TH2 cytokines with predominance of TH2, in urothelial carcinoma of bladder

Urologic Oncology: Seminars and Original Investigations 29 (2011) 58 – 65

Original article

A disproportion of TH1/TH2 cytokines with predominance of TH2, in urothelial carcinoma of bladder夡 Abhigyan Satyam, M.Sc.a, Prabhjot Singh, M.S.b, Nitika Badjatia, M.Sc.a, Amlesh Seth, M.Ch.b, Alpana Sharma, Ph.D.a,* a

Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India b Department of Urology, All India Institute of Medical Sciences, New Delhi, India

Received 11 April 2009; received in revised form 4 June 2009; accepted 11 June 2009

Abstract Objectives: Bladder cancer is a common tumor of the urinary tract, accounting for 6% to 8% of all male malignancies and 2% to 3% of all female malignancies. Urothelial carcinoma (UC) of bladder is the second most common urologic malignancy after prostate cancer. Earlier report has elucidated immunologic unreactivity in cancer patients. Cytokines play a pivotal role in the induction of cell mediated and humoral immunity. Quantification of cytokine response in cancer patients can give significant insights about the cellular immunologic potency against the neoplastic cells. In the present study, we aimed to assess alterations of Th1 and Th2 derived cytokines in progression of UC of bladder by determining their circulatory concentration in bladder cancer patients and healthy controls and to correlate the observations with grade and severity of the disease. Materials and methods: The study cohort consisted of 122 subjects; 72 patients with bladder UC (28, low grade; 17, high grade; 27, muscle invasive) and 50 healthy controls. The circulatory levels of various cytokines were measured using commercially available sandwich enzyme linked immunosorbent assay (ELISA) kit from BD Biosciences, San Diego, CA, and were statistically correlated according to the grade and the severity of disease. Results: The serum levels of typical Th1 cytokines: IL-2 and IFN-␥ were found to be significantly lower (P ⬍ 0.001) while levels of Th2 cytokines i.e., IL-4, IL-5, and IL-10 were significantly higher (P ⬍ 0.001) in patients than in controls. The levels of all the cytokines were correlated with the grade and severity of the disease. There were significant differences between the patients with low grade tumors and muscle invasive tumors for all cytokines (P ⬍ 0.001); except IL-10 (P ⬍ 0.626). Conclusions: The results of our study delineate that in bladder tumor patients a marked polarization exists towards the expression of Th2 type cytokines while Th1 remain suppressed. Furthermore, the levels of all the cytokines alter according to the grades of the tumor. This can give significant insights about the use of Th1 type cytokines for the administration of immunotherapy to bladder cancer patients. Development of new strategies attempting to manipulate the equilibrium between Th1 and Th2 cells would be beneficial in the management of UC of bladder in future. © 2011 Elsevier Inc. All rights reserved. Keywords: Urothelial carcinoma of bladder; TH1 cytokines; TH2 cytokines; Urinogenital cancer; T helper cells; TH1/TH2

1. Introduction Bladder cancer is a common tumor of the urinary tract, accounting for 6% to 8% of all male malignancies and 2% to 3% of all female malignancies [1]. Urothelial carcinoma

夡 This work was financially supported by the Indian Council of Medical Research, New Delhi, India. * Corresponding author. Tel.: ⫹0091-98-99061974; fax: ⫹0091-1126588641. E-mail address: [email protected] (A. Sharma).

