LPS-sensory peptide communication in experimental cystitis

Abstract

Stimulation of sensory nerves can lead to release of peptides such as substance P (SP) and consequently to neurogenic inflammation. We studied the role of bacterial lipopolysaccharide (LPS) in regulating SP-induced inflammation. Experimental cystitis was induced in female mice by intravesical instillation of SP, LPS, or fluorescein-labeled LPS. Uptake of fluorescein-labeled LPS was determined by confocal analysis, and bladder inflammation was determined by morphological analysis. SP was infused into the bladders of some mice 24 h after exposure to LPS. In vitro studies determined the capacity of LPS and SP to induce histamine and cytokine release by the bladder. LPS was taken up by urothelial cells and distributed systemically. Twenty-four hours after instillation of LPS or SP, bladder inflammation was characterized by edema and leukocytic infiltration of the bladder wall. LPS pretreatment enhanced neutrophil infiltration induced by SP, increased in vitro release of histamine, tumor necrosis factor-α, and interferon-γ, and significantly reduced transforming growth factor-β1 release. These findings suggest that LPS amplifies neurogenic inflammation, thereby playing a role in the pathogenesis of neurogenic cystitis.

Gene Expression Profiling of Mouse Bladder Inflammatory Responses to LPS, Substance P, and Antigen-Stimulation

“…LPS, SP, and Ag induced slightly different degrees of acute inflammation characterized by vasodilation, edema, and intense polymorphonuclear neutrophil (PMN) infiltration in the mucosa and submucosal layers (Table 1). Confirming our results obtained with LPS, 11 substance P, 17 and antigen stimulation, 12 both edema formation and PMN migration were observed as early as 4 hours and were stabilized 24 hours after stimulation (data not shown). In a separate study, we also examined additional time points at 48 and 72 hours after LPS stimulation to make sure the maximal PMN infiltration and edema was obtained at 24 hours.…”

“…[11][12][13]17 Briefly, sensitized female C57BL/6J mice were anesthetized (ketamine 40 mg/kg and xylazine 2.5 mg/ kg, i.p. ), then transurethrally catheterized (24-gauge; 3/4 inch; Angiocath, Becton Dickinson, Sandy, UT), and the urine was drained by applying slight digital pressure to the lower abdomen.…”

“…A semiquantitative score using defined criteria of inflammation severity was used to evaluate cystitis (11)(12)(13)17). A cross-section of bladder wall was fixed in formalin, dehydrated in graded alcohol and xylene, embedded in paraffin, and cut serially into four 5-m sections (8 m apart) to be stained with hematoxylin and eosin (H&E) and Giemsa.…”

“…Image analysis was performed using a MetaMorph Imaging System (Universal Imaging Corporation, West Chester, PA). The severity of lesions in the urinary bladder was graded [11][12][13]17 as follows: 1ϩ, mild (infiltra-tion of a 0 -10 neutrophils/cross-section in the lamina propria, and little or no interstitial edema); 2ϩ, moderate (infiltration of 10 -20 neutrophils/cross-section in the lamina propria, and moderate interstitial edema); 3ϩ, severe (diffuse infiltration of Ͼ20 neutrophils/cross-section in the lamina propria and severe interstitial edema). Identification of mast cells and quantification of their degree of degranulation was performed in Giemsa-stained sections.…”

“…It remains to be determined whether this down-regulation is part of a compensatory mechanism or a consequence of LPS-induced death and shedding of urothelial cells. 17 As the urothelium works with underlying afferent nerves to detect the presence of irritant stimuli, 25 it will be of value to determine whether CD14, integrin ␣ 7, and MMP11 are part of the unique set of urothelial genes contrasting with those found in the bladder detrusor muscle. Ongoing experiments in this laboratory are determining which genes are uniquely expressed by each of the urinary bladder layers.…”