title: Discriminators of mouse bladder response to intravesical Bacillus Calmette-Guerin (BCG).
abstract: BACKGROUND
  Intravesical Bacillus Calmette-Guerin (BCG) is an effective treatment for bladder superficial carcinoma and it is being tested in interstitial cystitis patients, but its precise mechanism of action remains poorly understood. It is not clear whether BCG induces the release of a unique set of cytokines apart from its pro-inflammatory effects. Therefore, we quantified bladder inflammatory responses and alterations in urinary cytokine protein induced by intravesical BCG and compared the results to non-specific pro-inflammatory stimuli (LPS and TNF-alpha). We went further to determine whether BCG treatment alters cytokine gene expression in the urinary bladder.
  
  
  METHODS
  C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-alpha. Morphometric analyses were conducted in bladders isolated from all groups and urine was collected for multiplex analysis of 18 cytokines. In addition, chromatin immune precipitation combined with real-time polymerase chain reaction assay (CHIP/Q-PCR) was used to test whether intravesical BCG would alter bladder cytokine gene expression.
  
  
  RESULTS
  Acute BCG instillation induced edema which was progressively replaced by an inflammatory infiltrate, composed primarily of neutrophils, in response to weekly administrations. Our morphological analysis suggests that these polymorphonuclear neutrophils are of prime importance for the bladder responses to BCG. Overall, the inflammation induced by BCG was higher than LPS or TNF-alpha treatment but the major difference observed was the unique granuloma formation in response to BCG. Among the cytokines measured, this study highlighted the importance of IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-10, IL-17, GM-CSF, KC, and Rantes as discriminators between generalized inflammation and BCG-specific inflammatory responses. CHIP/Q-PCR indicates that acute BCG instillation induced an up-regulation of IL-17A, IL-17B, and IL-17RA, whereas chronic BCG induced IL-17B, IL-17RA, and IL-17RB.
  
  
  CONCLUSIONS
  To the best of our knowledge, the present work is the first to report that BCG induces an increase in the IL-17 family genes. In addition, BCG induces a unique type of persisting bladder inflammation different from TNF-alpha, LPS, and, most likely, other classical pro-inflammatory stimuli.
authors.affiliation: College of Medicine, Department of Physiology, Oklahoma University Health Sciences Center, Ohlahoma City, OK 73104, USA. marcia-saban@ouhsc.edu <marcia-saban@ouhsc.edu>
authors.forename: Marcia R
authors.initials: MR
authors.lastname: Saban
authors.forename: Cindy
authors.initials: C
authors.lastname: Simpson
authors.forename: Carole
authors.initials: C
authors.lastname: Davis
authors.forename: Gemma
authors.initials: G
authors.lastname: Wallis
authors.forename: Nicholas
authors.initials: N
authors.lastname: Knowlton
authors.forename: Mark Barton
authors.initials: MB
authors.lastname: Frank
authors.forename: Michael
authors.initials: M
authors.lastname: Centola
authors.forename: Randle M
authors.initials: RM
authors.lastname: Gallucci
authors.forename: Ricardo
authors.initials: R
authors.lastname: Saban
mesh_terms: D000283:Administration, Intravesical; D000818:Animals; D001500:BCG Vaccine / Q000008:administration & dosage*; D047369:Chromatin Immunoprecipitation; D003556:Cystitis / Q000139:chemically induced* / Q000473:pathology / Q000652:urine*; D016207:Cytokines / Q000235:genetics / Q000378:metabolism* / Q000652:urine; D004195:Disease Models, Animal; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006099:Granuloma / Q000139:chemically induced / Q000473:pathology / Q000652:urine; D007150:Immunohistochemistry; D020381:Interleukin-17 / Q000235:genetics / Q000378:metabolism / Q000652:urine*; D008070:Lipopolysaccharides / Q000008:administration & dosage; D051379:Mice; D008810:Mice, Inbred C57BL; D009504:Neutrophils / Q000473:pathology / Q000648:ultrastructure; D014409:Tumor Necrosis Factor-alpha / Q000008:administration & dosage; D015854:Up-Regulation; D001743:Urinary Bladder / Q000187:drug effects* / Q000276:immunology / Q000648:ultrastructure
chemical_list: D001500:BCG Vaccine; D016207:Cytokines; D020381:Interleukin-17; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha