- Email: [email protected]
Nicotinic Signaling Ameliorates Acute Bladder Inflammation Induced by Protamine Sulfate or Cyclophosphamide
Nicotinic Signaling Ameliorates Acute Bladder Inflammation Induced by Protamine Sulfate or Cyclophosphamide Jonathan S. Starkman,* Magaly Martinez-Ferrer,* Juan M. Iturregui, Consolate Uwamariya, Roger R. Dmochowski† and Neil A. Bhowmick‡ From the Departments of Urologic Surgery and Pathology (JMI), Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee
Purpose: Nicotinic afferent pathways may be involved in the regulation of bladder inflammation. Based on that hypothesis we investigated the role of nicotinic signaling in a comparative analysis of 2 models of experimental bladder inflammation using protamine sulfate and cyclophosphamide. Materials and Methods: Protamine sulfate and cyclophosphamide were used to induce acute bladder inflammation. Nicotinic agonists and antagonists were given concomitant to the bladder inflammatory agents. Changes in bladder inflammation were measured histologically by a pathologist and through the expression of inflammatory genes. Results: Histologically cyclophosphamide induced more inflammatory changes than protamine sulfate during acute bladder inflammation. Antagonizing nicotinic signaling with mecamylamine induced further inflammatory changes on histology when used with cyclophosphamide but not with protamine sulfate. However, antagonizing nicotinic signaling in combination with protamine sulfate induced greater increases in mRNA expression of the inflammatory cytokine interleukin-6 compared to cyclophosphamide and mecamylamine combination treatments. The activation of nicotinic signaling attenuated acute bladder inflammation by protamine sulfate and cyclophosphamide independently through the down-regulation of increased interleukin-6 expression. Conclusions: Acutely cyclophosphamide treatment results in a greater frank bladder inflammation model in mice than protamine sulfate. However, cholinergic signaling can inhibit inflammation by either mechanism of induced bladder injury. Interleukin-6 gene expression is present and it can be regulated by afferent neuronal signaling even in the absence of observed histological changes in acute bladder inflammatory models. Key Words: bladder; cystitis, interstitial; receptors, nicotinic; mice; interleukin-6
nflammation is a physiological process that characterizes many bladder diseases, such as IC. IC is a chronic and debilitating bladder disease that affects approximately 1 million people in the United States.1 Recently the cost and disease burden associated with IC and painful bladder syndrome were analyzed by the Urologic Diseases in America Project and found to exceed $750 million dollars annually.2 Although to our knowledge the exact etiology and pathophysiology is unknown, potential mechanisms include inflammation, autoimmune disease, urinary toxic factors and defective urothelial cytoprotection, resulting in urinary frequency and suprapubic pain. IC represents only a fraction of patients with inflammatory bladder disease who have acute and chronic manifestations. An exogenous mediator of bladder inflammation includes the pharmaceutical use of CPX to treat cancer as well as various autoimmune diseases. However, the toxic CPX metabolite acrolein can accumulate
I
Submitted for publication September 2, 2007. Study received approval from the Vanderbilt University Medical Center institutional animal care and use committee. Supported by Public Health Service Grant DK069527 (NAB). * Equal study contribution. † Financial interest and/or other relationship with Allergan, Novartis and Pfizer. ‡ Correspondence: Department of Urologic Surgery, A-1302 MCN, Nashville, Tennessee 37232-2765 (telephone: 615-343-7140; FAX: 615-322-5869; e-mail: [email protected]).
0022-5347/08/1796-2440/0 THE JOURNAL OF UROLOGY® Copyright © 2008 by AMERICAN UROLOGICAL ASSOCIATION
in the bladder to cause acute inflammation and chronic hemorrhagic cystitis.3 Under normal physiological conditions the bladder remains impermeable to noxious substances due to an intact surface glycosaminoglycan layer and tight junctions between urothelial cells. A defective glycosaminoglycan layer leads to increased urothelial permeability, and exposure to urinary toxins and metabolites. Instilling PS into the bladder can mimic the loss of the glycosaminoglycan layer in model systems. Recently it has become increasingly evident that the urothelium provides more than a passive diffusion barrier. It has a role in sensory function and processing, neurotransmitter release and receptor expression, and signal transduction.4 Central and peripheral nicotinic acetylcholine receptors have an important role in lower urinary tract function and control. Interestingly several studies have recently shown that functional nicotinic acetylcholine receptors are expressed in rat and human bladder epithelial cells.5 Furthermore, results indicate that nicotinic acetylcholine receptor activation in the bladder afferent nerves can induce detrusor overactivity through capsaicin sensitive C fibers.6 Up-regulation of C-fiber afferents is known to have an important role in the neuro-inflammatory model of neurogenic detrusor overactivity secondary to spinal cord injury.
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Vol. 179, 2440-2446, June 2008 Printed in U.S.A. DOI:10.1016/j.juro.2008.01.082
NICOTINIC SIGNALING AMELIORATES ACUTE BLADDER INFLAMMATION The cholinergic anti-inflammatory pathway is a physiological mechanism in which the nervous system attenuates systemic inflammatory responses.7,8 The complex interrelationship between the nervous system and inflammatory cells has become increasingly recognized as contributing to the pathogenesis of a number of chronic inflammatory conditions, including IC. Recently activation of the nicotinic pathway was reported to down-regulate the production of inflammatory cytokines in mouse pancreatitis.9 A number of methodologies of inducing bladder inflammation have been described, including CPX, lipopolysaccharide and PS.3,10,11 Based on a hypothesis that nicotinic afferent pathways may be involved in the regulation of bladder inflammation, we investigated the role of nicotinic signaling in a comparative analysis of 2 models of experimental bladder inflammation using PS and CPX, which are models that disrupt urothelial integrity through different mechanisms. MATERIALS AND METHODS Animals and Models of Bladder Inflammation Female C57/B6 mice at ages 7 to 12 weeks were maintained in a pathogen-free environment and fed a standard diet with free access to food and water. All animals underwent ovariectomy 2 weeks before PS or CPX treatments. The purpose of removing the ovaries of the mice in our study was to have similar levels of estrogen among all treatments. Each group consisted of 4 mice. Following isoflurane (Minrad, Bethlehem, Pennsylvania) mediated anesthesia PS (30 mg/ml in saline) was instilled transurethrally via catheterization with an intravesical contact/ dwell time of 45 minutes. At 24 hours after PS administration the nicotinic receptor agonist anabasine (4 mg/kg in saline) or the nicotinic receptor antagonist mecamylamine (1 mg/kg in saline) was administered intravesically for 45 minutes. Control groups were given saline or the drugs individually. Animals were sacrificed and tissues were harvested 20 hours following the last intravesical administration. CPX treated mice were administered mecamylamine (1 mg/kg in saline) or anabasine (4 mg/kg in saline) 30 minutes before the injection of CPX (200 mg/kg in saline). CPX, mecamylamine and anabasine were injected intraperitoneally. Controls were administered intraperitoneal injections of saline, mecamylamine, anabasine or CPX alone. Animals were sacrificed and bladder tissue harvested 18 hours following the last intraperitoneal injection. All chemicals were obtained from Sigma®. The Vanderbilt University Medical Center institutional animal care and use committee approved the study.
