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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Mol Neurosci. Author manuscript; available in PMC 2009 November 1.
Published in final edited form as:
PMCID: PMC2695563
NIHMSID: NIHMS110576

Exaggerated Expression of Inflammatory Mediators in Vasoactive Intestinal Polypeptide Knockout (VIP−/−) Mice with Cyclophosphamide (CYP)-Induced Cystitis

Abstract

VIP is an immunomodulatory neuropeptide distributed in micturition pathways. VIP−/− mice exhibit altered bladder function and neurochemical properties in micturition pathways after cyclophosphamide (CYP)-induced cystitis. Given VIP's role as an anti-inflammatory mediator, we hypothesized that VIP−/− mice would exhibit enhanced inflammatory mediator expression after cystitis. A mouse inflammatory cytokines and receptors RT2 profiler array was used to determine regulated transcripts in the urinary bladder of WT and VIP−/− mice with or without CYP-induced cystitis (150 mg/kg; i.p.; 48 hour (h). Four binary comparisons were made: wildtype (WT) control versus CYP treatment (48 h); VIP−/− control versus CYP treatment (48 h); WT control versus VIP−/− control and WT with CYP treatment (48 h) versus VIP−/− with CYP treatment (48 h). The genes presented represent (1) greater than 1.5-fold change in either direction and (2) the p value is less than 0.05 for the comparison being made. Several regulated genes were validated using enzyme-linked immunoassays. The data suggest that in VIP−/− mice with bladder inflammation, inflammatory mediators are increased above that observed in WT with CYP. This shift in balance may contribute to increased bladder dysfunction in VIP−/− mice with bladder inflammation and altered neurochemical expression in micturition pathways.

Keywords: Micturition, Cytokines, Chemokines, Inflammation

Introduction

Recent studies with a chemically (cyclophosphamide, CYP)-induced bladder inflammation model have demonstrated alterations in neurochemical (Vizzard, 2000d, 2001), electrophysiological (Yoshimura and de Groat, 1999) and organizational (Vizzard and Boyle, 1999) properties of micturition reflex elements. These changes suggest considerable reorganization of reflex connections in the spinal cord and marked changes in micturition reflexes with CYP-treatment. These changes may be mediated by chemical mediators produced in the bladder, spinal cord or dorsal root ganglia with cystitis (Vizzard, 2000b, 2000d, 2001; Malley and Vizzard, 2002; Braas et al., 2006). Possible mechanisms underlying the neural plasticity following chronic CYP-induced cystitis (Vizzard and de Groat, 1996; Jennings and Vizzard, 1999; Yoshimura and de Groat, 1999; Vizzard, 2000d, 2000a, 2001) may involve alterations in neurotrophic factors and/or neural activity arising in the urinary bladder (Vizzard, 2000b). In addition, neuroimmune activation, including the production of cytokines, occurs after injury to the central or peripheral nervous system and cytokines are also likely to play a role in the development of pain, exacerbate pathology or may contribute to repair strategies (Cominelli and Pizarro, 1996; Wong et al., 1997; Hill et al., 1999; Anderson and Rao, 2001; Mason et al., 2001; Samad et al., 2001; Winkelstein et al., 2001). The concept that target organs can influence the neurons that innervate them now is widely accepted and readily demonstrated during embryonic or postnatal development (Unsicker et al., 1992; Vantini and Skaper, 1992; Eide et al., 1993; Korsching, 1993; McMahon et al., 1994; Snider and Silos-Santiago, 1996; Lentz et al., 1999).

