receptor in midlevel conducting airway slices of monkeys episodically exposed to FA, 0.5 ppm O₃, HDMA, or O₃+HDMA. The airway from a FA-exposed animal shows some NK-1R immunoreactivity in the epithelium (A) compared with no reactivity in the negative control (E). Relative to FA, animals exposed to O₃ (B) and HDMA (C) have increased NK-1R immunostaining. Animals exposed to O₃+HDMA (D) have slightly less staining than FA. The FA animals (F) show immunoreactivity for Nur77 in the apices of epithelium compared with the negative control (J). Relative to FA, animals exposed to O₃ (G), HDMA (H), or O₃+HDMA (I) showed increased immunostaining with the greatest reactivity in animals from the combined exposure. Arrows note location of inset image. Scale bar = 50 μm (inset scale bar = 10 μm).

Figure 4.

In vivo versus in vitro NK-1R protein expression. Immunohistochemistry for NK-1R in a midlevel conducting airway slice of a monkey episodically exposed to 0.5 ppm O₃ in vivo (A) and then an explant of an identically exposed monkey (B) subjected in vitro to 10 μM lipid ozonide. Explants were maintained in culture for 4 hours at 37°C. NK-1R immunoreactivity in airway epithelium is increased with more protein/positive cell after lipid ozonide oxidant challenge in culture (arrowheads mark similar regions). Scale bar = 50 μm.

Figure 5.

IL-8 gene expression in midlevel airway explants from monkeys exposed to 11 cycles of 0.5 ppm O₃, HMDA, or 0.5 ppm O₃+HDMA in vivo and subjected to oxidant exposure (1.2 mM H₂O₂) in vitro. Airways were cultured in vitro with NK-1R agonist (Sub P) or agonist and antagonist (Sub P+L119). All values were analyzed relative to filtered air sham control. IL-8 expression in airway explants from FA (A), O₃ (B), HDMA (C), or combined exposure (D) animals. n = 4 to 6 animals. Significance was considered at P < 0.05. *Different from matched Sub P group. ^†Different from matched sham group.