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Characterization of upper lamina propria interstitial cells in bladders from patients with neurogenic detrusor overactivity and bladder pain syndrome

Abstract The upper lamina propria (ULP) area of interstitial cells (IC) in bladder has been studied for more than a decade in several species including human beings. Nevertheless there is still lack of uniformity in terminology of this cell layer. The aim of the present study was to add new data to the morphological and immunohistochemical phenotype of these cells and to find out whether this phenotype is changed in bladders from patients with neurogenic detrusor overactivity (NDO) and bladder pain syndrome (BPS). Bladder tissue was obtained from a control group and from patients with NDO and BPS. Samples were processed for morphology, electron microscopy and immunohistochemistry. A morphological and immunohistochemical phenotype for the ULP IC was assessed and changes in this phenotype were looked for in samples from patients with NDO and BPS. The ULP IC were characterized ultrastructurally by the presence of actin filaments with densifications, many caveolae and abundant rough endoplasmic reticulum (RER); on immunohistochemistry ULP IC were immunoreactive for α-sma, vimentin, CD10 and podoplanin and categorized as interstitial Cajal-like cells (ICLC). In NDO and BPS bladders we found a phenotypical shift towards a fibroblastic phenotype which was even more pronounced in the NDO group. In both groups there was also an increased presence in ULP lymphocytes. The ULP area in the human bladder contains a population of ICLC with distinct ultrastructural morphology and immunohistochemical phenotype. Their unique α-sma+/desmin–/CD34– phenotype allows studying this population in various bladder disorders. In bladders form patients with BPS and NDO, we observed these ULP ICLC to shift towards a fibroblast phenotype.


Introduction
The urinary bladder serves as reservoir that alternates between urine storage and efficient urine expulsion at a convenient moment. These apparently simple functions are regulated by a complex control system that is located in the bladder wall, the peripheral ganglia, the spinal cord and the brain. Recent insights into the complex morphological and functional organization of bladder wall, suggest that not only neuronal [1], but also non-neu-ronal (sensory and motor) pathways play a crucial role in bladder (dys)function [2].
One of the non-neuronal cells that is suggested to coordinate local bladder processes is the interstitial cell (IC). In recent years several independent groups have reported the presence of these cells in animal and human bladder (for review see [3][4][5][6]). These cells have mainly been described in the upper lamina propria (ULP, directly underneath the urothelium, sometimes called suburothelium) and the intermuscular spaces in the detrusor. In the ULP these cells have been categorized heterogeneously as interstitial Cajal-like cells (ICLC) [4,7] or myofibroblasts [8,9], which are by definition two different types of cells. Reasons for this diverse nomenclature are mainly the use of different experimental methods (electron microscopy versus light microscopy and immunohistochemistry) and the use of different tissue hosts (human versus animals). A constant finding however is that these IC in the ULP are closely packed together, arranged in several consecutive layers and that these cells are immunoreactive for the intermediate filament vimentin.
Because of their particular organization just underneath the urothelium, ULP IC have attracted interest of many investigators because they could embody a structural and functional link between urothelial cells and sensory nerves and/or between urothelial cells and detrusor smooth muscle cells. Moreover these cells might be involved in the pathophysiology of functional bladder disorders, where local signalling processes are thought to play important roles. Two of the most frequent functional bladder disorders are neurogenic detrusor overactivity (NDO) and bladder pain syndrome (BPS). The aetiology of both pathologies is still far from being fully elucidated, but local alterations in the bladder wall are likely to be involved. In this paper, we studied the morphology of ULP IC in normal bladders and dysfunctional bladders from patients with NDO due to multiple sclerosis (MS) and BPS to describe the structural alterations that may disrupt normal bladder physiology.

Patient selection
The study protocol was in accordance with EU guidelines and approved by the institution's ethical committee. All

Tissue sampling and processing
Bladder tissue was obtained with two different procedures. Cold cup biopsies were taken during endoscopic procedures (diagnostic hydrodistention or ureteroscopy), whereas ex vivo biopsies were taken from cystectomy specimens immediately after surgery. All bladder biopsies were taken from the bladder dome (non-trigonal regions). One part of a each bladder biopsy was immediately fixed in formalin 6% and subsequently embedded in paraffin, whereas the other part was prepared for electron microscopy.