Neuropeptides are potential mediators or modulators of inflammation and are found in human micturition pathways (Chapple et al., 1992; Lasanen et al., 1992; Morgan et al., 1999). Changes in the expression of neuropeptides have been observed with bladder overactivity (Chapple et al., 1992; Lasanen et al., 1992) and in animal models of bladder inflammation (Vizzard, 2000d, 2001). We have recently demonstrated the contribution of vasoactive intestinal polypeptide (VIP) in afferent pathways to the urinary bladder by using wildtype and VIP−/− mice under control conditions or after induction of bladder inflammation. VIP is a member of the glucagon/secretin superfamily of hormones (Dickinson et al., 1999) and acts through two high affinity receptors, the VPAC1 and VPAC2 receptors (Harmar et al., 1998). VIP may exert excitatory or inhibitory actions in neural pathways controlling micturition and these functions may be altered with neural injury, disease or inflammation. Previous studies have demonstrated increased expression of phosphorylated cAMP response element binding protein (p-CREB) in bladder afferent cells in the lumbosacral dorsal root ganglia, increased NGF content in the urinary bladder and increased bladder hyperreflexia after CYP-induced cystitis compared to littermate, wildtype (WT) mice (Vizzard et al., 2007; Jensen et al., 2008). In addition to roles as neurotransmitter or neuromodulator in autonomic nervous system pathways, VIP exhibits considerable anti-inflammatory properties (Said, 1991; Voice et al., 2002; Szema et al., 2006) (Delgado et al., 2000). It has been shown that VIP inhibits the production of pro-inflammatory compounds (Chorny et al., 2006) whereas VIP upregulates production of anti-inflammatory cytokines (Delgado et al., 1999). We have suggested that differences in bladder hyperreflexia, pCREB expression and NGF expression in urinary bladder after bladder inflammation in VIP−/− mice may be attributed to a positive shift in the balance of proinflammatory mediators in the absence of VIP (Jensen et al., 2008).

Patients with interstitial cystitis (IC) or painful bladder syndrome (PBS), a painful, chronic urinary bladder inflammation syndrome, exhibit urinary frequency, urgency, suprapubic and pelvic pain and pain at low to moderate bladder pressure (Petrone et al., 1995). An involvement of C-fibers has been suggested (Chancellor and Yoshimura, 2004). In this study, we hypothesize that chemokines and cytokines and associated receptors expressed in the urinary bladder may contribute to the neuroplasticity of the lower urinary tract following bladder inflammation and contribute to inflammatory-induced changes including bladder hyperreflexia and changes in sensory processing. Furthermore, we hypothesize the VIP−/− mice will exhibit greater changes in proinflammatory mediators after CYP-induced cystitis. In this study, we: (1) examined a panel of inflammation-associated genes that may be regulated in urinary bladder of WT and VIP−/− with and without CYP-induced cystitis; and (2) selected several genes for validation using enzyme-lined immunoassays.

Materials and Methods

Animals

VIP−/− mice (Dr. James Waschek, University of California, LA, USA) (Colwell et al., 2003) were used in these experiments. The VIP−/− mouse model was prepared using a VIP gene disruption strategy with confirmation of targeted mutation in mice and subsequent backcrossing to the C57BL/6 strain for at least six generations (Colwell et al., 2003). Mice used were bred locally at The University of Vermont and lack of the VIP gene was confirmed by PCR genotyping of tailsnips. Mice were housed (12-hr light/dark cycle) in groups (5) in the UVM animal vivarium with water and food provided ad libitum. VIP−/− and wildtype (WT) controls from the same litter were analyzed in most cases (Jensen et al., 2008). When a WT littermate was unavailable, an age-matched control mouse from another C57BL/6 litter was used. Previous studies (Girard et al., 2006) have demonstrated that the closely related neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), does not compensate for VIP in VIP−/− mice. Thus, the VIP and PACAP systems appear distinct. All experimental protocols involving animal use were approved by the University of Vermont Institutional Animal Care and Use Committee (IACUC # 06-014). Animal care was under the supervision of the University of Vermont's Office of Animal Care Management in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and National Institutes of Health guidelines. All efforts were made to minimize the potential for animal pain, stress or distress.

