LPS-sensory peptide communication in experimental cystitis.

Stimulation of sensory nerves can lead to release of peptides such as substance P (SP) and consequently to neurogenic inflammation. We studied the role of bacterial lipopolysaccharide (LPS) in regulating SP-induced inflammation. Experimental cystitis was induced in female mice by intravesical instillation of SP, LPS, or fluorescein-labeled LPS. Uptake of fluorescein-labeled LPS was determined by confocal analysis, and bladder inflammation was determined by morphological analysis. SP was infused into the bladders of some mice 24 h after exposure to LPS. In vitro studies determined the capacity of LPS and SP to induce histamine and cytokine release by the bladder. LPS was taken up by urothelial cells and distributed systemically. Twenty-four hours after instillation of LPS or SP, bladder inflammation was characterized by edema and leukocytic infiltration of the bladder wall. LPS pretreatment enhanced neutrophil infiltration induced by SP, increased in vitro release of histamine, tumor necrosis factor-alpha, and interferon-gamma, and significantly reduced transforming growth factor-beta1 release. These findings suggest that LPS amplifies neurogenic inflammation, thereby playing a role in the pathogenesis of neurogenic cystitis.

Mast cell regulation of inflammation and gene expression during antigen-induced bladder inflammation in mice.

Mast cell numbers are significantly increased in bladder disorders including malignancy and interstitial cystitis, but their precise role has been difficult to determine. We characterized the role of mast cells on gene regulation associated with antigen-induced bladder inflammation in mice. For this purpose, we examined the responses in mast cell-deficient (Kit(W)/Kit(W-v)), congenic normal (+/+), and Kit(W)/Kit(W-v) mice that were reconstituted with bone marrow stem cells (BMR) to restore mast cells. All mice were actively sensitized and challenged intravesically with either saline or specific antigen. Bladder inflammation occurred in +/+ and BMR but not the Kit(W)/Kit(W-v) mice. Gene expression was determined using mouse cDNA expression arrays. Self-organizing maps, performed without preconditions, indicated gene expression changes dependent on the presence of mast cells. These genes were upregulated in bladders isolated from antigen challenge of +/+, not altered in Kit(W)/Kit(W-v), and were upregulated in BMR mice. Taken together these results demonstrate an important role for mast cells in allergic cystitis and indicate that mast cells can alter their environment by regulating tissue gene expression.

Time course of LPS-induced gene expression in a mouse model of genitourinary inflammation.

In this study, self-organizing map (SOM) gene cluster techniques are applied to the analysis of cDNA microarray analysis of gene expression changes occurring in the early stages of genitourinary inflammation. We determined the time course of lipopolysaccharide (LPS)-induced gene expression in experimental cystitis. Mice were euthanized 0.5, 1, 4, and 24 h after LPS instillation into the urinary bladder, and gene expression was determined using four replicate Atlas mouse cDNA expression arrays containing 588 known genes at each time point. SOM gene cluster analysis, performed without preconditions, identified functionally significant gene clusters based on the kinetics of change in gene expression. Genes were classified as follows: 1) expressed at time 0; 2) early genes (peak expression between 0.5 and 1 h); and 3) late genes (peak expression between 4 and 24 h). One gene cluster maintained a constant level of expression during the entire time period studied. In contrast, LPS treatment downregulated the expression of some genes expressed at time 0, in a cluster including transcription factors, protooncogenes, apoptosis-related proteins (cysteine protease), intracellular kinases, and growth factors. Gene upregulation in response to LPS was observed as early as 0.5 h in a cluster including the interleukin-6 (IL-6) receptor, alpha- and beta-nerve growth factor (alpha- and beta-NGF), vascular endothelial growth factor receptor-1 (VEGF R1), C-C chemokine receptor, and P-selectin. Another tight cluster of genes with marked expression at 1 h after LPS and insignificant expression at all other time points studied included the protooncogenes c-Fos, Fos-B, Fra-2, Jun-B, Jun-D, and Egr-1. Almost all interleukin genes were upregulated as early as 1 h after stimulation with LPS. Nuclear factor-kappaB (NF-kappaB) pathway genes collected in a single cluster with a peak expression 4 h after LPS stimulation. In contrast, most of the interleukin receptors and chemokine receptors presented a late peak of expression 24 h after LPS coinciding with the peak of neutrophil infiltration into the bladder wall. Selected cDNA microarray observations were confirmed by RNase protection assay. In conclusion, the cDNA array experimental approach provided a global profile of gene expression changes in bladder tissue after stimulation with LPS. SOM techniques identified functionally significant gene clusters, providing a powerful technical basis for future analysis of mechanisms of bladder inflammation.

