Allergic pulmonary inflammation in mice is dependent on eosinophil-induced recruitment of effector T cells

Abstract

The current paradigm surrounding allergen-mediated T helper type 2 (Th2) immune responses in the lung suggests an almost hegemonic role for T cells. Our studies propose an alternative hypothesis implicating eosinophils in the regulation of pulmonary T cell responses. In particular, ovalbumin (OVA)-sensitized/challenged mice devoid of eosinophils (the transgenic line PHIL) have reduced airway levels of Th2 cytokines relative to the OVA-treated wild type that correlated with a reduced ability to recruit effector T cells to the lung. Adoptive transfer of Th2-polarized OVA-specific transgenic T cells (OT-II) alone into OVA-challenged PHIL recipient mice failed to restore Th2 cytokines, airway histopathologies, and, most importantly, the recruitment of pulmonary effector T cells. In contrast, the combined transfer of OT-II cells and eosinophils into PHIL mice resulted in the accumulation of effector T cells and a concomitant increase in both airway Th2 immune responses and histopathologies. Moreover, we show that eosinophils elicit the expression of the Th2 chemokines thymus- and activation-regulated chemokine/CCL17 and macrophage-derived chemokine/CCL22 in the lung after allergen challenge, and blockade of these chemokines inhibited the recruitment of effector T cells. In summary, the data suggest that pulmonary eosinophils are required for the localized recruitment of effector T cells.

Figure 1.

Allergen-mediated pulmonary Th2 inflammatory responses and lymphocyte infiltration of the lung are attenuated in the absence of eosinophils. Wild-type and PHIL mice were subjected to the acute OVA sensitization/challenge (control animals received saline alone) protocol described in Materials and methods and Fig. S1 A. (A) Immunohistochemistry using an anti–mouse MBP rat mAb (black) demonstrated the absence of infiltrating eosinophils in OVA-treated PHIL mice relative to wild-type controls. Bar, 100 μm. (B) Allergen-induced increases in BAL Th2 cytokine levels (as assessed by ELISA) failed to occur in PHIL mice as compared with wild-type controls. (C) Assessments of BAL cellularity demonstrated that lymphocyte numbers are significantly reduced in OVA-treated PHIL mice relative to wild-type controls. Kinetic assessments (Fig. S1 B) of OVA-induced eosinophil (D) and lymphocyte (E) accumulation in the BAL of wild-type versus PHIL mice 1, 4, and 15 d after OVA challenge showed that in the absence of eosinophils, the early and sustained influx of lymphocytes into the lung after allergen challenge is abolished. Data presented are the means ± SE. *, P < 0.05.

Figure 2.

The OVA-induced accumulation of both CD4⁺ and CD8⁺ T cells is significantly reduced in the airways, lungs, and regional draining lymph nodes in the absence of eosinophils. Flow cytometric assessments of leukocytes derived from BAL, whole-lung digests, and lung draining lymph nodes showed that in the absence of eosinophils (i.e., PHIL), significant reductions of both CD4⁺ and CD8⁺ T cell subtypes are observed in all three pulmonary compartments after OVA provocation. This observation was shown to be lung specific, as assessments of the splenic cellularity of these mice showed no difference in either CD4⁺ or CD8⁺ T cell numbers between OVA-treated wild-type and PHIL mice. In each case, single-cell suspensions were counted, and the fractional composition of each suspension was assessed by flow cytometry based initially on scatter to establish a lymphocyte gate, and then stained with antibodies for the T cell markers CD3, CD4, and CD8. Data presented are the means ± SE. *, P < 0.05.

Figure 3.

