Chronic rhinosinusitis with nasal polyps is characterized by B-cell inflammation and EBV-induced protein 2 expression

Abstract

Background: Despite the high prevalence and morbidity of chronic rhinosinusitis (CRS), little is known about the mechanisms that underlie its pathogenesis. Recent studies have suggested that B cells might play an important role in CRS.

Objective: We sought to thoroughly characterize B lineage cells within sinus tissues of patients with CRS and healthy control subjects and to determine whether levels of EBV-induced protein 2, which is known to play an important role in the development of B-cell responses, were increased in patients with CRS.

Methods: Cells isolated from sinus tissues of patients with CRS and healthy control subjects were characterized by means of flow cytometry and immunohistochemistry. Local production of antibodies was measured in tissue extracts, nasal lavage fluid, and sera by using multiplex bead arrays and ELISA. Quantitative RT-PCR, ELISA, and Western blotting were used to assess gene and protein expression from tissue extracts.

Results: Nasal polyps (NPs) from patients with CRS had increased levels of both B cells and plasma cells compared with uncinate tissue from healthy control subjects (P<.05). NPs also contained significantly increased levels of several antibody isotypes compared with normal uncinate tissue (P<.05), but no differences in circulating antibody levels were found. Interestingly, levels of EBV-induced protein 2 were also increased in NPs (P<.05) and were positively correlated with expression of plasma cell markers (CD138 and B lymphocyte-induced maturation protein) in sinus tissue.

Conclusion: B cells and plasma cells are enriched in NPs, actively produce antibodies locally, and might contribute to chronic inflammation in patients with CRS. Elucidating the mechanisms that underlie this excessive local B-cell response might provide novel insights for the development of improved therapeutic strategies.

FIG 1

NPs contain increased numbers of inflammatory cells. Cells were isolated from sinus tissue biopsy specimens after 4 days and analyzed by using flow cytometry. A, Cell viability was assessed by using DAPI exclusion. B, Total cell yield was determined by dividing the number of cells recovered after 4 days by the original weight of the biopsy specimen. C, CD45⁺ cell numbers were calculated based on the total number of cells recovered and the frequency of CD45⁺ cells within the DAPI⁻ gate and divided by the original weight of the biopsy specimen. Data represent means ± SEMs. *P < .05 and **P < .01, Kruskal-Wallis test.

FIG 2

B cells and plasma cells were more abundant in NPs. Cells were isolated from sinus tissue biopsy specimens after 4 days and analyzed by means of flow cytometry. A, Representative flow cytometric plots. APC, Allophycocyanin; Pe-Cy7, phycoerythrin-cyanin 7. B, Numbers of B cells (CD19⁺) and plasma cells (PC, CD138⁺) were calculated as above from within the DAPl⁻ CD45⁺ population. C, Mean side scatter A (SSC-A) fluorescence intensity of CD19⁺ B cells. D, Representative ×20 immunofluorescence image of CD19⁺ (red) and CD138⁺ (green) cells in NP tissue. Data represent means ± SEMs. *P < .05, **P < .01, and ***P < .001, Kruskal-Wallis test.

FIG 3

Antibody levels were increased in NPs. A, Antibody isotype levels in tissue extracts were normalized to total protein. B, Antibody isotype levels measured in nasal lavage fluid. C, Gene expression of pIgR in tissue extracts. Data represent means ± SEMs. *P < .05, **P < .01, and ***P < .001, Kruskal-Wallis test.

FIG 4

Differential expression of IgA₁ and IgA₂ in NPs. Total IgA₁ and IgA₂ levels in tissue extracts (A) and nasal lavage fluid (B) are shown. sIgA₁ and sIgA₂ levels in tissue extracts (C) and nasal lavage fluid (D) are shown. Data represent means ± SEMs. *P < .05, **P < .01, and ***P < .001, Kruskal-Wallis test.

FIG 5

EBI2 was highly expressed in NPs. A, Gene expression analysis of EBI2 from sinus tissue biopsy specimens was normalized to β-glucuronidase. B, Representative Western blot showing EBI2 and β-actin. C, Average of EBI2 expression normalized to β-actin in sinus tissue. D, Correlations between normalized EBI2 gene expression and CH25H, CD138, and B lymphocyte–induced maturation protein (BLIMP-1; n = 60 in each plot). Data in Fig 5, A and C, represent means ± SEMs. *P < .05, **P < .01, and ***P < .001, Kruskal-Wallis test.