Expression of Toll-like receptor 9 in nose, peripheral blood and bone marrow during symptomatic allergic rhinitis

Abstract

Background: Allergic rhinitis is an inflammatory disease of the upper airway mucosa that also affects leukocytes in bone marrow and peripheral blood. Toll-like receptor 9 (TLR9) is a receptor for unmethylated CpG dinucleotides found in bacterial and viral DNA. The present study was designed to examine the expression of TLR9 in the nasal mucosa and in leukocytes derived from different cellular compartments during symptomatic allergic rhinitis.

Methods: The study was based on 32 patients with seasonal allergic rhinitis and 18 healthy subjects, serving as controls. Nasal biopsies were obtained before and after allergen challenge. Bone marrow, peripheral blood and nasal lavage fluid were sampled outside and during pollen season. The expression of TLR9 in tissues and cells was analyzed using immunohistochemistry and flow cytometry, respectively.

Results: TLR9 was found in several cell types in the nasal mucosa and in different leukocyte subpopulations derived from bone marrow, peripheral blood and nasal lavage fluid. The leukocyte expression was generally higher in bone marrow than in peripheral blood, and not affected by symptomatic allergic rhinitis.

Conclusion: The widespread expression of TLR9 in the nasal mucosa along with its rich representation in leukocytes in different compartments, demonstrate the possibility for cells involved in allergic airway inflammation to directly interact with bacterial and viral DNA.

Figure 1

Leukocyte gates on samples from bone marrow, peripheral blood and nasal lavage fluid. Flow cytometry data with dot plots showing gates for neutrophils, basophils, monocytes, lymphocytes, eosinophils and immature granulocytes in bone marrow, peripheral blood and nasal lavage fluid. Immature granulocytes were only found in bone marrow. In nasal lavage fluid only neutrophils could be clearly identified. A) FSC versus SSC with gate R1 representing nucleated leukocytes. B) CD45 versus SSC of cells gated from R1, representing basophils (R5), monocytes (R6) and lymphocytes (R7). C) CD45 versus CD16 of cells gated from R2, representing eosinophils (R8) and immature granulocytes (R9). D) FSC versus CD16 of cells gated from R3, representing neutrophils (R4).

Figure 2

TLR9 immunoreactivity in the nasal mucosa. Immunohistochemical localization of TLR9 in biopsies of nasal mucosa is depicted in (B-F) whereas (A) illustrates a representative picture of a control slide. A) No immunoreactivity was observed in control sections where an isotype-matched control antibody was used. B) In an adjacent section, immunoreactivity for TLR9 is seen in the apical part of the epithelial lining, in scattered intra- and subepithelial leukocytes and in elongated fibroblast-like cells in the subepithelial tissue (arrow). The epithelial TLR9 immunoreactivity varied from being foremost present within the apical region of the epithelium (B) to a more even distribution (C). D) A distinct TLR9 immunoreactivity was also present in endothelial cells (arrowhead). E) Bright field micrographs demonstrating TLR9-positive large non-granulated mononuclear cells (arrowhead) and mast cells (inset). F) TLR9-positive intraepithelial lymphocytes (arrows E-F). Scale bars: A-C = 50 μm, D-E = 20 μm, and F = 350 μm.

Figure 3

Expression of TLR9 in leukocytes from bone marrow, peripheral blood and nasal lavage fluid. Histogram plots of intracellular staining of TLR9 in neutrophils, eosinophils, basophils, monocytes, lymphocytes and immature granulocytes. Expression of TLR9 in leukocytes was analyzed by flow cytometry using mAbs against human TLR9 (open histograms). Cells were fixed and permeabilized prior to incubation with mAbs. Shaded histograms represent cells labeled with isotype-matched control Ab. The data shown were obtained from a control subject and they are representative of those from six independent experiments.

Figure 4

Expression of TLR9 in leukocytes in different compartments. Intracellular expression of TLR9, presented as MFIR, in bone marrow, peripheral blood and nasal lavage fluid. Expression of TLR9 in A) neutrophils (n = 23–28), B) eosinophils (n = 23–29), C) basophils (n = 23–27), D) monocytes (n = 23–29) and E) lymphocytes (n = 23–29). Data are presented as mean ± SEM. ** p < 0.01, *** p < 0.001

Figure 5

Expression of TLR9 in leukocytes during allergic rhinitis. Intracellular expression of TLR9, presented as MFIR, in different leukocytes in healthy controls (C), allergic patients outside season (O) and allergic patients during pollen season (P). Expression of TLR9 in neutrophils, eosinophils, basophils, monocytes, lymphocytes and immature granulocytes analyzed by flow cytometry. Expression of TLR9 in leukocytes in A) bone marrow (n = 23), B) peripheral blood (n = 27–29) and C) nasal lavage fluid (n = 27). Data are presented as mean ± SEM. ** p < 0.01