Redistribution of calcium-independent phospholipase A2 isoforms in IC/BPS urothelial cells

Background: Interstitial cystitis/bladder pain syndrome is a debilitating inflammatory bladder disease with currently unknown etiology. We determined the expression of calcium-independent phospholipase A2β (iPLA2β) and iPLA2γ isoforms in cultured immortalized urothelial cells isolated from normal or IC/ BPS patient bladders. Materials and methods: Immunoblot analysis was used to determine the iPLA2 isoform expression in immortalized urothelial cells isolated from normal and IC/BPS patients. iPLA2 activity was measured using a radiometric assay with (16:0, [3H]18:1) plasmenylcholine as substrate. Platelet-activating factor (PAF) accumulation was determined by labeling cells with [3H] acetic acid. Adherence of polymorphonuclear leukocytes (PMN) to urothelial cells was determined by measuring myeloperoxidase (MPO) activity. Results: We identified the two predominant mammalian iPLA2 isoforms, iPLA2β and iPLA2γ, in immortalized urothelial cells. IC/BPS urothelial cells demonstrated a decrease in iPLA2γ and an increase in iPLA2β immunoprotein when compared to normal cells. Selective pharmacologic inhibition demonstrated that the majority of iPLA2 activity in normal urothelium represents iPLA2γ whereas that in IC/BPS urothelium is iPLA2β. Greater PAF production and PMN adherence was observed in tryptase-stimulated IC/BPS urothelial cells when compared to normal urothelial cells. In both normal and IC/BPS urothelial cells, increased PAF production and PMN adherence was completely blocked when tryptase-stimulated cells were pretreated with (S)-bromoenol lactone (BEL) to inhibit iPLA2β. PMN adherence was blocked when PMNs were pretreated with ginkgolide B to block the PAF-receptor (PAFR). Conclusions: Urothelial cells derived from IC/BPS origin exhibit a redistribution of iPLA2 isoforms with iPLA2β being predominately expressed compared to normal urothelial cells. The redistribution of iPLA2β, increased PAF production and increased PMN adherence may represent a mechanism whereby IC/BPS patients’ exhibit increased susceptibility to bladder inflammation.


Introduction
The urothelium that lines the inside of the bladder was thought originally to function solely as a barrier restricting movement of urinary constituents into the underlying tissue. Subsequently, the urothelial cell has been shown to be instrumental in a variety of signaling pathways involved in inflammation, differentiation and imparting selective permeability [1,2]. Alterations in the normal signaling processes of the urothelial cell have been shown to be involved in tumor growth, reduced barrier function, and bladder inflammation. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory bladder disease associated with dysfunctional urothelial cell barrier function, altered signaling responses and increased mast cell activation [3,4]. Mast cells are the primary effectors of IgE-mediated immune responses, and upon activation, release a variety of preformed and newly synthesized inflammatory mediators. Tryptase stimulation of protease activated receptors (PARs) expressed on urothelial cells results in calcium-independent phospholipase A 2 (iPLA 2 ) activation and subsequent platelet activating factor (PAF) accumulation [5].
PAF synthesis is initiated via iPLA 2 -catalyzed hydrolysis of the sn-2 fatty acid from membrane phospholipids resulting in the release of a lysophospholipid which can be subsequently acetylated at the sn-2 position to form PAF [6]. The two major isoforms of iPLA 2 found in mammalian cells, iPLA 2 β and iPLA 2 γ, are responsible for a wide range of physiological responses. We have previously determined that endothelial cell iPLA 2 β is the predominant isoform responsible for PAF production [7]. PAF is a potent recruiter that facilitates transendothelial and transepithelial migration of circulating inflammatory cells. This action is accomplished by the interaction of endothelial or urothelial cell PAF with the inflammatory cell PAF-receptor (PAFR) resulting in tight adherence and subsequent migration through the paracellular space [8]. Increases in PAF accumulation result in enhanced inflammatory cell adherence to cultured endothelial cells [9]. This process could play a role in the increased presence of inflammatory cells in the bladder wall of patients with IC/BPS resulting in the pain and inflammation associated with the condition.
In the present study we determine the expression of iPLA 2 isoforms in normal and IC/BPS urothelial cells following tryptase stimulation. We further show that PAFR antagonism by ginkgolide B prevents adherence of inflammatory cells to cultured urothelial cells regardless of tryptase stimulated increases in iPLA 2 and subsequent increase in urothelial PAF accumulation. This work highlights two potential therapeutic targets for the management of inflammatory bladder conditions.

