Essential Contribution of CD4+ T Cells to Antigen-Induced Nasal Hyperresponsiveness in Experimental Allergic Rhinitis

Abstract

Nasal hyperresponsiveness (NHR) is a characteristic feature of allergic rhinitis (AR); however, the pathogenesis of NHR is not fully understood. In this study, during the establishment of an experimental AR model using ovalbumin-immunized and -challenged mice, augmentation of the sneezing reaction in response to nonspecific proteins as well as a chemical stimulant was detected. Whether NHR is independent of mast cells and eosinophils was determined by using mast cell- and eosinophil-deficient mice. NHR was suppressed by treatment with anti-CD4 antibody, suggesting the pivotal contribution of CD4+ T cells. Furthermore, antigen challenge to mice to which in vitro-differentiated Th1, Th2, and Th17 cells but not naïve CD4+ T cells had been adoptively transferred led to the development of equivalent NHR. Since antigen-specific IgE and IgG were not produced in these mice and since antigen-specific IgE-transgenic mice did not develop NHR even upon antigen challenge, humoral immunity would be dispensable for NHR. CD4+ T cells play a crucial role in the pathogenesis of AR via induction of NHR, independent of IgE-, mast cell-, and eosinophil-mediated responses.

Fig 1. Antigen-induced NHR in immunized mice.

Mice were immunized with 4-time i.p. injection of OVA plus alum. Two weeks after the last immunization, mice were challenged once a day with daily i.n. injection of OVA or BSA solution, or of saline on days 35–38 and 41–43. Then, these mice were challenged with OVA, BSA, or saline on day 44 (A). On days 41–44, the number of sneezes was counted for 5 min just after i.n. administration of OVA, BSA, or saline (B). The BSA- and histamine-evoked sneezing response was evaluated at 6 h after 0 (day 34)- to 4 (day 38)-time challenge with OVA or saline (C). Time course of histamine-evoked sneezing response after 4-time challenge on days 35–38 with OVA or saline was evaluated (D). Data are expressed as mean ± SEM for 4–10 animals. *p < 0.05, compared with saline-challenged control mice (Mann-Whitney U test).

Fig 2. Antigen-induced nasal inflammation in immunized mice.

Immunized mice were challenged 4 times with OVA or saline on days 35–38. Six hours after the last challenge, accumulation of lymphocytes, neutrophils, and eosinophils in NALF (A) and expression of IL-4, IL-5, IL-13, IFN-γ, IL-17, and EPO mRNA in the nasal tissue (B) were examined. Data are expressed as mean ± SEM for 4–9 animals. *p < 0.05, **p < 0.01, compared with saline-challenged control mice (Mann-Whitney U test). Lateral nose sections were stained with hematoxylin and eosin and observed under optical microscopy. Representative images from 3 independent experiments are shown in panel C. The lower panels are enlarged views of the squares in the upper panels. White and black bars indicate 500 and 50 μm, respectively. The number of infiltrated eosinophils and the grade of epithelial damage are evaluated from the histological images (D). Data are expressed as mean ± SEM. **p < 0.01, compared with saline-challenged control mice (Mann-Whitney U test).

Fig 3. Antigen-induced NHR in mast cell-deficient mice.

Immunized W/W^v and +/+ mice were challenged 7 times with OVA or saline, as shown in Fig 1. Six hours after the last challenge (day 43), the number of sneezes evoked by histamine (A), the accumulation of lymphocytes, neutrophils, and eosinophils in NALF (B), and the antigen-specific serum IgE levels were examined (C). Data are expressed as mean ± SEM for 4–9 animals. *p < 0.05, **p < 0.01, ***p < 0.001, compared with saline-challenged control mice (Mann-Whitney U test).

Fig 4. Antigen-induced NHR in eosinophil-deficient mice.

Immunized ΔdblGATA and WT mice were challenged 7 times with OVA or saline, as shown in Fig 1. Six hours after the last challenge (day 43), the number of sneezes evoked by histamine (A), the accumulation of lymphocytes, neutrophils, and eosinophils in NALF (B), and the antigen-specific serum IgE levels were examined. Data are expressed as mean ± SEM for 4–8 animals. *p < 0.05, **p < 0.01, compared with saline-challenged control mice (Mann-Whitney U test). N.D.: not detectable.

Fig 5. Effect of anti-CD4 mAb on antigen-induced NHR.

Immunized mice were challenged 4 times with OVA or saline, as shown in Fig 2. Anti-CD4 mAb or control rat IgG was administered twice, that is, at 9 and 6 days before the last challenge. Six hours after the last challenge, the CD3⁺CD4⁺ population in the spleen was determined by flow cytometry (A). Representative data from 3 independent experiments are shown. The number of sneezes evoked by histamine (B) and the accumulation of lymphocytes, neutrophils, and eosinophils in NALF (C) were also examined. Data are expressed as mean ± SEM for 4–6 animals. *p < 0.05, **p < 0.01, ***p < 0.01, compared with OVA-challenged control mice (Dunn’s test).

Fig 6. Antigen-induced NHR in T cell-transferred mice.

Twenty-four hours after transfer of Th1, Th2, or Th17-polarized cells, mice were challenged 3 times with OVA or saline. Six hours after the last challenge, the number of sneezes evoked by histamine (A), the accumulation of lymphocytes, neutrophils, and eosinophils in NALF (B), and the IFN-γ, IL-4, and IL-17 mRNA expression in the nasal tissue (C) were examined. Data are expressed as mean ± SEM for 4–8 animals. *p < 0.05, **p < 0.01, ***p < 0.001, compared with naïve T cell-transferred and OVA-challenged mice (Dunn’s test). N.D.: not detectable.

Fig 7. Defect in antigen-induced NHR in antigen-specific IgE-Tg mice.

IgE-Tg mice as well as antigen-immunized mice and T cell-transferred mice were challenged 3–4 times with OVA or saline, as shown in Figs 2 and 6. Six hours after the 3rd challenge, antigen-specific serum IgE, IgG, IgG₁, IgG_2a, IgG_2b, and IgG₃ were determined (A). The number of sneezes evoked by histamine (B) and accumulation of lymphocytes, neutrophils, and eosinophils in NALF (C) of IgE-Tg mice were examined 6 h after the 4th challenge. Data are expressed as mean ± SEM for 4–8 animals. *p < 0.05, **p < 0.01 (Dunn’s test). N.D.: not detectable.