Expression of Protease-Activated Receptor-1, -2, -3, and -4 in Control and Experimentally Inflamed Mouse Bladder
2003; Elsevier BV; Volume: 162; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63886-2
ISSN1525-2191
AutoresMichael R. D’Andrea, Marcia R. Saban, Ngoc-Bich Nguyen, Patricia Andrade‐Gordon, Ricardo Saban,
Tópico(s)Mast cells and histamine
ResumoInflammation underlines all major bladder pathologies and represents a defense reaction to injury involving a mandatory participation of mast cells and sensory nerves. Mast cells are particularly frequent in close proximity to epithelial surfaces where they are strategically located in the bladder and release their mediators in response to inflammation. Tryptase is specifically produced by mast cells and modulates inflammation by activating protease-activated receptors (PARs). We recently found that PAR-4 mRNA is up-regulated in experimental bladder inflammation regardless of the initiating stimulus. Because it has been reported that PAR-1, PAR-2, and PAR-3 may also be involved in the processes of inflammation, we used immunohistochemistry to characterize the expression of all known PARs in normal, acute, and chronic inflamed mouse bladder. We found that all four PARs are present in the control mouse bladder, and follow a unique distribution. All four PARs are co-expressed in the urothelium, whereas PAR-1 and PAR-2 are predominant in the detrusor muscle, and PAR-4 is expressed in peripheral nerves and plexus cell bodies. The strong expression of PARs in the detrusor muscle indicates the need for studies on the role of these receptors in motility whereas the presence of PAR-4 in nerves may indicate its participation in neurogenic inflammation. In addition, PARs are differentially modulated during inflammation. PAR-1 and PAR-2 are down-regulated in acute inflammation whereas PAR-3 and PAR-4 are up-regulated. Bladder fibroblasts were found to present a clear demarcation in PAR expression secondary to acute and chronic inflammation. Our findings provide evidence of participation of PARs in the urinary system, provide a working model for mast cell tryptase signaling in the mouse bladder, and evoke testable hypotheses regarding the roles of PARs in bladder inflammation. It is timely to understand the role of tryptase signaling and PARs in the context of bladder biology. Inflammation underlines all major bladder pathologies and represents a defense reaction to injury involving a mandatory participation of mast cells and sensory nerves. Mast cells are particularly frequent in close proximity to epithelial surfaces where they are strategically located in the bladder and release their mediators in response to inflammation. Tryptase is specifically produced by mast cells and modulates inflammation by activating protease-activated receptors (PARs). We recently found that PAR-4 mRNA is up-regulated in experimental bladder inflammation regardless of the initiating stimulus. Because it has been reported that PAR-1, PAR-2, and PAR-3 may also be involved in the processes of inflammation, we used immunohistochemistry to characterize the expression of all known PARs in normal, acute, and chronic inflamed mouse bladder. We found that all four PARs are present in the control mouse bladder, and follow a unique distribution. All four PARs are co-expressed in the urothelium, whereas PAR-1 and PAR-2 are predominant in the detrusor muscle, and PAR-4 is expressed in peripheral nerves and plexus cell bodies. The strong expression of PARs in the detrusor muscle indicates the need for studies on the role of these receptors in motility whereas the presence of PAR-4 in nerves may indicate its participation in neurogenic inflammation. In addition, PARs are differentially modulated during inflammation. PAR-1 and PAR-2 are down-regulated in acute inflammation whereas PAR-3 and PAR-4 are up-regulated. Bladder fibroblasts were found to present a clear demarcation in PAR expression secondary to acute and chronic inflammation. Our findings provide evidence of participation of PARs in the urinary system, provide a working model for mast cell tryptase signaling in the mouse bladder, and evoke testable hypotheses regarding the roles of PARs