In allergic airway inflammation, dendritic cells (DCs) are required for Th2 generation, recruitment, and activation in the respiratory tract. DCs have been shown to be necessary and sufficient for the induction of Th1 immune responses. In Th2 immunity and allergic airway inflammation, the ability of a DC to function as the sole APC has not been tested. We show that CD11c/Aβb mice with MHC class II expression restricted to CD11c-expressing DCs develop airway neutrophilia rather than allergic airway inflammation. Although CD11c/Aβb mice are capable of Th2 recruitment and activation in the lung, Th2 priming in CD11c/Aβb mice results in IFN-γ production. Effective Th2 generation and allergic airway inflammation was achieved in CD11c/Aβb mice after treatment with anti-IFN-γ. These studies show that DCs alone cannot drive the development of Th2 cells but require an additional MHC class II signal to stimulate effective Th2 immunity.

Asthma is characterized by the activation of CD4+ T cells in the respiratory tract resulting in airway eosinophilia, mucus hypersecretion, increased chitinase activity, and episodic airway obstruction (1, 2). The generation of Th2 cells and their recruitment into and activation in the lung require expression of MHC class II (MHC II)3 (3, 4). Dendritic cells (DCs) provide signals that direct the priming of naive CD4 T cells, including presentation of Ag in the context of MHC II, expression of costimulatory molecules, and secretion of cytokines. Indeed, Ag presentation by DCs has been shown to be sufficient for the development of Th1-dependent immune responses (5). However, the sufficiency of Ag presentation by DCs in the generation of Th2-dependent responses such as allergic airway inflammation is unknown.

In vivo depletion of DCs inhibits the priming of Th2 cells in response to Ag and the adjuvant alum (6). Similarly, DC depletion during secondary aerosolized Ag challenge blocks eosinophilic infiltration, mucus production, and bronchial hyperreactivity (4). Thus, DCs are necessary for the development of allergic airway inflammation. Yet, studies have shown that other cells, including B cells, mast cells, eosinophils, and basophils contribute to Th2 development and allergic airway inflammation, perhaps through the production of cytokines or expression of costimulatory molecules (7, 8, 9, 10, 11, 12). The in vivo requirement for Ag presentation by these accessory cell populations during Th2 generation, recruitment, and reactivation in the lung remains unclear.

To determine whether MHC II-dependent Ag presentation by DCs is sufficient to induce Th2 cell differentiation and allergic airway inflammation, we chose to take advantage of CD11c/Aβb mice with MHC II expression restricted to CD11c-expressing DCs.

CD11c/Aβb, Aβb+/−, Aβb−/−, and OT-II(Thy1.1+) mice were bred in our facility. C57BL/6 and TCRα−/− mice were purchased from The Jackson Laboratory. Mice 6–10 wk of age were used in all experiments. These studies were reviewed and approved by the Yale University Animal Care and Use Committee (New Haven, CT).

CD11c/Aβb, Aβb−/−, and TCRα−/− mice were reconstituted with 107 CD4 T cells isolated from syngeneic C57BL/6 mice and between 1 and 4 × 106 CFSE-labeled naive CD4 OT-II TCR transgenic (Tg) cells isolated by negative selection (13). The following day, mice were immunized i.p. with 50 μg of OVA (fraction V; Sigma-Aldrich) in 2 mg of alum. To induce allergic airway inflammation, mice were immunized i.p. on days 1 and 6 and challenged with inhaled 1% OVA in PBS using an ultrasonic nebulizer for 20 min daily for 3 days. Five hundred micrograms of (XMG1.2) or control rat IgG1 (Innovative Research) was administered i.p. on immunization days 1 and 6. For adoptive transfer of Th2 cells, Th2 cells were generated from OT-II mice (13). Cultured Th2 cells (2 × 106) were injected i.v. and the following day mice were challenged for 20 min daily for 7 days with inhaled 1% OVA in PBS. One day after the last exposure, mice were sacrificed for analysis of airway inflammation.

