Local immune response in bladder pain syndrome/interstitial cystitis ESSIC type 3C

This prospective cohort study assigned patients recruited at one urogynaecological centre in Switzerland from 2006 to 2011 to three comparison groups: BPS/IC ESSIC type 3C (group 1, n = 15), OAB (group 2, n = 11) and healthy controls (group 3, n = 8). Each woman reviewed and completed relevant study documentation approved by the local ethics commission including an informed consent form (reference 9900.013).

In order to detect markers, homogeneous groups of patients were required. Consequently, patients with predominant pain symptoms were selected based on their diagnosis with classic ulcerative IC or IC with Hunner’s lesions that conformed to the BPS/IC ESSIC type 3C criteria [4]. “Hunner’s lesions” were identified according to ESSIC [4] as distinctive, circumscript reddened mucosal areas with small vessels radiating towards a central scar that, upon bladder distension, will rupture and bleed. As a control group, we chose patients who had primary urgency symptoms and a cystoscopically non-inflamed bladder wall (“non-inflammatory OAB”). These patients represent a subgroup of the OAB condition since there are also reports of an association between OAB and inflammation [9‐11]. Healthy control patients were identified as women with no bladder symptoms and who were hospitalized for a hysterectomy shortly before enrolment in the study. Patients with urinary tract infections were excluded.

Two cold-cup biopsies were taken from each patient and, when present, from the transition zone lesion/non-lesion. Thus, bladder biopsies included various amounts of urothelium, submucosa and muscularis propria (detrusor). Histopathological testing of one of these tissue samples included mast cell quantification. Detrusor mastocytosis was defined according to ESSIC [4] as mast cell counts greater than 28 mast cells/mm². The other biopsied tissue was preserved in RNAlater (76104, QIAGEN, Valencia, CA, USA) and used for RNA isolation and quantification as described in the report of our pilot study [5]. Reverse transcription and RT-qPCR were done with materials and equipment from Applied Biosystems (Life Technologies, Zug, Switzerland) as follows: TaqMan® Reverse Transcription Reagents (N8080234) for reverse transcription, inventoried Human TaqMan® Gene Expression Assays (4331182) and TaqMan® Universal PCR Master Mix (4364340) for RT-qPCR on an Applied Biosystems 7500 Real-Time PCR System. The following TaqMan® Gene Expression Assays (4331182) were analysed: cytotoxic T-lymphocyte-associated protein 4 (CTLA4, Hs03044418_m1) as a T-cell marker; CD20 (membrane-spanning 4-domains, subfamily A, member 1 MS4A1, Hs00544819_m1) and CD79A molecule, immunoglobulin-associated alpha (CD79A, Hs00233566_m1) as B-cell markers; immunoglobulin heavy locus (IGH@, Hs00378230_g1) as a marker for antibody heavy chain gene expression; and uroplakin 1B (UPK1B, Hs00199583_m1), uroplakin 3A (UPK3A, Hs00199590_m1) and cytokeratin 20 (KRT20, Hs00300643_m1) as urothelial markers. RT-qPCR reactions were done in duplicates in a 25 μl volume. Standard cycling conditions were used (2 min 50 °C, 10 min 95 °C, 40 cycles 15 s 95 °C/1 min 60 °C). Relative gene expression (comparative Ct method quantitation) was calculated with the 7500 System SDS software version 1.3. Results were given as “relative expression” using glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs99999905_m1) as the endogenous control and healthy control patient 005 [5] as the calibrator.

