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From the Laboratory of Ocular Immunology, Inflammatory Diseases Research Unit, School of Medical Sciences, University of New South Wales, Sydney, Australia.
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METHODS. Normal human ocular tissues were evaluated for in vivo TLR4, MD-2, and CD14 mRNA and protein expression by RT-PCR and immunohistochemistry, respectively. The distribution patterns and phenotypes of the cells expressing these proteins were further characterized by confocal microscopy and double-label immunofluorescence studies.
RESULTS. Normal human uvea, retina, sclera, and conjunctiva constitutively expressed TLR4, MD-2, and CD14 mRNA. The protein expression of these molecules was restricted, however, to resident antigen-presenting cells (APCs) in the normal human uvea, consisting mainly of HLA-DR+ dendritic cells (DCs). These APCs endowed with the complete LPS receptor complex appeared to be strategically positioned in perivascular and subepithelial locations for surveying blood-borne or intraocular LPS. In contrast, other cell types of the normal human cornea, conjunctiva, retina, and sclera did not express TLR4/MD-2 protein in vivo as detectable by immunohistochemistry.
CONCLUSIONS. The present study demonstrates for the first time that resident APCs in the normal human uvea express TLR4 and its associated LPS receptor complex. This has significant implications for the understanding of normal ocular immunity as well as unraveling the potential role of Gram-negative bacteria in the pathogenesis of acute anterior uveitis (AAU).
Toll-like receptors (TLRs) are a recently discovered family of type I transmembrane, pattern-recognition receptors (PRRs) that are essential in the recognition of the highly conserved pathogen-associated molecular patterns (PAMPs) that are unique to microbes, such as LPS of Gram-negative bacteria, mannans of yeast cell wall, and viral double-stranded RNA. To date, 10 TLRs (TLR1 to -10) have been described, each recognizing a specific class or classes of PAMP.4 5 Stimulation of the TLR, by its respective and specific PAMP, results in the activation of an immunostimulatory and immunomodulatory cell-signaling pathway that is essential for innate immunity and for the activation of the adaptive arm of the immune response.4 6 It is now well established that TLR4 is the primary signaling receptor for LPS-specific recognition and cellular activation.5 7 CD14 is a glycosyl phosphatidylinositol-anchored cell surface protein that functions as a coreceptor for LPS, as it, unlike TLR4, is unable to activate cellular signal transduction due to the absence of an intracellular signaling domain.8 MD-2 is an accessory molecule that associates with the extracellular domain of TLR4 conferring on it LPS responsiveness and is an absolute requirement in TLR4-dependent LPS responses in vivo.9 10
TLR expression has been demonstrated on peripheral blood monocytes,11 dendritic cells (DCs),12 B cells,13 and dermal vascular endothelial cells,14 as well as in various human tissues including lymphoid tissues,15 intestinal epithelial cells,16 and skin.17 The TLRs have also been implicated in the pathogenesis of a variety of inflammatory or autoimmune human diseases such as rheumatoid arthritis,18 inflammatory bowel disease,16 and psoriasis.17 Song et al.19 have recently reported the expression of functional TLR4 and CD14 in cultured human corneal epithelial cells with implications for a role in the pathogenesis of Gram-negative bacterial keratitis.19 Although CD14 expression was demonstrated in fresh whole human corneas by immunohistochemistry, the in vivo corneal expression of TLR4 was not reported in that study. To date, no investigations have been conducted, to the best of our knowledge, to evaluate the in vivo expression of TLR4 in the normal human eye, including the uvea. Given the particular predilection for the involvement of the uveal tract in intraocular inflammatory disease and the implicated role of Gram-negative bacteria in their pathogenesis, the purpose of the present study was to determine the in vivo expression patterns of TLR4, MD-2, and CD14, collectively constituting the LPS-signaling receptor complex, in normal human ocular tissues. The results of this study demonstrate, for the first time, that resident antigen-presenting cells (APCs), including a significant subset of the DCs in the normal human uvea, particularly within the ciliary body and iris root, express all the components of the LPS-signaling receptor complex. This finding has important implications for our understanding of innate immunity and ocular immune privilege in the eye. The identification of TLR4 and its associated LPS receptor complex in the human uvea may also be of fundamental significance in unraveling the potential role of Gram-negative bacterial triggers of AAU.
