November 2001
Volume 42, Issue 12
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Immunology and Microbiology  |   November 2001
The Expression of Functional LPS Receptor Proteins CD14 And Toll-Like Receptor 4 in Human Corneal Cells
Author Affiliations
  • Peter I. Song
    From the Departments of Dermatology and
  • Tonya A. Abraham
    From the Departments of Dermatology and
  • Youngmin Park
    From the Departments of Dermatology and
  • Adam S. Zivony
    From the Departments of Dermatology and
  • Brad Harten
    From the Departments of Dermatology and
  • Henry F. Edelhauser
    Ophthalmology, Emory University School of Medicine, Atlanta, Georgia; the
  • Sherry L. Ward
    Gillette Medical Evaluation Laboratories, Needham, Massachusetts; and the
  • Cheryl A. Armstrong
    From the Departments of Dermatology and
    Veterans Affairs Medical Center, Atlanta, Georgia.
  • John C. Ansel
    From the Departments of Dermatology and
    Veterans Affairs Medical Center, Atlanta, Georgia.
Investigative Ophthalmology & Visual Science November 2001, Vol.42, 2867-2877. doi:
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      Peter I. Song, Tonya A. Abraham, Youngmin Park, Adam S. Zivony, Brad Harten, Henry F. Edelhauser, Sherry L. Ward, Cheryl A. Armstrong, John C. Ansel; The Expression of Functional LPS Receptor Proteins CD14 And Toll-Like Receptor 4 in Human Corneal Cells. Invest. Ophthalmol. Vis. Sci. 2001;42(12):2867-2877.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Gram-negative bacterial infections of the eye can lead to corneal bacterial keratitis, visual impairment, and blindness. Many of these pathologic changes may be mediated by bacterially derived products such as lipopolysaccharide (LPS). In this investigation, it has been established for the first time that human corneal cells are capable of expressing the functional LPS receptor complex proteins, CD14 and Toll-like receptor 4 (TLR4).

methods. CD14 and TLR4 mRNA expression in human corneal cells was determined by RT-PCR and Northern blot analysis, and cell surface expression of these proteins was measured by flow cytometry. LPS-mediated corneal cell activation was determined by measuring intracellular calcium mobilization. Cellular cytokine and chemokine secretion in response to LPS was measured by ELISA. The expression and localization of CD14 in whole human cornea was determined by immunohistochemistry.

results. Human corneal epithelial, stromal, and endothelial cells expressed CD14 mRNA and cell surface CD14. LPS binding to cornea CD14 resulted in a rapid intracellular calcium response and the secretion of multiple proinflammatory cytokines and chemokines. CD14 mRNA expression in corneal epithelial cells was upregulated by LPS. In addition to CD14, corneal epithelial cells expressed the functional LPS receptor–signaling protein TLR4, which was also augmented by LPS.

conclusions. The cornea expresses functional CD14 and TLR4 LPS receptor proteins. Understanding the function and biology of the corneal LPS receptor complex may lead to novel therapies for the management of ocular Gram-negative bacterial infections.