1078-1439/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2009.06.002

(UC) of bladder, accounting for about 90% of all bladder tumors [2], is the second most common urologic malignancy after prostate cancer [3]. UC of the urinary bladder presents a spectrum of tumors ranging from indolent nonprogressive to invasive cancer [4]. An earlier report has elucidated immunologic unreactivity in bladder cancer patients. This immunodeficiency is manifested by impaired cell mediated and humoral immunity as well as by impaired nonspecific host defense mechanisms [5]. Cytokines play a pivotal role in the induction of cell mediated and humoral immunity. Cytokines are re-

A. Satyam et al. / Urologic Oncology: Seminars and Original Investigations 29 (2011) 58 – 65

leased in response to a diverse range of cellular stresses, including carcinogen induced injury, infection, and inflammation [6,7]. Host reactions to cellular stresses can impact on several stages of cancer initiation and progression. Cytokines secreted by tumors and inflammatory/immune cells can either promote tumor development and tumor survival or exert antitumor effects. Tumor cells can exploit hostderived cytokines to promote growth, increase resistance to apoptosis, and to foster dissemination [6]. Thus, quantification of cytokine response in cancer patients can give significant insights about the cellular immunologic potency against the neoplastic cells [8]. It has been reported that qualitative as well as quantitative alterations in cytokine production can result in activation of inefficacious effector mechanisms and, therefore, complex and severe impairment in immune function [9]. The pivotal role played by T-helper cells in amplifying immune responsiveness is well established, and at least 4 main subsets have been described to date: Th1, Th2, Th17, and T regulatory cells (Tregs) [10]. Th1 cells produce type 1 cytokines: IL2, IL12, IL15, and IFN-␥. These cells promote cell mediated immunity and are required for effective responses to intracellular pathogens and tumor cells. Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13 which favor humoral immunity against extracellular pathogens and allergic responses [11,12]. The characteristic cytokine products of Th1 and Th2 cells are mutually inhibitory for the differentiation and effector functions of the reciprocal phenotype [13]; IFN-␥ inhibits Th2 cell functions whereas IL-4 and IL-10 are reported to inhibit Th1 response [14]. Th17 cells release IL-17 and IL-22, the information about the relevance of this cell subset in cancer biology is scant and contradictory [15]. Treg, which function as a protective mechanism against autoimmunity, may also mitigate the immune response against cancers, but the interactions between human tumors and Treg are unknown [16]. Th1 cytokines exert potent antitumor effects; IL-12, IL-2 and IFN-␥ activate cytotoxic lymphocyte (CTL) mediated and natural killer (NK) mediated cytolytic functions associated with effective antitumor defense mechanisms [17]. On the other hand, Th2 type cytokines were shown to down regulate tumor specific immune response by directly suppressing IFN-␥ and IL-12 production, thereby preventing activation of CTLs and NK cells [18], and by inhibiting tumor antigen presentation by antigen-presenting cells [19]. The generation of classic cell-mediated responses, where Th1 derived cytokines, cytotoxic CD8⫹ T cells, macrophages, and NK cells are appropriately activated, are all crucial for the generation of a potent antitumor immune response and tumor rejection [20,21]. However, a number of mouse and human tumors are associated with a more pronounced Th2 response with elevated Th2 cytokines and suppressed antitumor CTL responses. The macrophages associated with developing solid tumors also have a phenotype driven primarily by Th2 cytokines that supports a protumor microenvironment [22,23]. The shift from Th1 to

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Th2 cytokine production might facilitate the tumor progression by subverting cellular immune responses [19,24]. Though studies aiming to elucidate the Th1/ Th2 balance have been performed in several types of malignancies, the role of these two subsets in immunologic dysfunction in bladder cancer patients is not clearly defined. The present study was aimed to evaluate the role of Th1 and Th2 derived cytokines in the progression of urothelial cell carcinoma of bladder by determining the circulatory concentration of various cytokines in bladder cancer patients and healthy controls and to correlate the observations with grade and severity of the disease.