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Eyes (n = 2) used for total RNA isolation were enucleated within 4 hours of death, the corneas removed for grafting, and the remaining ocular tissue transported in RNA stabilization solution (RNAlater; Ambion Inc., Austin, TX) at 4°C until further processing in the laboratory. Iris, ciliary body, choroid, retina, cornea, sclera, and bulbar conjunctiva were dissected and snap frozen in liquid nitrogen (for RNA isolation) or were embedded in OCT medium (Sakura Finetek, Torrance, CA), snap frozen, and sectioned at 4- to 20-µm thickness (for immunohistochemical studies). These ocular tissues were stored at –80°C until used. Cryostat tissue sections were fixed for 10 minutes in acetone before performing immunohistochemical studies.
RNA Isolation and RT-PCR Analysis
Total RNA was isolated from the iris, ciliary body, choroid, retina, bulbar conjunctiva, and sclera (RNeasy Micro Kit; Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions and treated with RNase-free DNase I. The RNA was reverse transcribed to single-stranded cDNA using random hexamer-primed reverse transcriptase (Superscript II RNase H–; Invitrogen, Carlsbad, CA). PCR amplification was performed with Taq DNA polymerase (Platinum Taq; Invitrogen) using the following gene-specific primers: TLR420 sense, 5'-TGGATACGTTTCCTTATAAG-3' and antisense, 5'-GAAATGGAGGCACCCCTTC-3'; MD-221 sense, 5'-GAAGCTCAGAAGCAGTATTGGGTC-3' and antisense, 5'-GGTTGGTGTAGGATGACAAACTCC-3'; CD1420 sense, 5'-CCATGGAGCGCGCGTCCTGC-3' and antisense, 5'-GTCTTGGATCTTAGGCAAAGC-3'; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)22 sense, 5'-ACCACAGTCCATGCCATCAC-3' and antisense, 5'-TCCACCACCCTGTTGCTGTA-3'. Optimized PCR conditions were as follows: TLR4 (32 cycles of 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 45 seconds); MD-2 (28–32 cycles, depending on the tissue source, of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 25 seconds); CD14 (35 cycles of 95°C for 30 seconds, 62°C for 2 minutes, and 72°C for 3 minutes); and GAPDH (28 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 45 seconds). RT-PCR on RNA extracted from human peripheral blood monocytes that are known to express TLR4, MD-2, and CD14 mRNA served as positive controls for the gene-specific primers.11 Negative control reactions included performing the PCR under identical conditions except for the omission of the reverse transcriptase, the template cDNA, or the primers. PCR products were analyzed by electrophoresis on 2% agarose gels.
Single-Labeling Immunohistochemistry
Endogenous peroxidase activity in the cryostat tissue sections was blocked by 0.3% H2O2 and sodium azide for 5 minutes at room temperature. After three washes with tris-buffered saline (TBS) of 5 minutes each, sections were incubated with 20% normal goat or rabbit serum diluted in 2% bovine serum albumin (BSA)/TBS for 30 minutes at room temperature to block nonspecific binding. Sections were then incubated with the primary antibodies appropriately diluted in 2% BSA-TBS in a humidified chamber overnight at 4°C. The following primary monoclonal antibodies against the indicated specificity in human tissues were used: anti-TLR4/MD-2 complex, 1 µg/mL (clone HTA125, mouse IgG2a; Santa Cruz Biotechnology, Santa Cruz, CA); anti-CD14, 2.5 µg/mL (clone B365.1, mouse IgG1, 
; Biomeda Corp., Foster City, CA); anti-HLA-DR, 1 µg/mL (clone YD1/63.4.10, rat IgG2a; Cedarlane, Ontario, Canada); anti-CD68, 12.5 µg/mL (clone PG-M1, mouse IgG3, ; Dako, Glostrup, Denmark); and anti-von Willebrand Factor, 6.3 µg/mL (clone F8/86, mouse IgG1, ; Dako). After three washes with TBS, the sections were incubated with biotinylated rabbit anti-rat (3.3 µg/mL, Dako) or goat anti-mouse (7.5 µg/mL; Vector Laboratories, Burlingame, CA) secondary antibodies for 30 minutes at room temperature. After three further washes in TBS, the sections were incubated with horseradish peroxidase-conjugated streptavidin (Vector Laboratories) for 1 hour at room temperature. Immunolocalization was performed with the addition of the substrate 3-amino-9-ethylcarbazole (Sigma-Aldrich, St. Louis, MO) and the chromogen development monitored by light microscopy. The reaction was stopped once suitable color had developed or after a maximum of 10 minutes. Sections were counterstained with hematoxylin and mounted in crystal mounting medium (Biomeda Corp.).