Acute cyclophosphamide (CYP)-induced cystitis

To produce an acute inflammation of the bladder, the cytostatic drug, cyclophosphamide (CYP; Sigma-Aldrich, 40 mg/ml in saline), was injected (150 mg/kg; i.p.) (Qiao and Vizzard, 2002, 2004; Zvarova and Vizzard, 2006). After CYP-treatment, animal health status was observed daily. Control rats received volume-matched injections of saline (0.9%; i.p.) or no treatment and no difference among the control groups was observed. All injections were performed under isoflurane (2%) anesthesia. The CYP model of bladder inflammation produces an increase in voiding frequency with small micturition volumes and is associated with inflammatory cell infiltrates in the urinary bladder including mast cells, macrophages and neutrophils (Vizzard, 2000c; Hu et al., 2003; LaBerge et al., 2006). The animals were euthanized 48 hours (h) after treatment.

Tissue Harvest

After CYP treatment or in control situations (control, 4 h, 48 h; n = 7 – 9 for each), animals were deeply anesthetized with isoflurane (3–4%) and then euthanized via thoracotomy. The urinary bladder and was quickly removed under RNase-free conditions.

Quantitative real time polymerase chain reaction (qPCR)

The PCR Array System is a quantitative SYBR® Green-based real-time PCR for analyzing the expression of a focused panel of genes. The Mouse Inflammatory Cytokines and Receptors RT2 Profiler Array: PAMM-011 (Superarray, Gaithersburg, MD) is a 96 well plate containing RT2 qPCR Primer Assays for a set of 84 related genes, plus five housekeeping genes, and three controls. Each qPCR assay on the array is uniquely designed for use in SYBR Green real-time PCR analysis. The assay design criteria ensure that each qPCR reaction will generate single, gene-specific amplicons and prevent the co-amplification of non-specific products. The qPCR Assays used in PCR Arrays are optimized to work under standard conditions enabling a large number of genes to be assayed simultaneously.

Whole bladders from C57BL6 and VIP−/− mice with or without CYP treatment (48 h) (n = 3 for each group) were dissected under RNAse free conditions. Total RNA was extracted using the STAT-60 total RNA/mRNA isolation reagent (Tel-Test‘B’, Friendswood, TX, USA) as previously described. Quantity and quality of the total RNA for each preparation was determined using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). 1 µg of each experimental RNA sample was converted into PCR template with the RT2 First Strand Kit. The templates were then combined with RT2 SYBR Green qPCR Master Mix. Equal aliquots of this mixture (25 µl for 96-well plate) are loaded to each well of the same PCR array plate containing the pre-dispensed gene-specific primer sets, and PCR is performed on a 7900HT Sequence Detection System (Applied Biosystems, Norwalk, CT, USA). Sequence Detector 2.2 software (Applied Biosystems, Norwalk, CT, USA) was used to calculate the threshold cycle (Ct) values for all the genes on each PCR Array. Relative gene expressions were calculated by using the ΔΔCt method. The delta-delta method uses the normalized ΔCt value of each sample, calculated using a total of endogenous control genes. Examination of Ct value consistency for the housekeeping genes allowed gene selection and proper normalization method. For these analyses, three control genes that did not exhibit regulation with treatment or between mouse strains were used: hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1); heat shock protein 90kDa alpha (cytosolic), class B member 1 (Hsp90ab1); actin, beta, cytoplasmic (Actβ). An evaluation of the built-in RNA quality controls elements provides the relative levels of genomic DNA contamination and inhibitors of either the reverse transcription or the PCR itself. All plates were exempt of DNA contamination. Four binary comparisons were made: wildtype (WT) control versus CYP treatment (48 h); VIP−/− control versus CYP treatment (48 h); WT control versus VIP−/− control and WT with CYP treatment (48 h) versus VIP−/− with CYP treatment (48 h). Visual representations of the comparisons were prepared as two-dimensional volcano plots with log2 fold change as the abscissa and the p value as the ordinate. The genes presented in tables and volcano plots represent (1) greater than 1.5-fold change in either direction and (2) the p value is less than 0.05 for the comparison being made.

