Ten milliliters of heparinized blood samples were obtained intraoperatively by venous puncture from 10 patients undergoing paranasal sinus surgery and transferred to the laboratory. Lymphocytes were separated by density-gradient centrifugation (10 minutes, 1,000×g) at room temperature on equal amounts of Ficoll (Biochrom, Berlin, Germany), using a membrane-containing 10 mL cell tube (Greiner Bio-One, Frickenhausen, Germany). After washing twice in phosphate-buffered saline (PBS; Gibco BRL Life Technologies, Eggenstein, Germany), the cell number and the cell viability were determined using a Cell Counter+Analyser System (CASY TT; Innovatis, Reutlingen, Germany) according to the manufacturer’s protocol. After centrifugation with 1,600 rpm cells were frozen in –80℃ with 1 mL freezing medium, containing 10 parts fetal calf serum (Linaris, Dossenheim, Germany) and one part DMSO (Roth, Karlsruhe, Germany).

All tissue samples were collected intraoperatively from 10 patients undergoing standard paranasal sinus surgery. Additionally, two samples of normal nasal tissue as control group, where collected from two patients undergoing functional septorhinoplasty or uncinectomy because of a maxillary sinus cyst. The polyps and the nasal mucosae were cut into small fragments and mashed through a cell strainer (Greiner Bio-One) from 100 μm to 40 μm in PBS (Gibco). After washing twice in PBS, the cell number and cell viability were determined using a CASY system according to the manufacturer’s protocol. After centrifugation (5 minutes, 1,600 rpm) cells were frozen at –80℃ with 1 mL freezing medium.

The following antibodies were used: anti-CD45 Pacific Orange, anti-CD19 Brilliant Violet, anti-CD20 PE, anti-CD38 antigen-presenting cells, anti-CD24 PE.Cy7, anti-CD27 Per.CP-Cy5.5 and anti-HLA-DR Alexa 700 (all by BioLegend, San Diego, CA, USA). Viability Dye 780 (eBioscience, San Diego, CA, USA) was used to detect apoptotic cells. After blocking with 25 μg/mL normal mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes on ice, all cells underwent cell surface staining. All antibodies were used according to the manufacturers’ instructions. FACS analyses were performed using an LSR II flow cytometer (Becton Dickinson, San Diego, CA, USA), and the data were analyzed with FlowJo software (TreeStar, Ashland, OR, USA).

Data are presented as mean±standard deviation. Statistical significance was analysed by a two-tailed paired t-test using GraphPad Prism Software 6.0c (GraphPad Software Inc., La Jolla, CA, USA). For nonparametric distribution the Wilcoxon test was applied. The P-values <0.05 were considered statistically significant.

Ten patients were included in the study group (six males and four females). The mean age was 56±13 years. Inclusion criteria were a chronic rhinosinusitis with nasal polyps and the failure of medical treatment. Patients with established immunodeficiency, pregnancy, coagulation disorder, classic allergic fungal sinusitis, isolated antrochoanal polyps or cystic fibrosis were excluded from the study. All patients received intranasal topical steroids prior to the surgery according to the European guidelines [17]. This treatment started weeks before surgery and if the patients did not benefit from this medical therapy a surgical intervention was planned. Eosinophilic polyposis was described in the histological evaluation in most of the patients (8 of 10). Notably, in one histological finding with no eosinophilic infiltration numerous plasma blasts and plasma cells were visible. The characteristics are summarized in Table 1. Additionally, healthy nasal mucosa was obtained from two patients undergoing functional septorhinoplasty or uncinectomy because of a maxillary sinus cyst. The median age of these patients was 47±35 years.

