Host-Derived Nitric Oxide and Its Antibacterial Effects in the Urinary Tract.

Urinary tract infection (UTI) is one of the most common bacterial infections in humans, and the majority are caused by uropathogenic Escherichia coli (UPEC). The rising antibiotic resistance among UPEC and the frequent failure of antibiotics to effectively treat recurrent UTI and catheter-associated UTI motivate research on alternative ways of managing UTI. Abundant evidence indicates that the toxic radical nitric oxide (NO), formed by activation of the inducible nitric oxide synthase, plays an important role in host defence to bacterial infections, including UTI. The major source of NO production during UTI is from inflammatory cells, especially neutrophils, and from the uroepithelial cells that are known to orchestrate the innate immune response during UTI. NO and reactive nitrogen species have a wide range of antibacterial targets, including DNA, heme proteins, iron-sulfur clusters, and protein thiol groups. However, UPEC have acquired a variety of defence mechanisms for protection against NO, such as the NO-detoxifying enzyme flavohemoglobin and the NO-tolerant cytochrome bd-I respiratory oxidase. The cytotoxicity of NO-derived intermediates is nonspecific and may be detrimental to host cells, and a balanced NO production is crucial to maintain the tissue integrity of the urinary tract. In this review, we will give an overview of how NO production from host cells in the urinary tract is activated and regulated, the effect of NO on UPEC growth and colonization, and the ability of UPEC to protect themselves against NO. We also discuss the attempts that have been made to develop NO-based therapeutics for UTI treatment.

Localization of P2X receptor subtypes 2, 3 and 7 in human urinary bladder.

BACKGROUND: Voiding dysfunctions are a common problem that has a severe negative impact on the quality of life. Today there is a need for new drug targets for these conditions. The role of ATP receptors in bladder physiology has been studied for some time, primarily in animal models. The aim of this work is to investigate the localization of the ATP receptors P2X2, P2X3 and P2X7 and their colocalization with vimentin and actin in the human urinary bladder. METHODS: Immunohistochemical analysis was conducted on full-thickness bladder tissues from fundus and trigonum collected from 15 patients undergoing open radical cystectomy due to chronic cystitis, bladder cancer or locally advanced prostate cancer. Colocalization analyses were performed between the three different P2X subtypes and the structural proteins vimentin and actin. Specimens were examined using epifluorescence microscopy and correlation coefficients were calculated for each costaining as well as the mean distance from the laminin positive basal side of the urothelium to the vimentin positive cells located in the suburothelium. RESULTS: P2X2 was expressed in vimentin positive cells located in the suburothelium. Less distinct labelling of P2X2 was also observed in actin positive smooth muscle cells and in the urothelium. P2X3 was expressed in vimentin positive cells surrounding the smooth muscle, and in vimentin positive cells located in the suburothelium. Weaker P2X3 labelling was seen in the urothelium. P2X7 was expressed in the smooth muscle cells and the urothelium. In the suburothelium, cells double positive for P2X2 and vimentin where located closer to the urothelium while cells double positive for P2X3 and vimentin where located further from the urothelium. CONCLUSION: The results from this study demonstrate that there is a significant difference in the expression of the purinergic P2X2, P2X3 and P2X7 receptors in the different histological layers of the human urinary bladder.

Role of Tumor Pericytes in the Recruitment of Myeloid-Derived Suppressor Cells.