Preparation of Immunoassay Samples

Several genes were selected for further confirmation of regulated expression by immunoassay. Adult rats were euthanized as above, and the bladder (n = 4 – 6 for each treatment; n = 4 – 6 for control) was rapidly dissected and weighed. Individual bladders were solubilized in T-PER tissue protein extraction reagent (1 g tissue/20 ml; Pierce, Rockford, IL) with Complete (protease inhibitors cocktail tablets; Roche Diagnostics, Germany). Bladder tissue was disrupted with a Polytron homogenizer and then centrifuged (10,000 rpm for 5 min). The supernatants were used for IL-1α, IL-1β, IL-10, monocyte chemoattractant protein (MCP1/CCL2), or CXCL1 quantification. We used the Coomassie Plus protein assay reagent kit (Pierce) to determine total protein.

Enzyme-linked Immunoassays

Monoclonal antibodies against IL-1α, IL-1β, IL-10, MCP1/CCL2, or CXCL1 were adsorbed to microtiter (R&D Systems, Minneapolis, MN) plates. After addition of the sample or standard solution, the second antibody (polyclonal) was applied. Sample and standard solutions were run in duplicate. The antibody complex was detected with a horseradish peroxidase-labeled immunoglobulin. Enzyme activity was quantified by the change in optical density, using tetramethylbenzidine as substrate. The standards provided with this system generated linear standard curves from 5 to 2,000 pg/ml (R2 = 0.983–0.997, P ≤ 0.001) for IL-1α, IL-1β, IL-10 or CXCL1 and from 5 to 250 pg/ml (R2 = 0.983, P ≤ 0.001) for MCP1/CCL2. The absorbance values of standards and samples were corrected by subtraction of the background value (absorbance due to nonspecific binding). Samples were diluted to bring the absorbance values onto the linear portion of the standard curve. No samples fell below the minimum detection limits of the assay. Curve fitting of standards and evaluation IL-1α, IL-1β, IL-10, MCP1/CCL2, or CXCL1 content of samples was performed using a least squares fit. No significant cross-reactivity or interference is observed to other cytokines or chemokines with any immunoassay kit used in the present study according to the manufacturer.

Myeloperoxidase assay

Inflammation of the urinary bladder was also assessed with an assay for myeloperoxidase (MPO). Polymorphonuclear (PMN) cell infiltration is a characteristic of inflammation and MPO is a naturally occurring enzyme contained in the primary granules of the PMN cells (Vizzard, 2000a). Greater MPO activity in a tissue represents increased PMN cell infiltration in inflamed tissue. Thus an MPO assay was performed on freshly harvested urinary bladder tissue obtained from mice euthanized as described above. The MPO assay was performed as described previously (Vizzard, 2000a). Briefly, MPO was extracted from homogenized bladder tissue by suspending the material in 0.5% hexadecyltrimethylammonium bromide (Sigma-Aldrich) in 50 mM potassium phosphate buffer (pH 6.0) before sonication in an ice bath for 30 s. The specimens were freeze-thawed three times, and the sonication was repeated. Suspensions were then centrifuged (40,000 g) for 15 min, and the supernatant was assayed spectrophotometrically. Supernatant (0.1 ml) was mixed with 2.9 ml of potassium phosphate buffer containing 0.167 ml/ml of o-dianisidine dihydrochloride (Sigma-Aldrich) and 0.0005% hydrogen peroxide. The change in absorbency was measured at 460 nm. One unit of MPO activity was defined as that degrading one micromole of peroxide per minute at 25°C.

Materials

All standard chemicals were obtained from Sigma-Aldrich or Fisher and were either analytical or laboratory grade.

Statistical Analysis

All values are means ± SEM. Comparisons of MPO activity, cytokine or chemokine protein concentration in urinary bladder CYP-induced cystitis were made using analysis of variance. When F ratios exceeded the critical value (P ≤ 0.05), the Newman-Keuls post hoc test was used to compare the experimental means.