Bronchoalveolar lavage (BAL) and lung inflammatory cells were isolated (13). Cytospin preparations of BAL cells were stained with Diff-Quik (Baxter Healthcare) and differentials were performed on 200 cells based on morphology and staining characteristics. FACS analysis for CD4 and Thy1.1 was performed to identify the transferred OT-II TCR Tg cells. DCs were identified by FACS gating on low autofluorescent CD11c+ cells (14). Lungs were inflated and fixed with formalin and stained with H&E or period acid-Schiff. A histological mucus index was calculated using period acid-Schiff-stained lung sections (15). Chitinase bioactivity in BAL samples was determined using a fluorogenic substrate (2).

Spleen cells were isolated from immunized mice on day 13 and cultured (4 × 106/ml) with C57BL/6 APC (2 × 106/ml) and pOVA323–339 for 24 h. Cytokines in culture supernatants were assessed using multiplexed beads (Millipore) for IFN-γ, IL-4, IL-5, and IL-13. Intracytoplasmic staining was performed on lung cells stimulated with pOVA323–339 for 22 h followed by FACS analysis using Abs to CD4, Thy1.1, IFN-γ, and IL-4.

Data are reported as mean ± SE. Statistical significance was determined by an unpaired Student’s t test.

To determine whether DCs are sufficient to drive the development of allergic airway inflammation, we used CD11c/Aβb mice that have equivalent numbers and normal localization of DCs in the spleen and lymph nodes (LNs) (5). In CD11c/Aβb mice, CD11b+ and CD8α+ conventional DCs (cDCs) in LNs and spleen have similar expression of I-Ab compared with that of DCs from wild-type mice (5, 16), whereas lung DCs from naive CD11c/Aβb mice express modestly lower I-Ab (Fig. 1; mean fluorescence intensity: 1106 ± 28 vs 553 ± 5, p < 0.0001). CD11clow plasmacytoid DCs as well as B cells and macrophages lack expression of I-Aβb (5).

CD11c/Aβb mice lack MHC II expression on the cortical thymic epithelium and have no MHC-restricted CD4+ T cells, which are essential for the development of allergic airway inflammation (1, 5). We therefore reconstituted the mice with polyclonal CD4 T cells and OT-II CD4 (OVA TCR Tg) cells 1 day before i.p. immunization with OVA and alum. TCRα−/− mice were used as controls because they have wild-type expression of I-Aβb but, like CD11c/Aβb, would depend on transferred cells for an immune response to Ag.

Following OVA/alum immunization and inhaled OVA challenge, airway and lung eosinophilia were clearly present in Aβb+/− and TCRα−/− mice. The inflammatory response was MHC II dependent, as Aβb−/− mice had no airway infiltrates. Pulmonary eosinophilia in Aβb+/− and TCRα−/− mice was associated with mucus metaplasia and high chitinase activity in BAL fluid, characteristic of pulmonary Th2 responses (Fig. 2). CD11c/Aβb mice also developed airway inflammation; however, the infiltrating cells were predominantly neutrophils (Fig. 2 D). Lungs from OVA-immunized and -challenged CD11c/Aβb mice showed inflammation, but mucus metaplasia was minimal and chitinase activity was low. The reduced pulmonary inflammation in both CD11c/Aβb and TCRα−/− mice compared with Aβb+/− mice likely reflects a lack of endogenous CD4 T cells and limited reconstitution by the transferred CD4 T cells. These studies show that Ag presentation restricted to DCs can induce airway inflammation; however, the inflammation is neutrophil predominant.

The development of allergic airway inflammation in this model involves two steps. Intraperitoneal immunization with OVA/alum induces Th2 differentiation in the mesenteric LN and spleen, and inhaled OVA stimulates primed Th2 cells to be recruited to and activated in the respiratory tract. Both of these steps have been shown to require CD11c-expressing DCs (4, 6). We therefore asked whether CD11c/Aβb mice could effectively recruit to and activate effector Th2 cells in the respiratory tract and develop allergic airway inflammation. We generated OT-II Th2 cells in vitro with wild-type splenic APCs and transferred 2 × 106 effector Th2 cells into CD11c/Aβb, TCRα−/−, Aβb+/+, or Aβb−/− mice. Mice were then exposed to inhaled OVA. As expected, Aβb−/− mice did not exhibit lung inflammation. However, CD11c/Aβb, TCRα−/−, and Aβb+/+ all had comparable numbers of total inflammatory cells and OT-II CD4 T cells in the lung (not shown) and developed dramatic pulmonary eosinophilia and mucus metaplasia (Fig. 3). Thus, lung cDCs can effectively recruit Th2 cells to the respiratory tract, leading to robust allergic airway inflammation and other Th2 effector responses characteristic of asthma.