Lymphocytes of nasal polyps and peripheral blood were analysed from 10 patients by flow cytometry by gating on CD45⁺ cells (Fig. 1A) after eliminating all apoptotic cells (Fig. 1B). Then, B cells were identified by gating on CD19⁺ and CD20⁺ cells (Fig. 2A) and B cell subpopulations by gating on CD38 and CD24 (Fig. 2B). The 6.5% of all lymphocytes in nasal polyps were CD19⁺ CD20⁺ B cells (Table 2). The frequency of B cells in nasal polyps was significantly lower than in PBMCs (Fig. 3A) and B cell subpopulations in nasal polyps differ from PBMCs (Fig. 3B). In healthy nasal mucosa frequencies of B cells (4.77±0.93) were decreased compared to PBMCs and nasal polyps. CD19⁺ CD20^– plasma cells were significantly higher in nasal polyps compared to PBMC and in patients with CRSwNP (Table 3) and also compared to healthy nasal tissue (3.83±2.59). There was no significant difference between CD19⁺ CD20^– CD27^high HLA-DR^high plasma blasts and CD19⁺ CD20^– CD27^high HLA-1,58DR^low plasma cells (Table 3, Fig. 4). Compared to nasal polyps and PBMCs, the lowest frequencies of CD19⁺ CD20^– CD27^high HLA-DR^high plasma blasts and CD19⁺ CD20^– CD27^high HLA-DR^low plasma cells were found in healthy nasal mucosa (plasma blast, 2.49±1.48; plasma cells, 37.60 ±8.20).

CD19⁺ CD20⁺ CD38^high CD24^high B_reg (Fig. 2B) were significantly lower in nasal polyps compared to PBMC in patients with CRSwNP (Table 2), but in both groups they were the minor subpopulation with only a few cells (Fig. 3B). In healthy mucosa no B_reg were detected. Mature B cells described as CD19⁺ CD20⁺ CD38^int CD24^high B cells (Fig. 2B) were significantly lower in nasal polyps compared to peripheral blood (Table 2, Fig. 3B). In nasal mucosa the lowest frequencies of mature B cells (21.20±4.24) were measured. In PBMCs, mature B cells (Fig. 2B) were the highest frequency subpopulation (Fig. 3B). CD19⁺ CD20⁺ CD38^– CD24^high memory B cells (Fig. 2B) were the main subpopulation of B cells in nasal polyps and in healthy nasal mucosa (46.60±1.31). Memory B cells significantly increased in nasal polyps compared to PBMCs (Table 2, Fig. 3B).

In this study, a detailed quantification of subpopulations of peripheral and edaphic B lymphocytes in CRSwNP is presented. The majority of lymphocytes in polyp tissue are T cells, particularly CD8⁺ cytotoxic T cells [18]. Furthermore, B cells may also play an important role in the pathogenesis of nasal polyps. Some investigators have already shown a possible pathogenic role of B cells in CRS via the inflammatory reaction of mucosal nasal tissue. Kato et al. [19] described increased levels of the B cell activating factor of the tumor necrosis factor family (BAFF) in CRSwNP, IL-6 and B-lymphocyte chemoattractant (CXCL13) [20-22], and a production of BAFF by epithelial cells. In the present study, a significantly lower frequency of CD19⁺ CD20⁺ B cells was found in nasal polyps compared to PBMCs in patients with CRSwNP. Only 6.5% of edaphic lymphocytes in nasal polyps were B cells. Among the B cell subpopulations, B_reg and mature B cells were significantly lower in polypoid tissue. Memory B cells were significantly higher and represented the main subpopulation in nasal polyps compared to peripheral blood in patients with CRSwNP. In the present study, immunoglobulin-secreting plasma cells displayed a significantly higher frequency in nasal polyps compared to PBMC. Previous studies have also shown an increase in plasma cells as well, and a differentiation of B cells in nasal polyps [8] with high local production of IgE without higher serum IgE levels [9]. Memory B cells represent antigen-experienced B cells and are related to age: Weksler [23] showed changes in the B cell reservoir in elderly patients, which may contribute to their increased responsiveness to infections [24]. In patients with SLE, Jacobi et al. [14] found no correlation between frequency and total number of memory B cells and disease duration. Furthermore, they showed a statistically significant reduction in naïve and memory B cells, but no influence of immunosuppressive therapy on plasma cells in SLE. In the European Position Paper on Rhinosinusitis and Nasal Polyps [17], a surgical treatment of CRSwNP is only indicated after failure of medical treatment and if a local therapy with corticoids does not lead to an improvement in symptoms score. Patients included in the current study received topical or systemic corticosteroids preoperatively to reduce the size of nasal polyps according to the treatment guidelines of the university hospital. Therefore, it was not possible to include a control group without any pretreatment with corticosteroids. It is very likely that this therapy may have influenced the distribution of B cell subpopulations. A study design comparing patients with and without preoperative local or systemic immunosuppressive therapy would be of great interest.

