Objective
Localized provoked vulvodynia (LPV) may have inflammatory etiology. We wanted to find out whether the cell-mediated immune system becomes activated in the vestibular mucosa in LPV.
Study Design
This was a controlled cross-sectional study. Vestibular mucosal specimens were obtained from 27 patients with severe LPV and 15 controls. Detailed clinical history of the patients was obtained. For immunohistochemistry, antibodies against CD3 (T cells), CD20 (B cells), IgA (mucosal plasma cells), CD163 (dendritic cells [DCs]), CD68 (macrophages), and CD117 (mast cells) were employed. Mann-Whitney U test and χ 2 test were used for statistical analyses.
Results
More B lymphocytes and mature mucosal IgA-plasma cells were found in patients than in controls ( P < .001 and P < .001, respectively). In LPV samples, B and T cells were arranged into germinal centers representing local immune activation. Germinal centers were not seen in controls. Antigen-presenting DCs and macrophages were found both in patients and controls with similar densities. DCs were found to extend their dendrites into the luminal space through an intact epithelium. Similar amounts of mast cells were found evenly scattered throughout the stroma of vestibular mucosa of both patients and controls.
Conclusion
We demonstrate here local organized vestibule-associated lymphoid tissue analogous to mucosa-associated lymphoid tissue. Vestibule-associated lymphoid tissue may emerge as a response to local infection or inflammation in LPV.
Localized provoked vulvodynia (LPV) is a subset of vulvodynia, associated with induced pain by touch on vulvar mucosa in the absence of any other recognizable disease. LPV is further classified according to the location of pain. Vestibulodynia (formerly vulvar vestibulitis syndrome) represents the pain sensation in the vulvar vestibular mucosa and results in severe dyspareunia. This can have devastating consequences to the quality of life of affected young women. LPV prevalence rates can be up to 8-15% in premenopausal population.
The etiopathogenesis of LPV and the mechanisms resulting in the altered pain sensation are unknown. Different vulvovaginal infections, such as recurrent candidiasis as well as urinary tract infections have been considered as risk factors for the disease. Histopathology often reveals increased lymphocytic infiltrates in the vestibular mucosa but the characteristics of the inflammatory response have remained unclear. However, many studies have found no evidence of a classic active inflammation. Instead, a tendency to an exaggerated inflammatory response and dysregulation of inflammation in affected women have been suggested. The vestibular mucosa of LPV patients characteristically has an increased number of exceptionally superficial nerve endings. An increased number of mast cells producing heparanase enzyme has been suggested to boost nerve growth. However, the role of mast cells has not been fully proven. As the immune and neuronal systems are often closely interrelated, an increased inflammatory response may well predispose to the development of a pain syndrome.
LPV is a pain syndrome with a suspected inflammatory background but the detailed characteristics of the inflammatory response are unknown. In particular, the role of specific immune system cells has not been defined. In this study we performed a thorough immunohistochemical analysis of the vestibular mucosal tissue focusing on differences in the characteristics of the local immune system between LPV patients and controls.
Materials and Methods
Study subjects
We recruited 27 women with representative archival vestibulectomy specimens after surgically treated LPV during a period of 13 years from 1995 through 2007. The women were identified in the Helsinki University Central Hospital patient registry by matching the diagnosis (vulvar vestibulitis, vulvodynia, and dyspareunia) and the surgical procedure (posterior vestibulectomy). The total number of the original cohort was 70. It was not possible to reach 13 women, or they denied participating. Of the remaining 57 women we chose 27 with sufficient clinical data to form the patient population of this study. All the included patients were diagnosed as having localized provoked vestibulodynia with long disease history (mean, 5.3 years; 95% confidence interval [CI], 3.8–6.8; range, 2–18). The diagnoses in 8 patients were classified as primary (symptoms already at the first vaginal entry), in 15 patients as secondary (symptoms appearing later after an interval of painless intercourses), and in 4 patients the classification was unknown. Clinical data of these 27 LPV patients were collected from the patient records and by a face-to-face interview. Data on age, premenopausal status, general health, comorbidity, medications, and detailed disease history including duration and severity of symptoms, treatments before surgery, and response to surgery were systematically collected. As controls we recruited 15 premenopausal volunteers with no vulvar symptoms undergoing benign gynecological surgery under general anesthesia. No clinical data were collected. We obtained 4-mm punch biopsies from the posterior vestibule at 5 o’clock position from the controls. The study was approved by the local ethical committee. All participants provided informed consent.