in bladder inflammation. It is timely to understand the role of tryptase signaling and PARs in the context of bladder biology. Inflammation underlines all major bladder pathologies and represents a defense reaction to injury caused by physical damage, chemical substances, microorganisms, or other agents. In consequence to inflammation, products of mast cell degranulation such as tryptase can be found in the urine of both cancer and cystitis patients.1Okragly A Niles A Saban R Schmidt D Hoffman R Warner T Moon T Uehling D Haak-Frendscho M Elevated tryptase, NGF, NT-3, and GDNF levels in the urine of interstitial cystitis and bladder cancer patients.J Urol. 1999; 161: 434-441Abstract Full Text Full Text PDF Scopus (186) Google Scholar Recently, we presented experimental evidence suggesting the existence of a recursive molecular pathway supporting bladder inflammatory responses.2Saban M Nguyen N-B Hammond T Saban R Gene expression profiling of mouse bladder inflammatory responses to LPS, substance P, and antigen-stimulation.Am J Pathol. 2002; 160: 2095-2110Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar One such pathway involves the participation of serine proteases thrombin, trypsin, and tryptase that bladder cells secrete into the extracellular space to mediate processes such as cellular invasion, extracellular matrix degradation, angiogenesis, and tissue remodeling. Thrombin, tryptase, and trypsin responses are modulated by protease-activated receptors (PARs), a unique class of G protein-coupled receptors that use a fascinating mechanism to convert an extracellular proteolytic cleavage event into a trans-membrane signal: these receptors carry their own ligands that remain cryptic until unmasked by receptor cleavage.3Hollenberg D Compton S International Union of Pharmacology. XXVIII. Proteinase-Activated Receptors.Pharmacol Rev. 2002; 54: 203-217Crossref PubMed Scopus (410) Google Scholar, 4Vergnolle N Wallace JL Bunnett NW Hollenberg MD Protease-activated receptors in inflammation, neuronal signaling and pain.Trends Pharmacol Sci. 2001; 22: 146-152Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 5Vergnolle N Proteinase-activated receptors—novel signals for gastrointestinal pathophysiology.Aliment Pharmacol Ther. 2000; 14: 257-266Crossref PubMed Scopus (122) Google Scholar PARs may be involved in various biological responses, such as hemostasis, regulation of smooth muscle tone, and inflammation. To date, four PARs have been identified3Hollenberg D Compton S International Union of Pharmacology. XXVIII. Proteinase-Activated Receptors.Pharmacol Rev. 2002; 54: 203-217Crossref PubMed Scopus (410) Google Scholar, 6Ishihara H Connolly AJ Zeng D Kahn ML Zheng YW Timmons C Tram T Coughlin SR Protease-activated receptor 3 is a second thrombin receptor in humans.Nature. 1997; 386: 502-506Crossref PubMed Scopus (804) Google Scholar, 7Xu WF Andersen H Whitmore TE Presnell SR Yee DP Ching A Gilbert T Davie EW Foster DC Cloning and characterization of human protease-activated receptor 4.Proc Natl Acad Sci USA. 1998; 95: 6642-6646Crossref PubMed Scopus (759) Google Scholar, 8Asokananthan N Graham PT Fink J Knight DA Bakker AJ McWilliam AS Thompson PJ Stewart GA Activation of protease-activated receptor (PAR)-1, PAR-2, and PAR-4 stimulates IL-6, IL-8, and prostaglandin E2 release from human respiratory epithelial cells.J Immunol. 2002; 168: 3577-3585PubMed Google Scholar with distinct N-terminal cleavage sites and tethered ligand pharmacology.9Macfarlane S Seatter M Kanke T Hunter G Plevin R Proteinase-activated receptors.Pharmacol Rev. 2001; 53: 245-282PubMed Google Scholar Each receptor has been shown to modulate a variety of physiological processes such as cytokine and mediator release, vasodilation, platelet aggregation, cellular proliferation, and smooth muscle contraction or relaxation.3Hollenberg D Compton S International Union of Pharmacology. XXVIII. Proteinase-Activated Receptors.Pharmacol Rev. 2002; 54: 203-217Crossref PubMed Scopus (410) Google Scholar, 9Macfarlane S Seatter M Kanke T Hunter G Plevin R Proteinase-activated receptors.Pharmacol Rev. 2001; 53: 245-282PubMed Google Scholar PAR-1, PAR-3, and PAR-4 are activated by thrombin, whereas