OVA-treated PHIL mice display reduced accumulation of CD4⁺ T cell subtypes, intrinsic defects in allergen-specific T cell activities, and the loss of allergen-induced TARC and MDC expression. (A) Determinations of T cell subsets for whole-lung digests, regional draining lymph nodes, and spleen were completed by examining total cell numbers between OVA-treated wild-type versus PHIL mice. OVA-treated wild-type versus PHIL mice show that CD4⁺/CD25⁺ (T regulatory/proliferation) and CD4⁺/NK1.1⁺ (NKT) cells were elevated in the lungs of OVA-treated wild-type mice but not in OVA-treated PHIL mice. (B) Effector T cells (CD4⁺/CD62L^−/lo/CD44^hi) were reduced in lungs and caudal mediastinal (i.e., regional draining) lymph nodes. Furthermore, caudal mediastinal lymph nodes also had reduced numbers of memory T cells (CD4⁺/CD62L^med/hi/CD44^hi). Naive T cell (CD4⁺/CD62L^hi/CD44^−/lo) numbers remained unchanged between wild-type and PHIL mice in both the spleen and pulmonary compartments. (C) Splenocytes from sensitized and sensitized/challenged PHIL and wild-type mice were cultured for 96 h in the presence of media alone, 200 μg/ml OVA, or cross-linked anti-CD3/-CD28. Measurement of IL-13 in the supernatant revealed an inability for sensitized/challenged PHIL splenocytes to generate wild-type levels of this cytokine. (D) BAL chemokine levels were determined by ELISA from wild-type and PHIL mice subjected to the acute OVA sensitization/challenge protocol described in Fig. S1 A. Significantly, although substantive increases in MDC and TARC were observed in the BAL of OVA-treated eosinophil-sufficient wild-type animals (i.e., relative to saline control mice), increased levels of these Th2 cell chemokines did not occur in the lungs of OVA-treated PHIL mice (i.e., in the absence of eosinophils). Kinetic assessments (Fig. S1 B) of MDC and TARC in the BAL of post–OVA-challenged wild-type mice showed that the appearance of these Th2 cell chemokines correlated with the levels of OVA-induced airway eosinophilia. Data presented are the means ± SE. *, P < 0.05.

Figure 4.

Adoptive transfer of both OVA-specific Th2-polarized effector T cells (OT-II) and eosinophils are required to generate Th2 pulmonary pathologies in PHIL mice. (A) Schematic timeline of the acute OVA challenge protocol associated with the adoptive transfer studies of Th2-polarized OVA-specific T cells (OT-II) and eosinophils. (B) Th2 BAL levels of the cytokines IL-13 and IL-5 (IL-4 was undetectable) were assessed by ELISA. PHIL mice receiving either OT-II cells (group I) or eosinophils (group II) alone showed no pathologies, whereas adoptive transfer of OT-II cells in PHIL mice together with the restoration of the absent pulmonary eosinophilia (group III) promoted significant increases in the BAL levels of the Th2 cytokines IL-13 and IL-5. Data presented are the means ± SE. *, P < 0.05. (C) Assessments of goblet cell metaplasia and airway epithelial cell mucin accumulation by PAS staining of lung sections (purple) revealed that only the adoptive transfer of both OT-II cells and eosinophils to PHIL mice was capable of reconstituting this allergen-induced histopathology. PHIL/Eos, OVA-provoked PHIL mice after i.t. transfer of eosinophils; PHIL/OT-II, OVA-provoked PHIL mice after OT-II cell transfer; PHIL/OT-II/Eos, OVA-provoked PHIL mice after adoptive transfer of both OT-II cell (i.v.) and eosinophils (i.t.); WT/OT-II, OVA-provoked wild-type mice after OT-II cell transfer. Bar, 100 μm.

Figure 5.

Eosinophil-dependent expression of TARC and MDC is required for CD4⁺ T cell recruitment to the lungs of allergen-challenged PHIL mice. (A) Wild-type and PHIL mice were adoptively transferred with either OT-II cells (group I) or eosinophils (group II) alone, or with both eosinophils and OT-II cells (group III). Assessments of the CD4⁺ T lymphocyte cellularity in BAL and whole-lung digests showed that in contrast to adoptive transfer of either OT-II cells (group I) or eosinophils (group II) alone, adoptive transfer of OT-II cells in PHIL mice together with the restoration of the absent pulmonary eosinophilia (group III) promoted OVA-induced CD4⁺ T cell accumulation in the lung. (B) ELISA measurements of these samples showed that BAL TARC and MDC levels are restored to wild-type levels in PHIL mice receiving both eosinophils and OT-II cells (group III). (C) Groups of mice were treated as in A, with the exception that group III mice were additionally administered either control polyclonal goat antibody (Isotype Control Ab) or neutralizing antibodies to TARC and MDC (α-TARC/α-MDC) on days 0, 1, 2, and 3. Whole-lung digests were analyzed for CD4⁺ T cell populations by flow cytometry. Horizontal bars represent means. Data presented are the means ± SE. *, P < 0.05.