Gram-negative bacteria, such as Pseudomonas aeruginosa, are opportunistic ocular pathogens that can initiate a fulminating, highly destructive corneal infection in humans that may result in decreased visual acuity or blindness. 1 The incidence of Pseudomonas keratitis has greatly increased with the widespread use of contact lenses. Annually, 25,000 contact lens wearers have bacterial keratitis in the United States. 2 3 Contact lens corneal injury, most often seen with the use of extended-wear contact lens, is believed to facilitate the development of this infection. Although Gram-negative bacteria are capable of producing a large number of potential virulence factors, 4 5 6 lipopolysaccharide (LPS), a major component of the outer membrane of P. aeruginosa, is a principal initiator of host innate immune responses. 7 8 9 LPS exerts many of its biologic effects by stimulating host cells to produce bioactive inflammatory mediators. Monocytes and macrophages respond to LPS with the synthesis and release of proinflammatory cytokines, such as TNFα, IL-1, -6, and -8. 10 11 At low concentrations, LPS activates leukocytes to trigger a variety of host responses to eliminate invading bacteria. At higher concentrations, LPS may induce significant morbidity due to Gram-negative infections by its capacity to induce shock, fever, and severe inflammatory reactions during Gram-negative sepsis, through the release of endogenous mediators from host cells. 12  
CD14 is a 55-kDa glycosyl phosphatidylinositol (GPI)–anchored glycoprotein identified on the surface of monocytes, macrophages, and polymorphonuclear leukocytes(PMNs). 13 14 15 CD14 has been shown to be a pivotal membrane receptor for LPS-mediated cellular responses based on studies using monoclonal anti-CD14 antibodies, transfection of CD14 into Chinese hamster ovary (CHO) cells, and CD14 knockout mice. 16 17 18 During Gram-negative bacterial infections, LPS is released and complexes with the serum-derived LPS-binding protein (LBP), which acts as a lipid transfer protein that facilitates the binding of LPS monomers to the CD14 LPS receptor. 19 20 Although LBP mediates the transfer of LPS to CD14, LBP is not essential for this interaction, because LPS is capable of directly binding to CD14, although not as efficiently as LPS-LBP. 21 LPS binding to CD14 in a transfected cell line results in rapid intracellular Ca2+ mobilization, cellular tyrosine kinase phosphorylation, nuclear factor (NF)-κB activation, cytokine, and chemokine production. 22 Blockade of cell surface CD14 with anti-CD14 monoclonal antibodies prevents binding of LPS. This results in the inhibition of LPS-mediated production of TNFα by monocytes and prevents the activation of cellular adhesion molecule receptors of PMNs that normally occur in response to small amounts of LPS. 23  
Recent studies indicate that after LPS binding to CD14, additional cell membrane proteins are required for the initiation of transmembrane signaling. 24 25 26 27 28 29 30 31 32 The principal signaling component of the LPS receptor complex appears to be the Toll-like receptor 4 (TLR4) protein. 24 25 26 27 30 32 33 These studies indicate that CD14, in conjunction with TLR4, is necessary for the LPS-induced cellular responses. However, the precise way in which these proteins interact to confer cellular LPS responsiveness remains to be determined. 
Although there has been significant progress in our understanding of the expression and function of LPS binding and responses in leukocytes, little is currently known about LPS responses in nonleukocytes. It has been proposed that nonleukocytes primarily respond to LPS after it is complexed to LBP and soluble (s)CD14. 34 35 36 37 The cornea is avascular and thus, in contrast to most tissues, must initially respond to infectious agents without the assistance of circulating leukocytes. This early innate immunologic response of the cornea is poorly understood but may be critically important, allowing the cornea to rapidly respond and limit external ocular insults. Because responses of the cornea to external pathogens such as LPS cannot be predicted based on previous studies examining leukocyte responses, we examined the expression and function of both CD14 and TLR4 in human corneal cells. The results of these studies indicate that human corneal cells express both the functional CD14 and TLR4 LPS receptor proteins. This finding may have important implications for our understanding and treatment of innate immunologic responses to ocular microbial infections. 
