A role for CD44 in an antigen-induced murine model of pulmonary eosinophilia

Abstract

Previous studies established that IL-5-producing CD4(+) T cells play a pivotal role in allergic respiratory inflammation. It was also reported that CD4(+) T cells express higher levels of CD44 in the airway than in peripheral blood of patients with allergic respiratory diseases. We have used experimental pulmonary eosinophilia induced in mice by Ascaris suum (Asc) extract to investigate the role of CD44 in the development of allergic respiratory inflammation. Intraperitoneal administration of anti-CD44 mAb prevented both lymphocyte and eosinophil accumulation in the lung. Anti-CD44 mAb also blocked antigen-induced elevation of Th2 cytokines as well as chemokines (CCL11, CCL17) in bronchoalveolar lavage fluid (BALF). Treatment with anti-CD44 mAb inhibited the increased levels of hyaluronic acid (HA) and leukotriene concentrations in BALF that typically result from antigen challenge. Anti-CD44 mAb also blocked antigen-induced airway hyperresponsiveness. An anti-CD44 mAb (IM7) inhibited the HA-binding ability of splenocytes associated with decreased levels of CD44. Soluble CD44 levels in serum were increased in Asc-challenged IM7-treated mice, but not in KM201-treated mice, compared with Asc-challenged rat IgG-treated mice. Ab's that block CD44-HA binding reduced allergic respiratory inflammation by preventing lymphocyte and eosinophil accumulation in the lung. Thus, CD44 may be critical for development of allergic respiratory inflammation.

Figure 1

Anti-CD44 mAb blocks eosinophil and lymphocyte accumulation in BALF but does not affect numbers of eosinophils in bone marrow. The number of inflammatory cells in BALF (a) and percentages of eosinophils in bone marrow and peripheral blood (b) was determined 24 h after allergen challenge as described in Methods. Data represent mean ± SEM. The values shown are averaged from three to five independent experiments. ^#Significant differences (P < 0.05) between Asc-challenged mice (Asc) and PBS-challenged mice (PBS). *Significant differences (P < 0.05) between Asc-challenged anti-CD44 mAb-treatment mice (Asc/KM201, Asc/IM7, or Asc/IM7/30) and Asc-challenged RIgG-treatment mice (Asc/RIgG).

Figure 2

Anti-CD44 mAb blocks cytokine production in BALF. Concentrations of IL-4, IL-5, and IFN-γ in BALF were assessed 24 h after allergen challenge as described in Methods. Data represent mean ± SEM. The values shown are averaged from five independent experiments. ^#Significant differences (P < 0.05) between Asc-challenged mice (Asc) and PBS-challenged mice (PBS). *Significant differences (P < 0.05) between Asc-challenged anti-CD44 mAb-treatment mice (Asc/KM201 or Asc/IM7) and Asc-challenged RIgG-treatment mice (Asc/RIgG).

Figure 3

Effect of anti-CD44 mAb on chemokine production in BALF. Concentrations of eotaxin and TARC in BALF were assessed 24 h after allergen challenge as described in Methods. Data represent mean ± SEM. The values shown are averaged from five independent experiments. ^#Significant differences (P < 0.05) between Asc-challenged mice (Asc) and PBS-challenged mice (PBS). *Significant differences (P < 0.05) between Asc-challenged anti-CD44 mAb-treatment mice (Asc/KM201 or Asc/IM7) and Asc-challenged RIgG-treatment mice (Asc/RIgG).

Figure 4

Effect of anti-CD44 mAb on concentrations of HA and leukotriene in BALF. Concentrations of HA and leukotriene in bronchoalveolar lavage fluid (BALF) were measured 24 h after allergen challenge as described in Methods. Data represent mean ± SEM. The values shown are averaged from five independent experiments. ^#Significant differences (P < 0.05) between Asc-challenged mice (Asc) and PBS-challenged mice (PBS). *Significant differences (P < 0.05) between Asc-challenged anti-CD44 mAb-treatment mice (Asc/KM201 or Asc/IM7) and Asc-challenged RIgG-treatment mice (Asc/RIgG).

Figure 5

Effect of anti-CD44 mAb on allergen-induced airway hyperresponsiveness to methacholine. Airway responsiveness to aerosolized methacholine was measured in conscious mice as described in Methods, and sRaw values were determined. Expressed are the means ± SEM of the increase in sRaw values compared with sRaw values after PBS nebulization from five independent experiments. ^#Significant differences (P < 0.05) between Asc-challenged RIgG-treatment mice (Asc/RIgG) and PBS-challenged mice (PBS). *Significant differences (P < 0.05) between Asc-challenged anti-CD44 mAb-treatment mice (Asc/IM7 or Asc/KM201) and Asc-challenged RIgG-treatment mice (Asc/RIgG).

Figure 6

Expression and HA-binding ability of CD44 on spleen cells and alveolar macrophages in Asc-exposed mice treated with anti-CD44 mAb’s. Expression and HA-binding ability of CD44 on spleen cells (a) and alveolar macrophages (b) were assessed in mice 24 h after allergen challenge as described in Methods. Data represent mean ± SEM of percentages of positive cells compared with negative control in flow-cytometric analysis. The values shown are averaged from three to five independent experiments. *Significant differences (P < 0.05) between Asc-challenged anti-CD44 mAb-treatment mice (Asc/KM201 or Asc/IM7) and Asc-challenged RIgG-treatment mice (Asc/RIgG).

Figure 7

Soluble CD44 (sCD44) in serum and BALF. Amounts of sCD44 in serum and BALF were measured by ELISA as described in Methods. Data represent mean ± SEM. The values shown are averaged from five independent experiments. *Significant differences (P < 0.05) between Asc-challenged anti-CD44 mAb-treatment mice (Asc/KM201 or Asc/IM7) and Asc-challenged RIgG-treatment mice (Asc/RIgG).

Figure 8

Anti-CD44 mAb inhibits mite allergen–induced airway inflammation and airway hyperreactivity. The number of inflammatory cells in BALF (a) and airway responsiveness to aerosol methacholine (b) was determined 24 h after mite allergen challenge as described in Methods. Data represent mean ± SEM. The values shown are averaged from five independent experiments. ^#Significant differences (P < 0.05) between mite allergen–challenged (Der) RIgG-treatment mice (Der/RIgG) and PBS-challenged mice (PBS). *Significant differences (P < 0.05) between Der-challenged anti-CD44 mAb-treatment mice (Der/IM7) and Der-challenged RIgG-treatment mice (Der/RIgG).