Neutrophil Adherence to Bladder Microvascular Endothelial Cells following Platelet-Activating Factor Acetylhydrolase Inhibition

Interstitial cystitis (IC) is characterized by symptoms that include urinary frequency, urgency, and bladder and pelvic pain (for review, see Warren and Keay, 2002). The two main etiologic theories for IC are a defect in the cytoprotective layer of the bladder urothelium and an increase in numbers of activated mast cells that release chemical mediators and contribute to the pain, inflammation, and tissue damage of the bladder lining (Theoharides et al., 2001; Selo-Ojeme and Onwude, 2004). In a previous study, we showed that tryptase increases human bladder microvascular endothelial cell (HBMEC) calcium-independent PLA₂ (iPLA₂) activity, resulting in the production of multiple biologically active phospholipid metabolites that mediate the propagation of chronic inflammation (Rickard et al., 2004). These results suggest that inhibition of PLA₂ activity in the bladder may provide a useful therapeutic avenue for the relief of IC.

PLA₂-catalyzed hydrolysis of membrane phospholipids results in the stoichiometric production of a free fatty acid, most notably arachidonic acid, and a lysophospholipid. These phospholipid metabolites serve as precursors for inflammatory metabolites such as prostaglandins, leukotrienes, and platelet-activating factor (PAF). PAF is a highly potent phospholipid metabolite requiring concentrations as low as 10^-12 M to exert its physiological effects (Stafforini et al., 2003). As such, the synthesis and degradation of PAF are tightly regulated within the cell. PAF is synthesized by endothelial cells in response to stimulation by a variety of agonists and may contribute to the progression of IC and other inflammatory disorders by increasing vascular permeability and the expression of endothelial cell surface adhesion molecules that facilitate transmigration of polymorphonuclear leukocytes (PMN) across the endothelial cell monolayer (Lefer and Lefer, 1996). The synthesis of PAF in endothelial cells occurs via the remodeling pathway, activated during inflammation and hypersensitivity responses, as opposed to the de novo pathway in which PAF is synthesized for physiologic functions (Stafforini et al., 2003). In thrombin-stimulated endothelial cells, we have shown that the remodeling pathway begins with the activation of iPLA₂, which catalyzes the hydrolysis of membrane plasmenylethanolamine to yield lysoplasmenylethanolamine (lysoPlsEtn) and free arachidonic acid (Kell et al., 2003). The lysophospholipid then acts as an acceptor for an sn-2 fatty acid in a CoA-independent transacylation reaction with alkyl acyl glycerophosphoryl-choline to generate lyso-PAF. Finally, lyso-PAF is acetylated at the sn-2 position in a reaction catalyzed by lyso-PAF acetyltransferase. The biological activities of PAF can be rapidly terminated by PAF-acetylhydrolases (PAF-AH), a family of unique iPLA₂ enzymes that hydrolyze the acetyl group at the sn-2 position of PAF to generate biologically inactive lyso-PAF and acetate.

The efficient degradation of PAF and the abrupt abrogation of its inflammatory actions have led to the characterization of PAF-AH as an anti-inflammatory enzyme (Tjoelker et al., 1995). In addition, evidence suggests that the inhibition of PAF-AH activity contributes to PAF-mediated proinflammatory effects. Deficiency in PAF-AH is the mechanism implicated in several pathological conditions and has been shown to be caused by a number of genetic mutations, each resulting in inactivation of PAF-AH activity (Stafforini et al., 1996; Tjoelker and Stafforini, 2000; Prescott et al., 2002). In animal studies, administration of recombinant human PAF-AH resolved many of the PAF-induced inflammatory effects (Henderson et al., 2000). These observations highlight the tight regulation of PAF bioactivities mediated by PAF-AH.

Much effort has gone into designing PLA₂ inhibitors to be used therapeutically as anti-inflammatory agents. As more information about PLA₂ isoforms has expanded, some PLA₂ inhibitors originally thought to be selective for a single PLA₂ isoform have been found subsequently to inhibit more than one isoform. For example, methyl arachidonyl fluorophosphate (MAFP) was originally designed as a selective inhibitor of cytosolic PLA₂ (cPLA₂) and has subsequently been described as a potent and irreversible inhibitor of several PLA₂ isoforms, including iPLA₂ and PAF-AH (Lio et al., 1996; Kell et al., 2003). Furthermore, PLA₂ inhibitors may inhibit other enzymes involved in membrane phospholipid hydrolysis or remodeling. For example, bromoenol lactone (BEL), a selective inhibitor of iPLA₂, has been shown to inhibit phosphatidate phosphohydrolase (Zupan et al., 1993; Balsinde and Dennis, 1996a). Therefore, the development of a PLA₂ inhibitor as a potential anti-inflammatory agent must undergo rigorous evaluation.