Neurokinin-1 (NK-1) receptor is required in antigen-induced cystitis.

Interstitial cystitis (IC) is a debilitating disease that has been adversely affecting the quality of women's lives for many years. The trigger in IC is not entirely known, and a role for the sensory nerves in its pathogenesis has been suggested. In addition to inflammation, increased mast cell numbers in the detrusor muscle have been reported in a subset of IC patients. Experimentally, several lines of evidence support a central role for substance P and neurokinin-1 (NK-1) receptors in cystitis. The availability of mice genetically deficient in neurokinin-1 receptor (NK-1R(-/-)) allows us to directly evaluate the importance of substance P in cystitis. An unexpected finding of this investigation is that NK-1R(-/-) mice present increased numbers of mast cells in the bladder when compared with wild-type control mice. Despite the increase in mast cell numbers, no concomitant inflammation was observed. In addition, bladder instillation of wild-type mice with a sensitizing antigen induces activation of mast cells and an acute inflammatory response characterized by plasma extravasation, edema, and migration of neutrophils. Antigen-sensitized NK-1R(-/-) mice also exhibit bladder mast cell degranulation in response to antigen challenge. However, NK-1R(-/-) mice are protected from inflammation, failing to present bladder inflammatory cell infiltrate or edema in response to antigen challenge. This work presents the first evidence of participation of NK-1 receptors in cystitis and a mandatory participation of these receptors on the chain of events linking mast cell degranulation and inflammation.

Nuclear factor kappa B mediates lipopolysaccharide-induced inflammation in the urinary bladder.

PURPOSE: The proteins which constitute the final common pathway linking receptors on cell surfaces to the inflammatory cascade have recently been identified and cloned. Central to activation of this inflammatory cascade is translocation from cytosol to nucleus of the nuclear transcription factor known as nuclear factor-kappa B (NF-kappaB). The purpose of this study was to determine whether NF-kappaB cascade plays a role in lipopolysaccharide (LPS)-induced inflammation of the mouse urinary bladder. MATERIALS AND METHODS: Bladder inflammation was induced in anesthetized mice by intravesical instillation of lipopolysaccharide (LPS) and quantified by morphometric analysis. The NK-1 receptors for substance P were quantified by flow cytometry. LPS-induced degradation of inhibitory IkappaB subunit was quantified by Western blotting analysis and translocation of NF-kappaB protein from cytosol to the nucleus was determined by confocal microscopy and Western blotting analysis. In addition, we determine the effect of lactacystin, a proteosome inhibitor, on LPS-induced IkappaB degradation and NF-kappaB translocation, NK-1 receptor fluorescence intensity, and bladder inflammation. RESULTS: LPS instillation into the mouse bladder resulted in time dependent loss of the inhibitory IkappaB subunit of the NF-kappaB protein complex. IkappaB cleavage was followed by translocation of NF-kappaB from the cytosol to the nucleus. This was associated with increased expression of an NF-kappaB dependent inflammatory component, the NK-1 receptor. Pretreatment of mouse bladders with the NF-kappaB inhibitor, lactacystin, prevented cleavage of IkappaB in a dose-dependent manner. Lactacystin prevented increases in the NF-kappaB dependent inflammatory cascade components such as the NK-1 receptor. At the whole tissue level, the marked inflammatory infiltrate and mucosal breakdown associated with LPS administration was completely abolished by lactacystin. CONCLUSION: NF-kappaB mediates many features of urinary bladder inflammation induced by LPS. The NF-kappaB cascade is an important target for anti-inflammatory management of cystitis.

Nerve-mediated motility of ileal segments isolated from NK(1) receptor knockout mice.