BACKGROUND: Pericytes are members of the tumor stroma; however, little is known about their origin, function, or interaction with other tumor components. Emerging evidence suggest that pericytes may regulate leukocyte transmigration. Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with powerful inhibitory effects on T-cell-mediated antitumor reactivity. METHODS: We generated subcutaneous tumors in a genetic mouse model of pericyte deficiency (the pdgfb (ret/ret) mouse) and littermate control mice (n = 6-25). Gene expression profiles from 253 breast cancer patients (stage I-III) were evaluated for clinic-pathological parameters and survival using Cox proportional hazard ratios (HRs) and 95% confidence intervals (CIs) based on a two-sided Wald test. RESULTS: We report that pericyte deficiency leads to increased transmigration of Gr1(+)/CD11b(+) cells in experimentally induced tumors. Pericyte deficiency produced defective tumor vasculature, resulting in a more hypoxic microenvironment promoting IL-6 upregulation in the malignant cells. Silencing IL-6 expression in tumor cells attenuated the observed differences in MDSC transmigration. Restoring the pericyte coverage in tumors abrogated the increased MDSC trafficking to pericyte-deficient tumors. MDSC accumulation in tumors led to increases in tumor growth and in circulating malignant cells. Finally, gene expression analysis from human breast cancer patients revealed increased expression of the human MDSC markers CD33 and S100A9 with concomitant decreased expression of pericyte genes and was associated with poor prognosis (HR = 1.88, 95% CI = 1.08 to 3.25, P = .03). CONCLUSIONS: Our data uncovers a novel paracrine interaction between tumor pericytes and inflammatory cells and delineates the cellular events resulting in the recruitment of MDSC to tumors. Furthermore, we propose for the first time a role for tumor pericytes in modulating the expression of immune mediators in malignant cells by promoting a hypoxic microenvironment.

Shaping the murine macrophage phenotype: IL-4 and cyclic AMP synergistically activate the arginase I promoter.

Arginase I is a marker of murine M2 macrophages and is highly expressed in many inflammatory diseases. The basis for high arginase I expression in macrophages in vivo is incompletely understood but likely reflects integrated responses to combinations of stimuli. Our objective was to elucidate mechanisms involved in modulating arginase I induction by IL-4, the prototypical activator of M2 macrophages. IL-4 and 8-bromo-cAMP individually induce arginase I, but together they rapidly and synergistically induce arginase I mRNA, protein, and promoter activity in murine macrophage cells. Arginase I induction by IL-4 requires binding of the transcription factors STAT6 and C/EBPß to the IL-4 response element of the arginase I gene. Chromatin immunoprecipitation showed that the synergistic response involves binding of both transcription factors to the IL-4 response element at levels significantly greater than in response to IL-4 alone. The results suggest that C/EBPß is a limiting factor for the level of STAT6 bound to the IL-4 response element. The enhanced binding in the synergistic response was not due to increased expression of either STAT6 or C/EBPß but was correlated primarily with increased nuclear abundance of C/EBPß. Our findings also suggest that induction of arginase I expression is stochastic; that is, differences in induction reflect differences in probability of transcriptional activation and not simply differences in rate of transcription. Results of the present study also may be useful for understanding mechanisms underlying regulated expression of other genes in macrophages and other myeloid-derived cells in health and disease.

Localization and expression of inducible nitric oxide synthase in patients after BCG treatment for bladder cancer.

Treatment with Bacillus Calmette Guerin (BCG) bladder instillations is an established treatment modality for superficial urinary bladder cancer and carcinoma in situ (CIS), but the anti-tumor mechanisms following BCG instillations remain largely unknown. Previous data show increased nitric oxide (NO) concentrations in the urinary bladder from patients treated with BCG suggesting that NO-formation may be involved in the BCG mediated effect. In the present study we evaluated 11 patients with urinary bladder cancer who had received BCG treatment and 11 tumor free control subjects. We performed immunohistochemistry, Western blot and real-time polymerase chain reaction (PCR) on bladder biopsies to establish inducible nitric oxide synthase (iNOS) protein levels and localization as well as iNOS mRNA expression. Endogenous NO formation in the bladder was also measured. In patients with bladder cancer who had received BCG treatment iNOS-like immunoreactivity was found in the urothelial cells but also in macrophages in the submucosa. Furthermore, endogenously formed NO was significantly increased (p<0.001) in the BCG treated patients and they had a ten-fold increase in mRNA expression for iNOS compared to healthy controls (p=0.003). In conclusion iNOS was found to be localized to the urothelium and macrophages underlying it. Our study also confirms elevated levels of endogenously formed NO and increased mRNA expression and protein levels for iNOS in patients with BCG treated bladder cancer. These data further support the notion that NO may be involved in the anti-tumor mechanism that BCG exerts on bladder cancer cells.

