Induction of airway mucus production By T helper 2 (Th2) cells: a critical role for interleukin 4 in cell recruitment but not mucus production

Abstract

Airway inflammation is believed to stimulate mucus production in asthmatic patients. Increased mucus secretion is an important clinical symptom and contributes to airway obstruction in asthma. Activated CD4 Th1 and Th2 cells have both been identified in airway biopsies of asthmatics but their role in mucus production is not clear. Using CD4 T cells from mice transgenic for the OVA-specific TCR, we studied the role of Th1 and Th2 cells in airway inflammation and mucus production. Airway inflammation induced by Th2 cells was comprised of eosinophils and lymphocytes; features found in asthmatic patients. Additionally, there was a marked increase in mucus production in mice that received Th2 cells and inhaled OVA, but not in mice that received Th1 cells. However, OVA-specific Th2 cells from IL-4-deficient mice were not recruited to the lung and did not induce mucus production. When this defect in homing was overcome by administration of TNF-alpha, IL-4 -/- Th2 cells induced mucus as effectively as IL-4 +/+ Th2 cells. These studies establish a role for Th2 cells in mucus production and dissect the effector functions of IL-4 in these processes. These data suggest that IL-4 is crucial for Th2 cell recruitment to the lung and for induction of inflammation, but has no direct role in mucus production.

Figure 1

Cytokine production by Th1 or Th2-like cells before and after transfer. (A) At the time of transfer into recipient mice in vitro generated DO11.10 CD4 Th1, Th2 or freshly isolated CD4 T cells from naive BALB/c mice (N) were cultured with antigen presenting cells (2 × 10⁶ cells/ml) in the presence of pOVA^323-339. (B) After 7 d of exposure to inhaled OVA, BAL was performed on individual mice that received Th1 (Th1-OVA), Th2 (Th2-OVA), or naive (N) CD4 T cells. Total leukocytes recovered from BAL were restimulated in vitro with pOVA^323-339. BAL cells from mice that received naive CD4 T cells (N) and inhaled OVA contained <3% lymphocytes and were insufficient for cytokine analysis. Supernatants were collected at 48 h and cytokine ELISAs were performed. Cytokines production in BAL was adjusted for 10⁶ DO11.10 CD4 T cells per ml as determined by FACS^® analysis. Mean cytokine levels (±SEM) are shown (n = 4–5 mice per group). One experiment is shown and is representative of three experiments.

Figure 2

Lung histology in mice that received transfer of OVA-specific Th1, Th2, or naive CD4 T cells and 7 d of aerosolized OVA. (A1, B1, C1) = Th1 cell recipient. (A1) Lung showing moderate inflammation in peribronchial and perivascular location; H & E, 50×. (B1) Small airway showing neutrophil and mononuclear cellular infiltrate; H & E, 500×. (C1) Airways showing no staining for mucus; DPAS, 100×. (A2, B2, C2) = Th2 cell recipient. (A2) Lung showing moderate inflammation in peribronchial and perivascular location; H & E, 50×. (B2) Small airway showing infiltrate of eosinophils and small mononuclear cells; H & E, 500× (C2) Airways showing increased purple-staining mucus in bronchial epithelium; DPAS, 100×. (D) Mouse that received transfer of naive BALB/c CD4 T cells; H & E 50×.

Figure 3

Bronchoalveolar lavage cell recovery in mice after transfer of cells and exposure to inhaled OVA. Differential counts were performed on cytospins of cells recovered from BAL from individual mice. Mean cell counts (±SEM) are shown (n = 5 mice per group). One experiment is shown and is representative of three experiments. Statistical significance was determined by unpaired Student's t test. *P <0.0001 Th2 vs. Th1; ^‡ P <0.02 Th1 vs. Th2.

Figure 4

Immunohistochemical analysis of lungs from mice that received transfer of DO11.10 TCR transgenic cells. Localization in the lung of CD4 and donor TCR transgenic cells (A, B). Sequential lung sections from mice that received Th1 cells and exposure to aerosolized OVA were stained with (A) anti-CD4 and (B) KJ1-26 antibodies. KJ1-26 recognizes the DO11.10 TCR and was present on >99% of Th1 cells at the time of transfer. An airway and small vessel are shown with surrounding inflammatory cells (100×). A majority of the infiltrating cells stain positively for both CD4 and the transgenic TCR. (C, D) MHC Class II expression in the lung after transfer of Th1 or Th2 cells. Lung sections from mice that received (C) Th1 or (D) Th2 cells and exposure to inhaled OVA were stained with an anti–I-A antibody (212.A1). A small bronchiole is shown. Both mice that received transfer of Th1 or Th2 cells and aerosolized OVA exhibited increased MHC Class II expression on cells in the inflammatory infiltrates (red-stained cells) (200×). Mice that received transfer of Th1 cells had increased red-staining of bronchial epithelium, whereas mice that received Th2 cells had no evidence of Class II MHC expression on bronchial epithelial cells.

Figure 5

Mucus staining in airways of mice after transfer of Th1, Th2, or naive (N) CD4 T cells and exposure to inhaled OVA. An Histologic Mucus Index (HMI) was performed on lung sections stained with DPAS. Mean HMI (±SEM) is shown (n = 4 mice per group). One experiment is shown and is representative of three experiments. Statistical significance was determined by unpaired Student's t test. *P <0.0001, Th1 vs. Th2.

Figure 6

Mucus production and airway inflammation in mice after transfer of IL-4 +/+ or IL-4 −/− Th2 cells and exposure to inhaled OVA. BALB/c mice received transfer of OVA-specific IL-4 +/+ or IL-4 −/− Th2 cells and inhaled OVA. Controls received no cells and inhaled OVA. (A) HMI was performed on lung sections stained with DPAS. Mean HMI (±SEM) is shown. (B) BAL cells recovered from mice after exposure to 7 d of inhaled OVA. Differential counts were performed on cytospins of cells recovered from BAL of individual mice. Mean cell counts (±SEM) are shown (n = 4 mice per group). One experiment is shown and is representative of three experiments. Statistical significance was determined by unpaired Student's t test. *P <0.005, IL-4 +/+ Th2 vs. IL-4 −/− Th2. ^‡ P <0.0001, IL-4 +/+ Th2 vs. IL-4 −/− Th2.

Figure 7

Mucus production and airway inflammation in mice after transfer of cells and exposure to TNF-α and inhaled OVA. BALB/c mice received transfer of OVA-specific IL-4 +/+, IL-4 −/− Th2 cells or naive CD4 T cells (N), then exposure to TNF-α and 7 d of inhaled OVA. (A) HMI was performed on lung sections stained with DPAS. Mean HMI (±SEM) is shown. (B) BAL cells recovered from mice. Differential counts were performed on cytospins of cells recovered from BAL of individual mice. Mean cell counts (±SEM) are shown (n = 4–5 mice per group). One experiment is shown and is representative of three experiments. Statistical significance was determined by unpaired Student's t test. *P <0.02, IL-4 +/+ Th2 vs. IL-4 −/− Th2.

Figure 8

Mucus production and airway inflammation in mice after transfer of cells to IL-4 −/− recipient mice. IL-4 −/− mice received transfer of OVA-specific IL-4 +/+ or IL-4 −/− Th2 cells and exposure to TNF-α and 7 d of inhaled OVA. HMI was performed on lung sections stained with DPAS. Mean HMI (±SEM) is shown. (n = 3–4 mice per group). One experiment is shown and is representative of two experiments.