Tachykinins such as substance P (SP) and neurokinin A (NKA) acting on neurokinin (NK) receptors modulate the nonadrenergic noncholinergic (NANC) neurotransmission in the gastrointestinal tract of several species, but the information about the mouse small intestine is scanty. Both SP and NKA induced concentration-dependent contractions of ileal segments isolated from wild-type mice that were blocked by NK(1) and NK(2) antagonists, respectively. In contrast, segments isolated from NK(1) receptor (NK(1)-R) knockout mice responded only to elevated concentrations of SP. To reveal the inhibitory NANC (iNANC) responses, tissues were pretreated with atropine and guanethidine. Under these conditions, a tetrodotoxin-sensitive relaxation in response to electrical field stimulation (EFS) was observed. NK(1)-R knockout mice presented a trend toward an increase in iNANC responses, whereas the NK(1)-R antagonist significantly potentiated iNANC relaxation in tissues isolated from wild-type mice. N(G)-nitro-L-arginine methyl ester (100 microM) transformed the relaxant response to EFS into a tetrodotoxin-sensitive, frequency-dependent contraction characteristic of an excitatory NANC (eNANC) system. A NK(1)-R antagonist abolished the contractile responses of the mouse ileum to EFS, whereas a NK(2) receptor antagonist had a trend toward reducing EFS-induced contraction. The eNANC component was absent in NK(1)-R knockout mice. Measurement of SP-like immunoreactivity indicated similar amounts of SP per gram of tissue isolated from wild-type and NK(1)-R knockout mice, indicating that the observed differences in response to EFS were not due to a differential peptide content. It is concluded that, in the mouse ileum, both NK(1) and NK(2) receptors modulated the responses to exogenous tachykinins, whereas NK(1) is the primary tachykinin receptor involved in both iNANC and eNANC transmission.

Mast cells mediate the severity of experimental cystitis in mice.

PURPOSE: We hypothesized that experimental cystitis induced by substance P (SP) or E. coli lipopolysaccharide (LPS) would be less severe in mice rendered mast cell deficient by genetic manipulation. MATERIALS AND METHODS: Two strains of mast-cell deficient mice (WBB6F1- kitW/kitW-v or kitW/kitW-v and WCB6F1-Sl/Sld or Sl/Sld) and their congenic, normal (+/+) counterparts were used. Cystitis was induced in female mice by intravenous injection of SP (0.1 ml.; 10(-6) M) or E. coli LPS (0.1 ml.; 2 mg./ml.), and inflammation was assessed by Evans blue dye extravasation. In a separate group of kitW/kitW-v and congenic normal mice, cystitis was induced by intravesical infusion of SP (0.05 ml.; 10(-5) M) or E. coli LPS (0.05 ml.; 100 microg./ml.) and compared with intravesical pyrogen-free saline (0.05 ml.; 0.9%). Severity of cystitis was determined by histological evaluation of the bladder wall 24 hours after intravesical infusions. RESULTS: Intravenous SP or LPS stimulated increased plasma extravasation in congenic normal mice but not in mast cell-deficient mice. Intravesical SP or LPS resulted in increased edema, leukocytic infiltration, and hemorrhage within the bladder wall in congenic normal mice, but the only histological evidence of inflammation in the bladders of kitW/kitW-v mice was increased hemorrhage in response to LPS. CONCLUSIONS: This study indicates that mast cells modulate the inflammatory response of the bladder to SP and LPS in mice. Although clinical trials of the use of antihistamines to treat or prevent cystitis have not been successful, these results suggest that therapies directed toward preventing mast cell activation may yet prove effective in treating cystitis.

Kinetics of peptide-induced release of inflammatory mediators by the urinary bladder.

OBJECTIVE: To investigate the release of inflammatory mediators by the urinary bladder in response to exposure to pro-inflammatory peptides. MATERIALS AND METHODS: Isolated guinea pig urinary bladder was incubated with 10 mumol/L each of substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), octreotide acetate (a long-acting analogue of somatostatin, SOM), or bradykinin (BK), and the release of histamine, prostaglandin (PG) E2, PGF2 alpha and leukotriene B4 (LTB4) was determined during 0-5, 5-30 and 30-120 min after addition. RESULTS: Substance P, NKA, VIP and BK stimulated the release of histamine, while CGRP and SOM suppressed the release to below the spontaneous rates. All peptides, except CGRP and SOM, stimulated the release of PGE2 between 0 and 30 min, and only VIP failed to stimulate the release of PGF2 alpha within 5 min of exposure. Substance P, NKA, VIP and BK stimulated the release of LTB4 and this required > 5 min of exposure. CONCLUSION: These results indicate that the peptides evaluated induce an immediate and transient release of histamine and activation of cyclooxygenase and delayed activation of 5-lipoxygenase. These actions may directly regulate the participation of these peptides in the pathogenesis of cystitis.

Effect of octreotide, a somatostatin analogue, on release of inflammatory mediators from isolated guinea pig bladder.