2. Subjects and methods 2.1. Subjects The study included 72 patients with UC of bladder who were admitted to the Department of Urology at All India Institute of Medical Sciences, New Delhi, and 50 healthy age- and gender-matched controls. There were 45 patients with superficial bladder UC and 27 with muscle invasive UC of bladder. The superficial urothelial papillary tumors were graded according to the 2004 World Health Organization (WHO) grading system into low grade and high grade papillary neoplasms. There were 28 patients with low grade UC and 17 with high grade UC. The study protocol was approved by the ethical committee, and the patients and controls gave informed consent. The patient and control groups were of similar socioeconomic status. All the study subjects had normal serum albumin levels (⬎3.5 gm/dl) and were not anemic (hemoglobin ⬎ 12 gm/dl). None of the patients had any other significant disease or malignancies except bladder cancer, and only the newly diagnosed patients with no prior chemotherapeutic treatment were included in this study. The exclusion criteria were suffering from any inflammatory or chronic disease like diabetes, autoimmune disorders, or acute infections at the time of sampling. Total 4 ml venipuncture blood was collected from study subjects in plain endotoxin-free vials. The tubes were centrifuged for 10 minutes at 3000 rpm; serum was separated, and stored at ⫺20°C for further use. 2.2. Cytokine determinations The levels of various cytokines were measured by high sensitivity enzyme linked immunosorbent assay (ELISA) by using commercially available kits supplied by BD Biosciences (San Diego, CA). A monoclonal antibody specific for different cytokines (IL-2, IFN-␥, IL-4, IL-5, and IL-10) had been coated onto the wells of the microtiter strips provided during first incubation, IL-2/IFN-␥/IL-4/IL-5, and IL-10 present in the sample or standard and a monoclonal anti-IL-2/IFN-␥/IL4/IL-5, and IL-10 antibody conjugated to biotin were simul-

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Table 1 Demographic and clinical data for the bladder cancer patients and the control subjects Patients Total no. (n) Male/female Age (years) (Mean ⫾ S.D.) Clinical grading Low grade High grade Muscle iInvasive Controls Total no. (n) Male/female Age (years) (Mean ⫾ S.D.)

72 54/18 35–72 (53.9 ⫾ 10.48) 28 17 27 50 38/12 32–67 (49 ⫾ 6.45)

taneously incubated in respective microtiter plates. Following incubation, unbound IL-2/IFN-␥/IL-4/IL-5, and IL-10 are removed during a wash step. Streptavidin-HRP was added and bound to the biotinylated anticytokine antibody. After incubation and washing, substrate solution that reacts with HRP was added to the wells. A colored product was formed proportional to the amount of IL-2/IFN-␥/IL-4/IL-5, and IL-10 present in sample. The reaction was terminated by addition of acid and absorbance was measured at 450 nm. According to the manufacturer, the minimum detectable concentrations (MDCs) and intra-assay and inter-assay coefficient of variation (CV) were as follows: IFN-␥: MDC 1 pg/ml, intra-assay CV less than 4% and inter-assay CV less than 10%; IL-2: MDC 1 pg/ml, intra-assay CV less than 6% and inter-assay CV less than 4.5%; IL-4: MDC 2 pg/ml, intra-assay CV less than 7% and inter-assay CV less than 7.4%; IL-5: MDC 2 pg/ml; IL-10: MDC 2 pg/ml, intra-assay CV less than 2.7% and inter-assay CV less than 10.8%. 2.3. Statistical analysis Statistical assessment was carried out with the SPSS 12.0 for Windows (SPSS Inc., Chicago, IL) statistical software. Data are expressed as mean ⫾ SD. Differences in mean values of the parameters between bladder cancer patients of various groups and control subjects were analyzed statistically using one-way analysis of variance (ANOVA) test and the significance value between various groups was calculated using Bonferroni post hoc test. A P value ⬍ 0.05 was considered statistically significant. Similarly, the differences in Th1/Th2 ratio were analyzed using one way ANOVA and subsequently Bonferroni post hoc test to calculate inter-group significance value.

