Tryptase activation of immortalized human urothelial cell mitogen-activated protein kinase.

The pathogenesis of interstitial cystitis/painful bladder syndrome (IC/PBS) is multifactorial, but likely involves urothelial cell dysfunction and mast cell accumulation in the bladder wall. Activated mast cells in the bladder wall release several inflammatory mediators, including histamine and tryptase. We determined whether mitogen-activated protein (MAP) kinases are activated in response to tryptase stimulation of urothelial cells derived from human normal and IC/PBS bladders. Tryptase stimulation of normal urothelial cells resulted in a 2.5-fold increase in extracellular signal regulated kinase 1/2 (ERK 1/2). A 5.5-fold increase in ERK 1/2 activity was observed in urothelial cells isolated from IC/PBS bladders. No significant change in p38 MAP kinase was observed in tryptase-stimulated normal urothelial cells but a 2.5-fold increase was observed in cells isolated from IC/PBS bladders. Inhibition of ERK 1/2 with PD98059 or inhibition of p38 MAP kinase with SB203580 did not block tryptase-stimulated iPLA2 activation. Incubation with the membrane phospholipid-derived PLA2 hydrolysis product lysoplasmenylcholine increased ERK 1/2 activity, suggesting the iPLA2 activation is upstream of ERK 1/2. Real time measurements of impedance to evaluate wound healing of cell cultures indicated increased healing rates in normal and IC/PBS urothelial cells in the presence of tryptase, with inhibition of ERK 1/2 significantly decreasing the wound healing rate of IC/PBS urothelium. We conclude that activation of ERK 1/2 in response to tryptase stimulation may facilitate wound healing or cell motility in areas of inflammation in the bladder associated with IC/PBS.

Urothelial cell platelet-activating factor production mediated by calcium-independent phospholipase A2γ.

OBJECTIVES: To determine the effect of phospholipase A(2) (PLA(2)) inhibitors on urothelial cell platelet-activating factor (PAF) production in response to tryptase stimulation. METHODS: Urothelial cells isolated from normal human ureters were immortalized with the human papillomavirus type 16E6E7 cell line (TEU-2 cells). PLA(2) activity in TEU-2 cells was measured using (16:0, [(3)H]18:1) plasmenylcholine and phosphatidylcholine substrates in the presence and absence of calcium. [(3)H]PAF production was measured in TEU-2 cells prelabeled with [(3)H] acetic acid. PAF-acetylhydrolase activity was measured by determining the amount of [(3)H] acetate hydrolyzed from [(3)H]PAF incubated with TEU-2 cellular protein. Adherence of human polymorphonuclear leukocyte (PMN) to TEU-2 cells was assessed by measuring myeloperoxidase activity in adherent PMNs after incubation with TEU-2 cells. RESULTS: Most PLA(2) activity measured in TEU-2 cells was determined to be membrane-associated, calcium-independent PLA(2) and selective for plasmenylcholine substrate. Stimulation of TEU-2 cells with tryptase results in increased production of PAF and increased PMN adherence that were inhibited completely by pretreatment with the membrane-associated, calcium-independent PLA(2)γ-selective inhibitor (R)-bromoenol lactone. Pretreatment with the cytosolic PLA(2) inhibitor methyl arachidonyl fluorophosphonate resulted in potentiation of tryptase-stimulated PAF production and PMN adherence to TEU-2 cells that is a result of PAF-acetylhydrolase inhibition. CONCLUSIONS: Tryptase stimulation of TEU-2 cells results in activation of membrane-associated, calcium-independent PLA(2)γ, leading to an increase in PAF production and increased PMN adherence. Inhibition of TEU-2 cell PAF-acetylhydrolase activity with methyl arachidonyl fluorophosphonate potentiated tryptase-stimulated PAF production and PMN adherence.

Characterization of tight junction proteins in cultured human urothelial cells.