Methods
Reagents and Cell Lines
LPS derived from Pseudomonas aeruginosa (List Biological Laboratories, Inc., Campbell, CA) was used at a concentration of 50 ng/ml, based on our dose–response study and previous published studies by other investigators demonstrating the efficacy of this concentration of LPS in inducing physiologic responses in several different cell types. 38 39 40 Normal whole human corneas stored in preservative (Optisol; Chiron, Irvine, CA) were obtained from the Georgia Eye Bank and used within 4 days of enucleation. Fresh normal human corneal epithelial cells, stromal cells, and endothelial cells were isolated and cultured from donor corneas, as previously described. 41 42 For culturing normal human corneal epithelial cells, corneas were removed from the preservative and rinsed in Hanks’ balanced salt solution (HBSS). Corneas were divided into sections and placed epithelial side down in each well in collagen-coated six-well plates. Sectioned corneas were cultured for 5 days in keratinocyte growth medium (KGM; Clonetics, San Diego, CA) at 37°C in a humidified atmosphere containing 5% CO2. Cornea sections were then carefully removed from the wells to leave corneal epithelial cells attached to collagen matrix in the wells. The human corneal epithelial cell line 10.014 pRSV-T (HCE-T) was kindly provided by Sherry Ward (Gillette Medical Evaluation Laboratories, Gaithersburg, MD). 43 44 45 Human corneal stromal cells were maintained in DMEM/F12 cell culture media (Gibco BRL, Life Technology, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT). Cells were then maintained in monolayer cultures at 37°C in a humidified atmosphere containing 5% CO2 and harvested for use after passages 3 to 4. Cultured corneal epithelia–derived cells were grown in monolayers using serum-free medium (KGM; Clonetics) at 37°C in a humidified atmosphere containing 5% CO2. Cell culture media were changed every 2 to 3 days. The human B lymphoblastoid JY-1 cells (generously provided by Jack Strominger, Dana Farber Cancer Institute, Boston, MA) were cultured in RPMI 1640 medium (Gibco-Life Technologies) and supplemented with 10% FBS (HyClone), 100 U/ml penicillin, 0.25 mg/ml amphotericin B, and 10 μg/ml streptomycin (all from Gibco-Life Technologies). The U937 human monocyte cell line (American Type Culture Collection, Rockville, MD) was maintained in RPMI 1640 medium supplemented with 5% FCS (Gibco Life Technologies), 2 mM l-glutamine (Sigma, St. Louis, MO), and 0.1 mg/ml gentamicin (Sigma) and incubated at 37°C in a humidified atmosphere of 5% CO2
Detection of CD14 and TLR4 mRNA by Reverse Transcription–Polymerase Chain Reaction
CD14 mRNA was measured in cultured normal human corneal epithelial, stromal, and endothelial cells by RT-PCR, with or without the addition of LPS. Human corneal cells were cultured as described in the prior section, and mRNA was isolated with mRNA isolation kits (Roche Molecular Biochemicals, Indianapolis, IN). RT-PCR was performed as described in the manufacturer’s protocol. Oligonucleotide primers used to amplify human CD14 cDNA were based on the published sequences. 46 47 The CD14 primer sequences used were 5′-CGTGGGCGACAGGGCGTTCT-3′ for the sense primer and 5′-TAAAGGTGGGGCAAAGGGTT-3′ for the antisense primer. PCR amplification yielded a 777-bp product generated from human corneal cDNA templates. PCR was performed with 35 cycles performed as previously described. 48 TLR4 mRNA was also measured in cultured normal epithelial and HCE-T cells by RT-PCR, as just described. The oligonucleotide primers used to amplify the TLR4 cDNA was based on published sequences of this gene. 49 The TLR4 primer sequences used were 5′-GCTTACTTTCACTTCCAACAA-3′ for the sense primer and 5′-CAATCACCTTTCGGCTTTTAT-3′ for the antisense primer. PCR amplification yielded a 1139-bp product generated from human corneal cDNA templates. PCR was performed with 35 amplification cycles, as previously described. 48  
Determination of Surface CD14 and TLR4 Expression by Flow Cytometry