PAR-2 is activated by mast cell tryptase or trypsin.10Carr MJ Schechter NM Undem BJ Trypsin-induced, neurokinin-mediated contraction of guinea pig bronchus.Am J Respir Crit Care Med. 2000; 162: 1662-1667Crossref PubMed Scopus (38) Google Scholar In addition, PAR-1 mediates thrombin-dependent, cell-mediated renal inflammation in crescentic glomerulonephritis,11Cunningham MA Rondeau E Chen X Coughlin SR Holdsworth SR Tipping PG Protease-activated receptor 1 mediates thrombin-dependent, cell-mediated renal inflammation in crescentic glomerulonephritis.J Exp Med. 2000; 191: 455-462Crossref PubMed Scopus (187) Google Scholar and gastrointestinal inflammation.12Kawabata A Kuroda R Nagata N Kawao N Masuko T Nishikawa H Kawai K In vivo evidence that protease-activated receptors 1 and 2 modulate gastrointestinal transit in the mouse.Br J Pharmacol. 2001; 133: 1213-1218Crossref PubMed Scopus (76) Google Scholar In the urinary bladder, PAR-1-activating peptide (PAR-AP) stimulated Evans blue extravasation that is abolished by neurokinin-1 receptor antagonist.13de Garavilla L Vergnolle N Young SH Ennes H Steinhoff M Ossovskaya VS D'Andrea MR Mayer EA Wallace JL Hollenberg MD Andrade-Gordon P Bunnett NW Agonists of proteinase-activated receptor 1 induce plasma extravasation by a neurogenic mechanism.Br J Pharmacol. 2001; 133: 975-987Crossref PubMed Scopus (128) Google Scholar PAR-2 is expressed in the gastrointestinal tract, pancreas, kidney, liver, airways, prostate, ovary, and eye.14Nystedt S Emilsson K Wahlestedt C Sundelin J Molecular cloning of a potential proteinase activated receptor.Proc Natl Acad Sci USA. 1994; 91: 9208-9212Crossref PubMed Scopus (833) Google Scholar PAR-3 is the second thrombin receptor.6Ishihara H Connolly AJ Zeng D Kahn ML Zheng YW Timmons C Tram T Coughlin SR Protease-activated receptor 3 is a second thrombin receptor in humans.Nature. 1997; 386: 502-506Crossref PubMed Scopus (804) Google Scholar In humans it is expressed in the bone marrow, heart, brain, placenta, liver, pancreas, thymus, small intestine, stomach, lymph nodes, and trachea. The highest levels of PAR-4 mRNA have been detected in lung, pancreas, thyroid, testis, and small intestine. PAR-4 is relatively insensitive to thrombin and trypsin.7Xu WF Andersen H Whitmore TE Presnell SR Yee DP Ching A Gilbert T Davie EW Foster DC Cloning and characterization of human protease-activated receptor 4.Proc Natl Acad Sci USA. 1998; 95: 6642-6646Crossref PubMed Scopus (759) Google Scholar PAR-4 mRNAs were found up-regulated in response to cardiac inflammation15Hamilton JR Frauman AG Cocks TM Increased expression of protease-activated receptor-2 (PAR2) and PAR4 in human coronary artery by inflammatory stimuli unveils endothelium-dependent relaxations to PAR2 and PAR4 agonists.Circ Res. 2001; 89: 92-98Crossref PubMed Scopus (132) Google Scholar and a functionally active PAR-4 is present in smooth muscle and might contribute to thrombin-induced mitogenesis.16Bretschneider E Kaufmann R Braun M Nowak G Glusa E Schror K Evidence for functionally active protease-activated receptor-4 (PAR-4) in human vascular smooth muscle cells.Br J Pharmacol. 2001; 132: 1441-1446Crossref PubMed Scopus (52) Google Scholar In the urinary bladder, we found that PAR-4 mRNA was up-regulated in response to lipopolysaccharide (LPS), substance P (SP), or antigen (Ag) challenge.2Saban M Nguyen N-B Hammond T Saban R Gene expression profiling of mouse bladder inflammatory responses to LPS, substance P, and antigen-stimulation.Am J Pathol. 2002; 160: 2095-2110Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Another event tied to inflammation is the tissue repair process. Thrombin is known to play a role in tissue repair by enhancing neovascularization, collagen deposition, and wound healing.17Carney DH Mann R Redin WR Pernia SD Berry D Heggers JP Hayward PG Robson MC Christie J Annable C Fenton II, JW Glenn KC Enhancement of incisional wound healing and neovascularization in normal rats by thrombin and synthetic thrombin receptor-activating peptides.J Clin Invest. 1992; 89: 1469-1477Crossref PubMed Scopus (135) Google Scholar, 18Dabbagh K Laurent GJ McAnulty RJ Chambers RC Thrombin stimulates smooth muscle cell procollagen synthesis and mRNA levels via a PAR-1 mediated mechanism.Thromb Haemost. 