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cle were quantified in 10 high power fields at 400⫻ per sample. Individual slides were examined and photographed on an Olympus® BX 41TF microscope. RNA Isolation and RT-PCR An RNeasy® Mini Kit was used to isolate RNA from bladder tissue according to manufacturer instructions. cDNA was obtained from 1 g RNA using an iScript™ cDNA synthesis kit. Controls were performed in the absence of RT enzyme to ensure the absence of DNA in the RNA samples. Real-time quantitative PCR was performed using an iCycler™ and quantitated by iQ™ SYBR® Green detection. RT-PCR was performed at 95C for 15 seconds and at 60C for 1 minute. PCR efficiency and specificity were determined by serially diluting the template cDNA and melting curve analysis, respectively. Each mouse bladder sample was processed in duplicate and expression was normalized using GAPDH as an internal housekeeping gene. Quantitation of the results was determined by the relative standard curve method. The table lists primer sequences for GAPDH, IL-6, lipocalin 2 and cathepsin D. Immunohistochemistry For immunohistochemical analysis bladder sections were dewaxed in 2 changes of xylene and rehydrated in a series of descending concentrations of alcohol. Bladder antigens were retrieved by incubation in 10 mM citrate buffer (pH 6.0). Slides were incubated with antibody against IL-6 (15 g/ml dilution) (R & D Systems®) overnight in a humid chamber at 4C. Immunohistochemistry was detected using a Dako® Envision™⫹ System Peroxidase kit according to manufacturer instructions. Statistical Analysis Statistical analysis was performed using GraphPad® Prism® 4. ANOVA and Tukey’s multiple comparison test were used to compare means with p ⬍0.05 considered statistically significant. RESULTS Nicotinic Signaling Ameliorated CPX and PS Induced Histological Inflammatory Responses The acute effects of PS and CPX were measured with a 6-point bladder inflammation score based on the presence of PMNs, lymphocytes, tissue edema and epithelial architecture loss (see Appendix). As expected, control mice (ovariectomized mice treated with saline) had normal bladders show-
RT-PCR primer sequences
Tissue Collection and Histological Examination The bladder was transected from the urethra at the bladder neck and divided longitudinally into 3 individual rectangular sections for fixation and isolation of RNA. One bladder section was fixed in 4% paraformaldehyde and the others were frozen at ⫺80C. To determine inflammatory changes in the bladder tissue was embedded in paraffin and 5 m sections were stained with hematoxylin and eosin. Bladder inflammation was assessed using a 6-point scoring system (see Appendix) and determined by a pathologist in blinded fashion. Neutrophils present in the lamina propia and mus-
Gene GAPDH: Anti-sense Sense Lipocalin-2: Anti-sense Sense Cathepsin D: Anti-sense Sense IL-6: Anti-sense Sense
Primer Sequence 5=-AGTGGGAGTTGCTGTTGAAGTC-3= 5=-CGTGCCGCCTGGAGAAAAC-3= 5=-CGTCCTTGAGGCCCAGAGA-3= 5=-CCCCTGAACTGAAGGAACGTT-3= 5=-GACAGCTCCCCGTGGTAGTA-3= 5=-GGCATGGGCTACCCTTTTAT-3= 5=-GCATAACGCACTAGGTTTGCCGAG-3= 5=-AGCTGGAGTCACAGAAGGAGTGGC-3=
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NICOTINIC SIGNALING AMELIORATES ACUTE BLADDER INFLAMMATION
ing normal epithelium and lamina propria (fig. 1, A and B). PS caused a minimal inflammatory response with occasional scattered PMNs observed in the lamina propria (fig. 1, C and D). The addition of mecamylamine or anabasine did not significantly change the degree of inflammation vs that of PS alone, as observed during experimentation (fig. 1, E to H). In contrast, CPX administration induced a more robust histological inflammatory response with consistent infiltration of PMNs into the lamina propria (fig. 2, A and B). Furthermore, treatment with CPX and mecamylamine demonstrated consistent epithelial loss, edema and PMN infiltration into the lamina propria and muscularis propria compared to that in controls (fig. 2, C and D). Additional treatment with anabasine did not induce any histological changes compared with those of CPX alone (fig. 2, E and F). The extent of inflammation correlated with histological scores for the individual treatment conditions (fig. 3, A). Anabasine alone or mecamylamine alone did
FIG. 2. Photomicrographs demonstrate histological changes during acute bladder inflammation induced by CPX. Sections reveal bladder inflammation with predominance of PMN infiltrates induced with 200 mg/kg CPX (A and B), increased severity of bladder inflammation with epithelial loss, ulceration, PMNs, edema and infiltration of inflammatory cells into muscle when mice were treated with 200 mg/kg CPX and 1 mg/kg mecamylamine (C and D), and bladder inflammation in which addition of anabasine did not induce histological changes different from those of CPX (E and F). H & E, reduced from ⫻200 (A, C and E) and ⫻400 (B, D and F). Scale bar indicates 10.0 m (A, C and E) and 5.0 m (B, D and F).
FIG. 1. Photomicrographs show histological changes during acute bladder inflammation induced by PS. Sections reveal normal bladder histology with normal epithelium and lamina propria in control ovariectomized mice (A and B). Minimal inflammation was induced by PS (C and D). No histological changes were observed with additional treatment with mecamylamine (E and F) or anabasine (G and H). H & E, reduced from ⫻200 (A, C, E and G) and ⫻400 (B, D, F and H). Scale bar indicates 10.0 m (A, C, E and G) and 5.0 m (B, D, F and H).
