Toll-like receptor 4 expression and cytokine responses in the human urinary tract mucosa

Abstract

Mucosal pathogens trigger a local innate host response by activating epithelial cells. Bacterial adherence and Toll-like receptor 4 (TLR4) signaling have been implicated as key events in this process. This study addressed the molecular basis of the epithelial response to gram-negative infection in the human urinary tract. Mucosal biopsies were obtained from kidneys, ureters, and bladders of patients undergoing urinary tract surgery, and epithelial TLR4 and CD14 expression was examined by immunohistochemistry. TLR4 was detected in epithelial cells lining the entire urinary tract and in the renal tubular epithelium. CD14, in contrast, was completely absent from the epithelial tissue. The response of the epithelial cells to infection was studied by in vitro challenge of the biopsies with uropathogenic Escherichia coli bacteria. A rapid cytokine response was observed, with production of interleukin-1beta (IL-1beta), IL-6, and IL-8 but not of IL-4 or gamma interferon. Adhering, P- or type 1-fimbriated E. coli activated IL-6 and IL-8 production more efficiently than the nonfimbriated control, as shown by cellular staining and analysis of secreted cytokines. The results demonstrate that human uroepithelial cells possess the molecular machinery needed to respond to uropathogenic E. coli. This includes recognition receptors for fimbriae and TLR4 for transmembrane signaling. We speculate that the lack of membrane-bound CD14 allows the epithelium to regulate its sensitivity to lipopolysaccharide and to discriminate between more-virulent and less-virulent strains.

FIG. 1.

TLR4 expression in the human urinary tract epithelium. (Left panels) Biopsies from the human urinary tract were stained with monoclonal anti-human TLR4 antibody (IMG-320) and visualized with Fast-Red substrate. TLR4 (red) was detected in the epithelium (asterisks) lining the bladder, ureter, pelvis, and cortex. (Right panels) The isotype control antibody did not stain the sections. Magnification, ×200.

FIG. 2.

TLR4 and CD14 surface expression on excreted urinary tract epithelial cells. Urinary epithelial cells were harvested from healthy individuals, and the expression of TLR4 and CD14 was analyzed by flow cytometry. The shed cells stained strongly for TLR4 (A), but CD14 was not detected (D). Epithelial cells were identified by using the cytokeratin antibody MNF116 (not shown). The specificity of the anti-TLR4 antibody was confirmed by using HEK 293 cells that did not express tlr4 (B) and HEK 293 cells transfected with tlr4 (C). CD14-expressing blood monocytes were used as positive controls for anti-CD14 antibodies (E).

FIG. 3.

CD14 is not expressed by the human urinary tract epithelium. (Left panels) Biopsies were stained with the anti-CD14 antibody TÜK4 (epithelium is marked by asterisks). CD14 was not detected in the epithelium of the bladder, ureter, renal pelvis, or cortex. Weak CD14 staining was observed between the tubuli (G), probably reflecting the presence of serum. Occasional CD14-positive myeloid cells were detected under the epithelium. Similar results were obtained by staining with the CD14-specific antibody MY4 (not shown). (Right panels) Control sections were stained with the epithelial cell cytokeratin antibody MNF116. Magnification, ×200.

FIG. 4.

Epithelial cytokine response to bacterial challenge. Human biopsies were challenged with a uropathogenic E. coli strain. Sections were stained with monoclonal anti-IL-8 (A, D, and E), IL-6 (B and F), and IL-1β (C and G) antibodies. Tissue sections are shown before (left panels) or after (right panels) infection with E. coli Hu734. Magnification, ×200.

FIG. 5.

Cytokine response to in vitro infection of the biopsies. IL-1β-, IL-6-, and IL-8-positive epithelial cells were quantified before (white bars) and after (stippled bars) infection with E. coli Hu734. (The percentage of stained cells out of 200 epithelial cells scored is shown.) The biopsies were obtained from bladder (B) and pelvis (P).

FIG. 6.

Epithelial IL-6 response following in vitro infection with recombinant E. coli strains differing in fimbrial expression. Biopsies were infected with the P-fimbriated recombinant strain E. coli HB101(pPIL110-35) (pap⁺) (right panels). E. coli HB101 (pap) (left panels) was the nonfimbriated control. Magnification, ×200.