Cell culture
Urothelial cells were isolated from normal or IC/BPS patient bladders and immortalized with a retrovirus encoding the oncoproteins E6 and E7 of human papillomavirus type 16 and selected for stable integration of the retroviral pro virus with G418. Expanded cultures were grown in EpiLife Media (Cascade Biologics, Inc. Portland, OR) with calcium (0.06mM), growth factor supplements provided by the manufacturer and penicillin (20U/ml)/streptomycin (100mg/ml) (Sigma Chemical Company, St. Louis, MO) and incubated at 37˚C, with an atmosphere of 95% O 2 , 5% CO 2 . Confluent monolayers were differentiated by adding 1 mM calcium and 10% fetal bovine serum (Ca/FBS). Experiments were performed after 3 days of differentiation. Samples were collected according to an Oklahoma University Health Sciences Center Institutional Review Board approved protocol following informed written consent.
Regions of antibody binding were detected using enhanced chemiluminescence (Amersham, Arlington Heights, IL) after exposure to film (Hyperfilm, Amersham).

Measurement of PAF production
Urothelial cells grown to confluence were incubated with Hanks' balanced salt solution (135 mM NaCl, 0.8 mM MgSO 4 , 10 mM HEPES, pH 7.4, 1.2 mM CaCl 2 , 5.4 mM KCl, 0.4 mM KH 2 PO 4 and 6.6 mM glucose) containing 10 μCi of [ 3 H] acetic acid for 20 min at room temperature. Cellular lipids were extracted using the method of Bligh and Dyer [10] and the chloroform layer was dried under nitrogen. Total lipid extracts were resuspended in 9:1 CHCl 3 :MeOH and applied to TLC plates. Plates were developed in 100:50:16:8 chloroform, methanol, acetic acid and water. The region corresponding to PAF was scraped and measured by liquid scintillation counting.

Measurement of PMN adherence
Blood (80 ml) was obtained from healthy donors and PMNs were isolated using Polymorphprep (Axis-Shield, Oslo, Norway). PMNs (2x10 6 ) were added to urothelial cells grown to confluence in 34-mm dishes. At the end of incubation, non-adherent PMNs were removed by washing the well twice with Hanks' buffer. Urothelial cells and adherent PMNs were lysed with 0.2% Triton X-100 and myeloperoxidase (MPO) content was determined by adding 400 μL of cell lysate to a tube containing 1mL of PBS, 1.2mL Hanks buffer with bovine serum albumin, 200 μL of 0.125% 3,3'-dimethoxybenzidine, and 200 μl of 0.05% H 2 O 2 . After samples were incubated at 37°C for 15 min, the reaction was stopped by the addition of 200 μl of NaN 3 , and the absorbance of each tube was measured at 460 nm. The MPO content in 2x10 6 PMNs was determined and used as the value for 100% adherence.

Statistical analysis
Statistical analysis was performed using Students' t test. The doi: 10.7243/2055-0898-2-3 difference between groups was considered significant at a level of p<0.05. All assays were performed at least four times.