OBJECTIVE: Somatostatin has been demonstrated to inhibit inflammation under certain circumstances. We hypothesized that in vivo treatment with octreotide, a long-acting analogue of somatostatin analogue, would diminish the capacity of inflammatory peptides to stimulate in vitro release of inflammatory mediators by the bladder. METHODS: Female guinea pigs were injected with octreotide (20 mg./kg. i.m.) prior to euthanasia. Control guinea pigs received no treatment prior to euthanasia. Urinary bladders were removed and incubated with substance P (SP, 10 microM), neurokinin A (NKA, 10 microM), or bradykinin (BK, 10 microM) in the presence or absence of indomethacin (50 microM), and release of histamine, prostaglandins (PGE2 and PGF2 alpha), and leukotriene (LTB4) was determined. RESULTS: Sensory peptides and BK induced time-dependent release of histamine and eicosanoids from isolated urinary bladder. Blockade of cyclooxygenase with indomethacin (50 microM) abolished peptide-induced prostaglandin release but enhanced LTB4 release. In vivo octreotide pretreatment decreased peptide-induced histamine release, had no effect on PGE2 or PGF2 alpha release, and LTB4 release. However, octreotide prevented the increase in LTB4 release in tissues incubated with indomethacin. CONCLUSIONS: These results indicate that somatostatin has the capacity to suppress the release of histamine and prevents potentiation of LTB4 release by indomethacin by the guinea pig bladder in response to pro-inflammatory peptides, indicating that somatostatin may be useful in preventing or treating some forms of cystitis.

Morphometric analysis of oleic acid-induced permeability pulmonary edema: correlation with gravimetric lung water.

The technique used most commonly to quantitate pulmonary edema in in vivo animal models is postmortem gravimetric analysis (wet:dry) ratio. To determine whether lung water can be quantitated morphometrically, as accurately as by the commonly used gravimetric analysis, perivascular edema (cuff) area to vessel area ratio was correlated to wet:dry ratio. Anesthetized pigs were given either oleic acid (20 mg/kg/h, intravenously) or physiologic saline. At 4 h, lungs were excised and cuff:vessel and wet:dry ratio analysis was performed. The intermediate lobe was clamped across its main stem bronchus to maintain peak inspiratory inflation, excised, frozen in liquid nitrogen, and stored at -70 degrees C until cryostat sectioning and quantification of perivascular interstitial edema (cuff) area. Gravimetric analysis (wet:dry ratio) was performed on the remaining lung. Mean cuff:vessel and wet:dry analyzes showed that lung water increased significantly (p < .01) in the oleic-acid treated group (4.9 +/- .22 and 6.78 +/- .47, respectively), compared with the saline group (.03 +/- .02 and 2.55 +/- .27, respectively). The correlation coefficient between mean cuff:vessel and wet:dry ratios was .86 (p = .0016). This study demonstrates that cuff:vessel ratio analysis can be used to identify the distribution of edema fluid versus vessel diameter, and seems to be as effective a technique as gravimetric analysis to quantitate lung water changes in acute lung injury models. Moreover cuff:vessel ratio analysis can differentiate modest changes in pulmonary edema by direct quantitation, an important end-point not provided by wet:dry analysis. Therefore, it may be a more sensitive technique when investigating therapeutic interventions in in vivo models of acute lung injury.

Involvement of leukotrienes, TNF-alpha, and the LFA-1/ICAM-1 interaction in substance P-induced granulocyte infiltration.

Substance P (SP) has been shown to mediate granulocyte infiltration into the mouse skin by inducing mast cell degranulation. In this study, using a variety of specific inhibitors, we investigated the cascade of events involved in the response of neutrophils and eosinophils to SP. The prostaglandin inhibitor, indomethacin, had little effect on SP-induced leukocyte migration. In contrast, pretreatment with the leukotriene (LT) synthesis inhibitor, A-64077, completely blocked neutrophil but not eosinophil migration in response to SP. Participation of tumor necrosis factor alpha (TNF-alpha) and LFA-1/ICAM-1 interaction was confirmed by inhibition of SP-induced leukocyte migration by pretreatment of mice with monoclonal antibodies to TNF-alpha, LFA-1, and ICAM-1. Moreover, alteration in leukocyte migration by indomethacin was found to depend on the concentration of TNF-alpha used. Indomethacin did not alter the number of leukocytes induced by low concentrations of TNF-alpha (0.1 ng), but reduced the number of cells stimulated with high TNF-alpha concentrations (1.0 ng). These results support the concept that SP modulates in vivo neuroinflammatory responses, as measured by granulocyte migration, initiating a cascade of events that includes LT production, TNF-alpha secretion, and engagement of LFA-1 and ICAM-1.

In vitro passive sensitization of guinea pig, rhesus monkey and human bladders as a model of noninfectious cystitis.