Expression profiling of G-protein-coupled receptors in human urothelium and related cell lines.

UNLABELLED: What's known on the subject? and What does the study add? Urothelium emerged as a crucial integrator of sensory inputs and outputs in the bladder wall, and urothelial G-protein-coupled receptors (GPCRs) may represent plausible targets for treatment of various bladder pathologies. Urothelial cell lines provide a useful tool to study urothelial receptor function, but their validity as models for native human urothelium remains unclear. We characterize the mRNA expression of genes coding for GPCRs in human freshly isolated urothelium and compare the expression pattern with those in human urothelial cell lines. OBJECTIVES: To characterize the mRNA expression pattern of genes coding for G-protein-coupled receptors (GPCRs) in human freshly isolated urothelium. To compare GPCR expression in human urothelium-derived cell lines to explore the suitability of these cell lines as model systems to study urothelial function. MATERIALS AND METHODS: Native human urothelium (commercially sourced) and human urothelium-derived non-cancer (UROtsa and TERT-NHUC) and cancer (J82) cell lines were used. For mRNA expression profiling we used custom-designed real-time polymerase chain reaction array for 40 receptors and several related genes. RESULTS: Native urothelium expressed a wide variety of GPCRs, including α(1A), α(1D) and all subtypes of α(2) and ß adrenoceptors. In addition, M(2) and M(3) cholinergic muscarinic receptors, angiotensin II AT(1) receptor, serotonin 5-HT(2A) receptor and all subtypes of bradykinin, endothelin, cannabinoid, tachykinin and sphingosine-1-phosphate receptors were detected. Nerve growth factor and both its low- and high-affinity receptors were also expressed in urothelium. In all cell lines expression of most GPCRs was markedly downregulated, with few exceptions. In UROtsa cells, but much less in other cell lines, the expression of ß(2) adrenoceptors, M(3) muscarinic receptors, B(1) and B(2) bradykinin receptors, ET(B) endothelin receptors and several subtypes of sphingosine-1-phosphate receptors was largely retained. CONCLUSIONS: Human urothelium expresses a wide range of receptors which enables sensing and integration of various extracellular signals. Human urothelium-derived cell lines, especially UROtsa cells, show comparable mRNA expression to native tissue for several physiologically relevant GPCRs, but lose expression of many other receptors. The use of cell lines as model systems of human urothelium requires careful validation of suitability for the genes of interest.

Adenosine receptor expression in Escherichia coli-infected and cytokine-stimulated human urinary tract epithelial cells.

OBJECTIVE: To assess the expression and regulation of adenosine receptors in unstimulated, uropathogenic Escherichia coli (UPEC)-infected and cytokine-stimulated human urinary tract epithelial cells, and to examine the regulation of interleukin (IL)-6 secretion in response to A(2A) receptor activation. MATERIALS AND METHODS: Human urinary tract epithelial cells (A498, T24 and RT4) were grown in cell culture and stimulated with a mixture of pro-inflammatory cytokines (CM) or UPEC. The expression of adenosine receptors was evaluated using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunocytochemistry. IL-6 secretion was measured with an enzyme-linked immunosorbent assay. RESULTS: RT-PCR analysis showed the presence of transcripts for the A(1), A(2A) and A(2B) receptor subtypes but not for the A(3) receptor in A498 kidney epithelial cells. The expression of A(2A) receptor mRNA increased in A498 epithelial cells exposed to CM and UPEC, while A(1) and A(2B) receptor transcripts decreased or remained unchanged. Up-regulation of A(2A) receptors was confirmed at the protein level using Western blot analysis and immunocytochemistry. There was also an increase in A(2A) receptor mRNA in human bladder epithelial cells (T24 and RT4) and in mouse bladder uroepithelium in response to cytokines and UPEC. IL-6 secretion in UPEC-infected A498 cells was decreased by 38% when exposed to the A(2A) receptor agonist CGS 21680. CONCLUSION: Our data showed a subtype-selective plasticity among adenosine receptors in urinary tract epithelial cells in response to UPEC-infection and cytokines. There was a consistent up-regulation of A(2A) receptors in kidney and bladder epithelial cells. Functionally, A(2A) receptor activation reduced UPEC-induced IL-6 secretion. These findings suggest that adenosine might be a previously unrecognized regulator of the mucosal response in urinary tract infection.