In the present study, we pretreated HBMEC with PLA₂ inhibitors before stimulation with tryptase and measured their effect on PAF production. These data show that MAFP increases tryptase-stimulated PAF production because of its inhibition of PAF-AH and that this is associated with increased cell surface expression of P-selectin. Both of these responses can contribute to increased adherence of PMN to the endothelial cell monolayer.

Materials and Methods

Materials. Arachidonyl trifluoromethyl ketone (AACOCF₃, an inhibitor of cPLA₂ and iPLA₂), BEL (a selective inhibitor of iPLA₂), and MAFP were purchased from Cayman Chemical (Ann Arbor, MI). Recombinant human skin β-tryptase was purchased from Promega (Madison, WI). Richard Berney (Richard Berney Associates, LLC, Bethesda, MD) donated PX-18. [³H]acetic acid, [¹⁴C]acetic anhydride, and [acetyl-³H]PAF were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Cell Culture. HBMEC were grown in EGM-2MV medium (Cambrex Bio Science Baltimore, Inc., Baltimore, MD) and maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂. We extensively characterized these cells in a previous publication (Rickard et al., 2004).

PAF Production. HBMEC grown to confluence were incubated with Hanks' balanced salt solution (135 mM NaCl, 0.8 mM MgSO₄, 10 mM HEPES, pH 7.4, 1.2 mM CaCl₂, 5.4 mM KCl, 0.4 mM KH₂PO₄, 0.3 mM Na₂HPO₄, and 6.6 mM glucose) containing 10 μCi of [³H]acetic acid for 20 min at room temperature. Following incubation with 5 μM MAFP, BEL, or PX-18 for 10 min, cells were incubated with 20 ng/ml of tryptase or 5 μM lysoPlsEtn for an additional 10 min. Each incubation was terminated by the addition of methanol, and the cells were removed from the tissue culture well. Total lipids were extracted from the cells according to the method of Bligh and Dyer (1959). The chloroform layer was concentrated by evaporation under N₂. Isolated lipids were resuspended in a 9:1 v/v mixture of chloroform and methanol, applied to a silica gel 60 thinlayer chromatography plate, and developed in a 50:25:8:4 v/v mixture of chloroform, methanol, glacial acetic acid, and water. The region corresponding to PAF was scraped off the plate, and incorporated radioactivity was measured using a liquid scintillation counter. [¹⁴C]PAF was used as an internal standard to correct for the loss of PAF during the experimental procedure as described previously (McHowat et al., 2001).

PAF-AH Activity. PAF-AH activity was measured as described previously (McHowat et al., 2001). Briefly, HBMEC grown to confluence were incubated with the appropriate PLA₂ inhibitors for the required time, then removed from the tissue culture plate in 1.2 mM Ca²⁺ HEPES buffer, and sonicated on ice. Cellular protein (25 μg) was incubated with 0.1 mM [acetyl-³H]PAF (10 mCi/mmol) for 30 min at 37°C. The reaction was stopped by the addition of acetic acid and sodium acetate. Released [³H]acetic acid was isolated by passing the reaction mixture through a C18 silica gel column (J. T. Baker, Phillipsburg, NJ), and eluted radioactivity was measured using a liquid scintillation counter.

