Our data provide a global picture of the differences between mRNA levels of bladder biopsies from classic IC patients and from healthy controls. In the ni-vs-healthy comparison, non-ulcer tissue of classic IC patients (ni) and tissue from healthy controls (healthy) have been compared, and in the ulcus-vs-ni comparison, ulcer (ulcus) and non-ulcer tissues (ni) have been compared.
The ni-vs-healthy comparison yielded over 3,500 significant changes at an FDR of 8.7%, which means there is a possibility that a discrete gene could be a false positive. However, the majority of the detected changes can be attributed to genuine differences in non-ulcer and healthy tissue (see Additional file 2
). In contrast, the ulcus-vs-ni comparison yielded only 500 significant changes at an FDR of 57.2%, meaning the likelihood is only about 50% that a randomly picked "significant" gene is truly differentially expressed in ulcer vs. non-ulcer tissue (see Additional file 9
). This suggests that the ulcer and non-ulcer tissue of patients with Hunner's ulcer have only small differences in gene expression. An analysis with a significantly larger set of patients would probably have the statistical power to identify those changes more reliably. Given our limited set of patients, we obtained a rather unreliable set of approximately 500 differentially expressed genes, and therefore, our results of the associated GO categories should be regarded as an indication of how ulcer and non-ulcer biopsies may differ.
As the gene expression arrays used in our study cover all known human mRNAs (over 47,000 transcripts), previous gene expression data on IC can be compared with our results.
Keay and others have published microarray analysis data investigating gene expression differences in explanted epithelial cells from bladder biopsies [15
]. They focused on approximately 4,000 genes and compared differences between IC patients (NIDDK criteria) and asymptomatic controls, and differences between normal epithelial cells treated with APF, or with mock APF. One of the 13 genes discussed in their publication was significant in our ni-vs-healthy comparison. This was "neutral amino acid transporter B" which was, as in their results, down-regulated (see Additional file 4
). We anticipated only minor overlaps between the data from their paper and our results since histopathological data from our investigated bladder biopsies indicated that urothelial cells contribute only a minor part of all cell types (Figure ). Even if there were differences in urothelial gene expression between our two investigated subgroups, they might be too subtle to be detected. Furthermore, many of the 13 genes were ubiquitously expressed genes (e.g. cyclin D, stress-activated protein kinase JNK1, putative tRNA synthetase-like protein, ribosomal protein L27a), and as we primarily examined cell types other than epithelial cells, this must lead to different results.
Hurst and co-workers addressed the question of abnormalities in the urothelium of IC patients [42
]. With a set of immunostainings against proteoglycan core proteins and differentiation markers, they investigated biopsies of 27 IC patients (NIDDK criteria) and five controls having stress incontinence [42
]. They scored their data from -2 (most abnormal) to +2 (normal), and their findings suggested abnormal differentiation in the IC urothelium. Many of the proteins investigated in their study were present in both IC and control biopsies, but were localized at different sites (e.g. dense luminal staining vs. uniform distribution). Gene expression analyses of entire biopsies, as performed in our study, give no information on spatial distributions. Moreover, the amount of urothelium was reduced in our biopsies, and the presence of a protein and its respective mRNA will not necessarily need to correlate. Taking these three points into account, it is expected that genes encoding the proteins found by Hurst would not be significantly regulated in our analysis. Interestingly, two genes, keratin-20
were considerably up-regulated (20- and 11-fold, respectively) in the healthy controls, in agreement with the findings of Hauser et al. (keratin-20 or uroplakin staining in normal urothelium, no keratin-20 or weak uroplakin staining in most abnormal urothelium) [42
Generally, the presence of mRNA and the presence of the encoded protein do not need to correlate. Temporal and spatial differences in expression are likely. Regulation of mRNA translation, protein processing, protein stability and protein translocation are all factors that influence a direct correlation between an mRNA and its mature protein.
Inflammatory features in IC have been investigated by histological analysis. Peeker and Fall [4
] defined a clear picture of ulcerative or classic IC that included urothelial spongiosis and detachment; subepithelial, perineural and perivascular deposits of mononuclear cells; and a characteristic mast cell response, with an increase of mast cells in detrusor muscle and in the lamina propria. In addition, other groups reported extensive bladder inflammation of patients with ulcer [7
]. With our restriction to classic IC we were able to characterize a relatively homogenous group of patients. While our data agreed with reported histological findings, they additionally provided us with a series of potential gene expression markers for this subtype of the disease.
The generation of a gene expression profile for non-ulcerative IC is expected to be more difficult. In contrast to classic IC, literature regarding non-ulcerative IC described a heterogeneous picture with respect to inflammation. Peeker and Fall [4
] selected their patients according to the NIDDK criteria and reported no inflammatory signs and a less prominent mast cell involvement in non-ulcerative IC. Erickson et al. [44
] also evaluated their patients according to the NIDDK cystoscopic criteria. Severe inflammation was seen in 30% of the patients who met the criteria and in 23% who did not. Ulcers were reported for only four patients (corresponding to 11% of the patients who met the criteria). Also Denson et al. [21
], who used the NIDDK inclusion criteria, described mononuclear infiltration as the most consistent histological feature (while only 1% of their patients had a Hunner's ulcer, 70% showed mild to severe inflammation). In another study by Erickson et al. [7
], up to 35% of the non-ulcerative patients had severe inflammations.
We can conclude that patient evaluation and selection for a gene expression profile of non-ulcerative IC is crucial and challenging. Very likely, a careful subdivision of this heterogeneous patient group will be indispensable to obtain statistically significant results.
An interesting and unexpected result of our study was the severe inflammatory infiltration at non-ulcer ("ni") sites of a bladder with Hunner's ulcer (Figure , Additional file 3
). In the literature, many research groups have not focused on such tissue, but have instead analyzed biopsies of the most severely affected part of the bladder (e.g. [7
]). Factors released from an ulcer region, or, more generally, from an abnormal urothelium, may have paracrine influences on surrounding cells. For example, TNF alpha (6.5-fold overexpressed in our ni-vs-healthy comparison) plays a central role in pro-inflammatory paracrine signaling and mediates the recruitment of cells involved in inflammatory and wound healing responses [45
]. It was not clear to us why gene expression of biopsy 14 ni was similar to the healthy controls (Figure ). A potential reason might be a reduced paracrine signaling at the location of biopsy.
Compared to controls, mast cell counts were 6- to 8-fold higher in the detrusor muscle of classic IC, and 2- to 3-fold higher in non-ulcerative IC [9
Several reviews attribute a key role to mast cells in the mediation of chronic inflammation. Signals released from abnormal urothelium or penetrating through a damaged urothelium may stimulate submucosal sensory nerves, and recruit and activate inflammatory leukocytes including mast cells. Upon activation, mast cells release preformed mediators stored in granules (histamine, heparin, neutral proteases), generate mediators by enzymatic processing of membrane phospholipids (leukotrienes, prostaglandins), and produce cytokines and chemokines de novo (IL-6, IL-8, TNF and many others). Released mediators initiate and maintain the recruitment of inflammatory cells, promote angiogenesis and stimulate fibrosis [9
Upon histological analysis, mastocytosis in the deeper areas of the bladder wall was found in all of our classic IC patients. The concept outlined above describing the central role of mast cells in inflammation may explain many of our findings. Upon cystoscopy and bladder filling, the bladder wall was found to be very fragile at many points, not only at the sites of Hunner's ulcers (unpublished observation).