Because there was no evidence of a lung-specific immune defect in CD11c/Aβb mice, we next investigated whether the lack of allergic airway inflammation reflected altered T cell priming in the mesenteric LN and spleen after i.p. immunization with OVA in alum. Mice were reconstituted with polyclonal CD4 T cells and CFSE-labeled OT-II cells followed by immunization with OVA in alum. Three days later, OT-II T cell proliferation was comparable in mesenteric LNs (Fig. 4, A and B) and spleen (not shown) from CD11c/Aβb and TCRα−/− mice. We found that 60 ± 5% of OT-II cells in mesenteric LNs of CD11c/Aβb mice and 58 ± 10% of OT-II cells in mesenteric LNs of TCRα−/− mice had undergone between one and four cycles of proliferation. On day 13, CD11c/Aβb and TCRα−/− spleens had similar numbers of OT-II cells and cDCs and comparable expression of I-Ab (supplemental Fig. S1).4 Spleen cells were restimulated with pOVA323–339 and supernatants were assessed for cytokines. As expected, splenic cells from TCRα−/− mice produced IL-13, IL-4, and low IFN-γ (Fig. 4 C). Surprisingly, CD11c/Aβb splenic cells produced high levels of IFN-γ and minimal IL-4 and IL-13. Splenic cells from Aβb−/− mice produced very low levels of IFN-γ and no detectable IL-4 or IL-13. Thus in CD11c/Aβb mice a Th2 stimulus such as OVA in alum leads to DC activation of CD4 T cells and CD4 T cell proliferation, yet production of predominantly IFN-γ. Hence, when these cells are recalled to the lung by inhaled OVA, CD11c/Aβb mice do not develop allergic airway inflammation.

The fact that MHC II expression on cDCs is not sufficient to effectively prime Th2 cells but can activate naive CD4 T cells to proliferate suggests that another cell expressing MHC II must provide signals that promote Th2 development. Given the previous work demonstrating a critical role for CD11c+ DCs in Th2 induction by OVA in alum (6), these data suggest that cDCs act in concert with other MHC II-expressing cell populations to initiate Th2 immune responses.

A Th1 response to OVA/alum was also the outcome in mice deficient in IL-4, mice lacking Th2 signaling pathways, or mice that were treated with IFN-γ or IL-12 (17, 18, 19, 20, 21, 22). To define a mechanism controlling the Th1-predominant immune response to OVA in alum in CD11c/Aβb mice, we tested whether altering the cytokine milieu during CD4 T cell priming would restore a Th2 response. We treated mice with anti-IFN-γ or a control Ab followed by immunization with OVA/alum and challenge with inhaled OVA. CD11c/Aβb mice treated with anti-IFN-γ developed pulmonary eosinophilia, increased mucus (not shown), and chitinase activity, whereas CD11c/Aβb mice treated with control Ab did not (Fig. 5, A, and B). IFN-γ producing, OVA-responsive OT-II cells were increased and the ratio of IL-4 to IFN-γ was low in the lungs of CD11c/Aβb mice treated with control Ab, whereas CD11c/Aβb mice treated with anti-IFN-γ had a marked reduction in IFN- γ-producing, OVA-responsive OT-II cells and an increase in the ratio of IL-4 to IFN-γ to a level comparable to that of TCRα−/− mice (Fig. 5 C). These studies show that IFN-γ can regulate the Th1-predominent immune response to OVA/alum in CD11c/Aβb mice. Blockade of IFN-γ permits the generation of a Th2 response and the development of allergic airway inflammation in CD11c/Aβb mice. This suggests that MHC II-expressing cDC do not provide a cytokine milieu supportive of Th2 development.