As a positive control, human lymphoid tissues were stained for TLR4/MD-2, CD14, HLA-DR, and CD68.5 11 12 13 Negative controls included the replacement of the primary antibody with species- and isotype-matched monoclonal antibodies at the same concentrations or the omission of the primary antibody.
Double-Labeling Immunofluorescence Microscopy
Double immunofluorescence was performed by serially incubating the cryostat tissue sections with two primary antibodies of different species, using the same antibodies at the concentrations used for the immunoperoxidase staining. After blocking nonspecific binding as previously described with 20% normal rabbit serum, the sections were incubated with the first primary antibody, rat anti-human HLA-DR monoclonal antibody, for overnight at 4°C. After they were washed with TBS, sections were incubated with biotinylated rabbit anti-rat antibody for 30 minutes. After further washes with TBS, the sections were then incubated with Alexa 488-conjugated streptavidin (2 µg/mL, excitation-emission maxima of 495/519 nm; Molecular Probes, Eugene, OR) for 1 hour in the dark at room temperature. All subsequent steps were performed at room temperature with the sections protected from light.
The sections were washed with TBS and blocked with 20% normal goat serum for 30 minutes and then incubated for 3 hours with the second primary antibody, which was a mouse anti-human monoclonal antibody (anti-TLR4/MD-2, anti-CD14, or anti-CD68). After they were washed with TBS, sections were incubated with Alexa 568-conjugated goat anti-mouse antibody (10 µg/mL, excitation-emission maxima of 578/603 nm; Molecular Probes) for 1 hour. After further washing, slides were mounted in anti-fade medium (Vectashield; Vector Laboratories). Negative controls included the replacement of the first or second primary antibody or of both antibodies with the species- and isotype-matched irrelevant antibodies.
Slides were examined by microscope (BX60; Olympus, Tokyo, Japan) equipped with a 100-W mercury burner for epifluorescence illumination and wide-band interference filters for blue excitation-green emission (460–490-nm band-pass, 515-nm long-pass) as well as green excitation-red emission (520–550-nm band-pass, 590-nm long-pass). Images were captured with a digital camera (Spot Cooled Color Digital Camera; Diagnostic Instruments, Sterling Heights, MI) and the pairs of images were superimposed for colocalization analysis using image-management software (Photoshop ver. 7; Adobe Systems, Mountain View, CA).
Confocal Microscopy
Double immunofluorescence of tissue sections was also examined by inverted confocal laser scanning microscope (TCS; Leica Microsystems, Mannheim, Germany) fitted with helium and argon lasers (Leica).
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Double immunohistochemistry for TLR4/MD-2 and CD68 could not be performed, because these primary antibodies were from the same species and thus indistinguishable at the level of the secondary antibody. Therefore, the expression of TLR4/MD-2 by resident uveal macrophages could not be directly examined in this study. Previous studies have shown that monocytes/macrophages have high levels of expression of TLR45 12 27 and thus it is likely that a minor subset of the HLA-DR+ TLR4/MD-2+ cells demonstrated in this study were resident uveal macrophages, although this has not been definitively shown in the current experiments. Similarly, direct colocalization studies for TLR4/MD-2 and CD14 were unable to be performed as these antibodies also originated from the same species. However, double immunofluorescence for HLA-DR and CD14 demonstrated that HLA-DR+ APCs coexpress CD14 (Fig. 2G) . Thus, these results demonstrate that HLA-DR+ APCs coexpress both TLR4/MD-2 and CD14 that together constitute the complete LPS-signaling receptor complex.