Results

Using the established model of CYP-induced cystitis, we treated wildtype (WT) and VIP−/− mice with CYP (48 hr; 150 mg/kg, i.p.) to compare inflammation-induced responses in the urinary bladder. Separate groups of WT and VIP−/− mice were also used to establish baseline expression of inflammatory mediators in the urinary bladder in the absence of inflammation. The following comparisons were made: wildtype (WT) control versus CYP treatment (48 h); VIP−/− control versus CYP treatment (48 h); WT control versus VIP−/− control and WT with CYP treatment (48 h) versus VIP−/− with CYP treatment (48 h). When the data sets were filtered for fold changes ≥ 1.5 (either up or down) and p-values of ≤ 0.05, 24 target genes were identified for the WT control versus CYP treatment (48 h) comparison (Table 1), 31 genes for the VIP−/− control versus CYP treatment (48 h) comparison (Table 2), 14 genes for the WT control versus VIP−/− control comparison (Table 3) and 6 genes for WT with CYP treatment (48 h) versus VIP−/− with CYP treatment (48 h) (Table 4) comparison. In visual two-dimensional representations of the data in volcano plots, where log2 fold change for each gene was expressed as a function of the p-value, all of the upregulated genes were noted in the upper right quadrant and all of the downregulated genes were noted in the upper left quadrant (Fig. 1A–D).

Figure 1
Volcano plots demonstrate differential regulation of mouse inflammatory cytokines and receptors gene expression in urinary bladder. Criteria boundaries are shown for each plot. Transcripts upregulated are designated by diamonds located in the upper right ...
Table 1
Effect of 48 hour (h) cyclophosphamide (CYP) treatment on whole urinary bladder from wildtype mice. Regulated genes with fold changes ≥ 1.5 (either up or down) and p-values of ≤ 0.05 are presented expressed in urinary bladder after CYP ...
Table 2
Effect of 48 hour (h) cyclophosphamide (CYP) treatment on whole urinary bladder in VIP−/− mice. Regulated genes with fold changes ≥ 1.5 (either up or down) and p-values of ≤ 0.05 expressed in urinary bladder after CYP treatment ...
Table 3
Baseline comparison of whole bladder gene expression between wildtype and VIP−/− mice. Only genes with fold changes ≥ 1.5 (either up or down) and p-values of ≤ 0.05 are presented. n = 3 for each group.
Table 4
Comparative effect of 48 hour (h) cyclophosphamide (CYP) treatment on whole bladder between wildtype and VIP−/− mice. Regulated genes with fold changes ≥ 1.5 (either up or down) and p-values of ≤ 0.05 expressed in the urinary ...

Of the genes upregulated in the urinary bladder after CYP-treatment in WT and VIP−/− mice, several were selected for validation using enzyme-linked immunoassays. In addition, a myeloperoxidase (MPO) assay was used to determine the extent of neutrophil infiltration present in the urinary bladder after CYP-induced cystitis (48 h). As expected, MPO levels significantly increased in the urinary bladder after CYP treatment (48 h) in both WT and VIP−/− mice (Fig. 2A). CYP treatment of a shorter duration (4 h) only increased MPO levels in the urinary bladder of VIP−/− mice (Fig. 2A). MPO expression in the urinary bladder of VIP−/− mice at both 4 or 48 h CYP treatment was significantly (p ≤ 0.001) greater than that observed in WT mice (Fig. 2A). No differences in MPO expression in the urinary bladder were observed between WT and VIP−/− mice in the absence of inflammation (control) (Fig. 2A). This change in MPO expression with CYP-induced cystitis and the differences observed in MPO levels between WT and VIP−/− mice is consistent with the dramatic upregulation of the chemokines, CXCL5 and CXCL1, known to be involved in neutrophil recruitment in other organs systems following inflammation (Fong et al., 1998; Haskell et al., 2000).

Figure 2
Immunoassay validation of inflammatory mediator protein expression in urinary bladder of wildtype (WT) and VIP−/− mice after 48 hour (h) cyclophosphamide (CYP) treatment. A. Myeloperoxidase (MPO) expression in urinary bladder of WT and ...

