Toll-like receptor 4 and CD14 expression in human ciliary body and TLR-4 in human iris endothelial cells

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Abstract

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-α, interleukin-1β, 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-α, interleukin-1β, 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.

Introduction

Uveitis is an intraocular inflammation and a leading cause of visual impairment or blindness. Anterior uveitis describes a collection of different inflammatory diseases, which primarily affect the iris and ciliary body. It may be caused by infectious organisms or by an immune-mediated process as in sarcoidosis, spondyloarthritis, or juvenile idiopathic arthritis.

Gram-negative bacteria such as Klebsiella, Salmonella or Yersinia may be involved in the pathogenesis of anterior uveitis (Sprenkels et al., 1996; Baggia et al., 1997; Feltkamp and Ringrose, 1998). HLA B27-associated diseases, ankylosing spondylitis and reactive arthritis, are the most common systemic illnesses associated with uveitis in North America. Reactive arthritis is triggered by a variety of Gram-negative infections, including Salmonella, Shigella, Yersinia, or Campylobacter. In addition, Lewis rats challenged intraperitoneally or intravenously with Yersinia or Salmonella often develop a mild, transient anterior uveitis 7–14 days after the injection (Rosenbaum et al., 1999). Monoclonal antibodies directed against HLAB27 cross-react with gram-negative bacteria and vice versa (van Bohemen et al., 1984; Archer et al., 1985). Increased carriage of Klebsiella has been suggested to be a trigger for ankylosing spondylitis and uveitis (Ebringer, 1988), Gram-negative bacteria also contribute to the pathology of ulcerative colitis and Crohn's disease (Lyons and Rosenbaum, 1997; Landers et al., 2002).

Lipopolysaccharide (LPS) is a major cell wall component of Gram-negative bacteria and is implicated in their uveitogenicity. LPS activates monocytes, macrophages, and other cells types, such as endothelial and epithelial cells to produce several inflammatory cytokines including TNFα, IL-1, IL-6 and IL-12, which when in excess, lead to serious systemic disorders (Nomura et al., 2000). A link to ocular inflammation is apparent from the demonstration that systemic or intraocular injection of LPS induces uveitis in rodents (Rosenbaum et al., 1980).

Research over the past decade has established the importance of CD14 as an LPS receptor that is critical for the sensitivity of host cells to this bacterial glycolipid (Wright et al., 1990; Ulevitch and Tobias, 1995; Kitchens, 2000). Another protein, the lipopolysaccharide binding-protein (LBP) appears to function as a catalytic transfer protein that delivers LPS to CD14, a glycosylphosphatidylinositol-anchored membrane protein (Wright et al., 1990). Cells that lack membrane expression of CD14, including endothelial cells, utilize soluble CD14 for efficient activation (Kitchens, 2000). Recently, we have determined LBP secretion and mRNA expression by the rabbit's iris/ciliary body (Cano et al., paper in preparation). Despite the known function of CD14, this receptor lacks an intracellular signaling domain. Although CD14 is required to confer sensitivity in LPS recognition, other cellular molecules are required to complete the process of cellular recognition, intracellular-signaling capacity and response. Studies over the past few years have demonstrated that mammalian toll-like receptor protein 4 (TLR-4) as a member of a class of pattern-recognition receptors that participates in LPS induced signaling (Kopp and Medzhitov, 1999; Wright, 1999). The TLR family of proteins has been implicated in the defense system against pathogens in organisms as divergent as plants, flies and humans (Shimazu et al., 1999), and may also be important in human development and in the maintenance of normal organism functions, paralleling their roles in Drosophila (Frantz et al., 1999; Ohashi et al., 2000). Toll-like proteins (TRL1–TLR9) comprise a family of type I transmembrane receptors, which are characterized by an extracellular leucine-rich repeat (LRR) domain and an intracellular Toll/IL-1 receptor (TIR) domain (Rock et al., 1998). Toll-like receptors have a crucial role in the detection of microbial infection in mammals and insects. In mammals, these receptors have evolved to recognize conserved products unique to microbial metabolism. This specificity allows the TLR to detect the presence of infection and to induce activation of inflammatory and antimicrobial innate immune responses. TLR signaling can induce the production of proinflammatory cytokines and upregulate expression of costimulatory molecules, thereby activating not only innate but ultimately also adaptive immunity (Aderem and Ulevitch, 2000; Schuster and Nelson, 2000; Heuman and Roger, 2002). The remarkable conservation of the Toll signaling pathway reflects the evolutionary pressure for retaining and adapting efficient systems.

The likely importance of LPS in uveitis associated with HLA-B27 or colitis and the role of TLR-4 and CD14 in cellular responses to LPS lead us to determine the expression pattern of these proteins in the human eye.

Section snippets

Immunohistochemistry

Five micron paraffin tissue sections of five different human eyes (Pathology Service, Casey Eye Institute, Portland, OR, USA) were immunostained for TLR-4 and CD14 proteins using: (a) monoclonal antibody against human TLR-4 (10 μg ml−1 HTA-125 (Shimazu et al., 1999), a generous gift of Dr Kensuke Miyake, Saga Medical School, Japan), (b) antihuman CD14 (RPA-M1, 10 μg ml−1, Dako), or (c) an irrelevant mouse IgG1k (MOPC-21, 10 μg ml−1, Dako) followed by an alkaline phosphatase (AP) detection

Immunolocalization of TLR-4 and CD14 in whole human eyes

We used immunostaining to look for TLR-4 and CD14 proteins in sections of whole human eyes. In all five-donor eyes, TLR-4 was anatomically co-localized with CD14. Strong staining was observed on the non-pigmented epithelial cells of the human ciliary body for both proteins (Fig. 1, arrows and circles). Corneal epithelial cells showed weak staining for the endotoxin receptors, TLR-4 and CD14. The weakest staining was observed at the iris stroma, in one another cell that probably were resident

Discussion

It is now well known that CD14 is required, but not sufficient, to confer sensitivity to LPS. Other cellular molecules are required to complete the process of cellular recognition and response. Specifically, TLR-4 has been implicated as a gene product that regulates LPS response (Hoshino et al., 1999; Qureshi et al., 1999). In experiments to generate LPS-tolerant macrophages in mice, TLR-4 mRNA expression significantly decreased within a few hours of LPS pretreatment and returned to the

Acknowledgements

B.E.B. wants to thank Pfizer of Venezuela for funds for training support at The Casey Eye Institute, OHSU, Portland, OR, USA. Grants that support this work: EY06484.

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