Semi-quantitative analysis
Semi-quantitative analysis was done: measurement of the width of the ULP layer of IC, count of the amount of CD3 ϩ T-lymphocytes in the urothelium, count of the amount of CD3 ϩ T-lymphocytes in the ULP and count of the amount of CD20 ϩ lymphoid aggregates in the lamina propria. All analyses were done blinded on five high-power fields (

Phenotype of ULP IC in normal human bladder
When visualized with the light microscope the ULP reveals an IC population that is organized in 10-20 densely packed layers (Fig. 3) and characterized by a vesicular, rather round nucleus and scant eosinophilic cytoplasm. Remarkable regional differences in devel-opment of IC were present: within a single section areas of densely packed IC layers alternated with regions that contained only few ULP IC. Ultrastructurally the ULP cells contained caveolae in the peripheral cell membrane and well-developed rough endoplasmic reticulum, mitochondria and peripheral actin filaments (mostly organized in bundles as illustrated by the frequently observed densifications) in the cytoplasm (Figs 1 and 2, Table 2). The cells also showed several discontinuous plasmalemmal thickenings, but no clear fibronexus was seen. Intercellular connections consisted preferentially of intermediate junctions although occasional gap junctions were also observed (data not shown). Immunohistochemical characterization of ULP IC revealed the expression of vimentin, ␣-sma, podoplanin and CD10 (Figs 3 and 4). Anti-CD10 antibodies only labelled the upper cell layers. This cell    (Figs 3 and 6).

Phenotype of ULP IC in bladder from NDO patients
In bladders from NDO patients ULP IC were still present. However they appeared to be more slender with a broader space in between the cell layers and a less dense intercellular matrix (Fig. 1). Ultrastructurally the IC contained in their cytoplasm less actin filaments (with almost no organization in parallel bundles) and less caveolae in their peripheral membrane compared to those in normal bladders. In the perinuclear area several mitochondria and well-developed rough endoplasmic reticulum cisternae were obvious (Fig. 2). The immunohistochemical phenotype differed slightly: the IC were still vimentin ϩ but tend to express less ␣-sma (in line with the ultrastructural data, observed in all NDO bladders) (Fig. 7). Quantification of the reduced number of ␣-soma ϩ IC was difficult because the ULP area of IC was significantly enlarged in NDO bladders (Table 3, Fig. 6) (which means that comparison of cell counts/area with normal bladders was problematic). The ULP IC were also still immunoreactive for podoplanin and CD10 and negative for desmin and CD34. A significantly higher amount of CD20 ϩ lymphoid aggregates/HPF compared to control bladders was present in the lamina propria of NDO bladders (Fig. 7, Table 3). The amounts of CD3 ϩ T cells/HPF in the urothelium and in the ULP were not significantly increased compared to controls. A remarkable  observation on electron microscopy was the close apposition of lymphocytes to IC in the ULP, lying in the same direction, and thus creating couples of IC and lymphocytes (Fig. 1).

Phenotype of ULP IC in bladder from BPS patients
In bladders from BPS patients a remarkable loss of organization in layers of ULP IC was noticed (Fig. 1). Furthermore there was a heterogeneous population of interlaying inflammatory cells: lympho-cytes, mast cells, plasma cells and macrophages (Fig. 1). Ultrastructurally the IC contained in their cytoplasm actin filaments with densifications and caveolae at the peripheral cell membrane, in a smaller amount as compared to those in normal bladders. Several mitochondria and many rough endoplasmic reticulum cisternae were present (Fig. 2) 0.05, Fig. 7, Table 3). No higher numbers of CD20 ϩ lymphoid aggregates/HPF compared to control bladders were present in the lamina propria of NDO bladders (Fig. 8, Table 2). Another observation was the focal presence of urothelial denudation in all BPS bladders.