not demonstrate any significant inflammatory changes on bladder histology. Histologically acute PS treatments induced minimal inflammation and there were no significant differences among them. Inflammation caused by CPX and mecamylamine was 6.6-fold higher than that caused by control, anabasine and mecamylamine treatments (p ⬍0.05). Additional treatment with mecamylamine induced more inflammation when used with CPX but not with PS. Treatment with anabasine did not induced any histological changes with CPX or PS. To further analyze the inflammation level we quantified the number of neutrophils present in control bladders, and bladders treated with PS and CPX (fig. 3, B). Inflammation caused by CPX caused an increase in the number of neutrophils present in the bladder compared with that of control and PS treatments. Treatment with CPX and the nicotinic antagonist mecamylamine induced a 2.5-fold increase in the number of neutrophils compared with that of CPX alone (p ⬍0.05). Histologically CPX induced more inflammatory changes than PS during acute bladder inflammation. Differential Regulation of Inflammatory Genes by the 2 Bladder Inflammation Mechanisms The administration of PS alone or in combination with mecamylamine or anabasine did not induce any significant
NICOTINIC SIGNALING AMELIORATES ACUTE BLADDER INFLAMMATION
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duced a significant increase compared with that in controls (p ⬍0.05 and ⬍0.01, respectively). With respect to PS, the addition of mecamylamine did not result in altered IL-6 expression compared to that of PS alone, while anabasine resulted in IL-6 expression comparable to that of controls. In the CPX groups changes in IL-6 expression were not significant. To further analyze IL-6 expression bladders treated with saline, PS, CPX, anabasine and/or mecamylamine were examined by immunohistochemistry. Control bladders showed relatively no staining for IL-6 (fig. 5, A). We observed high IL-6 expression with acute PS induced inflammation (fig. 5, B). Additional treatment with anabasine resulted in a slight decrease in IL-6 expression compared with that of PS alone (fig. 5, C). Acute CPX induced inflammation induced IL-6 expression but to a lower extent compared with that of PS treatments (fig. 5, D). DISCUSSION
FIG. 3. Mean ⫾ SE values in 4 preparations per group. Acute CPX effects evaluated with 6-point bladder inflammation histological score based on infiltration of polymorphonuclear neutrophils, lymphocytes, edema and epithelial loss (A). Asterisk indicates p ⬍0.05 vs control, anabasine (Ana) and mecamylamine (Mec). Number of neutrophils in acute bladder inflammation induced by PS and CPX (B). CPX induced inflammation showed increased number of neutrophils in bladder compared with that of control and PS treatments. Asterisk indicates p ⬍0.001 vs control and PS, p ⬍0.05 vs CPX and p ⬍0.01 vs CPX plus anabasine.
changes in lipocalin-2 expression compared with that of control (fig. 4, A). However, mecamylamine administered with CPX demonstrated significantly higher lipocalin-2 mRNA expression than that of CPX alone or any other treatments (p ⬍0.05). Anabasine produced decreased lipocalin-2 expression in the CPX groups, although the result was not significant. Cathepsin D mRNA expression was not significantly increased over that in controls in PS and CPX treated animals (fig. 4, B). However, the addition of mecamylamine to CPX resulted in significantly higher cathepsin D expression than that of all other treatments (p ⬍0.05). Anabasine slightly decreased cathepsin D expression compared with that of PS or CPX treatment, although the change was not significant. IL-6 mRNA expression was increased following the administration of PS compared with that in controls (fig. 4, C). The administration of PS and PS with mecamylamine in-
Histological analysis comparing PS with CPX revealed a marked inflammatory response associated with CPX, resulting in greater infiltration of inflammatory cells in the lamina propria. In fact, using our scoring system minimal to no histological inflammation was observed following intravesical administration of PS. This observation is consistent with the mechanism of action of PS, which has been shown to selectively injure the urothelium, increasing permeability and exposure to urinary constituents. The selective effect of PS on urothelial permeability has been validated in animal models using fluorescein.12 Furthermore, differences in the urinary excretion of fluorescein have been noted in patients with documented IC vs healthy controls, suggesting that permeability defects of the urothelium are important for further understanding the relationship between IC and neuro-inflammatory pathways. While PS results in selective cytodestruction of the urothelium with structural breakdown of uroplakins and tight junctions, CPX mediates a much more robust inflammatory response, resulting in edema, hemorrhage and frank tissue destruction. This was observed in the current study since CPX led to higher histological inflammation scores on comparative analysis. Communication between the immune and nervous systems is important for regulating the inflammatory response with inflammatory cells serving a role in neuropeptide release and the stimulation of sensory nerves, which could potentially mediate the sequelae and symptoms of diseases such as IC. Recently the cholinergic anti-inflammatory pathway was found to have a role in modulating cytokine production and inflammation.7,9,13 Furthermore, manipulating this system with nicotinic acetylcholine receptor agonist and antagonists has been found to modulate the inflammatory response and improve survival in cases of experimentally induced sepsis.14 The demonstration of functional nicotinic receptors in the bladder urothelium suggests that this may be a novel pharmacological target to modulate inflammatory bladder processes.5 In the current study the nicotinic acetylcholine receptor antagonist mecamylamine augmented the histological inflammatory response associated with CPX but not with PS. This may imply that a certain threshold or minimum level of induced inflammation is necessary to up-regulate the re-
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NICOTINIC SIGNALING AMELIORATES ACUTE BLADDER INFLAMMATION
FIG. 4. Quantitative RT-PCR of inflammation markers. Mice were treated with PS, CPX, nicotinic receptor antagonist mecamylamine (Mec) and nicotinic receptor agonist anabasine (Ana) to determine effect of nicotinic signaling during acute bladder inflammation in 2 models. CPX induced more robust increase in lipocalin-2 expression, while mecamylamine increased IL-6 expression compared with that of CPX alone and anabasine decreased IL-6 expression (A). Asterisk indicates p ⬍0.05 vs all treatments. Cathepsin D expression was not significantly higher for PS, while for CPX it was significantly higher when used with mecamylamine (B). Asterisk indicates p ⬍0.05 vs all treatments. PS and PS with mecamylamine increased IL-6 expression (C). Single asterisk indicates p ⬍0.05 vs control. Double asterisks indicate p ⬍0.01 vs control.
sponse associated with histological inflammation. Because PS does not induce an aggressive histological inflammatory response with inflammatory cell recruitment, it would be unlikely to be amenable to up-regulation or down-regulation via manipulation with anabasine or mecamylamine. However, CPX results in the recruitment of an inflammatory cell infiltrate and these immune cells would be more likely to serve as a potential target for pharmacological manipulation via the nicotinic pathway. Expression of IL-6, lipocalin-2 and cathepsin-D mRNA were quantified by RT-PCR to investigate the molecular effects resulting from modulation of the nicotinic pathway. IL-6 is a proinflammatory cytokine produced by various cell
types, including endothelial cells, macrophages, fibroblasts and mast cells. In the current study PS treatment resulted in a significant increase in IL-6 expression compared with that of controls (p ⬍0.05). Although CPX treatment resulted in a 2 to 3-fold increase in IL-6 mRNA expression, the changes were not significant based on ANOVA. This suggests that even in the absence of histological inflammation (in the case of PS) up-regulation at the molecular level was occurring. The fact that IL-6 expression in the control, and the combined anabasine and PS treatment groups was statistically similar suggests that nicotinic signaling could suppress the significant PS mediated IL-6 up-regulation. In comparison, IL-6 up-regulation by CPX was not significant.
