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Physiologic Relevance of LL-37 Induced Bladder Inflammation and Mast Cells

Saturday, October 20, 2012: 10:15 AM
Grand Ballroom A/B (Hilton Riverside)
Siam Oottamasathien, M.D.1, Wanjian Jia, MD, PhD1, Lindsi McCoard, BS1, Jianxing Zhang, PhD2, Li Wang, MS1, Xiangyang Ye, MS3, A. Cameron Hill2, Justin Savage, PhD2, Wong Yong Lee, DVM, PhD2, AnnMarie Hannon, MSN1, Sylvia Milner, BSN1 and Glenn D. Prestwich, PhD2, (1)Urology, University of Utah, Salt Lake City, UT, (2)Medicinal Chemistry, University of Utah, Salt Lake City, UT, (3)Pharmacology, University of Utah, Salt Lake City, UT

Purpose: Inflammatory conditions that afflict the urinary bladder are of significant urologic health concern.  Novel inflammatory models to further interrogate mechanisms are needed.  We’ve previously shown the naturally occurring urinary anti-microbial peptide LL-37 can induce profound inflammation in a mouse model.  Our specific aim was to further establish the physiologic relevance of LL-37 induced bladder inflammation.  We first hypothesized human urinary LL-37 levels are elevated in pediatric spina bifida (SB) patients.  We further hypothesized within our mouse model that LL-37 induced inflammation occurs via urothelial binding and is dose dependent.  Finally, it was hypothesized LL-37 induced inflammation mechanistically involves mast cells.

Methods: To test our first hypothesis, urine samples were obtained from pediatric (<18 y/o) SB (n=56) and normal (n=24) patients.  Urinary dipstick ruled out acute infection.  Urinary LL-37 levels were measured by ELISA (Hycult Biotech).  Our second hypothesis was tested with adult female C57Bl/6 mice placed under anesthesia, catheterized, then challenged with LL-37 for 1 hr.  Six concentrations of LL-37 were tested (10, 20, 40, 80, 160, 320 μM; n=4 for each [ ]).  Controls consisted of sterile saline.  Animals were sacrificed after 24 hours, examined with gross and H&E histology, and tissue myeloperoxidase (MPO) assay to quantitate inflammation.  Fluorescently labeled LL-37 was instilled for 1 hour in separate experiments and tissues obtained either immediately after (t=0) or 24 hours (t=24) to further assess LL-37 activity.  To test our final hypothesis, tissues were subjected to immunohistochemistry (IHC) for mast cell tryptase.  Mast cell positivity was determined by examining 5 hpf’s for each bladder to yield a mean number of mast cells/mm2.  Statistical methods for the respective experiments included: Wilcoxon signed-rank, one-way ANOVA, Bonferroni adjustment (p<0.05 significance level, SAS v9.2 software).

Results: Urinary LL-37 levels were 89-fold higher in SB patients versus normals (20.43 ng/ml vs. 0.23 ng/ml, p<0.001).  In our mouse LL-37 dose escalation experiments, levels of inflammation rose both histologically and with MPO activity, with each successive increase in LL-37 concentration.  Fluorescently labeled LL-37 yielded global urothelial binding at t=0, but quickly vanished at t=24.  IHC for mast cell tryptase revealed significant infiltration of mast cells in both the urothelial/submucosa and detrusor layers.  Higher concentrations of LL-37 challenge led to significantly higher levels of mast cell infiltration (26-fold increase between 320 μM vs. 0 μM, p = 0.0158).

Conclusion:   Urinary LL-37 levels were significantly elevated in pediatric spina bifida patients.  We innovatively demonstrated within our mouse model that a naturally based biologic compound could elicit profound dose dependent bladder inflammation.  In addition, LL-37 appears to elicit inflammation through a urothelial mechanism.  Finally, the propagation of inflammation appears to intimately involve mast cells.  These findings represent the first biologic, non-infectious, non-chemically induced bladder inflammation model that is physiologically relevant.