Discussion
Wiseman et al. were the first to describe in detail the ultrastructure of the ULP layer of IC, which they categorized as myofibroblasts [8]. Others have questioned this myofibroblast phenotype due to   tions and gap junctions). These cells are immunoreactive for vimentin, ␣-sma, podoplanin and CD10 (superficial layers) and did not stain with antibodies directed to CD34, desmin, c-kit, synaptophysin and neurofilament. The ultrastructural phenotype found in the present study is highly similar to that reported by others [8]. However a straightforward classification is not so obvious. Features that do not match with the classical ICC are the presence of abundant RER, the numerous actin filaments and the lack of immunoreactivity with antibodies to c-kit and to CD34 [12]. Arguments against myofibroblast phenotype are the absence of fibronexus and the presence of caveolae [10,11]. The archetypical smooth muscle phenotype is also unlikely due to the abundant presence of RER and the immunohistochemical negativity for desmin. In the literature this problem with phenotyping is not new, and several authors have reported on the presence of a spectrum of spindle cell phenotypes, bearing to greater or lesser extent the ultrastructural features of myofibroblast, ICC, fibroblast or smooth muscle cell [10,12]. Putting together all morphological and immunohistochemical data we conclude that the phenotype of the main population of ULP IC lies somewhere in between that of a typical ICC and a typical myofibroblast, having features of both. Therefore we classify these cells as ICLC, a terminology which has been proposed before [13,14].
We were unable to demonstrate c-kit on ULP ICLC: the few c-kit ϩ cells appeared to be all mast cells given the similar staining pattern for tryptase. Literature on c-kit expression in bladder ICLC shows conflicting reports: publications mentioning c-kit on ICC [15][16][17] alternate with papers that state the opposite [18,19]. Almost no study on the expression of c-kit on bladder ULP ICLC used tryptase staining as a control for mast cells, which at least in our hands shows an almost full overlap. A recent paper mentioned the presence of c-kit ϩ /tryptase ϩ ICC in the lamina propria [17]. This report however deals with IC throughout the lamina propria, whereas our study was focused on ICLC directly beneath the urothelium (ϭ ULP) which are -in our hands -negative for c-kit.
The function of ULP ICLC has been under debate for several years. Because these cells are closely packed together underneath the urothelium, with intercellular connections and a distinct phenotype from the stromal cells covering the major part of the lamina propria, it is likely that these cells play a specific role in bladder physiology. Due to their localization, a communicating role between urothelium and detrusor and/or afferent nerves could be possible. A remarkable observation was the changing densities of ULP ICLC in a single biopsy, which could also have functional implications. Future studies should focus on the expression of specific ion channels or membrane receptors (such as TRP channels [20]), which are likely to give more insight into the function of this cell layer. The significance of CD10-and podoplanin immunoreactivity of ULP ICLC remains to be determined. Both markers have been studied on fibroblasts in the stroma surrounding tumour nests. Podoplanin was found to be up-regulated in cancer associated fibroblasts, both in colorectal [21] and lung [22] adenocarcinomas. CD10 expression in tumour-associated stromal cells has been described in several tumours [23][24][25]. Both markers could be related to a myofibroblast phenotype, because myofibroblasts are more and more seen as activated mesenchymal cells in tumour or inflammatory conditions [10]. In bladder both CD10 and podoplanin have a similar immunohistochemical expression pattern on ULP ICLC in normal and inflammatory conditions. However future quantitative studies might reveal differences in expression in pathologic conditions.
The ultrastructural and immunohistochemical phenotype of ULP ICLC showed modest changes in the pathologic conditions studied. In NDO bladders ULP ICLC showed less actin filaments together with a decreased expression of ␣-sma and also less caveolae. This observation suggests a trend towards a fibroblast phenotype. Furthermore the ULP area of ICLC was significantly broadened in NDO bladders with a remarkable less dense intercellular matrix and a broadened space between cell layers. These findings might also be due to a changed phenotype of the ULP ICLC. The functional implications of these observations remain to be determined, but it is likely that these are some of the many elements contributing to altered bladder function in NDO patients [26]. Also in bladders from BPS-patients the phenotype of the ULP ICLC showed a slight shift towards a fibroblast phenotype. The organization in dense parallel layers was clearly disturbed, resulting in a chaotic organized group of ICLC. The functional significance of these observations remains also unclear.
A remarkable finding was the enhanced inflammatory response in both NDO and BPS bladders, clearly with different characteristics. In NDO bladders frequent close apposition of lymphocytes and ULP ICLC was found, which might indicate a role of ICLC in antigen-presentation to T cells. Furthermore a significantly increased number of CD20 ϩ lymphoid follicles were found in the lamina propria of these bladders. In BPS bladders a dense infiltrate of CD3 ϩ lymphocytes was found in the area of ULP ICLC. This finding was clearly less pronounced in NDO bladders. The particular apposition of CD3 ϩ T-lymphocytes to ULP ICLC was not found. The inflammatory infiltrate here was denser and more heterogeneous with plasma cells and mast cells. An increased number of inflammatory cells in bladder mucosa, especially T cells, have been reported before [27,28]. It is however remarkable that this field is relatively unexplored. The further exploration of the functional significance of lamina propria inflammation in NDO and BPS is beyond the scope of this study, but the abundant presence of T cells in NDO and BPS bladders, both with a different presentation is likely to influence the physiological role of the ULP ICLC cell layer. Whether this inflammatory response is a primary or secondary phenomenon needs to be elucidated.
In conclusion we have defined the ULP IC as ICLC, with an ultrastructural an immunohistochemical phenotype somewhere between that of an archetypical myofibroblast and ICC. We further found obvious changes in the phenotype and/or topographical organization of these cells in NDO and BPS bladders, with a shift towards a fibroblast phenotype in both diseases. A remarkable high number of lymphocytes were found in the area of ULP ICLC, with a different topographical pattern in NDO and BPS bladders. Future studies should focus on specific receptor and ion channel expression profiles of these cells to elucidate the functional role of this intriguing cell layer in both normal and pathological bladders. Microdissection of this layer might therefore be a valuable experimental tool.