1998; 79: 405-409Crossref PubMed Scopus (66) Google Scholar, 19Stiernberg J Redin WR Warner WS Carney DH The role of thrombin and thrombin receptor activating peptide (TRAP-508) in initiation of tissue repair.Thromb Haemost. 1993; 70: 158-162PubMed Google Scholar The stimulatory effects of thrombin on growth factor production by fibroblasts can be reproduced by PAR-APs.20Chambers RC Dabbagh K McAnulty RJ Gray AJ Blanc-Brude OP Laurent GJ Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1.Biochem J. 1998; 333: 121-127Crossref PubMed Scopus (137) Google Scholar, 21Chambers RC Leoni P Blanc-Brude OP Wembridge DE Laurent GJ Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease-activated receptor-1.J Biol Chem. 2000; 275: 35584-35591Crossref PubMed Scopus (214) Google Scholar Another current study suggested that thrombin, through the activation of PAR, participates in tissue remodeling by regulating the release of matrix metalloproteinases,22Vliagoftis H Schwingshackl A Milne CD Duszyk M Hollenberg MD Wallace JL Befus AD Moqbel R Proteinase-activated receptor-2-mediated matrix metalloproteinase-9 release from airway epithelial cells.J Allergy Clin Immunol. 2000; 106: 537-545Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar a mechanism that involves activation of both MAP kinase and nuclear factor-κB.22Vliagoftis H Schwingshackl A Milne CD Duszyk M Hollenberg MD Wallace JL Befus AD Moqbel R Proteinase-activated receptor-2-mediated matrix metalloproteinase-9 release from airway epithelial cells.J Allergy Clin Immunol. 2000; 106: 537-545Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar As described above, PARs are widely distributed in a variety of tissues such as skin, intestine, blood vessels, myenteric plexus,4Vergnolle N Wallace JL Bunnett NW Hollenberg MD Protease-activated receptors in inflammation, neuronal signaling and pain.Trends Pharmacol Sci. 2001; 22: 146-152Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar and colonic myocytes23Corvera CU Dery O McConalogue K Bohm SK Khitin LM Caughey GH Payan DG Bunnett NW Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2.J Clin Invest. 1997; 100: 1383-1393Crossref PubMed Scopus (270) Google Scholar and mast cells.24D'Andrea MR Rogahn CJ Andrade-Gordon P Localization of protease-activated receptors-1 and -2 in human mast cells: indications for an amplified mast cell degranulation cascade.Biotech Histochem. 2000; 75: 85-90Crossref PubMed Scopus (110) Google Scholar However, the expression and relative distribution of PAR-1, -2, -3, and -4 in bladder tissue has not been extensively described in their histological context. The aim of this study was to characterize the expression of all PARs in normal and inflamed tissue using immunohistochemistry (IHC) and to correlate the relative alteration in PAR expression and distribution with the degree of acute and chronic inflammation. Our findings provide evidence of the presence of all four PARs in the mouse urinary bladder and that experimental inflammation leads to alteration in PAR expression. All animal experimentation described here was performed in conformity with the Guiding Principles for Research Involving Animals and Human Beings (OUHSC Animal Care & Use Committee protocol no. 00-109). Groups of 10- to 12-week-old female C57BL/6J mice were used in these experiments. Animals were maintained in housing facilities and allowed food and water ad libitum. One group of mice in this study was sensitized with intraperitoneal injections of 1 μg of dinitrophenol-human serum albumin (DNP4-HSA) in 1 mg of alum on days 0, 7, 14, and 21. This protocol induces sustained levels of IgE antibodies up to 56 days after sensitization25Haak-Frendscho M Saban R Shields RL Jardieu PM Anti-immunoglobulin E antibody treatment blocks histamine release and tissue contraction in sensitized mice.Immunology. 1998; 94: 115-121Crossref PubMed Scopus (20) Google Scholar and its specificity resides in the fact that this response is abolished by antibodies to IgE.25Haak-Frendscho M Saban R Shields RL Jardieu PM Anti-immunoglobulin E antibody treatment blocks histamine release and tissue contraction in sensitized mice.Immunology. 