FIG. 5. IL-6 expression in bladders treated with saline, PS, PS plus nicotinic receptor agonist anabasine and CPX. Immunohistochemical analysis of IL-6 showed IL-6 during acute inflammation induced by PS and CPX. Control bladders were negative for IL-6 (A). Cross-sections of mouse bladder treated with PS (B), PS with anabasine (C) and CPX (D) revealed IL-6. e, epithelium. lp, lamina propia. Reduced from ⫻200. Scale bar indicates 50.0 m.
NICOTINIC SIGNALING AMELIORATES ACUTE BLADDER INFLAMMATION This has specific implications for the pathophysiology of IC, in that PS treatment results in the disruption of urothelial integrity. Mast cells and their products, including IL-6, have been implicated in the pathophysiology of IC with increased levels identified in the urine of patients with IC.15 Lamale et al reported that IL-6 levels were increased in patients with IC vs controls16 and Erickson et al confirmed this observed correlation between IL-6 and IC, further establishing an association between IL-6 and symptom severity.17 Furthermore, Lotz et al confirmed that the cellular origin of increased IL-6 levels in IC is urothelial cells since increased levels of this molecular marker were present in spontaneously voided urine but not in urine obtained from urethral washings.18 To further study IL-6 expression in our models we used immunohistochemistry for IL-6 to determine its expression in the bladder. We identified IL-6 expression in cells in the lamina propia in the 2 inflammation models. However, the epithelium of bladders treated with PS had more IL-6 staining than that of bladders treated with CPX, suggesting that bladder epithelial cells treated with PS produce IL-6. Lipocalin-2 and cathepsin D were the other molecular markers studied. Lipocalin-2 has been described as a marker and potential positive modulator of acute inflammation.19 Cathepsin D is a ubiquitous enzyme involved in the recruitment and function of PMNs during acute inflammation.20 PS treatment did not have an effect on mRNA expression with respect to lipocalin-2 or cathepsin D. However, CPX and mecamylamine produced significantly increased lipocalin-2 and cathepsin D levels compared to those of CPX alone. These observed effects probably occurred because CPX induces a more consistent inflammatory infiltrate, which mediates the synthesis and secretion of these markers. The results have important implications for diseases such as IC, in which there is significant variability in the amount of observed inflammation, ranging from completely normal histology to gross inflammation with petechial hemorrhage and classic Hunner’s ulcerations. The inflammation may induce neuroplasticity with respect to neuronal density and morphology, which may explain the chronicity of pain and lower urinary tract symptoms long after the inciting inflammatory event. PS and CPX appear to be reliable models of inflammation that allow study of the role of the afferent nervous system in response to different inflammatory modulators.
APPENDIX Histology Score Based on Inflammation Score
Histological Characteristics
0
Morphologically unremarkable with no or very minimal inflammation or epithelial changes. Minimal inflammatory infiltrate composed of occasional neutrophils or lymphocytes within the lamina propria in the absence of inflammation in the muscularis propria, or significant edema, hemorrhage or urothelial changes. Minimal to mild inflammatory infiltrate within the lamina propria with scattered neutrophils or lymphocytes, accompanied by mild edema or hemorrhage, but in the absence of inflammation in the muscularis propria or significant urothelial changes. Mild or mild to moderate inflammatory infiltrate in the lamina propria and focal extension of the inflammation into the muscularis propria. Moderate inflammation with scattered to frequent neutrophils and lymphocytes in both the lamina propria and muscularis propria. Severe inflammation in the lamina propria and muscularis propria in association with other significant findings, such as urothelial ulceration, severe edema, hemorrhage and fibrin deposition.
1
2
3 4 5
Abbreviations and Acronyms CPX ⫽ cyclophosphamide GAPDH ⫽ glyceraldehyde-3-phosphate dehydrogenase IC ⫽ interstitial cystitis IL-6 ⫽ interleukin 6 PCR ⫽ polymerase chain reaction PMN ⫽ polymorphonuclear cell PS ⫽ protamine sulfate RT ⫽ reverse transcriptase
REFERENCES 1. 2.
3.
4. 5.
CONCLUSIONS 6.
CPX induced more histological inflammatory changes than PS during acute bladder inflammation. However, the otherwise chronic inflammatory cytokine IL-6 was significantly up-regulated with acute PS treatment compared to that in control bladders. Although the cholinergic anti-inflammatory pathway was more readily apparent with CPX induced bladder injury, histologically the PS bladder inflammation model highlighted the molecular effects clearly. Further studies of the nicotinic pathway require the 2 models of bladder inflammation in chronic long-term series to support the use of topical nicotinic agonists for inflammatory bladder diseases.
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7.
8. 9.
10.