Inhibition of phosphodiesterase 4 amplifies cytokine-dependent induction of arginase in macrophages.

Arginase is greatly elevated in asthma and is thought to play a role in the pathophysiology of this disease. As inhibitors of phosphodiesterase 4 (PDE4), the predominant PDE in macrophages, elevate cAMP levels and reduce inflammation, they have been proposed for use in treatment of asthma and chronic obstructive pulmonary disease. As cAMP is an inducer of arginase, we tested the hypothesis that a PDE4 inhibitor would enhance macrophage arginase induction by key cytokines implicated in asthma and other pulmonary diseases. RAW 264.7 cells were stimulated with IL-4 or transforming growth factor (TGF)-beta, with and without the PDE4 inhibitor rolipram. IL-4 and TGF-beta increased arginase activity 16- and 5-fold, respectively. Rolipram alone had no effect but when combined with IL-4 and TGF-beta synergistically enhanced arginase activity by an additional 15- and 5-fold, respectively. The increases in arginase I protein and mRNA levels mirrored increases in arginase activity. Induction of arginase II mRNA was also enhanced by rolipram but to a much lesser extent than arginase I. Unlike its effect in RAW 264.7 cells, IL-4 alone did not increase arginase activity in human alveolar macrophages (AM) from healthy volunteers. However, combining IL-4 with agents to induce cAMP levels induced arginase activity in human AM significantly above the level obtained with cAMP-inducing agents alone. In conclusion, agents that elevate cAMP significantly enhance induction of arginase by cytokines. Therefore, consequences of increased arginase expression should be evaluated whenever PDE inhibitors are proposed for treatment of inflammatory disorders in which IL-4 and/or TGF-beta predominate.

Induction of arginase I transcription by IL-4 requires a composite DNA response element for STAT6 and C/EBPbeta.

Arginine metabolism in macrophages during infection and inflammation is complex, owing to differential regulation of inducible nitric oxide synthase (iNOS) and arginases by cytokines and other agents. Changes in levels of Th2 cytokines such as interleukin-4 (IL-4) can play important roles in these conditions via effects on arginine metabolism. IL-4 alters macrophage arginine metabolism by inducing arginase I expression and inhibiting nitric oxide production. To determine the molecular basis for induction of arginase I, the promoter of the murine arginase I gene was cloned and analyzed by transfection in RAW 264.7 macrophage cells. IL-4 induction required a composite response element containing STAT6 and C/EBP sites located 2.86 kb upstream of the transcription start site. Competition experiments showed that STAT6 and C/EBPbeta bind to the STAT6 and C/EBP sites non-cooperatively. Elucidation of the mechanisms involved in regulation of arginase I transcription may provide a basis for developing strategies to modulate arginase expression in Th2 cytokine-predominant diseases.

The influence of uropathogenic Escherichia coli and proinflammatory cytokines on the inducible nitric oxide synthase response in human kidney epithelial cells.

PURPOSE: Nitric oxide (NO) is an antibacterial factor that is produced by the enzyme inducible NO synthase (iNOS). Uroepithelial cells express iNOS in experimental models of urinary tract infection but the stimulatory and regulatory mechanisms are still unclear. We investigated the influence of uropathogenic Escherichia coli strains with different fimbrial expression and the effect of proinflammatory cytokines on the host iNOS response. MATERIALS AND METHODS: A498 human kidney epithelial cells were stimulated with different uropathogenic E. coli strains, namely the P and type 1-fimbriated clinical isolate AD110, the recombinant P-fimbriated strain E. coli HB101(pPIL110-75) and the recombinant type 1-fimbriated strain E. coli AAEC191A(pPKL4). NO production was determined as nitrite production in cell culture medium. Studies of nuclear factor-kappaB (NF-kappaB) binding to the iNOS promoter and reverse transcriptase-polymerase chain reaction of iNOS mRNA were performed to investigate iNOS gene activation in response to uropathogenic E. coli. The effect of interleukin (IL)-6, IL-8 and transforming growth factor-beta on NO production was also examined. RESULTS: E. coli per se failed to induce NO production and iNOS mRNA in A498 cells. However, in combination with interferon-gamma AD110 and the type 1-fimbriated strain caused a small increase in NO production and iNOS mRNA. AD110 stimulated A498 cells demonstrated weak binding of NF-kappaB to a human iNOS promoter sequence. IL-6, IL-8 and transforming growth factor-a did not affect NO production in A498 cells. CONCLUSIONS: Uropathogenic bacteria are weak inducers of human uroepithelial iNOS, which may be related to insufficient binding of NF-kappaB to iNOS promoter. The uroepithelial iNOS response did not appear to be regulated by proinflammatory cytokines.

Signalling pathways regulating inducible nitric oxide synthase expression in human kidney epithelial cells.

The purpose of this study was to elucidate the signalling pathways involved in the cytokine-activated inducible nitric oxide synthase (iNOS) response in a human kidney epithelial cell line, A498. Unstimulated cells did not express iNOS. Exposure of A498 cells to a cytokine mixture consisting of interferon gamma, interleukin-1 beta and tumor necrosis factor-alpha (TNF-alpha) increased nitrite production, iNOS mRNA and protein expression. Pharmacological inhibition of tyrosine kinases, including janus kinase (JAK2), and protein kinase C (PKC) inhibited cytokine-mediated nitrite production and iNOS protein expression. The involvement of mitogen-activated protein kinases (MAPKs) was investigated. Inhibition of p38 MAPK, but not of an upstream activator of extracellular signal-regulated kinase (ERK), caused a decrease in iNOS expression and nitrite production in response to cytokines. Electrophoretic mobility shift assay of nuclear extract from cytokine-stimulated cells demonstrated a pronounced binding to a nuclear factor kappa B (NF-kappa B) sequence present in the human iNOS promoter. Furthermore, the NF-kappa B inhibitor pyrrolidinedithiocarbamate (PDTC) decreased cytokine-activated iNOS protein expression and nitrite production. The present study has demonstrated that cytokine-stimulated iNOS expression in human kidney epithelial cells involves activation of tyrosine kinases, including JAK2, PKC, p38 MAPK and NF-kappa B.

Human renal epithelial cells express iNOS in response to cytokines but not bacteria.

BACKGROUND: Epithelial cells form the mucosal barriers that prevent the entry of mucosal pathogens, and respond to bacterial infections by producing various host defense molecules. In this study, we examined the inducible nitric oxide synthase (iNOS) response of primary human renal tubular epithelial cells (HRTEC) following infection with uropathogenic Escherichia coli Hu734, or stimulation with lipopolysaccharide (LPS) or cytokines. METHODS: Induction of iNOS was examined by RT-PCR, Western blot, immunohistochemistry and nitrite measurements. The effects of endogenously produced nitric oxide (NO), and exogenously applied DETA/NO, SIN-1 and H2O2 on cell viability were analyzed using a respiration assay. RESULTS: HRTEC did not produce NO following infection with E. coli Hu734, LPS alone, or in combination with interferon-gamma (IFN-gamma), even though these agents caused a marked increase in iNOS expression by RAW 264.7, a macrophage cell line. In contrast, iNOS protein and mRNA expression by HRTEC increased after exposure to a cytokine mixture consisting of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma. This was due to the combination of IL-1beta and IFN-gamma, but the individual cytokines had no effect. Inducible NOS-expressing cell cultures showed reduced viability, and this effect was inhibited with the NOS inhibitor L-NMMA in RAW 264.7 cells, but not in HRTEC. HRTEC were more sensitive to oxidative stress induced by H2O2 than to nitrogen stress induced by DETA/NO. CONCLUSIONS: We conclude that uropathogenic E. coli that attach to HRTEC fail to directly activate iNOS expression, and that iNOS expression during bacterial infection is more likely to result from stimulation by local cytokines such as IL-1beta and IFN-gamma.