Pattern of Expression of the LPS Receptor Complex in Normal Human Ocular Tissues
Within the normal human uvea, there was a relatively rich network of TLR4/MD-2+ CD14+ APCs in the iris root and stroma of the ciliary body, compared to the iris or choroid in which only occasional positive-staining cells were found (Figs. 2 and data not shown). Some of these TLR4/MD-2+ CD14+ resident APCs within the uvea displayed a perivascular (Figs. 2A 2B 2J 2K) or subepithelial location (Figs. 2H 2I) . No other cell types in the normal human uvea expressed TLR4/MD-2 or CD14 protein. Notably, in contrast to other tissues such as the skin,17 28 TLR4/MD-2 complex was not expressed on the epithelial cells of the uvea, such as the ciliary or iris epithelium, nor on the vascular endothelium (Figs. 2) . Possible masking of positive chromogenic staining by the melanin granules in the pigmented epithelium of the iris and ciliary body was excluded by immunofluorescence microscopy (data not shown).
We were unable to demonstrate in vivo protein expression of TLR4/MD-2 complex in the normal human cornea, conjunctiva (Fig. 3) , retina, or sclera (Fig. 2J) as detectable by immunohistochemistry.
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Activation of TLR4 by its principal agonist, LPS, results in an immunostimulatory intracellular signaling pathway leading to the activation of the transcriptional factor, nuclear factor-B (NF-B), and consequently the induction of various proinflammatory cytokines, chemokines, and antimicrobial activities.4 5 6 29 30 The present study has shown that TLR4+ MD-2+ CD14+ APCs appear to be strategically placed in perivascular and subepithelial locations within the normal uvea and suggests that such uveal APCs endowed with the complete LPS-signaling receptor complex are optimally positioned to survey and respond to either blood-borne or intraocular LPS of Gram-negative bacteria. Thus, these innate immune cells in the uvea would be expected to be able to respond rapidly to LPS of Gram-negative bacteria, in contrast to the primary adaptive immune response that requires several days to become effective as this involves clonal expansion and maturation of naive effector cells. By becoming activated through its pattern recognition of the PAMP, LPS receptor-positive macrophages would acquire enhanced effector functions such as phagocytic activity,5 thus facilitating the rapid containment and eradication of the microbe with minimal tissue damage. In addition to their critical role in the innate immune response, TLRs have been recognized to be important in the efficient priming and initiation of the adaptive immune response by its activation of APCs.5 6 29 We propose that TLR4-dependent stimulation of resident DCs by LPS in the human uvea induces DC maturation with the induction of costimulatory molecules such as CD80 and CD86, the upregulation of major histocompatibility complex class II molecules, and the secretion of proinflammatory cytokines and chemokines that would recruit naive T cells and other inflammatory cells to the uvea for activation as has been shown in other settings.4 5 6 7 27 29 31 32 The perivascular location of TLR4+ DCs in the uvea places them in an unique position to activate the expression of vascular adhesion molecules and the initiation of the multistep process of leukocyte recruitment33 to the uvea through rolling, adhesion, and transendothelial migration toward the chemokines secreted by these LPS-activated perivascular DCs. Furthermore, LPS activation of DCs via TLR4 has been associated with a Th1 polarized immune response by inducing cytokines such as IL-12 and thus may have particular relevance to the pathogenesis of uveitis.29
The initiating factors in AAU are unknown despite intensive investigation. In addition to genetic factors, the most important of which is the strong association with HLA-B27, there is substantial evidence suggesting the role of environmental factors in its pathogenesis, particularly that of multiple Gram-negative bacteria.2 The development of AAU has been reported in patients after acute Yersinia or Salmonella infections.34 35 There have also been numerous studies demonstrating serologic evidence of infection with these implicated Gram-negative bacteria in patients with AAU.2 36 The inappropriate and/or exaggerated TLR4-mediated activation of the innate and adaptive immune responses within the uvea by LPS of the implicated Gram-negative bacteria may be a major contributing factor in the initiating mechanisms of AAU. LPS may also act as an adjuvant by activating APC maturation in the presence of