We evaluated IL-1α, IL-1β, IL-10, MCP1/CCL2, or CXCL1 expression in urinary bladder in WT and VIP−/− mice with and without CYP treatment (48 h). The expression patterns for CXCL1 and IL-1β in the urinary bladder of control and CYP-treated mice were similar (Fig. 2B, D). No differences in CXCL1 or IL-1β expression in urinary bladder of control WT or VIP−/− mice were observed (Fig. 2B, D). In contrast, CYP-treatment significantly (p ≤ 0.001) increased expression of CXCL1 and IL-1β in the urinary bladder of WT and VIP−/− (Fig. 2B, D) mice but expression in VIP−/− mice with CYP treatment was significantly (p ≤ 0.001) greater (4.2–13-fold increase) than that observed in WT urinary bladder (Fig. 2) (3.6–5-fold increase) (Fig. 2B, D). MCP1/CCL2 expression in the urinary bladder was significantly (p ≤ 0.001) increased after CYP-treatment (48 h) in both WT and VIP−/− mice; however, no differences in the magnitude of the change were observed between mouse strains (Fig. 2C). No differences in MCP1/CCL2 expression were observed in non-inflamed (control) urinary bladder of WT or VIP−/− mice (Fig. 2C). The changes in urinary bladder protein expression of CXCL1, CCL2 and IL-1β complement the increased urinary bladder transcript expression demonstrated in the superarray with CYP-treatment in WT and VIP−/− mice (Table 1, Table 2).

In contrast to robust changes in urinary bladder protein and transcript expression for CXCL1, CCL2 and IL-1β, no change in protein expression of IL-1α or IL-10 were observed in the urinary bladder with CYP treatment (48 h) in WT or VIP−/− mice. No differences in non-inflamed (control) urinary bladder expression of IL-1α or IL-10 were observed between WT or VIP−/− mice (data not shown). The absence of changes in protein expression in urinary bladder IL-1α or IL-10 in response to CYP treatment (48 h) complement the absence of effect on IL-1α and IL-10 urinary bladder transcript using the superarray. Changes in IL-1α and IL-10 transcripts in urinary bladder with CYP treatment in WT or VIP−/− mice did not meet the selection criteria described.

Discussion

The present studies demonstrate the increased expression of inflammatory gene transcripts and protein in urinary bladder of VIP−/− mice as demonstrated with a superarray and confirmed with enzyme-lined immunoassays. Previous studies have demonstrated that p-CREB expression in lumbosacral DRG was greater in VIP−/− mice under basal conditions and after bladder inflammation induced by cyclophosphamide (CYP) (Jensen et al., 2008). Urinary bladder from VIP−/− mice exhibit greater mass, increased thickness of detrusor smooth muscle and increased neurotrophic factor (e.g., nerve growth factor) content compared to WT mice (Jensen et al., 2008). With respect to urinary bladder function, VIP−/− mice exhibit more pronounced urinary bladder hyperreflexia in response to CYP-induced cystitis compared to WT mice (Vizzard et al., 2007). We have previously suggested that these differences may be due, in part, to differences in expression in urinary bladder inflammatory mediators under non-inflamed and inflamed conditions (Jensen et al., 2008). The present studies demonstrate more robust upregulation of numerous inflammatory mediator genes in VIP−/− mice treated with CYP compared to WT mice treated with CYP. Thus, these studies demonstrate a positive shift in the balance of proinflammatory mediators in urinary bladder of VIP−/− mice with bladder inflammation.