1998; 94: 115-121Crossref PubMed Scopus (20) Google Scholar One week after the last sensitization, cystitis was induced (see Induction of Cystitis) by intravesical challenge with Ag DNP4-ovalbumin (DNP-OVA) to induce bladder mast cell degranulation.2Saban M Nguyen N-B Hammond T Saban R Gene expression profiling of mouse bladder inflammatory responses to LPS, substance P, and antigen-stimulation.Am J Pathol. 2002; 160: 2095-2110Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 26Saban R Gerard N Saban M Nguyen N-B DeBoer D Wershil B Mast cells mediate substance P-induced bladder inflammation through a neurokinin-1 receptor-independent mechanism.Am J Physiol. 2002; 160: F2095-F2110Google Scholar, 27Saban R Saban M Nguyen N-B Lu B Gerard C Gerard N Hammond T Neurokinin-1 (NK-1) receptor is required in antigen-induced cystitis.Am J Pathol. 2000; 156: 775-780Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar Acute cystitis was induced as we described previously.2Saban M Nguyen N-B Hammond T Saban R Gene expression profiling of mouse bladder inflammatory responses to LPS, substance P, and antigen-stimulation.Am J Pathol. 2002; 160: 2095-2110Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 26Saban R Gerard N Saban M Nguyen N-B DeBoer D Wershil B Mast cells mediate substance P-induced bladder inflammation through a neurokinin-1 receptor-independent mechanism.Am J Physiol. 2002; 160: F2095-F2110Google Scholar, 27Saban R Saban M Nguyen N-B Lu B Gerard C Gerard N Hammond T Neurokinin-1 (NK-1) receptor is required in antigen-induced cystitis.Am J Pathol. 2000; 156: 775-780Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 28Saban M Hellmich H Nguyen N-B Winston J Hammond T Saban R Time course of LPS-induced gene-expression in a mouse model of genitourinary inflammation.Physiol Genomics. 2001; 5 (116): 147PubMed Google Scholar, 29Saban M Saban R Hammond T Haak-Frendscho M Steinberg H Tengowski M Bjorling D LPS-sensory peptide communication in experimental cystitis.Am J Physiol. 2002; 282: F202-F210Google Scholar Briefly, female mice were anesthetized (40 mg/kg i.p. ketamine and 2.5 mg/kg, i.p. xylazine), then transurethrally catheterized (24 Ga, 3/4 in, Angiocath; Becton Dickinson, Sandy, UT), and the urine was drained by applying slight digital pressure to the lower abdomen. The urinary bladders were instilled with 150 μl of one of the following substances: pyrogen-free saline, SP (10 μmol/L), Escherichia coli LPS strain 055:B5 (100 μg/ml; Sigma, St. Louis, MO), or Ag DNP4-OVA (1 μg/ml) in actively sensitized mice. Substances were infused at a slow rate to avoid trauma and vesicoureteral reflux.2Saban M Nguyen N-B Hammond T Saban R Gene expression profiling of mouse bladder inflammatory responses to LPS, substance P, and antigen-stimulation.Am J Pathol. 2002; 160: 2095-2110Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar To ensure consistent contact of substances with the bladder, infusion was repeated twice within a 30-minute interval and a 1-ml Tb syringe was maintained on the catheter end-retained intravesical solution for at least for 1 hour. After that the catheter was removed and mice were allowed to void normally. For acute inflammation, 24 hours after instillation, mice were sacrificed with pentobarbital (100 mg/kg, i.p.) and bladders were removed rapidly and placed in neutral buffered formalin. Chronic cystitis was induced by LPS (100 μg/ml) instillations performed every 24 hours for 4 days. Mice were sacrificed 24 hours after the last instillation. Control mice for this group received the same volume of pyrogen-free saline at the same time points and will be denoted as saline chronic hereafter. Previous results indicated that this protocol induces chronic inflammation characterized by a predominate infiltrate of macrophages/monocytes and lymphocytes into the bladder.2Saban M Nguyen N-B Hammond T Saban R Gene expression profiling of mouse bladder inflammatory responses to LPS, substance P, and antigen-stimulation.Am J Pathol. 