Reproducibility of a novel model of murine asthma-like pulmonary inflammation

Abstract

Sensitization to cockroach allergens (CRA) has been implicated as a major cause of asthma, especially among inner-city populations. Endotoxin from Gram-negative bacteria has also been investigated for its role in attenuating or exacerbating the asthmatic response. We have created a novel model utilizing house dust extract (HDE) containing high levels of both CRA and endotoxin to induce pulmonary inflammation (PI) and airway hyperresponsiveness (AHR). A potential drawback of this model is that the HDE is in limited supply and preparation of new HDE will not contain the exact components of the HDE used to define our model system. The present study involved testing HDEs collected from various homes for their ability to cause PI and AHR. Dust collected from five homes was extracted in phosphate buffered saline overnight. The levels of CRA and endotoxin in the supernatants varied from 7.1 to 49.5 mg/ml of CRA and 1.7-6 micro g/ml of endotoxin in the HDEs. Following immunization and two pulmonary exposures to HDE all five HDEs induced AHR, PI and plasma IgE levels substantially higher than normal mice. This study shows that HDE containing high levels of cockroach allergens and endotoxin collected from different sources can induce an asthma-like response in our murine model.

Fig. 1

Differential counting of the BAL fluid. Mice were immunized i.p. on day 0 with 1 : 10 diluted HDE mixed with TiterMax adjuvant. Mice were challenged intratracheally with 1 : 10 diluted HDE on days 14 and 21; 48 h following the last challenge mice were sacrificed and BAL fluid was collected. Differential counts were performed by counting 300 cells. *P < 0·05 compared to normal mice (NL). Each value is the mean ± s.e.m. for n = 7, except HDE 4, n = 4 due to limited supply.

Fig. 2

Plasma eotaxin and IgE levels. Mice were immunized once and challenged twice with 1 : 10 diluted HDE and sacrificed 48 h after the second challenge. Plasma was diluted 1 : 10 for ELISAs. *P < 0·05 compared to normal mice (NL). Each value is the mean ± s.e.m. for n = 7, except HDE 4, n = 4 due to limited supply.

Fig. 3

Bronchial hyperreactivity. Airway hyperresponsiveness to methylcholine challenge was measured in conscious animals in a whole body plethsymography system 24 h after the last airway challenge. Data are reported as a percentage increase in the average Penh for each dose of methylcholine compared to the average Penh of a PBS challenge. All mice challenged with HDE had significantly higher percentage increase at the highest methacholine dose compared to normal mice, P < 0·05. Each value is the mean ± s.e.m. for n = 7, except HDE 4, n = 4 due to limited supply.

Fig. 4

Bronchial hyperreactivity. Mice were challenged with 1 : 10 diluted HDE without prior immunization (thatched bars) and airway hyperresponsiveness to methylcholine challenge was measured 24 h later in a whole body plethysmography. The percentage increase above baseline for the 50 mg/ml methylcholine dose is reported. *P < 0·05 compared to mice immunized and challenged (solid bars) with the same HDE. Each value is the mean ± s.e.m. for n = 6, non-immunized groups, n = 7 immunized and challenged, except HDE 4 immunized, n = 4 due to limited supply.

Fig. 5

Bronchial hyperreactivity. Mice were immunized and challenged with HDE containing equivalent Bla g levels (grey bars) and airway hyperresponsiveness to methylcholine challenge was measured 24 h after the last pulmonary challenge. The percentage increase above baseline for the 50 mg/ml methylcholine dose is reported. *P < 0·05 compared to mice immunized and challenged with the same HDE diluted 1 : 10 (solid bars). Each value is the mean ± s.e.m. for n = 4, equivalent bla g groups, n = 7 HDE (1 : 10), except HDE 4 (1 : 10), n = 4 due to limited supply.