Frog urinary bladder epithelial cells express TLR4 and respond to bacterial LPS by increase of iNOS expression andl-arginine uptake

Abstract

As in mammals, epithelium of the amphibian urinary bladder forms a barrier to pathogen entry and is a first line of defense against penetrating microorganisms. We investigated the effect of Escherichia coli LPS on generation of nitric oxide (NO), a critically important mediator during infectious processes, by primary cultured frog (Rana temporaria) urinary bladder epithelial cells (FUBEC). It was found that FUBEC constitutively express Toll-like receptor 4 (TLR4), a receptor of LPS, and respond to LPS (10 μg/ml) by stimulation of inducible nitric oxide synthase (iNOS) mRNA/protein expression and NOS activity measured by nitrite produced in the culture medium and by citrulline assay. We characterized uptake of l-arginine, a precursor in NO synthesis, by FUBEC and showed that it is mediated mainly by the y+ cationic amino acid transport system. LPS stimulated l-arginine uptake, and this effect was blocked by the iNOS inhibitor 1400W. Arginase II was found to be expressed in FUBEC. Inhibition of arginase activity by (S)-(boronoethyl)-l-cysteine increased generation of NO, suggesting contribution of arginase to NO production via competing with NOS for the substrate. LPS altered neither total arginase activity nor arginase II expression. Among epithelial cells, phagocytic macrophage-like cells were observed, but they did not contribute to LPS-induced NO production. These data demonstrate that amphibian urinary bladder epithelial cells recognize LPS and respond to it by increased generation of NO via stimulation of iNOS expression and l-arginine uptake, which appears to be essential for the regulation of the innate immune response and the inflammation in bladder epithelium.

Comprehensive Transcriptome Profiling and Functional Analysis of the Frog (Bombina maxima) Immune System

“…28 In frog, the TLR4 is respond to LPS as a receptor in urinary bladder epithelial cells. 29 As the pathway prediction, it showed that TLR4 might be failing to respond to LPS through LBP binding to CD14 induced the pathway. Hence, the fact that TLR4 of B. maxima can recognize the LPS to induce the pathway remains to be elucidated.…”

Urotensin II upregulates migration and cytokine gene expression in leukocytes of the African clawed frog, Xenopus laevis

“…In amphibian immune studies, many of the genes known to be involved in mammalian innate immunity have been identified in X. laevis and Xenopus tropicalis (Robert and Ohta, 2009). In Xenopus species, inflammatory stimulation with bacteria and LPS induced increases in some important components (e.g., lectin, cytokines, nitric oxide, stress protein) of innate immune responses in various tissues and plasma (Cui et al, 2011;Morales et al, 2003;Nagata et al, 2013;Nikolaeva et al, 2012). However, few studies have examined the hormonal regulation of immune responses in amphibian leukocytes.…”

“…Recently, Ishii et al (2007) identified the putative TLR4, which is homologous to mammalian TLR4, from X. laevis. Furthermore, immunoblotting with TLR4 antibody demonstrated that the receptor expresses in frog leukocytes (Nikolaeva et al, 2012), although the function of the LPS receptor of the Xenopus TLR4 remains unclear. We also revealed that the UII-UTR system not only promoted the migration of monocytes and lymphocytes via the RhoA/Rho kinase signaling pathway but also upregulated the expression of some cytokine genes.…”

Colonization of frog Rana temporaria L. urinary bladder by Gram‐negative bacteria leads to decreased effect of arginine‐vasotocin on water reabsorption from the urinary bladder

“…In mammalian urinary bladder and inner medulla collecting duct epithelial cells, active TLR4/CD14‐complexes and signaling cascade of LPS were revealed (Schilling et al, ; Chassin et al, ; Song et al, ; Schnerberich and Hartinger, ). In frog urinary bladder epithelial cells, TLR4 mediates LPS‐induced expression of iNOS, suggesting the involvement of NO in the suppression of bacterial growth (Nikolaeva et al, ).…”

“…We have recently shown that R. temporaria urinary bladder epithelial cells are able to recognize bacterial LPS and to respond by TLR4 (toll‐like receptor 4)‐mediated increase of inducible NO‐synthase (iNOS) expression at the level of mRNA and protein, which appears to be a part of the innate immune response (Nikolaeva et al, ). The goals of the present work were to characterize the frequency of occurrence and pattern of Gram‐negative bacteria in urinary bladder tissue and isolated epithelial cells, and to study the relationship between the presence of bacteria in mucosal epithelium and AVT‐induced level of OWP.…”