Jones CA and Nyberg L: Epidemiology of interstitial cystitis. Urology 1997; 49: 2. Payne CK, Joyce GF, Wise M and Clemens JQ: Interstitial cystitis and painful bladder syndrome. J Urol 2007; 177: 2042. Gonzalez RR, Fong T, Belmar N, Saban M, Felsen D and Te A: Modulating bladder neuro-inflammation: RDP58, a novel antiinflammatory peptide, decreases inflammation and nerve growth factor production in experimental cystitis. J Urol 2005; 173: 630. de Groat WC: The urothelium in overactive bladder: passive bystander or active participant? Urology 2004; 64: 7. Bschleipfer T, Schukowski K, Weidner W, Grando SA, Schwantes U, Kummer W et al: Expression and distribution of cholinergic receptors in the human urothelium. Life Sci 2007; 80: 2303. Masuda H, Hayashi Y, Chancellor MB, Kihara K, de Groat WC, de Miguel F et al: Roles of peripheral and central nicotinic receptors in the micturition reflex in rats. J Urol 2006; 176: 374. Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S et al: Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 2003; 421: 384. Tracey KJ: The inflammatory reflex. Nature 2002; 420: 853. van Westerloo DJ, Giebelen IA, Florquin S, Bruno MJ, Larosa GJ, Ulloa L et al: The vagus nerve and nicotinic receptors modulate experimental pancreatitis severity in mice. Gastroenterology 2006; 130: 1822. Vemulakonda VM, Somogyi GT, Kiss S, Salas NA, Boone TB and Smith CP: Inhibitory effect of intravesically applied
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botulinum toxin A in chronic bladder inflammation. J Urol 2005; 173: 621. 11. Fraser MO, Chuang YC, Lavelle JP, Yoshimura N, de Groat WC and Chancellor MB: A reliable, nondestructive animal model for interstitial cystitis: intravesical low-dose protamine sulfate combined with physiological concentrations of potassium chloride. Urology 2001; 57: 112. 12. Eichel L, Scheidweiler K, Kost J, Shojaie J, Schwarz E, Messing E et al: Assessment of murine bladder permeability with fluorescein: validation with cyclophosphamide and protamine. Urology 2001; 58: 113. 13. Takahashi HK, Iwagaki H, Hamano R, Yoshino T, Tanaka N and Nishibori M: alpha7 Nicotinic acetylcholine receptor stimulation inhibits lipopolysaccharide-induced interleukin-18 and -12 production in monocytes. J Pharmacol Sci 2006; 102: 143. 14. Pavlov VA, Ochani M, Yang LH, Gallowitsch-Puerta M, Ochani K, Lin X et al: Selective alpha7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine
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endotoxemia and severe sepsis. Crit Care Med 2007; 35: 1139. Theoharides TC, Kempuraj D and Sant GR: Mast cell involvement in interstitial cystitis: a review of human and experimental evidence. Urology 2001; 57: 47. Lamale LM, Lutgendorf SK, Zimmerman MB and Kreder KJ: Interleukin-6, histamine, and methylhistamine as diagnostic markers for interstitial cystitis. Urology 2006; 68: 702. Erickson DR, Xie SX, Bhavanandan VP, Wheeler MA, Hurst RE, Demers LM et al: A comparison of multiple urine markers for interstitial cystitis. J Urol 2002; 167: 2461. Lotz M, Villiger P, Hugli T, Koziol J and Zuraw BL: Interleukin-6 and interstitial cystitis. J Urol 1994; 152: 869. Aigner F, Maier HT, Schwelberger HG, Wallnofer EA, Amberger A, Obrist P et al: Lipocalin-2 regulates the inflammatory response during ischemia and reperfusion of the transplanted heart. Am J Transplant 2007; 7: 779. Levy J, Kolski GB and Douglas SD: Cathepsin D-like activity in neutrophils and monocytes. Infect Immun 1989; 57: 1632.
Purpose: Nicotinic afferent pathways may be involved in the regulation of bladder inflammation. Based on that hypothesis we investigated the role of nicotinic signaling in a comparative analysis of 2 models of experimental bladder inflammation using protamine sulfate and cyclophosphamide. Materials and Methods: Protamine sulfate and cyclophosphamide were used to induce acute bladder inflammation. Nicotinic agonists and antagonists were given concomitant to the bladder inflammatory agents. Changes in bladder inflammation were measured histologically by a pathologist and through the expression of inflammatory genes. Results: Histologically cyclophosphamide induced more inflammatory changes than protamine sulfate during acute bladder inflammation. Antagonizing nicotinic signaling with mecamylamine induced further inflammatory changes on histology when used with cyclophosphamide but not with protamine sulfate. However, antagonizing nicotinic signaling in combination with protamine sulfate induced greater increases in mRNA expression of the inflammatory cytokine interleukin-6 compared to cyclophosphamide and mecamylamine combination treatments. The activation of nicotinic signaling attenuated acute bladder inflammation by protamine sulfate and cyclophosphamide independently through the down-regulation of increased interleukin-6 expression. Conclusions: Acutely cyclophosphamide treatment results in a greater frank bladder inflammation model in mice than protamine sulfate. However, cholinergic signaling can inhibit inflammation by either mechanism of induced bladder injury. Interleukin-6 gene expression is present and it can be regulated by afferent neuronal signaling even in the absence of observed histological changes in acute bladder inflammatory models. Key Words: bladder; cystitis, interstitial; receptors, nicotinic; mice; interleukin-6
nflammation is a physiological process that characterizes many bladder diseases, such as IC. IC is a chronic and debilitating bladder disease that affects approximately 1 million people in the United States.1 Recently the cost and disease burden associated with IC and painful bladder syndrome were analyzed by the Urologic Diseases in America Project and found to exceed $750 million dollars annually.2 Although to our knowledge the exact etiology and pathophysiology is unknown, potential mechanisms include inflammation, autoimmune disease, urinary toxic factors and defective urothelial cytoprotection, resulting in urinary frequency and suprapubic pain. IC represents only a fraction of patients with inflammatory bladder disease who have acute and chronic manifestations. An exogenous mediator of bladder inflammation includes the pharmaceutical use of CPX to treat cancer as well as various autoimmune diseases. However, the toxic CPX metabolite acrolein can accumulate
I
Submitted for publication September 2, 2007. Study received approval from the Vanderbilt University Medical Center institutional animal care and use committee. Supported by Public Health Service Grant DK069527 (NAB). * Equal study contribution. † Financial interest and/or other relationship with Allergan, Novartis and Pfizer. ‡ Correspondence: Department of Urologic Surgery, A-1302 MCN, Nashville, Tennessee 37232-2765 (telephone: 615-343-7140; FAX: 615-322-5869; e-mail: [email protected]).
0022-5347/08/1796-2440/0 THE JOURNAL OF UROLOGY® Copyright © 2008 by AMERICAN UROLOGICAL ASSOCIATION
in the bladder to cause acute inflammation and chronic hemorrhagic cystitis.3 Under normal physiological conditions the bladder remains impermeable to noxious substances due to an intact surface glycosaminoglycan layer and tight junctions between urothelial cells. A defective glycosaminoglycan layer leads to increased urothelial permeability, and exposure to urinary toxins and metabolites. Instilling PS into the bladder can mimic the loss of the glycosaminoglycan layer in model systems. Recently it has become increasingly evident that the urothelium provides more than a passive diffusion barrier. It has a role in sensory function and processing, neurotransmitter release and receptor expression, and signal transduction.4 Central and peripheral nicotinic acetylcholine receptors have an important role in lower urinary tract function and control. Interestingly several studies have recently shown that functional nicotinic acetylcholine receptors are expressed in rat and human bladder epithelial cells.5 Furthermore, results indicate that nicotinic acetylcholine receptor activation in the bladder afferent nerves can induce detrusor overactivity through capsaicin sensitive C fibers.6 Up-regulation of C-fiber afferents is known to have an important role in the neuro-inflammatory model of neurogenic detrusor overactivity secondary to spinal cord injury.
