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ORIGINAL ARTICLE
Year : 2022 | Volume
: 33
| Issue : 4 | Page : 199-204
Comparing concentration of urinary inflammatory cytokines in interstitial cystitis, overactive bladder, urinary tract infection, and bladder cancer
Michael B Chancellor1, Laura E Lamb2, Elijah P Ward3, Sarah N Bartolone3, Alexander Carabulea3, Prasun Sharma2, Joseph Janicki3, Christopher Smith4, Melissa Laudano5, Nitya Abraham5, Bernadette M M. Zwaans1
1 Department of Urology, Beaumont Health Spectrum Health System, Royal Oak; Department of Urology, Oakland University William Beaumont School of Medicine, Rochester, MI, USA
2 Department of Urology, Oakland University William Beaumont School of Medicine, Rochester, MI, USA
3 Department of Urology, Beaumont Health Spectrum Health System, Royal Oak, MI, USA
4 Department of Urology, Baylor College of Medicine; Michael E. DeBakey VA Medical Center, Houston, TX, USA
5 Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY, USA
Correspondence Address:
Michael B Chancellor
Beaumont Health Spectrum Health System, Royal Oak, MI; Oakland University William Beaumont School of Medicine, Rochester, MI
USA
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/UROS.UROS_26_22
Purpose: We sought to determine if urinary cytokine concentration profiles were different between various bladder conditions. Materials and Methods: Participants at three clinical sites completed a demographics survey and provided a urine sample in a collection cup containing a room-temperature urine preservative. Participants were divided into the following categories based on physician-documented diagnosis: asymptomatic control, nonulcerative interstitial cystitis (IC), overactive bladder with incontinence (OAB wet), urinary tract infection (UTI), and bladder cancer. Urinary cytokines were measured through Luminex multiplex assay. Results: Two hundred and seventy-seven urine samples were collected from three clinical sites. Urinary pro-inflammatory cytokines had an increasing trend in bladder disease versus control, with a significant increase for chemokine (C-X-C) ligand 1 growth-regulated protein alpha CXCL1 (GRO). Further analyses demonstrated that patients with UTI had significantly higher levels of GRO and interleukin-8 (IL-8) in comparison to control, nonulcerative IC, OAB wet, and bladder cancer. Both are chemokines that stimulate chemotaxis resulting in the rapid accumulation of immune cells such as neutrophils. IL-6 levels overall were at the lower limit of assay range but were significantly increased in urine of UTI patients versus IC patients. MCP-1 (CCL2) had the least separation among the control group and the various bladder diseases. Conclusion: Urinary concentrations of GRO were higher in disease state compared to control. Specifically, levels of GRO and IL-8 were higher in urine samples from patients with UTI compared to controls and other bladder conditions. Comparing and contrasting urinary cytokines may help improve our understanding of these important bladder diseases with great unmet needs.
Keywords: Bladder cancer, interstitial cystitis, overactive bladder, urinary tract infection
Interstitial cystitis (IC)/bladder pain syndrome (BPS) is a debilitating chronic disease that affects roughly 3–8 million women and 1–4 million men in the United States.[1],[2] Patients with IC/BPS are identified based on the presence of Hunner's lesions, though only an estimated 10% of IC/BPS cases present with these bladder lesions.[3] Diagnosis of IC/BPS is based on patient-reported symptoms and exclusion of other diseases with overlapping clinical presentation, such as overactive bladder with incontinence (OAB wet), urinary tract infection (UTI), and bladder cancer. Bladder cancer and IC patients, especially those IC with Hunner's lesion (HIC), are most often diagnosed by cystoscopy with hydrodistension.[4] However, this can be painful and requires anesthesia. Some patients may experience painful urination, hematuria, UTI, and rare risk of bladder wall perforation. Identifying these patients presents a therapeutic opportunity as bladder-directed therapies such as electrocautery, resection, or injection of these lesions with steroids can provide temporary symptom relief. Given the invasiveness and the side effects of cystoscopy with hydrodistension, there is a strong need for a noninvasive, rapid, diagnostic test to easily identify and diagnose bladder diseases, and to measure patients' response to therapy.