2002; 160: 2095-2110Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Mouse bladders were immediately placed in 10% neutral buffered formalin for a minimum of 48 hours and then placed in phosphate-buffered saline (PBS, pH 7.8) and dehydrated in graded alcohol and xylene. All bladders of the same group (n = 6) were then embedded together in paraffin as a multitissue block according to conventional methods. Five-μm sections were serially cut (8-μm apart), mounted onto SuperFrost Plus (Fisher Scientific, Pittsburgh, PA) microscopic slides, and dried overnight. Mast cells were staining according to Luna.30Luna L Histopathologic Methods and Color Atlas of Special Stains and Tissue Artifacts. American Histolabs, Inc., Publications Division, Gaithersburg1992: 311Google Scholar Briefly, paraffin tissue sections were routinely dewaxed and hydrated. After placing slides in dH2O, they were placed in 0.5% toluidine blue solution for 1 hour at room temperature. The slides were then briefly rinsed in 0.5% alcoholic hydrochloric acid until sections appeared colorless. The slides were dipped in 0.01% eosin ∼20 times and then were dehydrated and mounted for microscopic analysis. Mast cells appeared blue and were counted in all areas of the tissues. Histology slides were scanned using a Nikon digital camera (DXM1200; Nikon, Tokyo, Japan) mounted on a Nikon microscope (Eclipse E600, Nikon). Image analysis was performed using a MetaMorph Imaging System (Universal Imaging Corporation, West Chester, PA). The urinary bladder was evaluated for inflammatory cell infiltrates, mast cell numbers, and the presence of interstitial edema. A semiquantitative score using defined criteria of inflammation severity was used to evaluate edema formation as follows: 1+, mild (little or no interstitial edema); 2+, moderate interstitial edema; 3+, severe interstitial edema.2Saban M Nguyen N-B Hammond T Saban R Gene expression profiling of mouse bladder inflammatory responses to LPS, substance P, and antigen-stimulation.Am J Pathol. 2002; 160: 2095-2110Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar The protocols for routine single IHC have been described previously.31D'Andrea MR Derian CK Santulli RJ Andrade-Gordon P Differential expression of protease-activated receptors-1 and -2 in stromal fibroblasts of normal, benign, and malignant human tissues.Am J Pathol. 2001; 158: 2031-2041Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar Tissue sections on microscopic slides were dewaxed and rehydrated. Slides were microwaved in Target buffer (DAKO, Carpinteria, CA), cooled, placed in PBS (pH 7.4) and treated with 3.0% H2O2 for 10 minutes at room temperature. All incubations (30 minutes) and washes were performed at room temperature. Normal blocking serum (Vector Laboratories, Burlingame, CA) was placed on all slides for 10 minutes. After a brief rinse in PBS, sections were treated with the polyclonal antibodies that have been previously characterized [PAR-1 (Robert Wood Johnson Pharmaceutical Research & Development (RWJPRD), Spring House, PA31D'Andrea MR Derian CK Santulli RJ Andrade-Gordon P Differential expression of protease-activated receptors-1 and -2 in stromal fibroblasts of normal, benign, and malignant human tissues.Am J Pathol. 2001; 158: 2031-2041Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar); PAR-2 (RWJPRD32D'Andrea MR Derian CK Leturcq D Baker SM Brunmark A Ling P Darrow AL Santulli RJ Brass LF Andrade-Gordon P Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues.J Histochem Cytochem. 1998; 46: 157-164Crossref PubMed Scopus (293) Google Scholar); PAR-3 (Santa Cruz Biotechnology, Santa Cruz, CA33Lourbakos A Potempa J Travis J D'Andrea MR Andrade-Gordon P Santulli R Mackie EJ Pike RN Arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion.Infect Immun. 2001; 69: 5121-5130Crossref PubMed Scopus (201) Google Scholar); PAR-4 (Santa Cruz Biotechnology33Lourbakos A Potempa J Travis J D'Andrea MR Andrade-Gordon P Santulli R Mackie EJ Pike RN Arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion.Infect Immun. 