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Vol. 179, 2440-2446, June 2008 Printed in U.S.A. DOI:10.1016/j.juro.2008.01.082
NICOTINIC SIGNALING AMELIORATES ACUTE BLADDER INFLAMMATION The cholinergic anti-inflammatory pathway is a physiological mechanism in which the nervous system attenuates systemic inflammatory responses.7,8 The complex interrelationship between the nervous system and inflammatory cells has become increasingly recognized as contributing to the pathogenesis of a number of chronic inflammatory conditions, including IC. Recently activation of the nicotinic pathway was reported to down-regulate the production of inflammatory cytokines in mouse pancreatitis.9 A number of methodologies of inducing bladder inflammation have been described, including CPX, lipopolysaccharide and PS.3,10,11 Based on a hypothesis that nicotinic afferent pathways may be involved in the regulation of bladder inflammation, we investigated the role of nicotinic signaling in a comparative analysis of 2 models of experimental bladder inflammation using PS and CPX, which are models that disrupt urothelial integrity through different mechanisms. MATERIALS AND METHODS Animals and Models of Bladder Inflammation Female C57/B6 mice at ages 7 to 12 weeks were maintained in a pathogen-free environment and fed a standard diet with free access to food and water. All animals underwent ovariectomy 2 weeks before PS or CPX treatments. The purpose of removing the ovaries of the mice in our study was to have similar levels of estrogen among all treatments. Each group consisted of 4 mice. Following isoflurane (Minrad, Bethlehem, Pennsylvania) mediated anesthesia PS (30 mg/ml in saline) was instilled transurethrally via catheterization with an intravesical contact/ dwell time of 45 minutes. At 24 hours after PS administration the nicotinic receptor agonist anabasine (4 mg/kg in saline) or the nicotinic receptor antagonist mecamylamine (1 mg/kg in saline) was administered intravesically for 45 minutes. Control groups were given saline or the drugs individually. Animals were sacrificed and tissues were harvested 20 hours following the last intravesical administration. CPX treated mice were administered mecamylamine (1 mg/kg in saline) or anabasine (4 mg/kg in saline) 30 minutes before the injection of CPX (200 mg/kg in saline). CPX, mecamylamine and anabasine were injected intraperitoneally. Controls were administered intraperitoneal injections of saline, mecamylamine, anabasine or CPX alone. Animals were sacrificed and bladder tissue harvested 18 hours following the last intraperitoneal injection. All chemicals were obtained from Sigma®. The Vanderbilt University Medical Center institutional animal care and use committee approved the study.
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cle were quantified in 10 high power fields at 400⫻ per sample. Individual slides were examined and photographed on an Olympus® BX 41TF microscope. RNA Isolation and RT-PCR An RNeasy® Mini Kit was used to isolate RNA from bladder tissue according to manufacturer instructions. cDNA was obtained from 1 g RNA using an iScript™ cDNA synthesis kit. Controls were performed in the absence of RT enzyme to ensure the absence of DNA in the RNA samples. Real-time quantitative PCR was performed using an iCycler™ and quantitated by iQ™ SYBR® Green detection. RT-PCR was performed at 95C for 15 seconds and at 60C for 1 minute. PCR efficiency and specificity were determined by serially diluting the template cDNA and melting curve analysis, respectively. Each mouse bladder sample was processed in duplicate and expression was normalized using GAPDH as an internal housekeeping gene. Quantitation of the results was determined by the relative standard curve method. The table lists primer sequences for GAPDH, IL-6, lipocalin 2 and cathepsin D. Immunohistochemistry For immunohistochemical analysis bladder sections were dewaxed in 2 changes of xylene and rehydrated in a series of descending concentrations of alcohol. Bladder antigens were retrieved by incubation in 10 mM citrate buffer (pH 6.0). Slides were incubated with antibody against IL-6 (15 g/ml dilution) (R & D Systems®) overnight in a humid chamber at 4C. Immunohistochemistry was detected using a Dako® Envision™⫹ System Peroxidase kit according to manufacturer instructions. Statistical Analysis Statistical analysis was performed using GraphPad® Prism® 4. ANOVA and Tukey’s multiple comparison test were used to compare means with p ⬍0.05 considered statistically significant. RESULTS Nicotinic Signaling Ameliorated CPX and PS Induced Histological Inflammatory Responses The acute effects of PS and CPX were measured with a 6-point bladder inflammation score based on the presence of PMNs, lymphocytes, tissue edema and epithelial architecture loss (see Appendix). As expected, control mice (ovariectomized mice treated with saline) had normal bladders show-
RT-PCR primer sequences
Tissue Collection and Histological Examination The bladder was transected from the urethra at the bladder neck and divided longitudinally into 3 individual rectangular sections for fixation and isolation of RNA. One bladder section was fixed in 4% paraformaldehyde and the others were frozen at ⫺80C. To determine inflammatory changes in the bladder tissue was embedded in paraffin and 5 m sections were stained with hematoxylin and eosin. Bladder inflammation was assessed using a 6-point scoring system (see Appendix) and determined by a pathologist in blinded fashion. Neutrophils present in the lamina propia and mus-
Gene GAPDH: Anti-sense Sense Lipocalin-2: Anti-sense Sense Cathepsin D: Anti-sense Sense IL-6: Anti-sense Sense
Primer Sequence 5=-AGTGGGAGTTGCTGTTGAAGTC-3= 5=-CGTGCCGCCTGGAGAAAAC-3= 5=-CGTCCTTGAGGCCCAGAGA-3= 5=-CCCCTGAACTGAAGGAACGTT-3= 5=-GACAGCTCCCCGTGGTAGTA-3= 5=-GGCATGGGCTACCCTTTTAT-3= 5=-GCATAACGCACTAGGTTTGCCGAG-3= 5=-AGCTGGAGTCACAGAAGGAGTGGC-3=
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NICOTINIC SIGNALING AMELIORATES ACUTE BLADDER INFLAMMATION
ing normal epithelium and lamina propria (fig. 1, A and B). PS caused a minimal inflammatory response with occasional scattered PMNs observed in the lamina propria (fig. 1, C and D). The addition of mecamylamine or anabasine did not significantly change the degree of inflammation vs that of PS alone, as observed during experimentation (fig. 1, E to H). In contrast, CPX administration induced a more robust histological inflammatory response with consistent infiltration of PMNs into the lamina propria (fig. 2, A and B). Furthermore, treatment with CPX and mecamylamine demonstrated consistent epithelial loss, edema and PMN infiltration into the lamina propria and muscularis propria compared to that in controls (fig. 2, C and D). Additional treatment with anabasine did not induce any histological changes compared with those of CPX alone (fig. 2, E and F). The extent of inflammation correlated with histological scores for the individual treatment conditions (fig. 3, A). Anabasine alone or mecamylamine alone did
FIG. 2. Photomicrographs demonstrate histological changes during acute bladder inflammation induced by CPX. Sections reveal bladder inflammation with predominance of PMN infiltrates induced with 200 mg/kg CPX (A and B), increased severity of bladder inflammation with epithelial loss, ulceration, PMNs, edema and infiltration of inflammatory cells into muscle when mice were treated with 200 mg/kg CPX and 1 mg/kg mecamylamine (C and D), and bladder inflammation in which addition of anabasine did not induce histological changes different from those of CPX (E and F). H & E, reduced from ⫻200 (A, C and E) and ⫻400 (B, D and F). Scale bar indicates 10.0 m (A, C and E) and 5.0 m (B, D and F).
