Inhibiting pollen reduced nicotinamide adenine dinucleotide phosphate oxidase-induced signal by intrapulmonary administration of antioxidants blocks allergic airway inflammation

Abstract

Background: Ragweed extract (RWE) contains NADPH oxidases that induce oxidative stress in the airways independent of adaptive immunity (signal 1) and augment antigen (signal 2)-induced allergic airway inflammation.

Objective: To test whether inhibiting signal 1 by administering antioxidants inhibits allergic airway inflammation in mice.

Methods: The ability of ascorbic acid (AA), N-acetyl cystenine (NAC), and tocopherol to scavenge pollen NADPH oxidase-generated reactive oxygen species (ROS) was measured. These antioxidants were administered locally to inhibit signal 1 in the airways of RWE-sensitized mice. Recruitment of inflammatory cells, mucin production, calcium-activated chloride channel 3, IL-4, and IL-13 mRNA expression was quantified in the lungs.

Results: Antioxidants inhibited ROS generation by pollen NADPH oxidases and intracellular ROS generation in cultured epithelial cells. AA in combination with NAC or Tocopherol decreased RWE-induced ROS levels in cultured bronchial epithelial cells. Coadministration of antioxidants with RWE challenge inhibited 4-hydroxynonenal adduct formation, upregulation of Clca3 and IL-4 in lungs, mucin production, recruitment of eosinophils, and total inflammatory cells into the airways. Administration of antioxidants with a second RWE challenge also inhibited airway inflammation. However, administration of AA+NAC 4 or 24 hours after RWE challenge failed to inhibit allergic inflammation.

Conclusion: Signal 1 plays a proinflammatory role during repeated exposure to pollen extract. We propose that inhibiting signal 1 by increasing antioxidant potential in the airways may be a novel therapeutic strategy to attenuate pollen-induced allergic airway inflammation.

Clinical implications: Administration of antioxidants in the airways may constitute a novel therapeutic strategy to prevent pollen induced allergic airway inflammation.

FIG 1

Antioxidants inhibit RWE mediated oxidative stress in vitro. ID₅₀ of individual (A) and combination (B) of antioxidants. GSH, Glutathione; Toc, tocopherol.

FIG 2

Effect of antioxidants on RWE-induced intracellular ROS and 4-HNE protein adduct formation. A, Effect of increasing concentration of antioxidants on RWE-induced intracellular ROS in A549 cells. B, Effect of NAC on RWE-induced 4-HNE adduct formation in lungs. Each lane represents a different mouse. GO, Glucose oxidase.

FIG 3

Effect of AA+NAC on RWE-induced mucin production. A, RWE-sensitized BALB/c mice were challenged with PBS, RWE, or RWE+AA+NAC and killed at 72 hours. The PAS-stained lung sections show mucin content of airway epithelial cells. Arrow shows PAS-positive mucin globules. Magnification ×200. B, Morphometric quantification of mucin in airway epithelial cells. Results are means ± SEMs (n = 3–6 mice per group). C, Measurement of Clca3, IL-4, and IL-13 transcripts in the lungs by real-time quantitative PCR. **P < .01; ***P < .005.

FIG 4

Scavenging pollen NADPH oxidase–generated ROS inhibits allergic lung inflammation. Total inflammatory cells (A) and eosinophils (B) in BAL fluids of RWE-challenged mice. Results are means ± SEMs (n = 7–9 mice per group). ***P < .001. C, Effect of antioxidants on recruitment of eosinophils in peribronchial (br) and perivascular (v) regions (arrows). Magnification ×100. TOC, Tocopherol.

FIG 5

Effect of antioxidants on X+XO-mediated augmentation of allergic airway inflammation. Effect of antioxidants on X+XO-augmented total inflammatory cell (A) and eosinophil (B) recruitment in BAL fluids. Results are means ± SEMs (n = 7–9 mice per group). **P < .01; ***P < .001.

FIG 6

Scavenging ROS generated by RWE pollen NADPH oxidase and not by inflammatory cells inhibits allergic airway inflammation. A, Kinetics of neutrophil recruitment in the BAL fluid. RWE-sensitized mice were challenged with either PBS or RWE and killed kinetically, and BAL fluid was examined for cellular content. B, AA+NAC increased total antioxidant potential in the airways for 90 minutes. X-axis represents time after administration of antioxidants. Results are means ± SEMs (n = 3–6 mice per group). C, Analysis of eosinophil recruitment in the airways after administration of antioxidants at various time points. Results are means ± SEMs (n = 7–9 mice per group). *P < .05; ****P < .0001. D, BALB/c mice were sensitized with OVA and challenged with nebulized OVA. Mice were treated with either PBS or AA+NAC immediately before and just after nebulized OVA challenge. NS, Not significant.

FIG 7

Antioxidants inhibit second RWE challenge induced augmentation of allergic airway inflammation. A, AA+NAC prevents further increase in airway inflammation induced by second RWE challenge performed 72 hours after initial challenge. RWE-sensitized mice were challenged with PBS, RWE, or RWE+AA+NAC. A second similar challenge was performed 72 hours after the initial challenge; mice were killed 72 hours after last challenge, and BAL fluid was examined for cellular content. B, AA+NAC effectively inhibits RWE-induced airway inflammation during resolution phase. Mice were sensitized and challenged as described except the second challenge was performed 10 days after the first RWE challenge. *P < .05; **P < .01.