P-Selectin Surface Expression Assay. HBMEC were grown to confluence in 16-mm culture dishes. Cultures, including controls and those pretreated for 10 min with MAFP (5 μM), BEL (2 μM), or PX-18 (2 μM), were incubated with or without tryptase (20 ng/ml) for an additional 10 min. The medium was quickly removed and was replaced with a solution containing 127 mM NaCl, 5 mM KCl, 1.1 mM NaH₂PO₄, 0.4 mM KH₂PO₄, 2 mM MgCl₂, 5.5 mM glucose, 20 mM PIPES, and 1% paraformaldehyde, pH 6.5. Fixation was performed at 4°C overnight. After washing three times with phosphate-buffered saline, the cells were incubated in Tris-buffered saline containing 0.1% v/v Tween, 0.8% w/v bovine serum albumin, and 0.5% w/v fish gelatin for 1 h at 24°C. The cells were incubated with anti-P-selectin goat polyclonal antibody (1:50 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by incubation with horseradish peroxidase-conjugated rabbit anti-goat secondary antibody. After washing, each culture dish was incubated in the dark with 3,3′,5,5′-tetramethylbenzidine liquid substrate for 1 h. Reactions were terminated by the addition of 8 N sulfuric acid. Color development was measured with a microtiter plate spectrophotometer at 450 nm.

PMN Isolation and Adherence Assay. Human PMN were isolated from the peripheral blood of one healthy adult per experiment following dextran sedimentation on Polymorphprep solution (Axis-Shield, Oslo, Norway). After centrifuging at 400g for 30 min, the layer of PMN was removed, and contaminating red blood cells were lysed using 0.2% NaCl. Cell viability was determined to be >90% based on exclusion of 0.2% trypan blue. PMN were washed with Hanks' buffer and resuspended in Dulbecco's minimal essential medium supplemented with 20% bovine serum. Confluent HBMEC monolayers in 34-mm culture dishes were washed twice with Dulbecco's minimal essential medium. PMN (2 × 10⁶) were added to each dish, and control or PLA₂ inhibitor-treated cells were stimulated with ± tryptase (20 ng/ml) for 10 min.

At the end of stimulation, nonadherent PMNs were removed by washing the well twice with Hanks' buffer. After 0.2% Triton X-100 was added to each well to lyse the HBMEC and adherent PMN, the lysate was removed and sonicated on ice for 6 s. The myeloperoxidase (MPO) content of the adherent PMN was determined by adding 400 μl of cell lysate to a tube containing 1 ml of PBS, 1.2 ml of Hanks' buffer with bovine serum albumin, 200 μl of 0.125% 3,3′-dimethoxy-benzidine, and 200 μl of 0.05% H₂O₂. After samples were incubated at 37°C for 15 min, the reaction was stopped by the addition of 200 μl of NaN₃, and the absorbance of each tube was measured at 460 nm. MPO content in 2 × 10⁶ PMN was determined and used as the value for 100% adherence.

Microscopic Examination of Endothelial Cell-PMN Interaction. HBMEC were grown to confluence on transwell inserts and processed using standard procedures as described in detail previously (Ryerse, 1989; Farberman et al., 2005). Briefly, the cells were fixed with glutaraldehyde, postfixed with osmium tetroxide, dehydrated through graded ethanols, and embedded in PolyBed (Polysciences, Warrington, PA) resin for transmission electron microscopy or critical point dried and coated with gold-palladium in a sputter coater for scanning electron microscopy (SEM). Thick (0.25 μm) plastic sections were stained with toluidine blue for light microscopy, and thin (50-60 nm) sections were stained with uranyl acetate and lead citrate for transmission electron microscopy. Light micrographs were taken with an Olympus (Tokyo, Japan) digital camera; thin sections were viewed and photographed on a JEOL (Tokyo, Japan) 100CX EM; and the SEM samples were viewed and photographed with a JEOL 5800 SEM.

Results

To investigate the role of PLA₂ isoforms in the production of PAF in response to short intervals of tryptase stimulation, we pretreated HBMEC with MAFP (an inhibitor of cytosolic PLA₂ isoforms, 5 μM, 10 min) (Huang et al., 1994; Lio et al., 1996), PX-18 [a selective secretory phospholipase A₂ (sPLA₂) inhibitor, 5 μM, 10 min] (Franson et al., 1991; Franson and Rosenthal, 1997), or BEL (a selective iPLA₂ inhibitor, 5 μM, 10 min) (Zupan et al., 1993) before tryptase stimulation (20 ng/ml, 10 min). Pretreatment with BEL resulted in no change in PAF production in unstimulated HBMEC but completely inhibited tryptase-stimulated PAF production as shown previously (Fig. 1) (Rickard et al., 2004). Pretreatment with PX-18 partially inhibited tryptase-stimulated PAF production, whereas pretreatment with MAFP significantly potentiated PAF production in both unstimulated and tryptase-stimulated cells (Fig. 1).