the putative uveitogenic self-antigen(s) and thus mediate the breakdown of peripheral tolerance resulting in the induction of an autoimmune response. Current hypothesis on peripheral tolerance suggests that APCs that capture self-antigens present them to autoreactive T cells and induce T-cell tolerance by deletion or anergy, as these APCs are relatively immature.37 It is recognized that TLRs can convert tolerogenic signals to activating signals by promoting APC maturation.4 5 37 Thus, TLR4-mediated activation of resident uveal APCs may initiate breakdown in ocular immune privilege and the development of uveitis by various hypothesized autoimmune mechanisms such as that of molecular mimicry.2 In addition to the activation of TLR4 by LPS, its exogenous agonist, it has been more recently discovered that these receptors may also be stimulated by various endogenous agonists that are released at sites of inflammation and tissue injury, such as heat shock proteins and fibrinogen.5 38 This may be a mechanism for the perpetuation of uveitis and the progression to chronic inflammation as the products of tissue inflammation further stimulate, via TLR4, the immune cells that have infiltrated the eye.
Our finding that the epithelial and endothelial cells of the normal human iris and ciliary body do not express TLR4 and MD-2 proteins, in contrast to the corresponding cell types of other tissues such as the skin,14 17 may reflect the immunologically privileged nature of the eye. The strategic localization of the LPS receptor complex within the uvea may be designed to respond only to LPS of invasive organisms that have breached the blood-ocular barrier or the iris-ciliary epithelium, and thus minimizing the possibility of inappropriate and potentially sight-threatening ocular inflammatory responses to LPS.
TLR4/MD-2 protein was not detected at the normal ocular surface and this may again reflect the unique immune privileged state of the eye, particularly with respect to the cornea. Unique patterns of TLR expression appear to exist at different host-environment tissue interfaces. For example, TLR4 is strongly expressed by epithelial cells in the skin,17 whereas in the normal gastrointestinal tract, intestinal epithelial cells express minimal levels of TLR4, presumably to prevent inappropriate cellular activation in response to the constant exposure to LPS of commensal organisms in the gut lumen.16 We report herein that the normal human conjunctiva and cornea do not constitutively express the TLR4/MD-2 protein complex, and this may be important in the maintenance of clarity of the visual axis and prevention of inappropriate ocular surface inflammation in response to nonpathogenic LPS. Song et al.19 have previously demonstrated in vitro, cell surface expression of TLR4/MD-2 on cultured human corneal epithelial cells and have shown that this expression was found to increase after LPS treatment. The particular environment of cell culture mediums, including the presence of serum and/or LPS may modulate the in vitro expression of TLR4/MD-2. It is therefore possible that upregulation of these LPS receptor complexes from their low or undetectable constitutive levels may occur in response to the appropriate stimuli in various ocular surface inflammatory conditions, although these possibilities were not examined in this study.
In summary, the results of the present study demonstrate for the first time, the in vivo expression of TLR4 and its associated LPS-receptor complex in the normal human eye and their implications for ocular immunity in health and disease have been discussed. The preferential expression of these receptors on APCs within the uvea suggests a novel mechanism for the initiating factors and immunopathogenesis of uveitis, particularly HLA-B27-associated AAU, that have a particular predilection for affecting this middle vascular layer of the eye. Further studies, including functional studies, are indicated to investigate further the role of these receptors in the context of normal and pathologic ocular immunity.
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Acknowledgements |
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Footnotes |
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Submitted for publication October 7, 2003; revised January 15, 2004; accepted February 12, 2004.
Disclosure: J.H. Chang, None; P. McCluskey, None; D. Wakefield, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Denis Wakefield, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia; d.wakefield{at}unsw.edu.au.
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