3. Results 3.1. The study groups The study group comprised of 122 subjects, including 72 patients with UC of bladder (54 men, 18 women) and 50

healthy controls (38 men, 12 women). The age range was 35–72 years (mean 53.9 ⫾ 10.48) for the patients and 32– 67 years (mean 49 ⫾ 6.45) for the control subjects. According to 2004 WHO grading of superficial carcinoma of bladder, 28 patients were found to have low grade tumors, 17 were found to have high grade tumors, and 27 patients were having muscle invasive UC of bladder (Table 1). 3.2. The cytokine profile The mean levels of various cytokines analyzed in patients with UC of bladder and the control subjects are shown in Table 2. Highly significant differences were observed between the mean values of each cytokine level assayed in bladder cancer subjects compared with healthy controls (P ⬍ 0.001). 3.3. TH1 cytokine levels The mean value of Th1 cytokines; IFN-␥, and IL-2 were significantly reduced (P ⬍ 0.001) in patients (2.74 pg/ml and 2.92 pg/ml, respectively) compared with normal healthy controls (7.31 pg/ml and 6.30 pg/ml, respectively) (Table 2). Comparison of Th1 cytokine levels between the control group with different stages and grades of bladder UC is shown in Fig. 1. The decrease in levels of type 1 cytokines showed correlation with grade and stage of tumors. The circulatory levels of both IFN-␥ and IL-2 in low grade tumors were significantly lower than healthy controls (P ⬍ 0.001) and significantly higher than muscle invasive tumors (P ⬍ 0.001 for IFN-␥ and P ⬍ 0.003 for IL-2). No significant decrease in IFN-␥ and IL-2 levels was observed in high grade tumors compared with low grade tumors (P ⬍ 0.223 and P ⬍ 0.103, respectively, for IFN-␥ and IL-2). Also, the decrease was insignificant in muscle invasive tumor patients compared with high grade tumors for both Table 2 Levels (pg/ml) of cytokines in bladder cancer patients and healthy controls Cytokine levels IFN-␥ (pg/ml)* Mean ⫾ S.D. Median (range) IL-2 (pg/ml)* Mean ⫾ S.D. Median (range) IL-4 (pg/ml)* Mean ⫾ S.D. Median (range) IL-5 (pg/ml)* Mean ⫾ S.D. Median (range) IL-10 (pg/ml)* Mean ⫾ S.D. Median (range)

Patients (n ⫽ 72)

Controls (n ⫽ 50)

2.74 ⫾ 1.74 2.24 (1.2–9.40)

7.31 ⫾ 2.44 7.35 (2.95–14.25)

2.92 ⫾ 1.76 2.50 (1.25–9.40)

6.30 ⫾ 1.88 6.09 (2.05–10.20)

33.20 ⫾ 9.90 31.32 (16.5–59.3)

5.53 ⫾ 2.24 5.37 (2.0–10.8)

33.83 ⫾ 11.39 30.50 (18.4–62.4)

5.50 ⫾ 2.34 4.6 (2.2–11.3)

21.07 ⫾ 5.89 19.85 (10.4–38.5)

5.01 ⫾ 2.53 3.7 (2.2–14.3)

* Significance (P ⬍ 0.001) for all the cytokines in patients vs. controls.

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Fig. 1. Box plot representation of serum concentration of Typical Th1 Cytokine in pg/ml. (A) Levels of IFN-␥ and (B) levels of IL-2. Top and bottom lines of boxes are 25th and 75th percentile and the band in the middle of box is the median. The ends of whiskers represent 2nd and 98th percentile. Dots indicate outliers. LG ⫽ low grade; HG ⫽ high grade; MI ⫽ muscle invasive tumor patients; CTRL ⫽ healthy controls.