To determine whether MAFP inhibits PAF-AH in HBMEC, we measured PAF-AH activity in MAFP-treated cells. We found that pretreatment with MAFP resulted in a concentration-dependent inhibition of PAF-AH activity having an IC₅₀ of 0.3 μM (Fig. 2). This is similar to the concentrations of MAFP that inhibit cPLA₂ isoforms (Balsinde and Dennis, 1996b). At higher concentrations of MAFP (10 μM), PAF-AH activity is almost completely abrogated (Fig. 2). The results with MAFP significantly contrast those obtained with pretreatment of HBMEC with BEL, PX-18, or the reversible cPLA₂ and iPLA₂ inhibitor, AACOCF₃ (Ackermann et al., 1995), which did not have a significant effect on PAF-AH activity even when used at higher concentrations (10 μM; Fig. 2). We also stimulated HBMEC with up to 200 ng/ml of tryptase and observed that PAF-AH activity was not significantly affected (data not shown). These results show that MAFP potentiates the production of PAF by a mechanism that involves inhibition of PAF hydrolysis by PAF-AH.

To determine whether the remodeling pathway is involved in PAF production in HBMEC similar to that observed in previous endothelial cell experiments (McHowat et al., 2001), we pretreated cells with MAFP before tryptase or lysoPlsEtn stimulation. Incubation of HBMEC with tryptase or lysoPlsEtn resulted in similar increases in PAF production that were significant in comparison to unstimulated cells (Fig. 3, filled columns). Pretreatment with MAFP significantly increased PAF production in unstimulated, tryptase-stimulated, and lysoPlsEtn-stimulated HBMEC (Fig. 3, open columns). These results show that in HBMEC, the majority of PAF synthesized in response to tryptase stimulation is through the remodeling pathway.

Assessment of cell surface expression of P-selectin using spectrophotometric analysis showed a basal expression of P-selectin on unstimulated HBMEC (Fig. 4). Pretreatment with MAFP in unstimulated or tryptase-stimulated cells resulted in significant increases in absorbance compared with cells not pretreated with MAFP, indicating enhanced cell surface P-selectin expression. In contrast, pretreatment with BEL or PX-18 significantly inhibited the tryptase-induced cell surface expression of P-selectin (Fig. 4). To assess whether MAFP-dependent upregulation of P-selectin cell surface expression and increased PAF production resulted in enhanced PMN adherence, we pretreated HBMEC with MAFP, followed by stimulation with tryptase (20 ng/ml), and incubated with PMN. Adherence of leukocytes was quantified using the MPO assay as described under Materials and Methods. In unstimulated or tryptase-stimulated HBMEC pretreated with MAFP, there was approximately a 2-fold increase in the number of adherent PMN (Fig. 5). Pretreatment with BEL or PX-18 inhibited PMN adherence in tryptase-stimulated HBMEC. These results show that MAFP potentiates both PAF production and P-selectin cell surface expression resulting in increased leukocyte adherence to the endothelial cell monolayer.

Microscopy was used to show that neutrophils make contact with endothelial cells growing on a transwell membrane (Fig. 6). A confluent layer of HBMEC was incubated with 1 × 10⁶ PMN and MAFP (5 μM, 10 min) before processing for microscopy as described above. Neutrophils were observed adhering to the HBMEC monolayer in toluidine blue-stained thick plastic sections (Fig. 6A) and in samples processed for SEM (Fig. 6B). The neutrophils have cell surface ruffles and lamellipodia extending along the endothelial cell surface, both indicators of activated, motile leukocytes (Farberman et al., 2005). Transmission electron microscopy clearly shows that there is close apposition and cell contact between neutrophils and endothelial cells (Fig. 6C). The neutrophils develop extensions that seem to probe endothelial cells and make cell surface contact.

Discussion

PAF production is tightly regulated by its synthesis (mediated by iPLA₂) and degradation (mediated by PAF-AH). PAF-AH inactivates PAF by catalyzing the removal of the acetyl group at the sn-2 position of its glycerol backbone (Stafforini et al., 1997). We have shown that MAFP potentiates the production of PAF (Fig. 1) by inhibiting PAF-AH activity in a concentration-dependent manner (Fig. 2). This property seems unique to MAFP because pretreatment of HBMEC with other PLA₂ inhibitors tested did not affect PAF-AH activity.

