Interleukin-11 Attenuates Ifosfamide-Induced Hemorrhagic Cystitis

Objective: To investigate the possible protective effect of recombinant human interleukin-11 (rhIL-11) against ifosfamide (IFS)-induced hemorrhagic cystitis (HC). Materials and Methods: Male Swiss mice (20-30g) were pretreated with rhIL-11 (25-625 μg, subcutaneously.) 30 min before intraperitoneal injection of IFS (400 mg/kg) or with saline (control group). Twelve hours later, HC was evaluated by bladder wet weight (BWW) to quantify edema, Evans blue extravasation (EBE) to measure vascular permeability, and macroscopic and microscopic analysis. All bladders were assessed by histopathological analysis. Results: rhIL-11 (at 125 and 625 μg) attenuated the IFSinduced increase of BWW (37.48% and 45.44%, respectively, p < 0.05) and EBE (62.35% and 56.47%, respectively, p < 0.05). IFSinduced macroscopic edema and hemorrhage and microscopic alterations, were also prevented by rhIL-11 at 625 μg. (p < 0.05). Conclusion: Our results demonstrate a protective effect of rhIL-11 on experimental IFSinduced HC, not previously reported.


INTRODUCTION
Interleukin-11 (IL-11, thrombopoietin) is a pleiotropic 178-amino acid polypeptide with a molecular weight of 18 kDa which is expressed by a wide range of mesenchymal tissues (1,2) used in clinical practice as a megakaryocytopoiesis stimulator in patients with thrombocytopenia (3).It is well known that IL-11 protects the oral gastrointestinal mucosa against radiation (4) and 5-fluorouracil (5) damage by attenuating TNF-α and IL-1β expression in hamsters.In attempt to elucidate this anti-inflammatory effect, we investigated if IL-11 could protect against ifosfamide (IFS)induced hemorrhagic cystitis (HC).
IFS is an alkylating agent from the oxazaphosphorine group with a broad spectrum of antineoplasic activity.In the absence of adequate uroprotection, patients treated with IFS for malignant tumors or immunosuppression develop a dose-limiting HC with an average incidence of 40%.Such toxicity is attributed to the intravesical release of acrolein (ACR), an IFS highly urotoxic metabolite.It has been proposed that urothelial damage occurs by direct contact with ACR, causing edema, ulceration, neovascularization, hemorrhage and necrosis (6).Despite the preventive use of mesna, the occurrence of hematuria due to HC has been observed in 33% of patients (7).These facts increase the importance of studies to investigate novel therapies and elucidate the mechanisms involved in bladder lesion resulting from alkylating agent therapy.
Recombinant human IL-11 (rhIL-11, oprevelkin) has a number of biological activities which could impact HC including inhibition of pro-inflammatory cytokines such as tumor necrosis factor (TNF)α and interleukin-1β (IL-1β), nitric oxide (NO) synthesis and apoptosis (8,9), and stimulation of cell proliferation and differentiation and protection of connective tissue (8).For that reason, the present study investigated if rhIL-11 has a protective effect against IFS-induced HC in mice.

MATERIALS AND METHODS
Animals -Male Swiss mice (20-30 g), provided from the Central Bioterium of Pici, were kept in a temperature-controlled room with food ad libitum and water restriction 12 hours before they were sacrificed.All animal treatments and surgical procedures were performed in accordance with the Guide for Care and Use of Laboratory Animals, National Institutes of Health (Bethesda, MD, USA).
Effect of IL-11 on ifosfamide-induced hemorrhagic cystitis -Groups of 6 mice were pretreated with subcutaneous (s.c.) administration of sterile saline or rhIL-11 at 25, 125 or 625 µg 30 minutes before the induction of HC with intraperitoneal injection of IFS at 400 mg/kg.The control group received only sterile saline.Twelve hours after IFS treatment, ani-mals were euthanized, and the bladders were removed by careful dissection and emptied of urine.Bladder wet weight (BWW) was measured as a parameter of vesical edema and expressed as g/20 g body weight (mean ± SEM).
Macroscopic evaluation -Bladders were grossly examined for edema and hemorrhage.According to Gray´s scoring criteria (12), edema was considered severe (3+) when fluid was seen externally and internally in the bladder walls, moderate (2+) when confined to the internal mucosa, mild (1+) between normal and moderate and normal (0) when no edema was observed.Hemorrhage was scored as follows: 3+, intravesical clots; 2+, mucosal hematomas; 1+, telangiectasia or dilatation of bladder vessels; and 0, normal.
Microscopic evaluation -Bladders were fixed in formalin at 10%, embedded in paraffin and processed for hematoxylin and eosin (HE, Reagen) staining.Histopathological analysis was performed by a person (GACB) who was unaware of the treatments and group divisions, and scored as follows: (0) normal epithelium and absence of inflammatory cell infiltration and ulceration, (1) mild changes involving reduction of urothelial cells, flattening with submucosal edema, mild hemorrhage and few ulcerations, and (2) severe changes including mucosal erosion, inflammatory cell infiltration, fibrin deposition, hemorrhage and multiple ulcerations (10).
Evans blue extravasation assay -Vesical vascular permeability was evaluated by the Evans blue extravasation technique.Following the same previous group division (n = 6) and protocol, 2.5% Evans blue (25 mg/kg) was injected intravenously via the retro orbital plexus 30 minutes before the animals were sacrificed.Bladders were then excised, dissected and placed into glass tubes containing a formamide solution (1 mL/bladder) at 56 o C overnight to extract the stain.The total extracted dye was determined by measuring the absorbance change at 630 nm (ELISA).At the same time, an absorbance-concentration curve was determined.The results were then reported as in micrograms of Evans blue per bladder (mean ± SEM).
Statistical analysis -Data were reported as the mean ± SEM (bladder wet weight, Evans blue extravasation and NOS activity) or the median values followed by range values (macroscopic and histopathological scores) of groups of six mice.Statistical analysis was performed using one way analysis of variance (ANOVA) followed by Newman-Keuls test, when appropriate.Macroscopic and microscopic scores were evaluated by Kruskal Wallis non-parametric test followed by Dunn's multiple comparison.Statistical significance was set at p < 0.05.

