title: Immune activation enhances epithelial nerve growth in provoked vestibulodynia.
abstract: BACKGROUND
  Provoked vestibulodynia manifests as allodynia of the vulvar vestibular mucosa. The exact mechanisms that result in altered pain sensation are unknown. Recently, we demonstrated the presence of secondary lymphoid tissue, which is the vestibule-associated lymphoid tissue in the vestibular mucosa, and showed that this tissue becomes activated in provoked vestibulodynia.
  
  
  OBJECTIVE
  The purpose of this study was to examine whether expression of intraepithelial nerve fibers and nerve growth factor are related to immune activation in provoked vestibulodynia.
  
  
  METHODS
  Vestibular mucosal specimens were obtained from 27 patients with severe provoked vestibulodynia that was treated by vestibulectomy and from 15 control subjects. We used antibodies against the protein gene product 9.5, the neuron specific neurofilament, and nerve growth factor for immunohistochemistry to detect intraepithelial nerve fibers and nerve growth factor expressing immune cells in the vestibular mucosa. For intraepithelial nerve fibers, we determined their linear density (fiber counts per millimeter of the outer epithelial surface, protein gene product 9.5) or presence (neuron specific neurofilament). Nerve growth factor was analyzed by counting the staining-positive immune cells. Antibodies against CD20 (B lymphocytes) and CD3 (T lymphocytes) were used to identify and locate mucosal areas with increased density of lymphocytes and the presence of germinal centers (ie, signs of immune activation). B-cell activation index was used to describe the overall intensity of B-cell infiltration.
  
  
  RESULTS
  We found more protein gene product 9.5-positive intraepithelial fibers in vestibulodynia than in the control samples (6.3/mm [range, 0.0-15.8] vs 2.0/mm [range, 0.0-12.0]; P=.006). Neuron specific neurofilament -positive intraepithelial fibers were found in 17 of 27 vestibulodynia cases (63.0%) and in none of the control cases. Protein gene product 9.5-positive intraepithelial fibers were more common in samples with more pronounced immune activation. The density of these fibers was higher in samples with than without germinal centers (6.1/mm [range, 4.3-15.8] vs 3.0/mm [range, 0.0-13.4]; P=.020). A positive correlation between the fiber density and B-cell activation index score of the sample was found (Spearman's Rho, 0.400; P=.004; R2=0.128). No significant difference, however, was found in the density or presence of nerve fibers between samples with high and low T-cell densities. We identified areas of minor and major vestibular glands in 16 of the patient samples and in 1 control sample. Protein gene product 9.5-positive nerve fibers were found more often in glandular epithelium surrounded by B-cell infiltration than in glands without B cells (P=.013). Also, the presence of neuron specific neurofilament-positive fibers in glandular epithelium was associated with B-cell infiltrates (P=.053). Nerve growth factor-positive immune cells were more common in mucosal areas with than without B-cell infiltration and intraepithelial nerve fibers.
  
  
  CONCLUSIONS
  Excessive epithelial nerve growth in provoked vestibulodynia is associated with increased B-cell infiltration and the presence of germinal centers. This supports the fundamental role of immune activation in provoked vestibulodynia.
authors.affiliation: Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland. Electronic address: paivi.tommola@kolumbus.fi.
authors.forename: Päivi
authors.initials: P
authors.lastname: Tommola
authors.affiliation: Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
authors.forename: Leila
authors.initials: L
authors.lastname: Unkila-Kallio
authors.affiliation: Department of Pathology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
authors.forename: Anders
authors.initials: A
authors.lastname: Paetau
authors.affiliation: Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; Research Programs Unit, Program of Immunobiology, University of Helsinki, Helsinki, Finland.
authors.forename: Seppo
authors.initials: S
authors.lastname: Meri
authors.affiliation: Pain Clinic, Anesthesiology, Intensive Care, and Pain Medicine, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
authors.forename: Eija
authors.initials: E
authors.lastname: Kalso
authors.affiliation: Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
authors.forename: Jorma
authors.initials: J
authors.lastname: Paavonen
mesh_terms: D000293:Adolescent; D000328:Adult; D016022:Case-Control Studies; D004848:Epithelium / Q000276:immunology* / Q000294:innervation / Q000378:metabolism / Q000473:pathology; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008221:Lymphoid Tissue / Q000276:immunology* / Q000378:metabolism; D008875:Middle Aged; D009092:Mucous Membrane / Q000276:immunology* / Q000294:innervation / Q000378:metabolism / Q000473:pathology; D009412:Nerve Fibers / Q000276:immunology* / Q000473:pathology; D020932:Nerve Growth Factor / Q000276:immunology* / Q000378:metabolism; D014844:Vulva / Q000276:immunology / Q000294:innervation / Q000378:metabolism / Q000473:pathology; D056650:Vulvodynia / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D055815:Young Adult
chemical_list: D020932:Nerve Growth Factor
keywords: NF2F11; NGF; PGP9.5; germinal center; immune activation; immunohistochemistry; inflammation; nerve fibers; vestibulodynia; vulvar pain; vulvar vestibulitis; vulvodynia