PAF production in endothelial cells proceeds primarily via the remodeling pathway in which the initial step involves iPLA₂-catalyzed hydrolysis of plasmenylethanolamine. This initial reaction produces lysoPlsEtn that acts as an acyl group acceptor in a transacylation reaction to produce lysoPAF, which is finally acetylated to produce PAF. We have previously shown that PAF production proceeds via the remodeling pathway in thrombin-stimulated human umbilical artery endothelial cells (McHowat, 2001). The increase in PAF production after incubation with lysoPlsEtn in this study shows the involvement of the remodeling pathway for PAF synthesis in HBMEC. The inhibition of PAF-AH with MAFP resulted in a significant potentiation of PAF production after both tryptase and lysoPlsEtn stimulation.

MAFP is an active site-directed, irreversible inhibitor of cPLA₂ and iPLA₂. These enzymes do not require Ca²⁺ for catalytic activity, use a central serine for catalysis, and operate via the formation of an acyl-enzyme intermediate (Lio et al., 1996; Six and Dennis, 2000). Our results showing that MAFP inhibits PAF-AH suggest that MAFP may be a relatively nonselective inhibitor of multiple serine-dependent lipases. PAF-AH, which comprises the group VII and VIII iPLA₂ enzymes, resembles many neutral lipases in that a Ser-His-Asp triad characterizes the mechanism of catalysis (Tjoelker et al., 1995). In contrast, other PLA₂ inhibitors used in this study had no effect on PAF-AH activity. BEL is an active site-directed, irreversible inhibitor of group VI iPLA₂ (Zupan et al., 1993). PX-18 is a recently developed reversible inhibitor that shows selectivity for sPLA₂ (Franson et al., 1991; Franson and Rosenthal, 1997). AACOCF_3, a trifluoromethyl ketone analog of arachidonic acid, is a reversible inhibitor of both cPLA₂ and iPLA₂ isoforms (Ackermann et al., 1995). The mechanism of inhibition by AACOCF₃ is similar to MAFP in that both compounds compete with the endogenous phospholipid molecule for the active catalytic site on the PLA₂ enzymes.

The leukocyte-endothelial interaction involves surface expression of adhesion molecules that tether PMN to the endothelial cell membrane (Zimmerman et al., 1992). On stimulation by inflammatory mediators, including histamine and thrombin, Weibel-Palade bodies containing intracellular stores of P-selectin rapidly translocate to the plasma membrane and release their contents to expose P-selectin on the endothelial cell surface (McEver et al., 1989; Geng et al., 1990). In thrombin-stimulated endothelial cells, PAF is another proadhesive molecule that is coexpressed at the cell surface with P-selectin (Lorant et al., 1991). These two molecules act cooperatively to initiate a cascade of events that results in firm PMN adhesion and eventually to leukocyte extravasation. Endothelial cell surface expression of P-selectin mediates PMN rolling along the endothelium allowing for the newly synthesized PAF to interact with its receptor on the PMN and to signal upregulation of CD11/CD18 glycoproteins on the PMN plasma membrane. The induction of intercellular adhesion molecule-1 on the endothelium together with PMN CD11/CD18 expression supports firm leukocyte adherence to the endothelium. Finally, endothelial platelet-endothelial cell adhesion molecule-1 cell surface expression mediates effective PMN transmigration to extravascular sites (Lorant et al., 1991; Lefer and Lefer, 1996). In this study, we found that MAFP can stimulate both PAF synthesis and cell surface expression of P-selectin. Furthermore, we showed that in both unstimulated and tryptase-stimulated HBMEC, pretreatment with MAFP produced significant increases in PMN adherence.

It is of interest that MAFP induced the synthesis of PAF and cell surface expression of P-selectin in HBMEC concurrently. This observation agrees with recent work suggesting that in activated endothelial cells, the signal transduction mechanisms regulating PAF and P-selectin are interrelated. Rollin et al. (2004) showed that the synthesis of PAF is essential for the cell surface expression of P-selectin when endothelial cells were stimulated with vascular endothelial growth factor. In addition, studies in platelets indicate that cell surface expression of P-selectin is induced by exposure of the cells to PAF after the activation of at least three intracellular signal transduction pathways, including protein tyrosine phosphorylation, Na⁺/H⁺ exchange, and Ca²⁺ mobilization (Zeng et al., 1999). Thus, the evidence published to date suggests that the production of PAF and the cell surface expression of P-selectin are concurrent events that are dependent on similar intracellular pathways.