IFN-␥ and IL-2 (P ⬍ 1.0). But the decrease was highly significant in both the grades and muscle invasive disease compared with healthy individuals (P ⬍ 0.001). 3.4. TH2 cytokine levels Significantly enhanced values of typical Th2 cytokines IL-4, IL-5, and IL-10 were observed in patients (33.20 pg/ml, 33.83 pg/ml, and 21.07 pg/ml, respectively) compared with healthy volunteers (5.53 pg/ml, 5.5 pg/ml, and 5.01 pg/ml, respectively) (Table 2). Mean levels of all type 2 cytokines in low grade, high grade, and muscle invasive patients showed significant increase compared with controls (P ⬍ 0.001). Comparison of Th2 cytokine levels between the control group with different stages and grades of bladder UC is shown in Fig. 2. The circulatory levels of IL-4, IL-5, and IL-10 in patient with low grade tumors did not differ

significantly from high grade tumors (P ⬍ 0.1 for IL-4 and IL-10; P ⬍ 0.512 for IL-5), while a significant increase was observed for IL-4 and IL-5 in muscle invasive tumors when compared with low grade tumors (P ⬍ 0.014 and P ⬍ 0.001, respectively), but not for IL-10 (P ⬍ 1.0). No significant increase was observed in the levels of IL-4 and IL-10 in patients with muscle invasive tumors as against high grade tumors (P ⬍ 1.0) but the levels of IL-5 showed highly significant increase (P ⬍ 0.001). The statistical significance of the results in all study groups is clearly illustrated Table 3. 3.5. TH1/TH2 ratio A possible shift from Th1 to Th2-type activity was characterized by analyzing the ratio of IFN-␥ and IL-2 with various Th2 cytokine, i.e., IL-4, IL-5, and IL-10 for all

Fig. 2. Box plot representation of serum concentration of typical Th2 cytokine in pg/ml. (A) Levels of IL-4, (B) levels of IL-5, and (C) levels of IL-10. Top and bottom line of boxes are 25th and 75th percentile and the band in the middle of box is the median. The ends of whiskers represent 2nd and 98th percentile. Dots indicate outliers. LG ⫽ low grade; HG ⫽ high grade; MI ⫽ muscle invasive tumor patients; CTRL ⫽ healthy controls.

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Table 3 Statistical significance of results in all study groups Significance

IFN-␥

IL-2

IL-4

IL-5

IL-10

Low grade vs. controls Low grade vs. high grade Low grade vs. muscle invasive High grade vs. controls High grade vs. muscle invasive Muscle invasive vs. controls

0.001 0.223 0.001 0.001 1.000 0.001

0.001 0.103 0.003 0.001 1.000 0.001

0.001 1.000 0.014 0.001 1.000 0.001

0.001 0.512 0.001 0.001 0.001 0.001

0.001 1.000 0.626 0.001 1.000 0.001

study groups. The ratio of IFN-␥ vs. IL-4, IL-5, and IL-10 decreased significantly in patients compared with controls (P ⬍ 0.001). No significant difference in the ratio was observed amongst various groups of patient, i.e., low grade, high grade, or muscle invasive, indicating that this shift is not significant with respect to grade of tumor. Similar results were obtained for the ratio of IL-2 with IL-4, IL-5, and IL-10 as shown in Fig. 3.

4. Discussion The cancer immunoediting hypothesis combines 2 fundamental concepts of tumor immunology, immunosurveillance, and tumor immune escape to generate a microenvironment of tumor tolerance and resulting malignant growth [25]. It has been well established that malignant cells are able to escape immune surveillance using a number of strategies, among which the production and secretion of soluble factors capable of interfering with the function of the various immune cell population involved in mounting specific antitumor response appears to be a major mechanism [26]. Cytokines such as the interleukins have functions in regulating cancer immuno-response and are, therefore, studied extensively for cancer therapy [27]. An important con-

