Chronic inhaled ovalbumin exposure induces antigen-dependent but not antigen-specific inhalational tolerance in a murine model of allergic airway disease

Abstract

Sensitized mice acutely challenged with inhaled ovalbumin (OVA) develop allergic airway inflammation, characterized by OVA-specific IgE production, airway eosinophilia, increased pulmonary B and T lymphocytes, and airway hyperreactivity. In this study, a chronic exposure model was developed and two distinct patterns of response were observed. Discontinuous inhalational exposure to OVA (6 weeks) produced airway inflammation and hyperreactivity that were similar to acute (10 days) responses. Continuous inhalational exposure to OVA (6 or 11 weeks) resulted in attenuation of airway eosinophilia and hyperresponsiveness without reduction of OVA-specific IgE and IgG(1) levels. The inhibition of airway inflammation was dependent on continuous exposure to antigen, because continuously exposed mice with attenuated inflammatory responses redeveloped allergic airway disease if the OVA aerosols were interrupted and then restarted (11-week-discontinuous). Inhalational tolerance induced by continuous OVA exposure demonstrated bystander suppression of cockroach allergen-mediated airway eosinophilia. These findings may be attributed to changes in production of the anti-inflammatory cytokine interleukin-10 during continuous OVA aerosol exposure. The symptomatic and asymptomatic allergic responses in human asthmatics could be explained by similar variable or discontinuous exposures to aeroantigens.

Figure 1

Sensitization and aerosol challenge protocols. Mice were sensitized to OVA by three weekly intraperitoneal injections of 25 μg of OVA in alum. One week later, all mice were exposed to OVA aerosols (1% OVA in saline for 1 hour) daily for 10 days (heavily shaded blocks). Thereafter, 6-week-continuous mice were exposed to OVA 5 days a week for 4 weeks (lightly shaded blocks). Six-week-discontinuous mice were not exposed to OVA aerosols during this period (open blocks). Mice were then exposed to daily OVA aerosols for 10 days (heavily shaded blocks) and were sacrificed (S) 24 hours after the last aerosol. Additional 6-week-continuous mice were sensitized to cockroach antigen (CRA) during their continuous OVA aerosol exposure period and then were challenged with nasal CRA 14 days later in lieu of the final 10-day OVA period. In the 11-week studies, discontinuous mice were not exposed to OVA aerosols for 4 weeks after their initial exposure regimens, before being challenged with 10-day OVA aerosols. Penh (P) measurements were taken after 3 days of final aerosol exposure.

Figure 2

Comparison of methacholine reactivity in naive and acute OVA-challenged mice. Left: Acute mice were sensitized with three weekly injections of OVA/alum and then challenged with three daily OVA aerosols. Airway responsiveness to methacholine was measured before OVA aerosols (open symbols) and 12 hours after the third OVA aerosol (filled symbols). The Penh response to methacholine was potentiated after OVA aerosol challenge in acute sensitized mice. Right: Airway responsiveness was also assessed in naive, unexposed mice on the same days. Baseline responses (open symbols) were similar to the acute animals’ baselines, and the Penh response did not change after 3 days in naive unchallenged mice (filled symbols). Data represent mean ± SE levels of Penh responses in naive and acute mice (n = 5 in each group); *, P < 0.02 from baseline response.

Figure 3

Comparison of airway sensitivity in naive and 10-day-acute, 6-week-continuous, and 6-week-discontinuous OVA-challenged mice. Airway sensitivity to methacholine was determined from Penh measurements at baseline (B) and 12 hours after OVA aerosol challenges (C), as described in the text, with control measurements taken simultaneously in naive mice. Airway sensitivity was defined as the calculated concentration of methacholine associated with a Penh value of 2 units. Naive and continuous mice showed no changes in airway sensitivity between the two measurements. In contrast, acute and discontinuous mice demonstrated increased muscarinic sensitivity after OVA aerosol challenge (ie, decreased concentration of methacholine eliciting the Penh response). Circles and dashed lines, paired responses in individual animals; bars, mean values; *, P < 0.03 by paired t-test versus baseline.

Figure 4

Comparison of BAL cytokine concentrations in naive, 10-day-acute, 6-week-continuous, and 6-week-discontinuous mice. Mice were challenged with OVA aerosols acutely (acute, striped bars), continuously (cont, shaded bars), and discontinuously (disk, filled bars). BAL fluid was assayed for cytokine concentrations by enzyme-linked immunosorbent assay, as described in the text, and compared with BAL levels from naive mice (open bars). BAL levels of IL-13 were unmeasurable in naive mice and were less elevated in continuous than in acute or discontinuous mice. BAL levels of IL-5 were unmeasurable in naive mice and were most elevated in the continuous animals. BAL levels of TGF-β increased with acute OVA aerosol challenges, returned to baseline in continuous mice, and were again elevated in discontinuous animals. BAL levels of IL-10 were similar in naive, acute, and discontinuous mice but were significantly elevated in continuous animals. Data represent mean + SE measurements; *, P < 0.02 versus acute and discontinuous mice by analysis of variance; **, P < 0.05 versus naive and continuous mice by analysis of variance; n = 5 to 6 animals in each group.

Figure 5

Comparison of BAL leukocytes in 7-day CRA-challenged mice that were naive to OVA or subjected to 6 weeks of continuous OVA aerosol exposure. Mice were sensitized and challenged with CRA, as described in the text. One group of mice was naive to OVA; the other group had been subjected to the 6-week-continuous OVA exposure protocol before CRA administration. These mice continued to receive OVA aerosols during the CRA sensitization period. Note that although both groups of mice showed increased numbers of BAL macrophages, lymphocytes, and neutrophils relative to naive animals, the airway eosinophilic response to 7-day-CRA exposure was ablated in 6-week-continuous OVA-exposed mice. Data represent mean numbers of macrophages (shaded bars), eosinophils (filled bars), and lymphocytes (striped bars); n = 4 to 6 in each group.