These data highlight the necessity for highly specific PLA₂ inhibitors in order for them to be used therapeutically as anti-inflammatory agents. Although several causative factors have been proposed for IC, the etiology is unknown. In a recent review, possible causative considerations were infections, immunologic and neurogenic responses, and a defective glycosaminoglycan layer in the bladder (Nickel, 2004). Furthermore, IC may not be a single disease entity in all the patients based on the evidence of IC subtypes and its comorbidity with other unexplained clinical conditions (Buffington, 2004). IC is characterized by a defect in bladder cytoprotection by the urothelium and a pathophysiologic role for the mast cell (Theoharides et al., 2001). Whether this is because of the compromise in barrier function of the urothelium that allows released mast cell activators to penetrate the bladder wall and perpetuate inflammation remains to be clarified.

Because of the numerous etiologies for IC, the focus of current medical therapies is to provide relief of symptoms and to increase the overall quality of life for the patient. A potentially beneficial target for the treatment of IC and other inflammatory diseases is the use of pharmacological PLA₂ inhibitors that could be instilled into the bladder or concentrated in the urine, hence minimizing potential systemic side effects (Meyer et al., 2005). The use of these agents would inhibit the production of several membrane phospholipid-derived metabolites that play a role in inflammation, including lysoplasmalogens, PAF, and arachidonic acid and its metabolites. To date, the only designed inhibitors to have advanced into clinical trials are selective for the sPLA₂ enzymes (Tykka et al., 1985; Abraham et al., 2003; Scott et al., 2003). We showed in Fig. 1 that incubation of HBMEC with PX-18 resulted in a significant decrease in tryptase-stimulated PAF production of ∼50%, suggesting that sPLA₂ may also be involved in endothelial PAF production, although iPLA₂ seems to be the principal isoform that contributes significantly to the synthesis of PAF as evidenced by the complete inhibition of PAF production by BEL. This effect of PX-18 is independent of iPLA₂ activity because we did not measure a significant inhibition of iPLA₂ activity in the cytosolic (1.2 ± 0.1 versus 1.3 ± 0.1 nmol/mg protein/min, n = 6) or membrane fractions (6.1 ± 0.7 versus 6.7 ± 0.8 nmol/mg protein/min, n = 6) of HBMEC following PX-18 treatment (5 μM, 10 min). As more information becomes available regarding the role of PLA₂ enzymes in different organs and cells, our ability to custom design inhibitors for a single disease process is enhanced. There are relatively few data available that highlight the role of PLA₂ in the bladder in inflammatory diseases such as IC, but PLA₂ inhibitors may prove to be useful in alleviating the morbidity of this disease.

In summary, we have shown that in HBMEC, MAFP potentiates PAF production in both unstimulated and tryptase-stimulated cells by inhibiting PAF-AH activity. Further, we have shown that MAFP augments cell surface P-selectin expression and PMN adherence to the endothelial monolayer in unstimulated and tryptase-stimulated cells. However, pretreatment with BEL or PX-18 significantly inhibited PAF production, cell surface expression of P-selectin, and PMN adherence to the HBMEC monolayer in response to tryptase stimulation. These data suggest that there may be an interaction between iPLA₂ and sPLA₂ in tryptase-stimulated HBMEC that results in increased PAF production. In addition, inhibition of HBMEC iPLA₂ with BEL or sPLA₂ with PX-18 may represent a potential anti-inflammatory therapeutic avenue in IC.

PAF signaling is terminated by the rapid degradation by PAF-AH, thereby providing an immediate defense mechanism to prevent proinflammatory effects. In endothelial cells, the dysregulation of this mechanism would lead to the diminished ability to terminate the biological activities of PAF, resulting in accumulation of PAF and subsequent PMN activation. Thus, maintaining appropriate PAF-AH activity is essential to suppress the effects of PAF in active inflammation. Our data using MAFP highlight the key role of PAF-AH in the regulation of PAF-induced inflammatory responses in bladder endothelium.