Urine cytokines have been of high interest as potential biomarkers for IC, and there is a strong suggestion that urinary cytokines are elevated with IC.[5] A valid biomarker for diseases with similar symptomatology such as IC, bladder cancer, UTI, or OAB wet would be advantageous for both clinicians and patients. Our research group has previously identified three urinary cytokines, interleukin-6 (IL-6), IL-8, and GRO, to be elevated in IC/BPS patients with Hunner's lesions in comparison to patients without Hunner's lesions or healthy controls.[6] In addition, IL-8 and IL-6 were found to be significantly higher in urine from nonulcerative IC/BPS patients versus healthy controls.[6] However, bladder inflammation is also common in other bladder conditions. In a study by Kumari et al., 12 urinary cytokines, including IL-8, were identified to be more abundant in patients with bladder cancer versus healthy controls.[7] Likewise, increased urinary cytokines have been identified during active UTI and in patients with OAB.[8],[9],[10] Thus, we saw a need for a study to directly compare urinary cytokines in several confusable bladder conditions.
The objective of this study was to compare the levels of four urinary cytokines that we previously identified as being elevated in IC/BPS to determine if they can be used to differentiate different types of bladder diseases.[6] We collected urine samples from patients with clinically diagnosed nonulcerative IC/BPS, bladder cancer, OAB wet, or UTI, and healthy controls. We sought to understand if there were differences in urinary cytokine concentration between these conditions.
Materials and MethodsStudy population
Urine samples were collected with local Internal Review Board approval from three academic urology centers from different regions of the United States, Montefiore Medical Center Albert Einstein College of Medicine in Bronx, New York (IRB #2019-10949), Baylor College of Medicine/Michael E. DeBakey VA Medical Center in Houston, Texas (IRB #H-49346), and Beaumont Health, Royal Oak, Michigan (IRB #2019-266). This study was also approved by the Department of Defense Human Resource Protect Office (E00749) and aimed at enrolling participants with IC and without IC including asymptomatic/normal controls, bladder cancer, current UTI, and OAB wet (OABw) patients. All participants had confirmed physician diagnosis for nonulcerative IC, bladder cancer, UTI, and/or OAB wet. Urine collection was carried out by trained clinical research staff. A urine preservative (Norgen Biotek, Thorold, ON, Canada) was added to all urine samples immediately after collection to allow for storage of urine samples at room temperature up to 1 year. Urine samples and corresponding surveys were deidentified and marked with a unique study identification number before shipping to Beaumont through FedEx.
Patients meeting study inclusion and exclusion criteria as described in [Table 1] were recruited by the study physicians at their respective clinic. Candidates with IC were given the diagnosis based on NIDDK criteria and satisfied the inclusion and exclusion criteria. All physicians involved in this study are fellowship trained and have years of experience evaluating women with IC. Patients with OABw, UTI, and bladder cancer were recruited in the clinic if they met all inclusion and exclusion criteria [Table 1].
Visual Analog ScaleA 10-point VAS for maximum daily pain was utilized where 0 is no pain and 10 is maximum pain. Extensive research supports the validity of the VAS for assessment of pain intensity.[11]
Multiplex Luminex assay
Samples were aliquoted and stored at room temperature and ten 1.0 mL aliquots were stored at −80°C. Samples were randomized and assigned an experimental ID number to run and analyze samples in a blinded fashion. Expression of a panel of cytokines (GRO, IL-6, IL-8, and MCP-1) was determined using the MILLIPLEX MAP Human Cytokine/Chemokine Multiplex Immunoassay (Millipore) following manufacturer's protocol and as previously described.[6]
Statistical analysis
Two hundred and seventy-seven urine samples were collected from three clinical sites. To compare cytokine levels between control and disease groups, a two-tailed nonparametric independent t-test was performed using Mann–Whitney U-test. To determine statistical difference between control and different disease states, we performed a Kruskal–Wallis nonparametric test followed by Dunn's multiple comparison test. P < 0.05 was considered statistically significant.