Reduced numbers and/or function of regulatory T cells were demonstrated to be responsible for different types of autoimmune disorders and chronic infections [25]. Blair et al. [12] recently showed a similar population within B cells with regulatory functions (B_reg). They also demonstrated that B_reg could suppress T_H1 differentiation by secretion of IL-10 when anti-CD80 and CD86 mAb are added in vitro. In SLE, B cells isolated from peripheral blood lacked this suppressive effect. In the present study, a statistically significant decrease in B_reg in nasal polyps compared to peripheral lymphocytes was shown. Future studies should focus on the functional influence of B_reg with respect to the immunosuppressive effects on T helper cells as in SLE [12].

As previously mentioned, autoreactive B cells play an important role in SLE, and preliminary attempts have been made to use B cell subset composition as a marker for disease activity [14]. Based on these findings, Tan et al. [26] attempted to examine autoreactive B cells in CRSwNP. They found production of autoantibodies in nasal polyps and concluded that these autoantibodies lead to autoimmunity. Furthermore, the investigators hypothesized that autoreactive memory B cells persist in inflammatory nasal tissue after surgery and are responsible for recurrence of nasal polyps. High frequencies of memory B cells in the present study suggest that B cell autoreactivity might be important for the development of CRSwNP. In the present study, the total number of B cells was significantly lower than in PBMCs. Although the total number of edaphic B cells was low in our observations, data from the literature indicates a key role of B cells in the maintenance of CRSwNP [8-10,20].

A detailed characterization of B cell subpopulations, such as in the present study, is necessary to gain further insight into the pathophysiology of nasal polyps. The development of targeted therapies addressing CRS requires a comprehensive overview on the immunological mechanisms of this disease. Increased levels of local IgE in nasal polyps have already led to the introduction of Omalizumab as an anti-IgE treatment in clinical trials [27]. Other B cell-directed therapies, e.g., rituximab in autoimmune disease, are also promising [28], and future studies should focus on special targets like anti-dsDNA or BAFF, which are already being investigated in clinical trials in SLE [26].

The small number of subjects limits the results of this study. A control group of healthy nasal mucosa was tried to establish. But, one problem of analysis of B cells of healthy mucosa is the very small amount of cells, so a meaningful statistical analysis is difficult. For multicolour FACS analysis more cells are necessary in the opinion of the authors. Frequencies of two samples are shown in this study to show the difference between the groups. The study is declared to be a pilot project with a limited amount of samples. Since there is no statistically significant control group with healthy mucosa, interpretation of the data is affected. For example, a significantly amount of B_reg in nasal polyps compared to PBMC in patients with CRSwNP was observed. But, if the number of B_reg is much lower in healthy control tissue than in polyps this would indicate that there is an accumulation of B_reg in polyps. Without a control group of healthy mucosa, it is difficult to provide further mechanistic insight. Unfortunately, analysis of B cells from healthy nasal mucosa was tried to perform but failed due to low amounts of these cells in this noninflammatory tissue, for example only a total number of 100 B cells were found in the first sample of healthy mucosa. In the opinion of the authors, high cell amounts are necessary for this multicolour flow cytometry analysis and for a significant analysis of this data. When assuming that most of the lymphocytes in healthy nasal mucosa were circulating intravascular cells, a control group of PBMC seemed to be equivalent, and, furthermore, a comparison of isolated T cells from tissue with PBMC is well established in the literature [4,29,30].

In this study, a detailed contemporary characterization of B cell subpopulations is presented. Memory B cells are the main subpopulation in nasal polyps. B_reg and mature B cells are significantly decreased in nasal polyps compared to PBMC in CRSwNP. B_reg are necessary to suppress the T helper cell activation and T and B cell interaction. The question arises as to whether the reduced B_reg frequencies cause T cell dysfunction and/or the high frequencies of memory B cells. Such principle pathophysiological relationships need to be clarified in future studies and further functional studies are necessary to gain further insight into the genesis of CRSwNP [29,30].