Smooth Muscle Hypocontractility and Airway Normoresponsiveness in a Mouse Model of Pulmonary Allergic Inflammation

Abstract

The contractility of airway smooth muscle (ASM) is labile. Although this feature can greatly modulate the degree of airway responsiveness in vivo, the extent by which ASM's contractility is affected by pulmonary allergic inflammation has never been compared between strains of mice exhibiting a different susceptibility to develop airway hyperresponsiveness (AHR). Herein, female C57BL/6 and BALB/c mice were treated intranasally with either saline or house dust mite (HDM) once daily for 10 consecutive days to induce pulmonary allergic inflammation. The doses of HDM were twice greater in the less susceptible C57BL/6 strain. All outcomes, including ASM contractility, were measured 24 h after the last HDM exposure. As expected, while BALB/c mice exposed to HDM became hyperresponsive to a nebulized challenge with methacholine in vivo, C57BL/6 mice remained normoresponsive. The lack of AHR in C57BL/6 mice occurred despite exhibiting more than twice as much inflammation than BALB/c mice in bronchoalveolar lavages, as well as similar degrees of inflammatory cell infiltrates within the lung tissue, goblet cell hyperplasia and thickening of the epithelium. There was no enlargement of ASM caused by HDM exposure in either strain. Unexpectedly, however, excised tracheas derived from C57BL/6 mice exposed to HDM demonstrated a decreased contractility in response to both methacholine and potassium chloride, while tracheas from BALB/c mice remained normocontractile following HDM exposure. These results suggest that the lack of AHR in C57BL/6 mice, at least in an acute model of HDM-induced pulmonary allergic inflammation, is due to an acquired ASM hypocontractility.

Keywords: airway responsiveness; asthma; mouse models; resistance; respiratory mechanics.

FIGURE 1

Experimental protocol to induce pulmonary allergic inflammation. While 60 C57BL/6 mice were exposed to saline (n = 20) or either 4 (n = 20) or 6 mg/mL (n = 20) of house dust mite (HDM), 60 BALB/c mice were exposed to saline (n = 20) or either 2 (n = 20) or 3 mg/mL (n = 20) of HDM once daily for 10 consecutive days. At day 11, the degree of in vivo airway responsiveness to methacholine was measured in half of the mice within each group (n = 10) using the flexiVent. The same mice were used to collect the bronchoalveolar lavages in order to measure cellular inflammation. The trachea was collected in the other half of mice within each group (n = 10), also at day 11, in order to measure the contractile capacity of airway smooth muscle in response to incremental concentrations of methacholine and potassium chloride. The left lung of the same mice was collected and processed for histology to quantify the infiltration of inflammatory cells within the tissue, the content of airway smooth muscle, the number of goblet cells and the thickness of the epithelium.

FIGURE 2

The degree of in vivo airway responsiveness in C57BL/6 (left panels) and BALB/c (right panels) mice exposed to either saline or one of two doses of HDM. Phosphate-buffered saline and incremental doses of methacholine were delivered by nebulization and the changes in several parameters were used to evaluate the degree of airway responsiveness, including: (A) respiratory system resistance (Rrs); (B) respiratory system elastance (Ers); (C) Newtonian resistance (R_N); (D) tissue damping (G); and (E) tissue elastance (H). *, #, and $ designate significant differences in BALB/c mice for saline vs. HDM 3 mg/mL, for saline vs. HDM 2 mg/mL, and for HDM 3 vs. 2 mg/mL, respectively (P < 0.05). Data are shown as means ± SD (some error bars seem absent because their length is smaller than the symbol). n = 10 mice per group.

FIGURE 3

The contractile capacity of excised tracheas derived from C57BL/6 (left panels) and BALB/c (right panels) mice exposed to either saline or one of two doses of HDM. The graphs in (A,B) show the isometric force generated by the tracheas in response to increasing concentrations of methacholine and potassium chloride, respectively. *, #, and $ designate significant differences in C57BL/6 for saline vs. HDM 6 mg/mL, for saline vs. HDM 4 mg/mL, and for HDM 6 vs. 4 mg/mL, respectively (P < 0.05). Data are shown as means ± SD. n = 10 mice per group.

FIGURE 4

Inflammatory cells in bronchoalveolar lavages of C57BL/6 (left panels) and BALB/c (right panels) mice exposed to either saline or one of two doses of HDM. The scatter plots in (A,B) show the number of total cells/mL and the differential cell counts in percentages, respectively. Note that the scale on the y-axes in (A) is twice greater for C57BL/6 than BALB/c mice. *Designates significantly different from saline-treated mice within the same mouse strain (P < 0.05). Data are shown as means ± SD. n = 10 mice per group. Macro, macrophages; Lympho, lymphocytes; Neutro, neutrophils; and Eosino, eosinophils.

FIGURE 5

Inflammatory cell infiltrates within the lung tissue. The images in (A) show representative lung sections from C57BL/6 (upper panels) and BALB/c (lower panels) mice exposed to incremental doses of house dust mite (HDM) (from left to right). The zone enclosed by the gray square on images of the third row is zoomed on the next image on the right. The scale bar is 250 μm for the six first images from the left and 25 μm for the last two images on the right. An inflammatory score was assigned to each of these images and average results for each mouse in all groups are presented in the scatter plot shown in (B). * and # designate significantly different from saline-treated mice within the same mouse strain and from the other mouse strain exposed to the same treatment, respectively (P < 0.05). Data are shown as medians with 95% confidence intervals. n = 10 mice per group.

FIGURE 6

The content of smooth muscle within the airway wall. The images in (A) show representative lung sections from C57BL/6 (upper panels) and BALB/c (lower panels) mice exposed to incremental doses of house dust mite (HDM) (from left to right). The scale bar is 100 μm. For each bronchus analyzed, the area occupied by the airway smooth muscle (ASM) was divided by the square of the basement membrane (BM) perimeter and average results for each mouse in all groups are presented in the scatter plot shown in (B). Data are shown as means ± SD. n = 10 mice per group.

FIGURE 7

Goblet cell counts and epithelium thickness. The images in (A) show representative lung sections from C57BL/6 (upper panels) and BALB/c (lower panels) mice exposed to incremental doses of house dust mite (HDM) (from left to right). The scale bar is 25 μm. For each of these images, the number of goblet cells/basement membrane perimeter and the area occupied by the epithelium/basement membrane perimeter were calculated and average results for each mouse in all groups are presented in scatter plots shown in (B,C), respectively. *Designates significantly different from saline-treated mice within the same mouse strain (P < 0.05). Data are shown as means ± SD. n = 10 mice per group.