Studies of human bladder inflammation have been limited to examination of urine, bladder biopsy, or examination of autopsy material. We have developed an in vitro bladder passive sensitization technique which can measure type I responses of isolated human bladder tissue. We have compared these results using human tissue to those obtained with bladder tissue from guinea pigs and Rhesus monkeys. In our studies, bladder tissue was passively sensitized in vitro for 20 hours with immunoglobulin-containing serum. Subsequent antigen challenge of the passively sensitized tissue resulted in a time-dependent contraction that was accompanied by tissue histamine release. Contractions of guinea pig, monkey and human bladder tissue reached 79%, 100% and 78% of the maximal contraction induced by potassium chloride. In contrast, adjacent strips of unsensitized tissue had no detectable response to antigen challenge. The responses were reduced in the presence of histamine H1 receptor blockade with pyrilamine and abolished in the presence of a concomitant blockade of leukotriene synthesis with nordihydroguaiaretic acid (NDGA). Blockade of cyclooxygenase activity with indomethacin increased the contraction of the sensitized guinea pig bladder in response to antigen challenge. These findings demonstrate that in vitro passive sensitization of human bladder tissue can be used to investigate basic mechanisms of noninfectious bladder inflammation in humans.

Response of the isolated guinea pig bladder to exogenous and endogenous leukotrienes.

Noninfectious urinary bladder inflammation is a poorly understood phenomenon, and the participation of leukotrienes (LTs) in the pathogenesis of bladder inflammation is unclear. Leukotrienes are synthesized by the bladder, and exogenous LTs induce contraction of isolated bladder segments. LTD4 and LTC4 were more potent contractile agents than LTE4. Leukotriene-induced contractions were blocked by ICI 198,615 (10(-6) M. and 10(-7) M.) a specific LT receptor antagonist. In the presence of indomethacin (5 x 10(-6) M.), bladder contraction in response to LTD4 was increased. Endogenous LT release was studied using an experimental model of cystitis. Antigen (ovalbumin 10(-6) to 10(-2) mg./ml.) challenge of bladder segments isolated from actively sensitized animals induced release of LT, prostaglandin D2 and histamine. A-64077 (Zileuton), a 5-lipoxygenase (5-LO) inhibitor, significantly reduced contraction of sensitized bladder tissue in response to antigen challenge in a concentration-dependent manner and abolished LT release. These data indicate that the guinea pig urinary bladder produces sulfidopeptide-LTs that can be released upon specific stimulation. Furthermore, LTs activate specific receptors promoting bladder contraction. Our findings suggest that specific 5-LO inhibitors or LT-receptor antagonists might be useful in treating or preventing bladder inflammation.

Differential release of prostaglandins and leukotrienes by sensitized guinea pig urinary bladder layers upon antigen challenge.

The relative contributions of mucosal/submucosal and detrusor layers to the release of inflammatory mediators were investigated in isolated, ovalbumin (OVA) sensitized guinea pig urinary bladders. Ovalbumin challenge of sensitized mucosa induced release of prostaglandins (PG): PGE2, PGD2 and PGF2 alpha, in that order of magnitude. The total release of PGs was significantly higher from the mucosa/submucosa than from the detrusor/serosa. Under the same conditions, net release of leukotriene (LT) was observed predominantly from the detrusor. The total amount of histamine released from the mucosa was greater than that from the muscle layer. These results indicate differential production and release of inflammatory mediators from the mucosal/submucosal and detrusor smooth muscle layers. These results may have serious implications in disorders, such as interstitial cystitis, involving bladder mucosal damage. The cytoprotective effect of PGE2 is likely to be lost when the mucosa is damaged, and LT release from deeper layers may contribute significantly to symptoms of bladder inflammation.

Neurogenic inflammation of Guinea-pig bladder.

Capsaicin, substance P, and ovalbumin, instilled into the bladders of naive and ovalbumin (OVA) sensitized guineapigs caused inflammation, as indicated by increased vascular permeability. Histological changes after exposure to these compounds progressed with time from intense vasodilatation to marginalization of granulocytes followed by interstitial migration of leukocytes. In vitro incubation of guinea-pig bladder tissue with substance P and ovalbumin stimulated release of prostaglandin D(2) and leukotrienes. In vitro incubation of bladder tissue with capsaicin, OVA, prostaglandin D(2), leukotriene C(4), histamine, or calcium ionophore A-23587 all stimulated substance P release. These data suggest that bladder inflammation initiated by a variety of stimuli could lead to a cyclic pattern of release of inflammatory mediators and neuropeptides, which could result in amplification and persistence of cystitis after the inciting cause has subsided.