Corneal cell surface CD14 and TLR4 were examined by flow cytometry (FACScan; Becton Dickinson, Raleigh, NC), as previously described. 48 Cells were incubated with the mouse anti-human CD14 monoclonal antibody RMO52 (Immunotech, Marseille, France), anti-human TLR4 monoclonal antibody HTA125 (eBioscience, San Diego, CA), or isotype control mouse anti-human IgG (H+L; Jackson ImmunoResearch, West Grove, PA) at a final concentration of 10 μg/ml for 1 hour on ice. Cells were then washed twice and incubated with FITC-conjugated affinity-purified goat F(ab′)2 anti-mouse IgG (H+L; Jackson ImmunoResearch) at a final concentration of 10 μg/ml for 1 hour on ice. Cells were then again washed twice and analyzed by a flow cytometer equipped with the manufacturer’s software (CellQuest; Becton Dickinson) for data acquisition and analysis. For these studies, JY-1 cells and phorbol 12-myristate 13-acetate (PMA)–differentiated U937 cells were used as negative and positive control cultures, respectively. 50 51  
Intracellular Calcium Mobilization Studies
Intracellular calcium mobilization was determined to assess the functional response of corneal CD14 and TLR4 by LPS. Cultured human corneal epithelial cells were grown on glass coverslips to approximately 50% to 70% confluence. Cells then were washed twice with HBSS without Ca2+ and Mg2+ containing 10 mM HEPES (Gibco BRL, Grand Island, NY) and then incubated for 45 minutes at 37°C in HBSS containing 10 mM HEPES and 2 μM of the fluorescent calcium probe fura-2/acetylmethyl (AM) ester (Molecular Probes, Eugene, OR). After three washes with this same washing buffer, cells were treated either with 50 ng/ml Pseudomonas LPS or were pretreated for 30 minutes with 0.5 μg/ml anti-CD14 monoclonal antibody (Leu-M3; Becton Dickinson Immunocytometry Systems, San Jose, CA) or anti-human TLR4 monoclonal antibody HTA125 (eBioscience) before LPS. Fluorescence was measured with a spectrofluorometer (model LS50; Perkin Elmer, Branchburg, NJ) with excitation wavelengths of 340 and 380 nm, and an emission wavelength of 510 nm. From the ratio of measured fluorescence at the two excitation wavelengths, the intracellular free calcium was calculated as previously described. 52  
Determination of CD14 and TLR4 mRNA Expression by Quantitative RT-PCR
CD14 and TLR4 mRNA was measured in cultured human corneal cells after exposure to LPS by quantitative RT-PCR. Human corneal epithelial cells were cultured as described earlier, and mRNA was isolated using mRNA isolation kits (Roche Molecular Biochemicals). Quantitative RT-PCR was performed as described in the manufacturer’s protocol (SYBR Green PCR Core Reagents; PE-Applied Biosystems, Foster City, CA). Oligonucleotide primers used to amplify human CD14 and TLR4 cDNA were designed by use of the manufacturer’s software (Primer Express 1.0; PE-Applied Biosystems) based on the published sequences. 47 49 The CD14 primer sequences used were 5′-CGCTCCGAGATGCATGTG-3′ for the sense primer and 5′-AACGACAGATTGAGGGAGTTCAG-3′ for the antisense primer. The TLR4 primer sequences used were 5′-TGGTGGAAGTTGAACGAATGG-3′ for the sense primer and 5′-AGGACCGACACACCAATGATG-3′ for the antisense primer. 
Determination of TLR4 mRNA Expression in HCE-T Cells by Northern Blot Analysis
HCE-T cells were cultured at a density of 1 × 106 cells/ml, and mRNA was obtained with an mRNA isolation kit (Roche Molecular Biochemicals). Northern blot analysis was performed as previously described. 48 The TLR4 probe used for the Northern blot analysis studies was a synthesized oligonucleotide (5′-ACAGAGACTTTATTCCCGGTGTGGCCATTG-3′) based on published TLR4 cDNA sequences. 49  
Determination of Cytokine and Chemokine Secretion by Cultured HCE-T Cells by ELISA
To quantify cytokine and chemokine secretion, HCE-T cells were plated in tissue culture flasks and after reaching confluence were either left untreated or were preincubated with the anti-CD14 monoclonal antibody Leu-M3 (0.5 μg/ml) for 2 hours followed by the addition of LPS (50 ng/ml). To determine the specificity of these CD14 antibody studies, control experiments were performed in which cells were preincubated with an antibody directed against a monomorphic determinant of human class I HLA molecules (HLA-A,B,C; 0.5 μg/ml) followed by exposure to LPS (50 ng/ml). After 24 hours, the culture supernatants were harvested and tested by ELISA (R&D Systems, Minneapolis, MN), as previously described, 53 to measure secreted IL-6, -1α, and -8 and TNFα. 