FIG. 1. Photomicrographs show histological changes during acute bladder inflammation induced by PS. Sections reveal normal bladder histology with normal epithelium and lamina propria in control ovariectomized mice (A and B). Minimal inflammation was induced by PS (C and D). No histological changes were observed with additional treatment with mecamylamine (E and F) or anabasine (G and H). H & E, reduced from ⫻200 (A, C, E and G) and ⫻400 (B, D, F and H). Scale bar indicates 10.0 m (A, C, E and G) and 5.0 m (B, D, F and H).
not demonstrate any significant inflammatory changes on bladder histology. Histologically acute PS treatments induced minimal inflammation and there were no significant differences among them. Inflammation caused by CPX and mecamylamine was 6.6-fold higher than that caused by control, anabasine and mecamylamine treatments (p ⬍0.05). Additional treatment with mecamylamine induced more inflammation when used with CPX but not with PS. Treatment with anabasine did not induced any histological changes with CPX or PS. To further analyze the inflammation level we quantified the number of neutrophils present in control bladders, and bladders treated with PS and CPX (fig. 3, B). Inflammation caused by CPX caused an increase in the number of neutrophils present in the bladder compared with that of control and PS treatments. Treatment with CPX and the nicotinic antagonist mecamylamine induced a 2.5-fold increase in the number of neutrophils compared with that of CPX alone (p ⬍0.05). Histologically CPX induced more inflammatory changes than PS during acute bladder inflammation. Differential Regulation of Inflammatory Genes by the 2 Bladder Inflammation Mechanisms The administration of PS alone or in combination with mecamylamine or anabasine did not induce any significant
NICOTINIC SIGNALING AMELIORATES ACUTE BLADDER INFLAMMATION
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duced a significant increase compared with that in controls (p ⬍0.05 and ⬍0.01, respectively). With respect to PS, the addition of mecamylamine did not result in altered IL-6 expression compared to that of PS alone, while anabasine resulted in IL-6 expression comparable to that of controls. In the CPX groups changes in IL-6 expression were not significant. To further analyze IL-6 expression bladders treated with saline, PS, CPX, anabasine and/or mecamylamine were examined by immunohistochemistry. Control bladders showed relatively no staining for IL-6 (fig. 5, A). We observed high IL-6 expression with acute PS induced inflammation (fig. 5, B). Additional treatment with anabasine resulted in a slight decrease in IL-6 expression compared with that of PS alone (fig. 5, C). Acute CPX induced inflammation induced IL-6 expression but to a lower extent compared with that of PS treatments (fig. 5, D). DISCUSSION
FIG. 3. Mean ⫾ SE values in 4 preparations per group. Acute CPX effects evaluated with 6-point bladder inflammation histological score based on infiltration of polymorphonuclear neutrophils, lymphocytes, edema and epithelial loss (A). Asterisk indicates p ⬍0.05 vs control, anabasine (Ana) and mecamylamine (Mec). Number of neutrophils in acute bladder inflammation induced by PS and CPX (B). CPX induced inflammation showed increased number of neutrophils in bladder compared with that of control and PS treatments. Asterisk indicates p ⬍0.001 vs control and PS, p ⬍0.05 vs CPX and p ⬍0.01 vs CPX plus anabasine.
changes in lipocalin-2 expression compared with that of control (fig. 4, A). However, mecamylamine administered with CPX demonstrated significantly higher lipocalin-2 mRNA expression than that of CPX alone or any other treatments (p ⬍0.05). Anabasine produced decreased lipocalin-2 expression in the CPX groups, although the result was not significant. Cathepsin D mRNA expression was not significantly increased over that in controls in PS and CPX treated animals (fig. 4, B). However, the addition of mecamylamine to CPX resulted in significantly higher cathepsin D expression than that of all other treatments (p ⬍0.05). Anabasine slightly decreased cathepsin D expression compared with that of PS or CPX treatment, although the change was not significant. IL-6 mRNA expression was increased following the administration of PS compared with that in controls (fig. 4, C). The administration of PS and PS with mecamylamine in-
Histological analysis comparing PS with CPX revealed a marked inflammatory response associated with CPX, resulting in greater infiltration of inflammatory cells in the lamina propria. In fact, using our scoring system minimal to no histological inflammation was observed following intravesical administration of PS. This observation is consistent with the mechanism of action of PS, which has been shown to selectively injure the urothelium, increasing permeability and exposure to urinary constituents. The selective effect of PS on urothelial permeability has been validated in animal models using fluorescein.12 Furthermore, differences in the urinary excretion of fluorescein have been noted in patients with documented IC vs healthy controls, suggesting that permeability defects of the urothelium are important for further understanding the relationship between IC and neuro-inflammatory pathways. While PS results in selective cytodestruction of the urothelium with structural breakdown of uroplakins and tight junctions, CPX mediates a much more robust inflammatory response, resulting in edema, hemorrhage and frank tissue destruction. This was observed in the current study since CPX led to higher histological inflammation scores on comparative analysis. Communication between the immune and nervous systems is important for regulating the inflammatory response with inflammatory cells serving a role in neuropeptide release and the stimulation of sensory nerves, which could potentially mediate the sequelae and symptoms of diseases such as IC. Recently the cholinergic anti-inflammatory pathway was found to have a role in modulating cytokine production and inflammation.7,9,13 Furthermore, manipulating this system with nicotinic acetylcholine receptor agonist and antagonists has been found to modulate the inflammatory response and improve survival in cases of experimentally induced sepsis.14 The demonstration of functional nicotinic receptors in the bladder urothelium suggests that this may be a novel pharmacological target to modulate inflammatory bladder processes.5 In the current study the nicotinic acetylcholine receptor antagonist mecamylamine augmented the histological inflammatory response associated with CPX but not with PS. This may imply that a certain threshold or minimum level of induced inflammation is necessary to up-regulate the re-
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FIG. 4. Quantitative RT-PCR of inflammation markers. Mice were treated with PS, CPX, nicotinic receptor antagonist mecamylamine (Mec) and nicotinic receptor agonist anabasine (Ana) to determine effect of nicotinic signaling during acute bladder inflammation in 2 models. CPX induced more robust increase in lipocalin-2 expression, while mecamylamine increased IL-6 expression compared with that of CPX alone and anabasine decreased IL-6 expression (A). Asterisk indicates p ⬍0.05 vs all treatments. Cathepsin D expression was not significantly higher for PS, while for CPX it was significantly higher when used with mecamylamine (B). Asterisk indicates p ⬍0.05 vs all treatments. PS and PS with mecamylamine increased IL-6 expression (C). Single asterisk indicates p ⬍0.05 vs control. Double asterisks indicate p ⬍0.01 vs control.