COMMENTS
The present study demonstrates the protective effect of rhIL-11 on IFS-induced hemorrhagic cystitis (HC).Our results show evidence that pretreatment with rhIL-11 in a dose-dependent manner prevents the increase of bladder wet weight, (parameter used to measure edema) and Evans blue extravasation in bladder (parameter used to measure vascular permeability), induced by IFS at 400 mg/kg.The results obtained were confirmed by macroscopic and microscopical analysis, in which only 625 µg, but not 125 µg dose, was effective in protecting against IFSinduced macroscopic edema and hemorrhage and microscopic alterations.Consequently, our preclinical Recently, some important mechanisms of experimental ifosfamide-induced hemorrhagic cystitis have been elucidated.Pro-inflammatory cytokines such as platelet-activating factor (PAF), TNF-α and IL-1β have been implicated in its pathogenesis.Previously, we demonstrated the participation of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) pathway in the induction of nitric oxide (NO) production as an important pathway in the pathogenesis of lesion in hemorrhagic cystitis induced by alkylating agents.NO was demonstrated to be the final mediator of urothelial damage and hemorrhage in that type of HC (11).TNF-α and IL-1β have been shown to be important mediators of NO synthesis, since treatment with anti-TNF-α and anti-IL-1β decreased cyclophosphamide vesical damage as well the rise of inducible NO synthase expression and activity (12,13).
A previous study from our laboratory proved that dexamethasone alone was ineffective in preventing experimental IFS-induced hemorrhagic cystitis, but when combined with mesna, this corticosteroid potentiated its protective effect (14).More recently, we showed that amifostine and glutathione prevent ifosfamide and acrolein-induced hemorrhagic cystitis in mice (15).Therefore, it is becoming important to establish novel therapies for hemorrhagic cystitis, for the reason that, as previously demonstrated, 33% of patients using alkylating agents for the treatment of cancer develop hematuria (hemorrhagic cystitis sign) despite the preventive use of mesna (7).An experimental study in rats reinforced that the notion that there a high percentage of non protection despite preventive mesna usage (14).
Interleukin-11 (IL-11) has been extensively investigated as a protective agent for inflammatory events, such as inflammatory bowel disease, psoriasis, autoimmune joint disease, and many other inflammatory diseases (16).rhIL-11 has been shown to down regulate macrophage production in vitro of IL-1β, TNF-α, IL-12 and nitric oxide, without inducing antiinflammatory cytokines such as IL-10, TGF-b, and IL-6 (8).It was also observed that rhIL-11 attenuates Th1 cytokines production in human psoriasis lesions, noting that its effect is not restricted to macrophages (17).More recent works demonstrate the rhIL-11 property of inhibiting NF-κB and AP-1 activation in islets to prevent streptozotocin-induced diabetes (18).Other studies have successfully used IL-11 for the prevention of intestinal ischemia-reperfusion lesion (19) and intestinal mucosa damage in response to chemotherapy and radiation due to diminished cell apoptosis and consequent death (9).On the other hand, other authors demonstrated that IL-11 does not prevent methotrexate-induced intestinal cell apoptosis but reduces the damage by compensatory crypt cell proliferation (20).Our data introduce a novel anti-inflammatory effect of IL-11 and are in accordance with previous reports in the literature.
In conclusion, the present study demonstrated that the pleiotropic cytokine rhIL-11 partially prevents IFS-induced experimental HC, an inflammatory event that depends on TNF-α, IL-1β and nitric oxide release.Possibly, rhIL-11 acts on an inhibitory pathway of these inflammatory mediators.Taking into account that rhIL-11 is already being used in clinical practice, it is possible to propose a clinical trial to investigate its effect on human HC.

Figure 1 -
Figure 1 -Protective effect of rhIL-11 against ifosfamide-induced bladder wet weight increase.Ifosfamide at 400 mg/kg induced an increase in BWW when compared to the control group (C), and rhIL-11 at 125 µg and 625 µg protected against this side effect.* p < 0.05 compared with control group.# p < 0.05 compared to ifosfamide only group.ANOVA followed by Newman-Keuls.

Figure 2 -
Figure 2 -Histological analysis of cross sections of representative bladder walls.A) Control group which received only saline.B) Ifosfamide at 400 mg/kg induced urothelium desquamation, edema, pronounced hemorrhage and fibrin deposition and leukocyte infiltration when compared to control, shown in A. C) and D) Pretreatment with rhIL-11 at 625 µg was capable of reducing these side effects.(HE, A, B and C X100 and D X400).

Figure 3 -
Figure 3 -Protective effect of rhIL-11 against ifosfamide-induced Evans blue extravasation in bladder.Ifosfamide at 400 mg/kg induced an increase in Evans blue extravasation when compared to the control group (C), thus demonstrating an increase in vascular permeability.rhIL-11 at 125 µg and 625 µg reduced ifosfamide-induced Evans blue extravasation.* p < 0.05 compared with control group.# p < 0.05 compared to ifosfamide only group.ANOVA followed by Newman-Keuls.

Table 1 -
Macroscopic and microscopic analysis of rhIL-11 effect on ifosfamide-induced hemorrhagic cystitis.