cept in tumor immunology is that 2 distinct cytokine patterns may be generated by Th1 and Th2 lymphocytes, which have opposing effects on each other [28]. Evidence has accumulated that cancer patients have an imbalance in the Th1/Th2 axis, with the latter predominating over the former. Differences in costimulatory molecule expression, extinction of IL-12 signaling, modification of promoter binding proteins, and soluble factors such as prostaglandins have been reported to modulate the Thl/Th2 axis [14]. The cytokines produced by Th1 and Th2 cell subsets of CD4⫹ play a vital role in determining the functionality and the immune response of cytotoxic T-lymphocytes (CTL) because these can regulate the differentiation of these cells. Th1 cytokines are required for the generation of effector CTLs that can cause lysis of tumor cells. This is consistent with the finding that over expression of Th2 type cytokines, with high IL-10 and low IL-2 and IFN-␥ can impair immune responses against tumors [24]. We analyzed circulatory levels of cytokines, and our study showed a significant decline in Th1-type cytokines and significant rise in the levels of typical Th2 cytokines analyzed in all bladder cancer patients compared with controls in correlation with the severity of disease. The imbalance observed in the present study is in accordance with other reports in patients with non-small cell lung cancer [14], oral cancer [29], cervical cancer [19,30], and bladder cancer [24]. The significant decline in interferon-␥ and IL-2 observed in the present study might be an important factor contributing to tumor development as reported by previous findings [19,24,30]. IFN-␥ working synergistically with TNF-␣ prevent bladder tumor proliferation as well as up-regulate the expression of major histocompatibility complex class I and II antigens and adhesion molecules on bladder cancer cells following tumor resection [31]. The result of this is a change in the surface phenotype, which increases the probability of

Fig. 3. Cytokine ratios (Th1/Th2) in controls and bladder cancer patients of various grade. (A) Ratio of IFN-␥ vs. various Th2 cytokines. (B) Ratio of IL-2 vs. various Th2 cytokines. LG ⫽ low grade; HG ⫽ high grade; MI ⫽ muscle invasive tumor patients.

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tumor cell to be recognized by the CD4⫹ T cells or CD8⫹ cytotoxic T lymphocytes [32]. IFN-␥ is able to induce the expression of Fas receptor on bladder cancer cells, providing a mechanism for apoptotic destruction by Fas ligand positive activated T cells, usually associated with a Th1 response [24]. Also, high levels of urinary IFN-␥ is associated with better prognosis in bladder cancer patients [33]. Thus, lowering of IFN-␥ observed in our study might be responsible for augmented proliferation of bladder tumor cells and inability of tumor cells to be recognized by CTLs. Another Th1 type cytokine, interleukin 2 (IL 2), found to be lowered in our study, is a true biologic response modifier. Unlike interferons, which act directly, IL2 mediates its antitumor activity through complex indirect effects on the immune system. IL-2 used alone shows documented antitumor activity, which is both dose- and schedule-related [34]. In a study done by Kaempfer et al. in bladder cancer patients, it was shown that patients whose lymphocytes show a depressed ability to make IL-2 mRNA have poor clinical response [35]. Also, according to the study by Jackson et al., patients with high urinary IL-2 levels show more potent antitumor response [33]. Thus, in the present study, decreased amount of Th1 cytokines indicates that the proliferation and sustenance of tumor cells in patients with bladder cancer might be due to immune-compromised state of the patients due to lowering of key Th1 cytokines that have vital antitumor activities. IL-10 has a variety of suppressive effects that include inhibiting Th1 cytokine production, down-regulating APC and NK cell function, and lowering overall T-cell proliferation. Especially under the influence of IL-4, tumor cells apparently up-regulate IL-10 that suppresses nearby killer cells [36]. High IL-10 levels have been detected in sera from patients with a wide variety of solid and hematopoietic tumors [14,19,24,26,37,38]. IL-10 production contributes to malignant tumor cell survival by generating a microenvironment conducive to tumor growth and metastases. Additional evidence for the involvement of IL-10 in tumor immunosuppressive networks has come from studies employing IL-10 transgenics, IL-10 knockouts, and IL-10 transfectants. Whereas IL-10 transgenic mice are extremely sensitive to growth of tumors and demonstrate impaired development of effective immune responses against tumors, IL-10 knockout mice are resistant to moderately immunogenic transplantable tumors that readily grow in wild type mice [39]. Our result demonstrating higher levels of IL-10 in cancer patients than controls is consistent with the findings of Agarwal et al. in bladder cancer [24], Galizia et al. in colon cancer [26], Sharma et al. [19], and Bais et al. [30] in cervical cancer. In all these studies, elevated expression of IL-10 was found compared with healthy individuals, clearly indicating the role played by IL-10 as a tumor enhancing agent. IFN-␥ and other Th1 cytokines are typically lowered in advanced cancer patients, while the Th2 markers like IL-4 can be higher or unchanged [40]. Nodules of non-small cell