Tissues
All vestibular tissues were routinely embedded in paraffin after a maximum of 24 hours fixation in 10% buffered formalin. We first stained 5-μm sections with hematoxylin–eosin for evaluation to exclude dermatological diseases, to confirm the quality of samples, and to grade lymphocytic inflammation. Immunohistochemistry on consecutive sections (5 μm in thickness; 10 μm for dendritic cells [DCs] and macrophages) was performed in the Helsinki University Central Hospital–Huslab tissue laboratory with routine staining procedures according to the manufacturers’ instructions. We employed antibodies against the following antigens: CD3 (clone 2GV6, RTU; Roche Diagnostics Ltd, Rotkreuz, Switzerland) for T lymphocytes, CD20 (clone L26, RTU; Roche Diagnostics Ltd) for B lymphocytes, CD68 (clone PG-M1, 1:1000; Dako, Glostrup, Denmark) for macrophages, CD117 (polyclonal, 1:1000; Dako) for mast cells, IgA (polyclonal, 1:2000; Dako) for mucosal plasma cells, CD163 (clone 10D6, 1:100; Leica Biosystems, Nussloch, Germany) for dendritic cells. For the first 4 antibodies above, we used a biotin-free polymer-based detection kit Ultraview (Roche Diagnostics Ltd). For the last 2 antibodies, we used a polymer-based detection kit Envision (Dako). All sections were counterstained with hematoxylin.
Tissue analyses
The entire material was analyzed blinded to clinical data. Histopathological inflammation was analyzed in hematoxylin–eosin sections and graded as none, mild, or moderate (R.B.) based on the amount of mononuclear infiltrates in stromal tissue beneath the vestibular epithelium at low-power magnification (×10).
Immunohistochemical scoring of the representative sections was performed under light microscopy (Nikon Eclipse E800). The stainings of all antigens were analyzed for localization (stromal, epithelial, both) and for densities of individual cell types in the vestibular mucosa. The scoring of each section was based on a consensus of 2 investigators (P.T. and J.P. or L.U-K. or S.M.) and disagreements were resolved by a joint review.
All immune cells, except T lymphocytes, were quantified from the most representative areas of inflammation by calculating the mean number of identified positive cells per field from 2-4 high-power fields (hpf) (×40 objective). T lymphocytes were scored by the overall cell density both in the epithelium and stroma. A single number score was given from 1-3 (1 = low density, <50 cells/hpf; 2 = moderate density, 50-100 cells/hpf; 3 = high density, >100 cells/hpf). Germinal centers were identified and counted from each section. For IgA, the overall staining intensity was additionally scored as 1 (mild intensity), 2 (moderate intensity), or 3 (strong intensity).
For statistical purposes and to reflect the overall level of B-cell infiltration we created a B-cell activation index (BAI) as follows. The most typical density of B cells in each section scored points from 0 (no B cells) to 4 (high amount of B cells), separately in the epithelium and in the stroma. The existence of at least 1 germinal center scored 4 points and absence scored 0 points. The sum of these 3 values gave a BAI from 0-12.
Statistical analyses of the data were performed with software (SPSS, version 20; IBM Corp, Armonk, NY). For comparisons between patients and controls, between patients with primary and secondary LPV, and between patients with different clinical characteristics, we used Mann-Whitney U test or Kruskal-Wallis test for continuous data and χ 2 analysis or Fisher exact test for categorical data when appropriate. For correlations Spearman rho test for nonparametric data was used. A P value of < .05 was considered significant in a 2-tailed analysis.