Immunolocalization of CD14 in Whole Human Cornea
Human corneas stored in preservative were fixed for 4 hours in 10% formaldehyde solution and embedded in paraffin. Specimens were prepared for immunohistochemistry, as previously described. 54 Specimens were then incubated overnight at 4°C with the anti-human CD14 monoclonal antibody (Biomeda Corp., Foster City, CA). Subsequently, sections were incubated for 1 hour at room temperature with biotin-conjugated anti-mouse IgG. As a control, the same specimens were incubated with an isotype-matched antibody using identical experimental conditions. After three washes in PBS buffer, the samples were incubated with streptavidin-horseradish peroxidase avidin-biotin complex (StreptABComplex/HRP, Dako Corp., Carpinteria, CA) for 15 to 30 minutes at room temperature. Slides were then incubated with substrate 3,3′ diaminobenzidine tablets (DAB; Sigma) for 5 minutes. The sections were counterstained with hematoxylin for 1 to 4 minutes. The immunolocalization of CD14 was observed and photographed by light microscopy (Olympus Optical Co., Ltd., Tokyo, Japan). Four human corneas were examined by immunohistochemistry, and one representative example was prepared for this study. 
Statistical Analysis
Results are expressed as mean ± SE. For statistical analysis, ANOVA with probabilities were performed for both the overall significance (P) and the pair-wise comparison, indicated by asterisks. P < 0.05 was considered to be significant. 
Results
Expression of CD14 mRNA
To determine whether CD14 mRNA was constitutively expressed in human corneal cells, we prepared mRNA from freshly isolated corneal epithelium, stromal cells, and endothelium, and performed RT-PCR followed by partial DNA sequencing of the PCR product. A specific CD14 PCR amplification product of 777 bp was generated from mRNA isolated from human corneal epithelium, stromal cells, and endothelium using primers specific for human CD14 (Figs. 1A 1B 1C) . The B-lymphoblastoid cell line JY-1, which does not express CD14, was used as a negative control, and the CD14-expressing U937 human monocyte cell line was used as a positive control (Fig. 1D) . The identity of amplified gene products in human corneal cells was confirmed by nucleotide sequencing as shown in Figure 1E . This nucleotide sequence was identical with the GeneBank sequence reported for human leukocyte CD14 (GeneBank is provided by the National Center for Biotechnology Information, Bethesda, MD, and is available in the public domain at http://www.ncbi.nlm.nih.gov/genebank/). 47 These results demonstrate that CD14 mRNA is constitutively expressed in normal human corneal epithelium, stromal cells, and endothelial cells. 
Expression of Cell Surface CD14
We next examined the cell surface expression of CD14 in normal human corneal cells by flow cytometry. CD14 cell surface expression was detected on human corneal epithelial cells (Fig. 2B) , stromal cells (Fig. 2C) , and endothelial cells (Fig. 2D) . In these studies, cultures of U937 cells and JY-1 cells served as positive and negative controls, respectively (Fig. 2A) . Thus, normal corneal epithelial cells, stromal cells, and endothelial cells not only expressed CD14 mRNA but also expressed cell surface CD14. 
LPS-Induced CD14-Dependent Intracellular Ca2+ Response
Corneal epithelial cells represent the critical initial barrier to Gram-negative corneal infections. For this reason subsequent studies were focused on this cornea cell type. We tested the functional competence of corneal epithelial cell CD14 to respond to LPS. For these studies we used the HCE-T human corneal epithelial cell line. HCE-T cells have been demonstrated to be biologically similar to freshly isolated human corneal epithelial cells. 43 44 45 Previous studies in other laboratories indicate that LPS-triggered CD14 activation in monocytes results in a rapid increase in intracellular Ca2+ levels. 22 The addition of Pseudomonas LPS to HCE-T resulted in a rapid intracellular Ca2+ response (Fig. 3B) , whereas the addition of human serum alone (0.1%) did not affect Ca2+ mobilization (Fig. 3A) . The peaks represent the simultaneous response of different cells. When HCE-T cells were preincubated with the anti-CD14 monoclonal antibody Leu-M3 (0.5μ g/ml) for 30 minutes before the addition of LPS, the intracellular Ca2+ response was abrogated (Fig. 3C) . Similarly, when HCE-T cells were treated with LPS plus polymyxin B (300 ng/ml) to confirm that this response was due to LPS (not to contaminants), no intracellular calcium fluctuation was detected (Fig. 3D) . Therefore, these results demonstrate that LPS can trigger a rapid induction of corneal epithelial cell intracellular Ca2+ by a CD14-dependent mechanism. 