2001; 69: 5121-5130Crossref PubMed Scopus (201) Google Scholar)]. We also used the following antibodies to further characterize the mouse bladders: smooth muscle actin (SMA, DAKO), proliferating cell nuclear protein (PCNA, DAKO), myeloperoxidase (DAKO), and macrophage marker MAC-3 (Pharmingen, San Diego, CA). In addition, we used the following antibodies to characterize nerve elements: neuronal-specific enolase (Chemicon, Temecula, CA), S100, a Schwann cell marker (Sigma, St. Louis, MO), and glial fibrillary acid protein (DAKO). Slides were then washed in PBS and treated with goat anti-rabbit biotinylated secondary antibodies (Vector Laboratories). After washing in PBS, the avidin-biotin-horseradish peroxidase complex reagent (Vector Laboratories) was added. Slides were washed and were treated with 3,3′-diaminobenzidine (Biomeda, Foster City, CA) two times for 5 minutes, rinsed in dH2O, and counterstained with hematoxylin. Negative controls included replacement of the primary antibody with the antibody diluent (Zymed Laboratories, South San Francisco, CA) or use of the nonimmune serum (Vector Laboratories). Preabsorption controls including preincubation of the primary antibody (overnight at 4°C) with its specific Ag (10-fold titer excess) have been previously described for the PAR antibodies.31D'Andrea MR Derian CK Santulli RJ Andrade-Gordon P Differential expression of protease-activated receptors-1 and -2 in stromal fibroblasts of normal, benign, and malignant human tissues.Am J Pathol. 2001; 158: 2031-2041Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 32D'Andrea MR Derian CK Leturcq D Baker SM Brunmark A Ling P Darrow AL Santulli RJ Brass LF Andrade-Gordon P Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues.J Histochem Cytochem. 1998; 46: 157-164Crossref PubMed Scopus (293) Google Scholar, 33Lourbakos A Potempa J Travis J D'Andrea MR Andrade-Gordon P Santulli R Mackie EJ Pike RN Arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion.Infect Immun. 2001; 69: 5121-5130Crossref PubMed Scopus (201) Google Scholar Urinary bladders isolated from control, acutely inflamed [LPS, SP, and Ag (in sensitized mice)], and chronic LPS-stimulated mice were scored for relative intensity of PAR-1, PAR-2, PAR-3, and PAR-4 immunolabeling in the urothelium and submucosa, detrusor smooth muscle, stromal fibroblasts, and inflammatory cells. For each tissue, the presence of PAR immunoreactivity was ranked under a ×20 objective according to the following criteria: 1) no immunoreactivity (score = 0); 2) weak, light brown immunoreactivity (score = 1); 3) moderate brown immunolabeling (score = 2); and 4) intense, dark brown immunoreactivity (score = 3). The statistical analysis of histological data and IHC was performed using Wilcoxon's rank sum test. Results are expressed as mean ± SEM. The n values reported refer to the number of animals used for each experiment. In all cases, a value of P < 0.05 was considered indicative of significant difference. We examined whether the mouse bladder elicits an inflammatory response to all three stimuli by morphological analysis of tissue edema, detection of polymorphonuclear neutrophils (PMNs) (anti-myeloperoxidase), and macrophages (anti-MAC-3) using IHC, and staining of mast cells using the Luna's toluidine blue method (Figure 1, A and B). In addition, the effect of inflammation on urothelial and stromal cell proliferation was determined by PCNA immunolabeling (Figure 1C). LPS (acute and chronic conditions)-, Ag-, and SP-challenged conditions induced slightly different degrees of acute inflammation characterized by vasodilation, edema, and intense PMN infiltration in the mucosa and submucosal layers (Figure 1A). Confirming our results obtained with LPS,2Saban M Nguyen N-B Hammond T Saban R Gene expression profiling of mouse bladder inflammatory responses to LPS, substance P, and antigen-stimulation.Am J Pathol. 2002; 160: 2095-2110Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 28Saban M Hellmich H Nguyen N-B Winston J Hammond T Saban R Time course of LPS-induced gene-expression in a mouse model of genitourinary inflammation.Physiol Genomics. 2001; 5 (116): 147PubMed Google Scholar Ag,2Saban M Nguyen N-B Hammond T Saban R Gene expression profiling of mouse bladder inflammatory responses to LPS, substance P, and antigen-stimulation.Am J Pathol. 2002; 160: 2095-2110Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 27Saban R Saban M Nguyen N-B Lu B Gerard C Gerard N Hammond T Neuroki
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