Cytotoxic T lymphocytes mediate chronic inflammation of the nasal mucosa of patients with atypical allergic rhinitis

Abstract

Background: The prevalence of chronic rhinitis is increasing rapidly; its pathogenesis is to be further understood; immune inflammation is one of the possible causative factors. Antigen specific CD8+ T cells play a critical role in the induction of chronic inflammation.

Aims: This study aimed to investigate the role of antigen specific CD8+ T cells in the pathogenesis of chronic atypical allergic rhinitis.

Material and methods: Nasal mucosal epithelial surface scratching samples were obtained from patients with chronic obstruction atypical allergic rhinitis. Exosomes were purified from the scratching samples and examined by immune gold electron microscopy. The effect of exosomes on modulating dendritic cell's properties, the effect of exosome-pulsed dendritic cells on naïve T cell differentiation and the antigen specific CD8+ T cell activation were observed by cell culture models.

Results: Exosomes purified from patients with chronic atypical allergic rhinitis carried microbial products, Staphylococcal enterotoxin B (SEB), and airborne antigen, Derp1. Dendritic cells pulsed by SEB/Derp1-carrying exosomes showed high levels of CD80, CD86 and the major histocompatibility class I (MHCI). Exosome-pulsed dendritic cells could induce the naïve CD3+ T cells to differentiate into CD8+ T cells. Upon the exposure to a specific antigen, the CD8+ T cells released granzyme B and perforin; more than 30% antigen specific CD8+ T cells proliferated.

Conclusions: Antigen specific CD8+ T cells play an important role in the pathogenesis of chronic obstruction atypical allergic rhinitis.

Keywords: Antigen specific response; CD8 T lymphocytes; Epithelium; Rhinitis.

Fig. 1

Exosomes carrying microbial products and protein antigens are localized in the nasal mucosa. The nasal epithelial layer was sampled from patients with chronic rhinitis as described above. The samples were processed for EM. The representative pictures show exosomes stained with antibodies of SEB and Derp1. The larger gold particles (12 nm) indicate the labels of avb6; the smaller gold particles (6 nm) indicate the labels of Derp1. C-D, the gels show the immune blots of Derp1 (C) and SEB (D). The blots of beta-actin were used as internal loading controls. The data represent 3 experiments.

Fig. 2

Generation of nasal mucosal cell-derived exosomes. A-B, the representative EM pictures show the nasal epithelial cell-derived exosomes carrying Derp1 and SEB (A) Panel B is an isotype control. C, proteins were extracted from purified exosomes and analyzed by Western blotting. The gel pictures show immune blots of Derp1 or SEB. The β-actin immune blots were used as internal control. D, bars indicate the summarized data of panel C. The data were presented as mean ± SD (from 3 experiments).

Fig. 3

DCs can capture the nasal epithelial cell-derived exosomes. DCs were generated from isolated PBMC and cultured in RPMI1640 medium. Avb6, or Derp1, or SEB/Derp1-carrying exosomes were added to the culture. The cells were collected 6 h later and analyzed by flow cytometry and immunocytochemistry respectively. A1-B1, the flow cytometry dot plots show SEB+ or Derp1+ DCs. A2-B2, the representative confocal images show SEB+ staining (in green) and Derp1+ cells (in red). The yellow color was merged by green and red colors. The data represent 3 experiments.

Fig. 4

Nasal epithelial cell-derived exosomes modulate the properties of DCs. Human peripheral DCs were cultured in the presence of the nasal epithelial cell-derived exams at the indicated concentrations for 5 days. The cells were analyzed by flow cytometry. The bars indicate the percentage of cells of CD80 (A), CD86 (B) and MHCI (C). *, p<0.05, compared with the “0 ng” group. The data were presented as mean ± SD from 3 experiments. LPS: DCs were stimulated by LPS at 50 ng/ml in culture.

Figure 5

Exosome-pulsed DCs facilitate antigen specific CD8+ T cell differentiation. A-C. Human peripheral DCs were prepared from PBMC as described in the text. The DCs were pulsed with SEB/Derp1-carrying exosomes at the concentrations as indicated in panels A-C. Peripheral CD3+ CD25- T cells were isolated from PBMC. The DCs were cultured with CD3+ CD25- T cells at a ratio of 1:10 for 3 rounds (3 days/round). Cells were collected at the end of culture and analyzed by flow cytometry. A, bars indicate the frequency of CD8+ T cells. B-C, bars indicate the levels of granzyme B (B) and porforin (C) in culture medium. D. The cell culture procedures in panel D were the same as that in A-C except the CD3+ CD8+ T cells were labeled with CFSE prior to the culture. The specific antigen, Derp1, was added to the culture in the last culture round. The cells were collected at the end of culture and analyzed by CFSE-dilution assay. The bars indicate the frequency of proliferated CD8+ T cells. The data were presented as mean ± SD from 3 experiments. *, p<0.05, compared with the “0 ng” group in panels A-C and the saline group in panel D.