Toll-like receptor 4 and CD14 expression in human ciliary body and TLR-4 in human iris endothelial cells.
We investigated the expression of the functional endotoxin receptor proteins Toll-like receptor-4 and CD14 in human eyes. Toll-like receptor-4 and CD14 proteins were detected by immunohistochemical analysis of sections of whole human eyes embedded in paraffin with monoclonal antibodies against human toll-like receptor-4 (HTA-125), human CD14 (RPA-M1), or as a control, an irrelevant mouse IgG1k (MOPC-21). Incubation of explants with a neutralizing anti-toll-like receptor-4 monoclonal antibody was used to determine if lipopolysaccharide stimulation of tumor necrosis factor or interleukin-6 secretion was dependent on Toll-like receptor-4 activity. Reverse transcription-polymerase chain reaction was used to detect mRNAs for toll-like receptor-4, tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-8, 3 hr after stimulation of cultured iris microvascular endothelial cells. By immunohistochemistry, human ciliary body non-pigmented epithelial cells showed strong expression of the endotoxin receptor proteins, toll-like receptor-4 and CD14. Toll-like receptor-4 antibodies significantly inhibited lipopolysaccharide-stimulated tumor necrosis factor secretion by the ciliary body. Toll-like receptor-4 mRNA was constitutively expressed in iris endothelial cells and slightly down-regulated by endotoxin. mRNA levels for tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-8 were all increased by endotoxin treatment. This is the first report that shows intraocular (ciliary body and iris) expression of toll-like receptor-4, other than in cornea. Our results show that the ciliary body also expresses CD14, which is anatomically colocalized with toll-like receptor-4. This suggests a potential interaction between both molecules during endotoxin activation of ciliary body cells. The juxtaposition of toll-like receptor-4 and CD14 in the anterior uveal tract helps to explain the sensitivity of the iris/ciliary body to bacterial endotoxin as seen in the standard animal model of endotoxin-induced uveitis.
Brito
Beatriz E
BE
Cellular and Molecular Pathology Laboratory, Experimental Medicine Center, Venezuelan Institute for Scientific Research (IVIC), Apdo 21827, Caracas 1020-A, Venezuela. bbrito@medicina.ivic.ve
Zamora
David O
DO
Bonnah
Robert A
RA
Pan
Yuzhen
Y
Planck
Stephen R
SR
Rosenbaum
James T
JT
D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology; D002478:Cells, Cultured; D002924:Ciliary Body / Q000378:metabolism*; D016207:Cytokines / Q000096:biosynthesis; D042783:Endothelial Cells / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007498:Iris / Q000378:metabolism*; D018950:Lipopolysaccharide Receptors / Q000378:metabolism*; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008875:Middle Aged; D012333:RNA, Messenger / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
D000911:Antibodies, Monoclonal; D016207:Cytokines; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors