Signal transducer and activator of transcription 6 controls chemokine production and T helper cell type 2 cell trafficking in allergic pulmonary inflammation

Abstract

Antigen-specific CD4 T helper type 2 (Th2) cells play a pivotal role in the induction of allergic asthma, but the mechanisms regulating their recruitment into the airways are unknown. Signal transducer and activator of transcription factor (Stat)6 is a transcription factor essential for Th2 cell differentiation. Here we show that Stat6 also controls Th2 cell recruitment and effector function in allergic inflammation in vivo. To isolate the role of Stat6 in regulating Th2 cell trafficking and effector function from its role in Th2 cell differentiation, we used a murine model of asthma in which in vitro-differentiated Stat6(+/+) antigen-specific Th2 cells were adoptively transferred into naive Stat6(-/-) and Stat6(+/+) mice followed by aerosol antigen challenge. We found that all of the features of asthma, including Th2 cell accumulation, Th2 and eosinophil-active chemokine production, and airway eosinophilia, mucus production, and hyperresponsiveness seen in Stat6(+/+) mice, were dramatically absent in Stat6(-/)- mice that received Stat6(+/)+ antigen-specific Th2 cells. Our findings establish Stat6 as essential for Th2 cell trafficking and effector function and suggest that interruption of Stat6 signaling in resident cells of the lung is a novel approach to asthma therapy.

Figure 1

Attenuated eosinophilic airway inflammation and mucus cell production in Stat6^{− / −} mice after Th2 cell transfer and aerosol OVA challenge. (a) BAL cell counts. OVA-specific Stat6^+/+ Th2 cells were transferred intravenously into Stat6^{+ / +} and Stat6^{− / −} mice and exposed to daily challenges with aerosolized OVA or PBS for 7 d. 18 h after the last OVA challenge, leukocytes were recovered from the BAL and total and differential counts were performed. Data represent mean number of BAL cells (± SEM; n = 10 for OVA-Stat6^{+ / +} and OVA-Stat6^{− / −} mice; n = 3 for PBS-Stat6^{+ / +} and PBS-Stat6^{− / −} mice). *P < 0.0001; **P = 0.0003; and ***P = 0.02 in OVA-Stat6^{+ / +} versus OVA-Stat6^{− / −} mice. (b) Hematoxylin and eosin (i and ii) and PAS (iii and iv) stained formalin-fixed lung sections isolated from Stat6^{+ / +} (i and iii) and Stat6^{− / −} mice (ii and iv) after transfer of OVA-specific Th2 cell and aerosol OVA challenge described above. (i) Stat6^{+ / +}. Characteristic intense peribronchial inflammatory cell infiltrate comprised of lymphocytes and eosinophils. Enlarged multinucleated giant cells are seen within the inflammation and the alveolar spaces. In the peribronchial location, they are loosely organized into a granuloma. (ii) Stat6^{− / −}. By contrast, Stat6^{− / −} mice show a marked attenuated inflammatory response around the airways with notable and marked decreases in eosinophils. There is almost complete absence of multinucleated giant cells and granulomas. (iii and iv) PAS staining of lung sections revealed characteristic mucin staining in bronchial epithelium (arrow) of Stat6^{+ / +} (iii) mice, which is almost completely absent in Stat6^{− / −} mice (iv). Similarly treated PBS-challenged Stat6^{+ / +} and Stat6^{− / −} had no pulmonary inflammation or mucus production (data not shown). Note presence of subepithelial eosinophils (arrowheads) in Stat6^{+ / +} (iii) but not Stat6^{− / −} (iv) mice. Original magnifications: (i and ii) ×125; (iii and iv) ×600.

Figure 1

Attenuated eosinophilic airway inflammation and mucus cell production in Stat6^{− / −} mice after Th2 cell transfer and aerosol OVA challenge. (a) BAL cell counts. OVA-specific Stat6^+/+ Th2 cells were transferred intravenously into Stat6^{+ / +} and Stat6^{− / −} mice and exposed to daily challenges with aerosolized OVA or PBS for 7 d. 18 h after the last OVA challenge, leukocytes were recovered from the BAL and total and differential counts were performed. Data represent mean number of BAL cells (± SEM; n = 10 for OVA-Stat6^{+ / +} and OVA-Stat6^{− / −} mice; n = 3 for PBS-Stat6^{+ / +} and PBS-Stat6^{− / −} mice). *P < 0.0001; **P = 0.0003; and ***P = 0.02 in OVA-Stat6^{+ / +} versus OVA-Stat6^{− / −} mice. (b) Hematoxylin and eosin (i and ii) and PAS (iii and iv) stained formalin-fixed lung sections isolated from Stat6^{+ / +} (i and iii) and Stat6^{− / −} mice (ii and iv) after transfer of OVA-specific Th2 cell and aerosol OVA challenge described above. (i) Stat6^{+ / +}. Characteristic intense peribronchial inflammatory cell infiltrate comprised of lymphocytes and eosinophils. Enlarged multinucleated giant cells are seen within the inflammation and the alveolar spaces. In the peribronchial location, they are loosely organized into a granuloma. (ii) Stat6^{− / −}. By contrast, Stat6^{− / −} mice show a marked attenuated inflammatory response around the airways with notable and marked decreases in eosinophils. There is almost complete absence of multinucleated giant cells and granulomas. (iii and iv) PAS staining of lung sections revealed characteristic mucin staining in bronchial epithelium (arrow) of Stat6^{+ / +} (iii) mice, which is almost completely absent in Stat6^{− / −} mice (iv). Similarly treated PBS-challenged Stat6^{+ / +} and Stat6^{− / −} had no pulmonary inflammation or mucus production (data not shown). Note presence of subepithelial eosinophils (arrowheads) in Stat6^{+ / +} (iii) but not Stat6^{− / −} (iv) mice. Original magnifications: (i and ii) ×125; (iii and iv) ×600.

Figure 3

Decreased recruitment of OVA-specific Th2 cells in the BAL despite activation in paratracheal LNs of Stat6^−/− mice. (a and b) Decreased OVA-specific CD4⁺ cells in the BAL of OVA-Stat6^−/− mice. FACS^® analysis was performed on BAL cells isolated from mice described above. Cells were stained with anti-CD4 and KJ1-26 (OVA-TCR specific) antibodies. (a) Percentage of CD4 and OVA-specific CD4 cells in the BAL of Stat6^+/+ and Stat6^−/− mice. *P < 0.0001 in OVA-Stat6^+/+ versus OVA-Stat6^−/− mice. (b) Total number of OVA-specific cells in the BAL = (% KJ⁺ cells in the lymphocyte gate) × (total number of lymphocytes calculated by differential counts). *P < 0.0001 in OVA-Stat6^+/+ versus OVA-Stat6^−/− mice. (c and d) Analysis of paratracheal LNs of OVA- and PBS-challenged Stat6^+/+and Stat6^−/− mice after Th2 cell transfers. (c) Total LN cells recovered from paratracheal LNs (n = 10 for OVA-Stat6^+/+ and OVA-Stat6^−/− mice; n = 3 for PBS-Stat6^+/+ and PBS-Stat6^−/− mice). *P < 0.0001 in OVA-Stat6^+/+ versus PBS-Stat6^+/+ mice and OVA-Stat6^−/− versus PBS-Stat6^−/− mice. (d) Percentage of CD4 and OVA-specific cells in paratracheal LNs (mean of five mice/group, representative of two experiments). There were too few CD4⁺ cells in the BAL of PBS-Stat6^+/+ and PBS-Stat6^−/− mice for FACS^® analysis. *P = 0.004 in Stat6^+/+ versus Stat6^−/− mice; **P < 0.0001 in Stat6^+/+ versus Stat6^−/− mice.

Figure 2

Stat6^−/− mice have attenuated AHR after transfer of Th2 cells and OVA challenge. Airway reactivity to increasing concentrations of methacholine was measured in OVA-Stat6^+/+ (n = 8), OVA-Stat6^−/− (n = 8), PBS-Stat6^+/+ (n = 4), and PBS-Stat6^−/− (n = 4) mice. Data represent mean P _enh values (± SEM). *P < 0.001 in OVA-Stat6^+/+ versus OVA-Stat6^−/− mice.

Figure 4

Cytokine levels in the lung and BAL of Stat6^+/+ and Stat6^−/− mice. (a) Cytokine mRNA expression was determined by RPA analysis. Each lane represents a single mouse. Levels of IL-4 (b), IL-5 (c), and IL-13 (d) were measured in the BAL of OVA- and PBS-challenged Stat6^+/+ and Stat6^−/− mice by ELISA. Data are presented as mean cytokine level (± SEM; n = 5 mice for IL-4 and IL-5; n = 10 for IL-13; n = 3 for all PBS-challenged controls). *P = 0.004 in OVA-Stat6^+/+ versus PBS-Stat6^+/+ mice; **P = 0.0002 in OVA-Stat6^+/+ versus OVA-Stat6^−/− mice; and P = 0.001 in OVA-Stat6^−/− versus PBS-Stat6^−/− mice. No significant differences were detected in IL-13 levels.

Figure 5

Chemokine induction in the lungs of OVA- and PBS-challenged Stat6^+/+ and Stat6^−/− mice. Chemokine mRNA expression was determined by Northern blot analysis. Each lane contains RNA from a different mouse. Blots were sequentially hybridized with the cDNA probes for the indicated chemokine and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control for RNA loading. Blots were exposed for 72 h except for TCA-3, which was exposed for 2 wk.