Hypothesis / aims of study
Hunner type interstitial cystitis (HIC) is a chronic inflammatory disorder of the urinary bladder characterized by persistent pelvic/bladder pain, urinary frequency and/or urgency. Histologically, HIC bladder manifests subepithelial chronic inflammatory changes such as lymphoplasmacytic infiltration, epithelial denudation, and stromal oedema, neovascularization and hyperemia. To date, few animal models which reproduce the histological and clinical correlates of HIC have been yet established. Although the aetiology of HIC remains elude, recent evidence suggests that enhanced immune responses may underlie the pathophysiology of HIC (1). The higher prevalence of comorbid systemic autoimmune disorders is a well-known fact in patients with HIC. Based on this evidence, we aimed to develop a novel animal model for HIC via autoimmunity to the bladder urothelium in this study.
Study design, materials and methods
A transgenic model (URO-OVA) mouse, that expresses the membrane form of the model antigen ovalbumin (OVA) on the bladder urothelium as a self-antigen, was used in this study (2). Antigen OVA-specific lymphocytes were generated by immunization of C57BL/6 mice with complete Freund’s adjuvant-emulsified OVA protein (100µg) subcutaneously. Splenocytes (a mixture of T and B cells) of the OVA-immunized C57BL/6 mice were prepared 14 days after immunization and immediately injected to the URO-OVA mice intravenously, or cultured at 1 x 105 cells/well in a 48-well plate for 3 days in the absence or presence of OVA, OVA257-264 peptide (a MHC class I-restricted peptide epitope), OVA323-339 peptide (a MHC class II-restricted peptide epitope), or ras control peptide for subsequent ELISA analysis of IFN-γ levels in the culture supernatants. Bladder histology (Hematoxylin-Eosin staining and immunohistochemistry for CD3-positive T lymphocytes and CD19-positive B lymphocytes), gene expression levels of IFN-γ, TNF-α, and pre-substance P in the bladder tissues, pain behaviour (von Frey filament test), and voiding function were evaluated at 7, 14, 21, 28 days after adoptive transfer of OVA-primed splenocytes. Age-matched normal URO-OVA mice that were not underwent adoptive transfer served as controls. Each timepoint contained 3 to 5 mice for both groups.
Results
The URO-OVA mice transferred with OVA-primed splenocytes developed cystitis from day 7 exhibiting histological chronic inflammatory changes such as remarkable mononuclear cell infiltration, increased vascularity, and mucosal hyperemia in the bladder with a peak at day 21, which perpetuated until day 28. Immunohistochemical detection revealed that infiltrating mononuclear cells were predominantly composed of T and B lymphocytes in the cystitis bladder. Meanwhile, the control mice did not show any histological changes (Fig. 1). In parallel, the cystitis URO-OVA mice demonstrated significantly increased pelvic pain and urinary frequency, and reduced micturition volume compared to the controls from day 7 to 28 (Fig. 2). The gene expression levels of IFN-γ, TNF-α, and substance P precursor in the bladder were significantly up-regulated in the cystitis URO-OVA mice compared to the controls (Fig. 3a and 3b). In vitro stimulation of the cultured OVA-primed splenocytes by OVA, OVA257-264, and OVA323-339 proteins resulted in increased IFN-γ production by these cells (Fig. 3c).
Interpretation of results
The results indicate that autoimmunity to the urothelium antigen induced bladder chronic inflammation with increased nociceptive responses and voiding dysfunction perpetuating more than 4 weeks, which mimics the characteristic features of human HIC.