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lung cancer freshly removed from patients expressed a marked imbalance toward Th2 [14]. In prostate cancer patients, IL-2 was low and IL-10 high [41]. This shows that in tumor tissue there is a marked suppression of Th1 cytokines while Th2 cytokines are over expressed. T lymphocyte priming in presence of high concentrations of IL-4 and IL-10 favors maturation of Th2 cells. Thus, type 2 secreting tumor cells could induce a preferential priming of type 2 cytokine secreting lymphocytes with generation of selfamplifying loop [19]. The strong bias towards Th2 cytokines might be a strategy used by neoplastic cells to escape immune surveillance the by host immune system. With both IL-4 and IL-10 proven to be inhibitors of Th1 and promoters of Th2 activity, the recognized capability of cancerous tissue to suppress immunity is readily rationalized [36]. We have observed a significant lowering of Th1/Th2 cytokine ratio in bladder cancer patients compared with healthy controls, though this lowering was insignificant between various groups of patients. Possibly as result of imbalance in Th1/Th2 cytokine production, there is a dysregulation in the functionality of Th1 and Th2 cells. There is only a single previous report by Agarwal et al. [24] about the Th1/Th2 imbalance in peripheral blood mononuclear cells of 41 patients with superficial papillary TCC of bladder. Our results are in accordance with that of Agarwal et al.; moreover we have also included muscle invasive patients in our study and correlated our data with respect to grades of the tumor. Furthermore, inherent oxidative stress in cancer patients can also be one of the reasons that can result in imbalance in Th1/Th2 axis, as oxidative stress plays a major role in inflammation [36]. It is possible that immune activity can have Th1 or Th2 character depending on the relative antioxidant status of the cells directing the process [42– 44]. It can be concluded from previous reports that antioxidant status at the level of the immune cell and its microenvironment can markedly affect the ultimate pattern of immune response. Our group has previously shown that in the same bladder cancer patients [45] and cervical cancer patients [46], a marked antioxidant depletion and increased lipid peroxidation is present in circulation, which also correlates with the severity of the disease. Thus, the presence of oxidative stress might be responsible for a polarized Th2 type response observed in the present study. To allow the generation of a specific and productive immune response, which destroys the tumor, it is vital to elucidate how a shift in cytokine balance can be achieved that can provide a therapeutic benefit to bladder cancer patients. Thus, immunotherapy in bladder cancer patients should be directed towards the modulation of a Th2-like immunosuppressive response that possibly exists in these patients towards a Th1-like protective cell-mediated immune response [24,47]. In conclusion, the results of our study delineate that in bladder tumor patients, a marked polarization exists towards

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the expression of Th2 type cytokines, while Th1 remains suppressed. Furthermore, the levels of these cytokines alter according to the grades of the tumor, indicating that tumor cells themselves are capable of modulating the cytokine levels to an immunosuppressive state, favoring their survival, proliferation, and ability to escape immune surveillance. The strength of the present work lies in the fact that it depicts a striking imbalance in Th1/Th2 axis, with the Th2 response being favored over the former in 72 bladder cancer patients of different stage and grade as against 50 healthy controls. This can give significant insights about the use of Th1 type cytokines for the administration of immunotherapy to bladder cancer patients. Development of new strategies attempting to manipulate the equilibrium between Th1 and Th2 cells would be beneficial in the management of UC of bladder in future.

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