Tight junctions (TJs) are essential for normal function of epithelia, restricting paracellular diffusion and contributing to the maintenance of cell surface polarity. Superficial cells of the urothelium develop TJs, the basis for the paracellular permeability barrier of the bladder against diffusion of urinary solutes. Focusing on the superficial cell layer of stratified cell cultures of an immortalized human ureteral cell line, TEU-2 cells, we have examined the presence of TJ and TJ-associated proteins. TEU-2 cells were treated with calcium chloride and fetal bovine serum culture conditions used to induce stratification that resembles the normal transitional epithelial phenotype. Cultures were examined for TJ and TJ-associated proteins by confocal immunofluorescence microscopy and evaluated for TJ mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). TEU-2 cultures exhibited immunoreactivity at intercellular margins for claudins 1, 4, 5, 7, 14, and 16 whereas claudins 2, 8, and 12 were intracellular. RT-PCR corroborated the presence of these claudins at the mRNA level. The TJ-associated proteins occludin, JAM-1, and zonula occludens (ZO-1, ZO-2, and ZO-3) were localized at cell margins. We have found that numerous TJs and TJ-associated proteins are expressed in stratified TEU-2 cultures. Further, we propose TEU-2s provide a useful ureteral model for future studies on the involvement of TJs proteins in the normal and pathological physiology of the human urinary system.

Potential mechanism for recruitment and migration of CD133 positive cells to areas of vascular inflammation.

OBJECTIVE: Mast cells are found in large numbers in atherosclerotic plaques. The present study was conducted to determine whether tryptase stimulation of human coronary artery endothelial cells (HCAEC) would lead to an increase in transmigration of CD133 positive cells (CD133+). In vitro these cells can differentiate into mast cells under the influence of specific cytokines and growth factors. METHODS AND RESULTS: CD133+ cells were isolated from umbilical cord blood. They express mRNA for several adhesion molecules that are also utilized in neutrophil migration and can migrate across an HCAEC monolayer. Migration increased significantly when HCAEC were stimulated with tryptase and decreased when CD133+ cells were pretreated with CV3988, a platelet activating factor receptor (PTAFR) antagonist. Following long-term cell culture, these cells stained positively for the presence of tryptase, a mast cell enzyme. CONCLUSION: CD133+ cells can be utilized as a mast cell precursor population. The transendothelial migration is facilitated by the presence of tryptase and may utilize the PAF/PTAFR interaction in a manner similar to that involved in neutrophil transmigration. Following transmigration, a subset of these progenitor cells may mature into mast cells in the subendothelial space and play a role in propagation of the inflammatory process in atherosclerosis.

Loss of prostaglandin E2 release from immortalized urothelial cells obtained from interstitial cystitis patient bladders.

Interstitial cystitis (IC) is associated with increased activated mast cell numbers in the bladder and impairment of the barrier function of the urothelium. We stimulated immortalized urothelial cells derived from the inflamed region of IC bladders (SR22A or SM28 abn) or from healthy bladders (PD07i or PD08i) with tryptase and measured phospholipase A(2) (PLA(2)) activity and the resultant release of arachidonic acid and prostaglandin E(2) (PGE(2)). Tryptase stimulation of either PD07i or SR22A resulted in similar increases in PLA(2) activity and arachidonic acid release. However, tryptase stimulation of SR22A and SM28 abn did not result in a significant increase in PGE(2) release compared with the increase in PGE(2) release from tryptase-stimulated PD07i and PD08i cells. Expression of mRNA for cyclooxygenase-2 and PGE synthase was lower and mRNA for 15-hydroxyprostaglandin dehydrogenase was higher in SR22A compared with PD07i, suggesting that both decreased synthesis and increased metabolism are responsible for the lack of a PGE(2) response in tryptase-stimulated SR22A cells. Since PGE(2) is a cytoprotective eicosanoid, the failure to produce this metabolite in cells isolated from the IC bladder may represent an increased susceptibility to damage by proinfammatory stimuli.

Prostacyclin production in tryptase and thrombin stimulated human bladder endothelial cells: effect of pretreatment with phospholipase A2 and cyclooxygenase inhibitors.

PURPOSE: Thrombin and tryptase stimulation of human bladder microvascular endothelial cells (Cambrex Bioscience, Walkersville, Maryland) results in the production of multiple membrane phospholipid derived inflammatory mediators via the activation of a calcium independent phospholipase A2 that may have important implications in bladder inflammatory conditions, such as interstitial cystitis. We examined the effect of multiple phospholipase A2 and cyclooxygenase inhibitors on the immediate release of prostacyclin from human bladder microvascular endothelial cells. MATERIALS AND METHODS: We stimulated confluent human bladder microvascular endothelial cell monolayers with thrombin or tryptase and measured the immediate release of prostacyclin. Human bladder microvascular endothelial cells were pretreated with several selective phospholipase A2 and cyclooxygenase inhibitors before thrombin or tryptase stimulation to determine which combination of phospholipase A2/cyclooxygenase isoforms was involved in this process. Phospholipase A2 activity was measured using (16:0, [3H]18:1) plasmenylcholine substrate in the absence of calcium. [3H] arachidonic acid release was measured in the surrounding medium from prelabeled human bladder microvascular endothelial cell monolayers. Prostacyclin release into the surrounding medium was measured using a commercially available immunoassay kit. RESULTS: The immediate increase in prostacyclin release from thrombin or tryptase stimulated human bladder microvascular endothelial cells depended on the activation of membrane associated calcium independent phospholipase A2, resulting in an increase in arachidonic acid production. Constitutively active cyclooxygenase-1 was then responsible for further metabolism of free arachidonic acid to prostacyclin. CONCLUSIONS: These results show that the search for a suitable anti-inflammatory agent that selectively target specific phospholipase A2 isoforms requires rigorous testing in several cell types in response to various stimuli.

