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

Abstract

Purpose We established the physiological relevance of LL-37 induced bladder inflammation. We hypothesized that 1) human urinary LL-37 is increased in pediatric patients with spina bifida, 2) LL-37 induced inflammation occurs in our mouse model via urothelial binding and is dose dependent and 3) LL-37 induced inflammation involves mast cells. Materials and Methods To test our first hypothesis, we obtained urine samples from 56 pediatric patients with spina bifida and 22 normal patients. LL-37 was measured by enzyme-linked immunosorbent assay. Our second hypothesis was tested in C57Bl/6 mice challenged with 7 LL-37 concentrations intravesically for 1 hour. At 24 hours tissues were examined histologically and myeloperoxidase assay was done to quantitate inflammation. In separate experiments fluorescent LL-37 was instilled and tissues were obtained immediately (time = 0) and at 24 hours (time = 24). To test our final hypothesis, we performed immunohistochemistry for mast cell tryptase and evaluated 5 high power fields per bladder to determine the mean number of mast cells per mm2. Results Urinary LL-37 was 89-fold higher in patients with spina bifida. Mouse LL-37 dose escalation experiments revealed increased inflammation at higher LL-37 concentrations. Fluorescent LL-37 demonstrated global urothelial binding at time = 0 but was not visible at time = 24. Immunohistochemistry for tryptase revealed mast cell infiltration in all tissue layers. At higher concentrations the LL-37 challenge led to significantly greater mast cell infiltration. Conclusions Urinary LL-37 was significantly increased in pediatric patients with spina bifida. To our knowledge we report for the first time that LL-37 can elicit profound, dose dependent bladder inflammation involving the urothelium. Finally, inflammation propagation involves mast cells.

Topical cathelicidin (LL‐37) an innate immune peptide induces acute olfactory epithelium inflammation in a mouse model

“…Specifically, LL-37 had robust epithelial barrier binding in addition to high levels seen in the lamina propria and serous/mucus glands seen at 1 hour after inoculation, consistent with prior investigations demonstrating rapid penetration within the bladder submucosa and uroepithelium. 30 The majority of LL-37 was cleared after 24 hours, with trace amounts seen in the serous/mucus glands of the lamina propria. This suggests that the inflammatory insult extends beyond the initial exposure as evidenced by increased neutrophils and mast cells in the tissue.…”

“…9,23 This pathway is not unique to the sinuses, as the RAGE pathway has been elucidated in many other chronic inflammatory diseases, including, but not limited to, rosacea, 27 arthritis, 42 and interstitial cystitis. 30 Further investigations are needed to define LL-37-RAGE induced upper airway inflammation.…”

“…Reactions were stopped by immersion into distilled H2O, after which the tissues were counterstained and dehydrated, as previously described. 30 Immunoflourescence experiments were done with DyLight® 633 secondary antibody (goat anti-rabbit IgG, 1:800 dilution).…”

LL-37 induced cystitis and the receptor for advanced glycation end-products (RAGE) pathway

“…Using our previously established model, we created bladder inflammation using the cathelicidin LL-37[8, 40], which has been found to be elevated in urine samples obtained from pediatric patients with either cystitis or pyelonephritis[42]. Profound bladder inflammation was observed after LL-37 exposure yielding global erythema, edema, hemorrhage, and hypervascularity.…”

“…LL-37 induced bladder inflammation was performed as previously described[8, 40]. Briefly, 8 to 12-week old female C57BL/6 mice were catheterized, bladders emptied, then washed with 150 μL of 0.9% sodium chloride and emptied.…”