ResultsPatient population
Patient demographics among the groups are listed in [Table 2]. Among the three sites, a total of 277 urine samples were collected: 31 urine samples were collected of healthy controls and 246 of patients with OAB wet (n = 84), nonulcerative IC/BPS (n = 83), bladder cancer (n = 61), and UTI (n = 18). For all conditions, more women were enrolled in the study, except for bladder cancer. The age ranges of study participants were similar among the five groups, though the average age for bladder cancer was the highest followed by UTI (73.4 and 69.3, respectively). The VAS score indicated that the patients with IC/BPS and UTI reported higher pain scores than control, OAB wet, and bladder cancer groups.
Increase in cytokine levels in bladder disease
Urinary cytokine levels were measured through Luminex multiplex assay. Urinary concentrations of IL-6, IL-8, MCP-1, and GRO were measured. Initially, cytokine levels were compared between control and disease treatment groups [Figure 1]. Of the four cytokines investigated, all cytokines were higher in disease state versus control. However, only the increase in GRO (CXCL1) was statistically significant. We further divided the disease states into nonulcerative IC, OABw, UTI, and bladder cancer to identify potential disease-specific changes in cytokine levels [Table 3] and [Figure 2]. Overall, average cytokine levels had a higher trend in all four diseases states studied, but only urine samples from patients with UTI had significantly higher levels of GRO and IL-8 compared to control samples and other bladder conditions studied. IL-6 levels were also significantly higher in UTI samples versus IC samples, though in most samples, IL-6 concentrations were below the lower level of detection of the assay (3.2 pg/ml). All samples were adjusted for creatinine.
Figure 1: Urinary chemokine GRO is significantly increased in bladder disease versus normal control. Protein concentrations of (a) GRO, (b) interleukin-6, (c) interleukin-8, and (d) MCP-1 were measured in urine samples of patients with bladder disease and healthy controls using multiplex Luminex assay. Statistical analysis was carried out using Mann–Whitney U-test. ****P < 0.0001
Figure 2: Urinary tract infections result in increased cytokines that are distinct from other bladder conditions. Protein concentrations of (a) GRO and (b) interleukin-8 were measured in urine samples of healthy controls and patients with IC, OABw, UTI, or bladder cancer using multiplex Luminex assay. Statistical analysis was carried out using Kruskal–Wallis nonparametric test followed by Dunn's multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 Discussion In this study, we sought to determine if urinary GRO, IL-6, IL-8, and MCP-1 were increased in patients with bladder disease in comparison to healthy control samples, and if there was a distinction in their urinary levels between different bladder conditions (nonulcerative IC, OABw, UTI, and bladder cancer). These four cytokines were specifically chosen as our group has previously identified these cytokines as potential biomarkers for the diagnosis of IC.[6],[12]
Chemokines are divided into four families (CXC, CC, C, and CX3C) based on the relative position and the number of conserved N-terminal cysteine residues as well as the absence (CC) or presence of intervening amino acid(s) between the cysteine residues (CXC). The CC class of chemokines, such as MCP-1, provokes mast cell activation and has chemotactic activity for monocytes but not for neutrophils.[13] Bacterial infection is reported to induce higher levels of chemokines from the CXC family versus the CC family.[14]
Previous in vitro studies using detrusor muscle cells have shown that low levels of chemokines and cytokines are secreted in a healthy bladder state. It has been further demonstrated that basal levels of cytokines and chemokines are responsible for the autocrine, paracrine, and endocrine signals in the detrusor, and that these chemokines are produced at higher levels during inflammation.[13],[15] In the urine samples of asymptomatic control study participants, we have detected low levels of GRO, IL-8, and MCP-1, suggesting that these cytokine levels reflect the amount needed to maintain basal immune surveillance [Table 3].