Expression of iPLA 2 isoforms in normal and IC/BPS urothelial cells
We identified the two predominant mammalian iPLA 2 isoforms, iPLA 2 β and iPLA 2 γ, in cultured immortalized urothelial cells from normal and IC/BPS patients. As shown in Figure 1, iPLA 2 β immunoprotein expression was significantly greater in IC/ BPS-derived urothelial cells when compared to cells derived from normal bladders. In contrast, expression of iPLA 2 γ was significantly decreased in IC/BPS urothelial cells (Figure 1). In the representative blots shown in Figure 1, the presence of a lower molecular weight band is detected in the normal group however this band is present in both normal and IC/ BPS derived urothelial cells following longer exposure time.
To determine if the redistribution of iPLA 2 isoform immunoprotein resulted in alterations in iPLA 2 activity, we measured activity using (16:0, [ 3 H]18:1) plasmenylcholine in the absence of calcium (Figure 2). To determine the relative contribution of iPLA 2 β and iPLA 2 γ to total iPLA 2 activity, we pretreated urothelial cell membrane fractions with bromoenol lactone (BEL) prior to incubation with the substrate. Racemic BEL inhibits both iPLA 2 γ and iPLA 2 β, whereas (R)-BEL and (S)-BEL inhibit iPLA 2 γ and iPLA 2 β respectively, with an approximate 10fold selectivity [11]. Treatment of immortalized urothelial cells  isolated from normal bladders demonstrated PLA2 activity in the absence of calcium that was inhibited by pretreating with racemic BEL or (R)-BEL in a concentration dependent manner (Figure 2, upper panel). iPLA 2 activity in normal urothelial cells was relatively resistant to pretreatment with (S)-BEL, suggesting that the majority of iPLA 2 activity could be contributed to iPLA 2 γ (Figure 2, upper panel). In contrast, urothelial cells isolated from IC/BPS patients demonstrated a much greater inhibition of iPLA 2 activity with (S)-BEL pretreatment (Figure 2, lower panel) than that observed at corresponding concentrations in normal urothelial cells (Figure 2, upper panel). Inhibition of iPLA 2 activity in IC/BPS urothelial cells was comparable when using both racemic BEL and (R)-BEL, suggesting a lesser role for iPLA 2 γ in these cells (Figure 2, lower panel). Taken together, our data indicate a redistribution of iPLA 2 β and iPLA 2 γ in IC/PBS urothelial cells demonstrated by immunoblot analysis and measurement of iPLA 2 activity.

PAF production in normal and IC/BPS urothelial cells
To determine if the redistribution of iPLA 2 isoforms in IC/BPS urothelial cells resulted in a difference in PAF production, we used a radiometric assay to quantify PAF accumulation in cultured urothelial cells. When normal uro thelial cells were stimulated with tryptase, we observed a significant increase in PAF production compared to unstimulated controls (Figure 3). The observed increase in PAF production in response to tryptase was even greater in urothelial cells derived from IC/ BPS patients (Figure 3). Since PAF production is initiated by iPLA 2 activation, we pretreated urothelial cells with BEL prior to tryptase stimulation. As shown in Figure 3, pretreatment of both normal and IC/BPS derived urothelial cells with BEL prior to tryptase stimulation blocked PAF production completely.

PMN adherence to normal and IC/BPS urothelial cells
PAF facilitates transepithelial migration via interaction with the PAF-receptor (PAFR) expressed on the inflammatory cell surface. Modulation of this PAF-PAFR interaction could serve as a beneficial means of managing bladder inflammation. Corresponding to the increased accumulation of PAF in urothelial cells, we observed a significant increase in PMN adherence to tryptase-stimulated urothelial cells that was greater in IC/BPS-derived urothelial cells when compared to normal (Figure 4) PMN adherence to both normal and IC-BPS-derived urothelial cells was blocked by pretreating the urothelial cells with BEL, further demonstrating the role of iPLA 2 and PAF in modulating inflammation (Figure 4, gray bars). Pretreatment of PMN with ginkgolide B to block the PAFR also inhibited cell adherence to tryptase-stimulated urothelial cells (Figure 4, hatched bars).