Interstitial cystitis (IC)/painful bladder syndrome (PBS) is a chronic inflammatory bladder disease syndrome characterized by urinary frequency, urgency, suprapubic and pelvic pain (Petrone et al., 1995; Driscoll and Teichman, 2001). Although the etiology and pathogenesis of IC are unknown, numerous theories including; infection, autoimmune disorder, toxic urinary agents, deficiency in bladder wall lining and neurogenic causes have been proposed (Petrone et al., 1995; Ho et al., 1997; Johansson et al., 1997; Driscoll and Teichman, 2001; Sant and Hanno, 2001). We have hypothesized that pain associated with IC involves an alteration of visceral sensation/bladder sensory physiology. Altered visceral sensations from the urinary bladder (i.e., pain at low or moderate bladder filling) that accompany IC/PBS (Petrone et al., 1995; Ho et al., 1997; Johansson et al., 1997; Driscoll and Teichman, 2001; Sant and Hanno, 2001) may be mediated by many factors including changes in the properties of peripheral bladder afferent pathways such that bladder afferent neurons respond in an exaggerated manner to normally innocuous stimuli (allodynia). These changes may be mediated, in part, by inflammatory changes in the urinary bladder. Among potential mediators of inflammation, neurotrophins (eg., nerve growth factor) (Woolf and Doubell, 1994; Dray, 1995; Baba et al., 1999) as well as pro-inflammatory cytokines have been implicated in this sensitization process. Specifically, cytokines cause sensitization of polymodal C-fibers and facilitate A-beta input to the spinal cord (Woolf and Doubell, 1994; Dray, 1995; Baba et al., 1999). The present studies suggest that cytokines produced in the urinary bladder after CYP-induced cystitis may also contribute to this sensitization process. Sources of cytokines in the CYP-induced cystitis model may include a variety of cell types (resident and/or infiltrating) in the urinary bladder: lymphocytes, macrophages, mast cells, microglial cells, urothelial cells, urinary bladder smooth muscles cells, and fibroblasts (Bartfai and Schultzberg, 1993; Gadient and Otten, 1997; Parkin and Cohen, 2001). Previous studies from this laboratory (Malley and Vizzard, 2002) have suggested that cytokines produced in the urinary bladder after CYP-induced cystitis may also contribute to this sensitization process.

Changes in synthesis and/or release of cytokines by target organs may also have an impact on the changes in lower urinary tract function as well as reorganization of reflex connections in the spinal cord after CYP-induced cystitis. Saban et al. (Saban et al., 2001a; Saban, 2001; Saban et al., 2001b) were the first to demonstrate gene-regulation during inflammatory bladder responses in the mouse (lipopolysaccharide, substance P or antigen-induced inflammation) using cDNA expression array technology. Consistent with the results of the present study using the CYP-induced cystitis model in the mouse, Saban et al. (Saban et al., 2001a; Saban, 2001; Saban et al., 2001b) have also demonstrated urinary bladder upregulation of cytokines. Interestingly, several cytokines (i.e., IL-1β, IL-6, IL-8 among others) have also been reported to be upregulated during active states of inflammatory bowel disease (Sartor, 1991, 1994; Cominelli and Pizarro, 1996; MacDermott, 1996; Debreceni et al., 2001). A significant body of literature exists to support the concept that cytokines (e.g., IL-1β) are key signals that are released in the periphery to signal the central nervous system that infection/inflammation has occurred (Lindholm et al., 1987; Dinarello, 1998; Poole et al., 1998; Poole and Woolf, 1998; Maier and Watkins, 1999). IL-1β has also been implicated in inflammatory hyperalgesia (Ferreira et al., 1988; Poole et al., 1998). Several lines of evidence further suggest that IL-1β may cause hyperalgesia by releasing cyclooxgenase products through stimulation of cyclooxygenase-2 (COX-2) (Geng et al., 1995; Samad et al., 2001) and phospholipase A2 (Burch et al., 1993) or by inducing arachidonic acid release (Dinarello, 1997), respectively. In the present study, more robust upregulation of IL-1β transcript in urinary bladder was demonstrated in VIP−/− mice with CYP-induced cystitis compared to WT. Similarly, greater IL-1β protein expression in the urinary bladder was demonstrated in VIP−/− mice treated with CYP compared to WT mice treated with CYP. Thus, inflammatory processes in visceral organs and associated changes in neurochemistry (Vizzard, 1997, 2000d, 2001), and reflex organization (Vizzard, 2000a) may involve cytokines.