Results
Antigen-presenting cells
DCs can capture foreign antigens and process them for presentation to CD4 + helper T cells. CD163 immunostain revealed the typical morphology of DCs with the antigen-capturing dendrites. DCs were occasionally found to extend their dendrites through the epithelial surface to the luminal space ( Figure 1 , A and B). DCs were located both in the epithelium and stroma and tended to be more numerous in controls than in patients ( Table 1 ).
| Cell type | LPV (n = 27) Mean (95% CI) a | Controls (n = 15) Mean (95% CI) a | P value b |
|---|---|---|---|
| Dendritic cells | 98 (86–110) c | 129 (101–157) d | .053 |
| Macrophages | 23 (17–29) c | 19 (15–24) d | .780 |
| B lymphocytes | 121 (97–145) | 38 (15–60) | < .001 |
| Germinal centers e | 14 (51.9) | 0 (0) | .001 |
| Plasma cells | 41 (31–52) c | 11 (8–15) f | < .001 |
| Mast cells | 35 (31–40) | 33 (26–39) f | .512 |
a Values are mean cell counts per microscopy field, analyzed from 2–4 fields (×40 objective)
e No. of samples (%) with ≥1 germinal center
Staining for macrophages demonstrated the chubby shape of the cell body and revealed the typical intracellular cytoplasmic granules ( Figure 1 , C and D). Macrophages were evenly scattered throughout the vestibular stroma with similar densities in patients and controls ( Table 1 ).
T and B lymphocytes and plasma cells
T-cell staining (using the cytoplasmic pan T-cell marker CD3) visualized collectively both helper (CD4 + ) and killer (CD8 + ) T cells. These were expressed both in the stromal and epithelial layers of the vestibular mucosa with no significant difference in densities between patients and controls ( Figure 2 , A and B). T-cell density was high in 26.9% (7/26) of patients vs 21.4% (3/14) of controls, moderate in 30.8% (8/26) vs 57.1% (8/14), and low in 42.3% (11/26) vs 21.4% (3/14) of patients and controls, respectively ( P = .280). T-cell densities were similar in primary and secondary LPV ( P = .512; data not shown).
CD20 antigen is expressed on B cells at all stages of development, except the early pro-B cells and mature plasma cells capable of secreting antibodies. Anti-CD20 antibody showed that B lymphocytes were present mainly in the stromal layer of the mucosa. Small amounts were also found in the epithelium in the most populated areas ( Figure 2 , C and D). B lymphocytes were more numerous in patients than in controls ( Table 1 ).
In areas with highest lymphocyte densities, T cells and B cells clearly formed lymphoid follicle-like structures. These were identified as germinal centers representing secondary lymphoid tissue. These germinal centers were visualized by both the CD3 ( Figure 3 , A through C) and CD20 ( Figure 3 , D through F) antibodies showing the typical zonewise arrangement of the individual cell types. Germinal centers were found only in LPV patients, but not in controls. The number of germinal centers in patients varied from 1-4 centers in 14 sections (52%), but was 0 in 13 sections. BAI was significantly higher in patients than in controls (median 5.0, range 1.0–9.0 and median 1.0, range 0–4.0, respectively; P = .001).
Mature mucosal plasma cells were stained on the basis of their ability to produce IgA-class antibodies. Compared to B cells, the plasma cells were bigger, had oval shape, showed high nucleus-to-cytoplasm ratio, and their nuclei had typical round cartwheel appearance. IgA-plasma cells were found in the subepithelial layer of the stroma ( Figure 4 ). These were more numerous in patients than in controls ( Table 1 ). In addition to the plasma cells’ cytoplasm, IgA was also seen diffusely in the epithelial surfaces and stromas of mucosal samples. In a semiquantitative analysis no difference in the densities of IgA between patients and controls was found (data not shown).
Mast cells
Mast cells are tissue granulocytes. Typically they are prominent near the boundaries between the outside and inside environments, such as underneath mucosal surfaces, and adjacent to blood vessels. CD117 + mast cells were located in the stroma mostly as evenly distributed single cells, often adjacent to small capillaries ( Figure 5 ) showing similar densities in patients and controls ( Table 1 ).
Clinical correlates of immunological parameters
Histopathological inflammation was more pronounced in LPV than in controls and tended to be more severe in secondary than in primary LPV ( Table 2 ). The grade did not associate with severity (visual analog scale score for preoperative dyspareunia) ( P = .550) or duration of symptoms ( P = .650).