Neutrophil adherence to bladder microvascular endothelial cells following platelet-activating factor acetylhydrolase inhibition.

Interstitial cystitis (IC) is an inflammatory bladder condition of unknown etiology. Tryptase released from elevated numbers of activated mast cells is a proposed mediator of the inflammatory process in IC. We have previously shown that tryptase increases human bladder microvascular endothelial cell (HBMEC) calcium-independent phospholipase A(2) (iPLA(2)) activity, resulting in the production of multiple biologically active phospholipid metabolites, including platelet-activating factor (PAF), that can mediate inflammation. Because the design of selective PLA(2) inhibitors may provide a useful therapeutic strategy to reduce the inflammatory process in IC, we tested several frequently used PLA(2) inhibitors on PAF production in tryptase-stimulated HBMEC. Among the inhibitors tested, methyl arachidonyl fluorophosphonate (MAFP) was found to be a potent inhibitor of PAF-acetylhydrolase activity. Pretreatment of HBMEC with MAFP significantly increased PAF production in both unstimulated and tryptase-stimulated cells. In addition, MAFP pretreatment of tryptase-stimulated HBMEC increased both surface expression of P-selectin and polymorphonuclear leukocyte adherence to the HBMEC monolayer. These effects suggest that MAFP has a proinflammatory effect, irrespective of its ability to inhibit PLA(2).

Protease-activated receptor stimulation activates a Ca2+-independent phospholipase A2 in bladder microvascular endothelial cells.

Increased mast cell numbers and mast cell activation represent one of the prevalent etiologic theories for interstitial cystitis, an inflammatory condition in the bladder. This study was designed primarily to determine whether increased mast cell tryptase in the bladder wall may play a role in activating bladder endothelial cell phospholipase A(2) (PLA(2)), leading to increased inflammatory phospholipid metabolite accumulation, which may propagate the inflammatory process. We stimulated human bladder microvascular endothelial cells with thrombin or tryptase and measured the activation of PLA(2) and the production of multiple membrane phospholipid-derived inflammatory mediators. Thrombin and tryptase stimulation resulted in activation of a Ca(2+)-independent PLA(2), leading to increased release of arachidonic acid and prostacyclin and increased production of platelet-activating factor. These responses were blocked completely by pretreatment of human bladder microvascular endothelial cells with the Ca(2+)-independent PLA(2)-selective inhibitor bromoenol lactone. The combination of increased prostacyclin and platelet-activating factor in the bladder circulation may result in vasodilation and increased polymorphonuclear leukocyte adherence to the endothelium and may facilitate recruitment of polymorphonuclear leukocytes to the bladder wall of patients with interstitial cystitis.

Phospholipid metabolite production in human urothelial cells after protease-activated receptor cleavage.

Our laboratory demonstrated previously that stimulation of protease-activated receptors (PARs) on the human urothelial carcinoma cell line RT4 results in activation of a calcium-independent phospholipase A(2) (iPLA(2)), leading to arachidonic acid and PGE(2) release. In this study, we have examined PAR activation in normal human urothelial cells (HUR) leading to the production of inflammatory or cytoprotective phospholipid metabolites. The presence of both PAR-1 and PAR-2 on HUR was confirmed by immunoblotting. Stimulation of PAR-1 with thrombin or PAR-2 by tryptase leads to activation of a membrane-associated iPLA(2) and the production of platelet-activating factor, arachidonic acid, and PGE(2). These responses were all blocked by pretreatment with the iPLA(2)-selective inhibitor bromoenol lactone. Thus stimulation of PAR-1 or PAR-2 on HUR leads to iPLA(2)-catalyzed phospholipid hydrolysis, resulting in the production of metabolites that may mediate inflammation or provide cytoprotection to the bladder.