As described in our previous study, biopsied tissue was embedded in paraffin and sections were stained with either haematoxylin and eosin or Giemsa and van Gieson elastin [5]. For immunohistochemistry testing, biopsy sections were treated according to the standard protocol with the Leica BOND-MAX automated system using Leica Novocastra reagents (Biosystems, Nunningen, Switzerland). The standard protocol for immunohistochemistry was as follows: Dewaxing (AR9222), Epitope Retrieval Solution 2 (AR9640) and Bond Polymer Refine Detection Kit (DS9800) or Bond Polymer Refine Red Detection Kit (DS9390). The primary antibodies were 1F6 mouse monoclonal antibody to cluster of differentiation 4 (CD4, NCL-CD4-1F6, Novocastra) diluted 1:50; MJ1 mouse monoclonal antibody to CD20 (NCL-CD20-MJ1, Novocastra) diluted 1:100; 11E3 mouse monoclonal antibody to CD79A (NCL-CD79a-225, Novocastra) diluted 1:125; AU-1 mouse monoclonal antibody to uroplakin 3 (UPK3, 345 M-14, Cell Marque, Rocklin, CA, USA) diluted 1:25; and PW31 mouse monoclonal antibody to KRT20 (NCL-L-CK20-561, Novocastra) diluted 1:200. Microphotographs were taken at ×20 magnification with a UC30 colour camera mounted on a BX43 Olympus System Microscope. Images were processed by the CellF image analysis software (Olympus, Volketswil, Switzerland).

Blood was collected in VACUETTE® Heparin tubes (Greiner Bio-One, St. Gallen, Switzerland) one day before biopsy. IgG and IgA concentrations were determined on a cobas® 6000 analyzer (Roche Diagnostics AG, Rotkreuz, Switzerland).

Urine was obtained through urinary catheterization one day before the biopsy. Standard laboratory techniques included a routine urine status and a urine culture. Sterile urine was immediately centrifuged (5 min, 900 g), and the supernatant was frozen at −20 °C and subsequently stored at −80 °C. After thawing, protease inhibitor (Complete 04693116001, Roche Diagnostics AG, Rotkreuz, Switzerland) was added and 300 μl-aliquots were stored at −80 °C. Concentration of urine creatinine was determined with the CREA plus kit from Roche/Hitachi on a Roche/Hitachi Modular System P Chemistry Analyzer (Roche Diagnostics AG, Rotkreuz, Switzerland) at Ilamed AG, Institute for Laboratory Medicine (Frauenfeld, Switzerland). For immunoglobulin quantification, urine aliquots were thawed and centrifuged for 15 min at 2,000 g. Supernatants were adjusted to pH 7.4 by adding a modified phosphate-buffered saline (PBS) buffer (final 1× concentration: 137 mM NaCl, 2.7 mM KCl, 40 mM phosphate buffer, pH 7.4). To quantify the protein concentration, three different dilutions were measured per urine sample. IgG and IgA concentrations were determined on a BD FACSCalibur flow cytometry instrument using Human Total IgG CBA Flex Set (No. 558679) and Human Total IgA CBA Flex Set (No. 558681) kits in accordance with the manufacturer’s protocol (BD Biosciences, Allschwil, Switzerland).

Statistical analyses were done in IBM® SPSS® Statistics (version 19). A p value of less than 0.05 was considered to indicate statistical significance. Tests of normality (Shapiro-Wilk) and homogeneity of variance (Levene statistic) were done with all continuous variables. The Kruskal-Wallis test was used for non-parametric data. Post hoc tests (Mann-Whitney U) identified differences between two groups, and the Bonferroni correction was applied. As such, a p value of 0.016 (0.05/3) indicated significance. For normally distributed data, analysis of variance (ANOVA) followed by Tukey’s HSD post hoc tests were used to detect differences between the groups. Non-parametric receiver-operating characteristic (ROC) curves were generated for all biomarkers to plot the sensitivity against the false-positive rate (1-specificity). To accomplish this, biomarker values for BPS/IC ESSIC type 3C were compared to values of OAB combined with the healthy control group. The optimal cut-off for each biomarker was selected to maximize the sum of sensitivity and specificity [Youden index = max. (selectivity + specificity-1] [12]. The accuracy of a biomarker to predict BPS/IC, defined as the average of sensitivity and specificity, was also calculated.