When comparing each of the cytokines in disease versus healthy state, an increase in cytokines appears to be a common characteristic among different bladder conditions. GRO (CXCL1) specifically is elevated in the urine of patients with IC, OABw, UTI, and bladder cancer. Preclinical studies have shown that increased urine levels of Th2-polarizing factor MCP-1 and GRO accompany bladder inflammation.[16] GRO is a chemokine that acts as a chemoattractant, hereby playing an important role in inflammation and attracting neutrophils to the site of infection. Neutrophils subsequently produce cytokines and inflammatory factors themselves that help regulate the immune response and aid in the resolution of inflammation.[17] Initially, it was thought that the role of neutrophils was limited to an antimicrobial function, but this role has been widely expanded.[17] The elevated levels of GRO in bladder conditions combined suggest that neutrophils may play a role in each of these bladder conditions.
When analyzing the bladder conditions separately, GRO was elevated significantly in urine samples of UTI and OABw patients, though GRO levels for OABw were significantly lower than those seen in UTI. Bacterial infection is reported to induce higher levels of chemokines from the CXC family (e.g., GRO) versus the CC (e.g., MCP-1) family, which we also observe in this study.[14] The presence of increased levels of neutrophil-specific chemokine GRO in urine from UTI and OABw patients, therefore, suggests an infiltration of neutrophils in the bladder tissue. Given the role of neutrophils in fighting bacterial infections, the high levels of GRO in UTI are not surprising. A recent study identified that the neutrophil-to-lymphocyte ratio is increased in the serum of patients with OAB.[18] Thus, the increased levels of GRO in OABw could indicate that neutrophils play an important role in mediating OAB symptoms. The finding that UTI and OABw have different cytokine profiles suggests that different inflammatory pathways are activated following UTI versus OAB. The quantitative differences in the elevation of specific chemokines in UTI and OAB may assist in differentiating two disease categories.
Like GRO, IL-8 levels are significantly higher in patients with UTI versus control, nonulcerative IC, OABw, and bladder cancer [Table 3] and [Figure 2]. Elevated seminal plasma levels of IL-8 have been linked to the symptoms of chronic prostatitis/chronic pelvic pain syndrome and nonspecific urethritis.[19] IL-8 plays an important role in bladder inflammation and is secreted by infiltrating immune cells and resident immune cells present in both the urothelium and detrusor.[13],[15],[20] Elevated levels of IL-8 have also been reported in bladder cancer, where neoplastic cells are capable of secreting IL-8.[21],[22]
A weakness of the study is that the stage of disease was not taken into consideration. As observed in [Table 3], the standard deviations vary greatly between patients, which could be explained by disease stage. For IC, it was previously shown that IL-6, IL-8, and GRO were increased in IC patients with Hunner's lesions versus those without lesions.[6] Our IC cohort included urine samples of both types of IC, though the number of patients with Hunner's lesions enrolled (n = 10) was too low to draw any conclusions. In addition, there may be differences between patients going through an IC flare-up versus those who are not. In bladder cancer, higher urinary levels of IL-8 are associated with increased risk of recurrence.[22] However, our analysis did not take stage of bladder cancer or ongoing treatment into consideration.
ConclusionUrinary concentrations of GRO were higher in disease state compared to control. Specifically, levels of GRO and IL-8 were higher in urine samples from patients with UTI compared to controls and other bladder conditions. These findings provide preliminary data for further studies. Comparing and contrasting urinary cytokines may help improve our understanding of these important bladder diseases with great unmet needs.
Acknowledgments
The authors gratefully acknowledge the IC community that contributed to this study and the Interstitial Cystitis Association (ICA; http://www.ichelp.org/) for assistance with participant recruitment and supporting IC research.
Financial support and sponsorship
This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Technology/Therapeutic Development Research Program under Award No. W81XWH-19-1-0288. The opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense. This work was also supported by Oakland University William Beaumont School of Medicine's EMBARK capstone program. The American Urological Association also supported this work through 2021 Summer Medical Student Fellowship Award.
Conflicts of interest
LEL, JJ, and MBC have intellectual property associated with methods for diagnosing IC. All other authors declare no competing interests.
Prof. Michael B. Chancellor, an editorial board member at Urological Science, had no role in the peer review process of or decision to publish this article. The other authors declared no conflicts of interest in writing this paper.
References
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