Discussion
The urothelium that lines the inside of the bladder provides a barrier that prevents leakage of urinary constituents into the underlying tissue that can lead to pain and inflammation. Disruption of the barrier function can occur during times of inflammation, infection or mechanical damage and result in activation of resident mast cells and can directly depolarized nerve and muscle leading to pain and further inflammation [2,12]. Mast cells release a variety of inflammatory mediators upon degranulation, including tryptase. Tryptase has been detected in the urine of patients with IC/BPS indicating mast cell activation [13]. Previous studies have shown that tryptase stimulation of human urothelial cells, through PAR-2 cleavage, results in the activation of iPLA 2 and subsequently PAF production [5]. Platelet activating factor (PAF) is a potent inflammatory mediator whose formation is facilitated by iPLA 2 mediated hydrolysis of the sn-2 fatty acid from membrane phospholipids resulting a free fatty acid and a lysophospholipid, which can be acetylated subsequently to produce PAF [6]. The majority of iPLA 2 activity in mammalian cells is contributed by iPLA 2 β and iPLA 2 γ [14]. In this study, we show that there is a redistribution of iPLA 2 isoforms in urothelial cells derived from IC/BPS patients, with a decrease in iPLA 2 γ and increased iPLA 2 β. We have demonstrated previously that pharmacological inhibition or genetic ablation of iPLA 2 β results in attenuated PAF production and significantly reduced inflammatory cell adherence to endothelial cells [7]. Similarly, in this study, the increased iPLA 2 β immunoprotein expression and contribution to total iPLA 2 activity observed in IC/BPS-derived urothelial cells is associated with increased PAF production and PMN adherence. PAF is known to be involved in many cellular processes, including doi: 10.7243/2055-0898-2-3 angiogenesis, tumor growth, platelet aggregation and inflammation. A significant increase in PAF production in tryptase-stimulated urothelial cells provides evidence that this response could be involved in bladder inflammation mediated by mast cell activation, such as occurs in IC/BPS patients. A redistribution of iPLA 2 isoforms could have a profound impact on membrane phospholipid derived inflammatory mediators resulting in altered signaling responses. In IC/BPS derived urothelial cells, the increase in iPLA 2 β activity could render the bladders from these patients more susceptible to inflammation via increased PAF production and inflammatory cell retention. As iPLA 2 β is known to be the predominant isoform responsible for PAF production and the role of PAF in inflammation is well established, the work detailed in the paper provides evidence of altered inflammatory signaling in IC/BPS patients that could predispose to increased susceptibility to bladder inflammation. Increased PAF accumulation on urothelial cells could propagate the inflammatory response by facilitating the urothelial cell PAF-inflammatory cell PAFR interaction, retaining the inflammatory cells in the bladder wall, instead of allowing for clearance into the urine, furthering perpetuating and prolonging the inflammatory response. PAF released from activated urothelial cells could directly activate residing mast cells [15], act in an autocrine manner to promote further urothelial cell PAF production or stimulate endothelial cell PAFR-mediated iPLA 2 activation. These data represent a potential signaling pathway that is up regulated in IC/BPS derived urothelial cells that could be targeted for management of bladder inflammation and potentially as a means of preventing recurrence of symptoms ( Figure 5). Mast cell accumulation and activation has been accepted as a hallmark characteristic in IC/BPS [3]. The observation that tryptase results in accumulation of urothelial cell PAF, which is potentiated in IC/BPS derived urothelial cells, suggests that the PAF-PAFR interaction would be an important event in bladder inflammation, particularly in the scenario of mast cell degranulation and IC/BPS. Our studies provide two potential therapeutic targets for the management of IC/ BPS symptoms: inhibition of urothelial cell iPLA 2 β activity or blocking the PAFR on bladder wall resident inflammatory cells. To date there is no cure or universal management strategy for IC/ BPS and treatment options are aimed at symptom management ( Figure 5). Due to the ambiguity of IC/BPS pathogenesis, development of treatment options has been very difficult. Gingko biloba has been used to manage symptoms of several diseases including memory loss, circulatory diseases and asthma [8,16,17]. Ginkgolide B is an extract of the Gingko biloba plant, that accumulates unchanged in the urine [18], making it an exciting potential therapeutic approach for managing bladder inflammation. Our data suggest that targeting the inflammatory cell PAFR with a PAFR antagonist such as Ginkgo biloba may alleviate the symptoms of IC/BPS, particularly in acute inflammatory flare-ups, by managing the recruitment and retention of inflammatory cells in the bladder wall.

Conclusion
The data shown above demonstrate that IC/BPS is associated with an increase in iPLA 2 β activity, increased PAF production and PMN adherence. A redistribution of iPLA 2 isoforms in IC/BPS may represent a novel therapeutic target to manage IC/BPS. These data show that targeting of urothelial iPLA 2 β or PAFR antagonism could be beneficial therapeutic targets for the management of bladder inflammation.