The present study also demonstrates upregulation of transcripts and protein for numerous chemokines and associated receptors in urinary bladder after CYP-induced cystitis. Chemokines, chemotactic cytokines, are another class of neuromodulatory agents that may contribute to inflammatory-induced changes (Wieseler-Frank et al., 2004; Marchand et al., 2005). Chemokine expression has been demonstrated at sites of inflammation (Garcia et al., 2000; Raychaudhuri et al., 2001; Yamashita et al., 2003; Ahn et al., 2004; Nagarsekar et al., 2005; Sung et al., 2005) and chemokines are potential mediators of nociceptive facilitation (Johnston et al., 2004; Milligan et al., 2004; Verge et al., 2004; Lindia et al., 2005). Some classes of chemokines are upregulated in the periphery by tumor necrosis factor-alpha (TNF-α), interleukin-1β, and lipopolysaccharide (Bazan et al., 1997; Garcia et al., 2000; Ahn et al., 2004; Sung et al., 2005). Chemokines have numerous roles including adhesion molecules, chemotactic agent to T cells and monocytes (Fong et al., 1998; Haskell et al., 2000), and involvement in the establishment and/or maintenance of inflammatory pain (Marchand et al., 2005), neuropathic pain (Verge et al., 2004), thermal hyperalgesia and mechanical allodynia (Watkins et al., 2001; Watkins and Maier, 2002; Milligan et al., 2004; Verge et al., 2004). In the present study, robust changes in CXCL5 and CXCL1 transcript expression were demonstrated in VIP−/− mice with CYP-induced cystitis. CXCL5 was also robustly expressed in urinary bladder of WT mice with CYP-induced cystitis. The magnitude of change for both CXCL5 and CXCL1 transcripts was greater in the VIP−/− mouse. Both CXCL5 and CXCL1 cause neutrophil infiltration in various organ systems and the demonstration of increased urinary bladder MPO, an indicator of neutrophil presence, is consistent with this potent chemotactic role (Fong et al., 1998; Haskell et al., 2000). Immunoassay for CXCL1 in urinary bladder validated the transcript data obtained with the superarray. MCP1/CCL2 is another chemokine with a role in the recruitment of inflammatory cells into tissue during inflammation (Castellani et al., 2007). MCP1/CCL2 exhibited more robust transcript expression in VIP−/− mice compared to WT with CYP-induced cystitis. However, no difference in the magnitude of MCP1/CCL2 protein expression in the urinary bladder between WT and VIP−/− mice was demonstrated. Inhibition of MCP1 is currently being explored as a possible therapeutic intervention that may provide benefits in different clinical scenarios including cancer, inflammation, and autoimmune diseases (Castellani et al., 2007).

In summary, the present study has demonstrated upregulation of numerous inflammatory genes in the urinary bladder of WT and VIP−/− mice after CYP-induced cystitis (48 h). Several inflammatory genes were downregulated in the urinary bladder of both mouse strains with CYP-induced cystitis but these downregulated genes were fewer in number compared to upregulated genes. Furthermore, the magnitude of upregulation for inflammatory genes in urinary bladder common to both WT and VIP−/− mice was more robust in the VIP−/− mouse. This difference in magnitude of change may be due to the absence of the anti-inflammatory influence of VIP to the context of bladder inflammation. We selected several transcripts for validation in immunoassays and the protein expression generally complemented the transcript expression. Future studies will address the contribution of robustly regulated inflammatory genes and protein to urinary bladder hyperreflexia and altered sensory processing in the CYP-induced cystitis model of bladder inflammation (Vizzard et al., 2007).

Acknowledgements

This work was funded by NIH grants DK051369, DK060481, and DK065989. The authors gratefully acknowledge the technical expertise and support provided for the superarray by the VT Cancer Center DNA Analysis Facility.

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