These data show that effective Th2 immunity requires class II MHC expression on cells other than cDCs. Because prior studies indicate that cDCs are required to generate Th2 immune responses to OVA/alum (6), it is likely that an alternative APC interacts with the naive T cell in conjunction with cDC to activate Th2 immunity. cDCs were also shown to be insufficient to induce Th2 immunity in the gut mucosal surface, suggesting that alternative APCs may be necessary universally in Th2 generation (23). MHC II expression on this accessory APC may insure specific targeting, but its major role may be to provide a cytokine milieu supportive of Th2 development, because the signal for T cell proliferation can be provided by cDCs. Another explanation is that an alternative APC is sufficient on its own to stimulate an immune response. Potential candidates that express MHC II and have the capacity to stimulate Th2 development include basophils, eosinophils, macrophages, and B cells (8, 10, 23, 24, 25). Traditional theory suggests that cDCs can provide all of the necessary signals for CD4 T effector cell priming. These data show that effective Th2 immunity requires class II MHC expression on cells in addition to cDCs.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grant R01–64040 (to L.C.) and the Seltzer Family Translational Research Fund (to L.C.).

3

Abbreviations used in this paper: MHC II, MHC class II; BAL, bronchoalveolar lavage; DC, dendritic cell; cDC, conventional DC; LN, lymph node; Tg, transgenic.

4

The online version of this article contains supplemental material.