sponse associated with histological inflammation. Because PS does not induce an aggressive histological inflammatory response with inflammatory cell recruitment, it would be unlikely to be amenable to up-regulation or down-regulation via manipulation with anabasine or mecamylamine. However, CPX results in the recruitment of an inflammatory cell infiltrate and these immune cells would be more likely to serve as a potential target for pharmacological manipulation via the nicotinic pathway. Expression of IL-6, lipocalin-2 and cathepsin-D mRNA were quantified by RT-PCR to investigate the molecular effects resulting from modulation of the nicotinic pathway. IL-6 is a proinflammatory cytokine produced by various cell
types, including endothelial cells, macrophages, fibroblasts and mast cells. In the current study PS treatment resulted in a significant increase in IL-6 expression compared with that of controls (p ⬍0.05). Although CPX treatment resulted in a 2 to 3-fold increase in IL-6 mRNA expression, the changes were not significant based on ANOVA. This suggests that even in the absence of histological inflammation (in the case of PS) up-regulation at the molecular level was occurring. The fact that IL-6 expression in the control, and the combined anabasine and PS treatment groups was statistically similar suggests that nicotinic signaling could suppress the significant PS mediated IL-6 up-regulation. In comparison, IL-6 up-regulation by CPX was not significant.
FIG. 5. IL-6 expression in bladders treated with saline, PS, PS plus nicotinic receptor agonist anabasine and CPX. Immunohistochemical analysis of IL-6 showed IL-6 during acute inflammation induced by PS and CPX. Control bladders were negative for IL-6 (A). Cross-sections of mouse bladder treated with PS (B), PS with anabasine (C) and CPX (D) revealed IL-6. e, epithelium. lp, lamina propia. Reduced from ⫻200. Scale bar indicates 50.0 m.
NICOTINIC SIGNALING AMELIORATES ACUTE BLADDER INFLAMMATION This has specific implications for the pathophysiology of IC, in that PS treatment results in the disruption of urothelial integrity. Mast cells and their products, including IL-6, have been implicated in the pathophysiology of IC with increased levels identified in the urine of patients with IC.15 Lamale et al reported that IL-6 levels were increased in patients with IC vs controls16 and Erickson et al confirmed this observed correlation between IL-6 and IC, further establishing an association between IL-6 and symptom severity.17 Furthermore, Lotz et al confirmed that the cellular origin of increased IL-6 levels in IC is urothelial cells since increased levels of this molecular marker were present in spontaneously voided urine but not in urine obtained from urethral washings.18 To further study IL-6 expression in our models we used immunohistochemistry for IL-6 to determine its expression in the bladder. We identified IL-6 expression in cells in the lamina propia in the 2 inflammation models. However, the epithelium of bladders treated with PS had more IL-6 staining than that of bladders treated with CPX, suggesting that bladder epithelial cells treated with PS produce IL-6. Lipocalin-2 and cathepsin D were the other molecular markers studied. Lipocalin-2 has been described as a marker and potential positive modulator of acute inflammation.19 Cathepsin D is a ubiquitous enzyme involved in the recruitment and function of PMNs during acute inflammation.20 PS treatment did not have an effect on mRNA expression with respect to lipocalin-2 or cathepsin D. However, CPX and mecamylamine produced significantly increased lipocalin-2 and cathepsin D levels compared to those of CPX alone. These observed effects probably occurred because CPX induces a more consistent inflammatory infiltrate, which mediates the synthesis and secretion of these markers. The results have important implications for diseases such as IC, in which there is significant variability in the amount of observed inflammation, ranging from completely normal histology to gross inflammation with petechial hemorrhage and classic Hunner’s ulcerations. The inflammation may induce neuroplasticity with respect to neuronal density and morphology, which may explain the chronicity of pain and lower urinary tract symptoms long after the inciting inflammatory event. PS and CPX appear to be reliable models of inflammation that allow study of the role of the afferent nervous system in response to different inflammatory modulators.
APPENDIX Histology Score Based on Inflammation Score
Histological Characteristics
0
Morphologically unremarkable with no or very minimal inflammation or epithelial changes. Minimal inflammatory infiltrate composed of occasional neutrophils or lymphocytes within the lamina propria in the absence of inflammation in the muscularis propria, or significant edema, hemorrhage or urothelial changes. Minimal to mild inflammatory infiltrate within the lamina propria with scattered neutrophils or lymphocytes, accompanied by mild edema or hemorrhage, but in the absence of inflammation in the muscularis propria or significant urothelial changes. Mild or mild to moderate inflammatory infiltrate in the lamina propria and focal extension of the inflammation into the muscularis propria. Moderate inflammation with scattered to frequent neutrophils and lymphocytes in both the lamina propria and muscularis propria. Severe inflammation in the lamina propria and muscularis propria in association with other significant findings, such as urothelial ulceration, severe edema, hemorrhage and fibrin deposition.
1
2
3 4 5
Abbreviations and Acronyms CPX ⫽ cyclophosphamide GAPDH ⫽ glyceraldehyde-3-phosphate dehydrogenase IC ⫽ interstitial cystitis IL-6 ⫽ interleukin 6 PCR ⫽ polymerase chain reaction PMN ⫽ polymorphonuclear cell PS ⫽ protamine sulfate RT ⫽ reverse transcriptase
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4. 5.
CONCLUSIONS 6.
CPX induced more histological inflammatory changes than PS during acute bladder inflammation. However, the otherwise chronic inflammatory cytokine IL-6 was significantly up-regulated with acute PS treatment compared to that in control bladders. Although the cholinergic anti-inflammatory pathway was more readily apparent with CPX induced bladder injury, histologically the PS bladder inflammation model highlighted the molecular effects clearly. Further studies of the nicotinic pathway require the 2 models of bladder inflammation in chronic long-term series to support the use of topical nicotinic agonists for inflammatory bladder diseases.
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8. 9.
10.
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