LPS-Induced Upregulation of CD14 mRNA Expression
In monocytes, LPS is capable of upregulating the expression of CD14. We performed studies to determine whether LPS would have a similar effect in corneal epithelial cells. CD14 mRNA expression after LPS treatment was measured in freshly isolated human corneal epithelial cells as well as HCE-T cells by quantitative RT-PCR, and results were normalized to human 18S rRNA mRNA expression for each experimental condition. Freshly isolated human corneal epithelial cells constitutively expressed low levels of CD14 mRNA that were increased after the addition of LPS (50 ng/ml; Fig. 4A ). When we analyzed the kinetics of this effect in HCE-T cells, CD14 mRNA expression was increased at 6 hours and peaked 12 hours after the addition of LPS (Fig. 4B) . A similar induction of CD14 mRNA was observed in human corneal stromal cells after the addition of LPS (data not shown). In a dose–response study, 10 ng/ml of LPS induced increased CD14 mRNA expression, which became maximally elevated after the addition of 50 ng/ml LPS (Fig. 4C) . Therefore, in the current study LPS was capable of upregulating the expression of corneal CD14 that could in turn facilitate and amplify further LPS–corneal interactions. 
LPS-Induced Production of Cytokines and Chemokines
To examine biological relevance of the HCE-T CD14 receptor in the initiation of innate inflammatory responses to LPS, we measured the effect of Pseudomonas-derived LPS on human corneal epithelial proinflammatory cytokine and chemokine production. The effect of LPS on IL-6 secretion by HCE-T cells was measured by ELISA. The cells secreted increased amounts of IL-6 at 24 and 48 hours after exposure to LPS (Fig. 5A) . In other experiments we found that LPS also induced HCE-T IL-1α and TNFα secretion (data not shown). We then examined the effect of LPS on the secretion of the CXC chemokine, IL-8, in HCE-T cells. Our studies demonstrated that the cells secreted increased quantities of IL-8 at 24 and 48 hours after the addition of LPS (Fig. 6A)
To determine whether the induction of HCE-T IL-6 and -8 by LPS was primarily mediated by binding to CD14, HCE-T cells were preincubated with the LPS blocking anti-CD14 monoclonal antibody Leu-M3 for 2 hours before the addition of LPS. Treatment with Leu-M3 abrogated the induction of HCE-T cell IL-6 and -8 by LPS (Figs. 5B 6B , respectively). In contrast, when HCE-T cells were preincubated with an irrelevant antibody directed against a monomorphic determinant of major histocompatibility complex (MHC) class I HLA, no inhibition of HCE-T cell IL-6 and -8 secretion was observed after the addition of LPS (Figs. 5B 6B) . Thus, these data demonstrate that LPS was capable of directly activating corneal epithelial cells to secrete proinflammatory cytokines and chemokines by a CD14 mediated mechanism. 
Immunolocalization of CD14 in Whole Human Cornea
We next determined whether CD14 is immunolocalized in fresh whole human cornea (Fig. 7) . In normal human corneal tissue, immunoreactive CD14 staining was detected in corneal epithelium (Figs. 7B 7D) , stromal cells (Figs. 7F 7H) , and corneal endothelial cells (Figs. 7F 7H) . In contrast, little staining was found in tissue treated with an isotype-matched antibody (Figs. 7A 7C 7E 7G) . Thus, CD14 was found to be expressed in cells of the epithelium, stroma, and endothelium in human corneas. 