1
Cohn, L., J. A. Elias, G. L. Chupp.
2004
. Asthma: mechanisms of disease persistence and progression.
Annu. Rev. Immunol.
22
:
789
-815.
2
Zhu, Z., T. Zheng, R. J. Homer, Y. K. Kim, N. Y. Chen, L. Cohn, Q. Hamid, J. A. Elias.
2004
. Acidic mammalian chitinase in asthmatic Th2 inflammation and IL-13 pathway activation.
Science
304
:
1678
-1682.
3
Lambrecht, B. N., B. Salomon, D. Klatzmann, R. A. Pauwels.
1998
. Dendritic cells are required for the development of chronic eosinophilic airway inflammation in response to inhaled antigen in sensitized mice.
J. Immunol.
160
:
4090
-4097.
4
van Rijt, L. S., S. Jung, A. Kleinjan, N. Vos, M. Willart, C. Duez, H. C. Hoogsteden, B. N. Lambrecht.
2005
. In vivo depletion of lung CD11c+ dendritic cells during allergen challenge abrogates the characteristic features of asthma.
J. Exp. Med.
201
:
981
-991.
5
Lemos, M. P., L. Fan, D. Lo, T. M. Laufer.
2003
. CD8α+ and CD11b+ dendritic cell-restricted MHC class II controls Th1 CD4+ T cell immunity.
J. Immunol.
171
:
5077
-5084.
6
Kool, M., T. Soullie, M. van Nimwegen, M. A. Willart, F. Muskens, S. Jung, H. C. Hoogsteden, H. Hammad, B. N. Lambrecht.
2008
. Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells.
J. Exp. Med.
205
:
869
-882.
7
Karasuyama, H., K. Mukai, Y. Tsujimura, K. Obata.
2009
. Newly discovered roles for basophils: a neglected minority gains new respect.
Nat. Rev. Immunol.
9
:
9
-13.
8
Hernandez, H. J., Y. Wang, M. J. Stadecker.
1997
. In infection with Schistosoma mansoni, B cells are required for T helper type 2 cell responses but not for granuloma formation.
J. Immunol.
158
:
4832
-4837.
9
Georas, S. N., J. Guo, U. De Fanis, V. Casolaro.
2005
. T-helper cell type-2 regulation in allergic disease.
Eur. Respir J.
26
:
1119
-1137.
10
Jacobsen, E. A., S. I. Ochkur, R. S. Pero, A. G. Taranova, C. A. Protheroe, D. C. Colbert, N. A. Lee, J. J. Lee.
2008
. Allergic pulmonary inflammation in mice is dependent on eosinophil-induced recruitment of effector T cells.
J. Exp. Med.
205
:
699
-710.
11
Hammad, H., B. N. Lambrecht.
2006
. Recent progress in the biology of airway dendritic cells and implications for understanding the regulation of asthmatic inflammation.
J. Allergy Clin. Immunol.
118
:
331
-336.
12
Galli, S. J., S. Nakae, M. Tsai.
2005
. Mast cells in the development of adaptive immune responses.
Nat. Immunol.
6
:
135
-142.
13
Niu, N., M. K. Le Goff, F. Li, M. Rahman, R. J. Homer, L. Cohn.
2007
. A novel pathway that regulates inflammatory disease in the respiratory tract.
J. Immunol.
178
:
3846
-3855.
14
Vermaelen, K., R. Pauwels.
2004
. Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: methodology and new insights.
Cytometry A
61
:
170
-177.
15
Cohn, L., R. J. Homer, A. Marinov, J. Rankin, K. Bottomly.
1997
. Induction of airway mucus production By T helper 2 (Th2) cells: a critical role for interleukin 4 in cell recruitment but not mucus production.
J. Exp. Med.
186
:
1737
-1747.
16
Lemos, M. P., F. Esquivel, P. Scott, T. M. Laufer.
2004
. MHC class II expression restricted to CD8α+ and CD11b+ dendritic cells is sufficient for control of Leishmania major.
J. Exp. Med.
199
:
725
-730.
17
Brewer, J. M., M. Conacher, A. Satoskar, H. Bluethmann, J. Alexander.
1996
. In interleukin-4-deficient mice, alum not only generates T helper 1 responses equivalent to Freund’s complete adjuvant, but continues to induce T helper 2 cytokine production.
Eur. J. Immunol.
26
:
2062
-2066.
18
Tomkinson, A., A. Kanehiro, N. Rabinovitch, A. Joetham, G. Cieslewicz, E. W. Gelfand.
1999
. The failure of STAT6-deficient mice to develop airway eosinophilia and airway hyperresponsiveness is overcome by interleukin-5.
Am. J. Respir. Crit. Care Med.
160
:
1283
-1291.
19
Gavett, S. H., D. J. O'Hearn, X. Li, S. K. Huang, F. D. Finkelman, M. Wills-Karp.
1995
. Interleukin 12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in mice.
J. Exp. Med.
182
:
1527
-1536.
20
Rempel, J. D., M. Wang, K. T. HayGlass.
1997
. In vivo IL-12 administration induces profound but transient commitment to T helper cell type 1-associated patterns of cytokine and antibody production.
J. Immunol.
159
:
1490
-1496.
21
Kips, J. C., G. J. Brusselle, G. F. Joos, R. A. Peleman, J. H. Tavernier, R. R. Devos, R. A. Pauwels.
1996
. Interleukin-12 inhibits antigen-induced airway hyperresponsiveness in mice.
Am. J. Respir. Crit. Care Med.
153
:
535
-539.
22
Hsieh, C. S., S. E. Macatonia, A. O'Garra, K. M. Murphy.
1993
. Pathogen-induced Th1 phenotype development in CD4+ αβ-TCR transgenic T cells is macrophage dependent.
Int. Immunol.
5
:
371
-382.
23
Perrigoue, J. G., S. A. Saenz, M. C. Siracusa, E. J. Allenspach, B. C. Taylor, P. R. Giacomin, M. G. Nair, Y. Du, C. Zaph, N. van Rooijen, et al
2009
. MHC class II-dependent basophil-CD4+ T cell interactions promote TH2 cytokine-dependent immunity.
Nat. Immunol.
10
:
697
-705.
24
Sokol, C. L., N. Q. Chu, S. Yu, S. A. Nish, T. M. Laufer, R. Medzhitov.
2009
. Basophils function as antigen-presenting cells for an allergen-induced T helper type 2 response.
Nat. Immunol.
10
:
713
-720.
25
Yoshimoto, T., K. Yasuda, H. Tanaka, M. Nakahira, Y. Imai, Y. Fujimori, K. Nakanishi.
2009
. Basophils contribute to TH2-IgE responses in vivo via IL-4 production and presentation of peptide-MHC class II complexes to CD4+ T cells.
Nat. Immunol.
10
:
706
-712.