Expression of TLR4 mRNA
A number of recent studies indicate that TLR4 is the signaling component of the LPS cellular receptor. 24 25 26 27 30 32 55 56 To determine whether TLR4 is expressed in HCE-T cells, we prepared mRNA from these cells and performed RT-PCR. A PCR amplification product of 1139 bp was generated from HCE-T cDNAs, by using primers specific for human TLR4 (Fig. 8A) . 49 We confirmed that HCE-T express TLR4 mRNA by Northern blot analysis (Fig. 8B) . As indicated, TLR4 mRNA was constitutively expressed in HCE-T cells and was augmented by LPS. The induction of HCE-T TLR4 mRNA by LPS was abrogated by pretreatment of cells with the anti-CD14 monoclonal antibody Leu-M3. The relative intensity of HCE-T cell TLR4 expression was determined by densitometric analysis. Using quantitative RT-PCR, we confirmed that TLR4 mRNA is also expressed in freshly isolated corneal epithelial cells, and increased expression was detected after the addition of LPS (Fig. 8C) . Thus, in these studies TLR4 mRNA was expressed in human corneal epithelial cells. 
Expression of Cell Surface TLR4
We next examined the cell surface expression of TLR4 in HCE-T cells by flow cytometry. TLR4 was constitutively expressed on human corneal epithelial cells and increased after LPS treatment (Fig. 9B) . Human monocytic leukemia (THP-1) cells, and human B-lymphoblastoid (JY-1) cells served as positive and negative controls, respectively (Fig. 9A) . Thus, these results indicate that human corneal epithelial cells not only expressed TLR4 mRNA but also cell surface TLR4, which was increased by LPS treatment. 
LPS-Induced TLR4-Dependent Intracellular Ca2+ Response
We tested the functional competence of HCE-T TLR4 to respond to LPS. The addition of Pseudomonas LPS to HCE-T cells resulted in a rapid intracellular Ca2+ response (Fig. 10A) . On the contrary, when HCE-T cells were preincubated with the anti-TLR4 monoclonal antibody HTA125 (0.5 μg/ml) for 30 minutes before the addition of LPS, the intracellular Ca2+ response was abrogated (Fig. 10B) . The peaks represent the simultaneous response of different cells. Therefore, these results indicate that LPS could trigger a rapid induction of corneal epithelial cell intracellular Ca2+ by a TLR4- as well as a CD14-dependent mechanism. 
Discussion
LPS exerts many of its biologic effects by binding to specific cell surface receptors. A number of candidate proteins have been proposed to be components of the LPS receptor complex. 16 30 57 58 59 60 61 CD14 appears to be the principal binding element of the LPS receptor complex. LPS can either bind directly to CD14 or in conjunction with LBP, which facilitates transfer and binding to CD14. 20 21 62 In previous investigations CD14 expression has been primarily described on myeloid-derived cells, such as monocytes and macrophages. 13 14 15 63 Our studies indicate that the cornea is also capable of expressing functional CD14. When LPS or LPS-LBP engages monocyte CD14, a series of cellular activation events occur that result in the production and release of proinflammatory factors that are capable of initiating host innate immune responses. 19 22 23 Similarly, our data support the existence of a similar cellular response when the cornea encounters LPS that may play an important role in innate immune responses in corneal diseases such as Gram-negative bacterial keratitis. 
Because CD14 is a GPI-anchored membrane protein that has no intrinsic intracytoplasmic signaling sequences, other intracellular molecules are required for cellular responses. It has been proposed that CD11/CD18 may serve as the cell-signaling component of CD14 32 . Recently two strong candidates for the LPS signaling protein have been identified in myeloid cells and have been termed TLR2 and TLR4. 24 25 26 27 28 29 30 31 32 61 64 Several studies report that TLR4 may be the predominant cell surface–signaling molecule for cellular LPS responses 24 30 55 56 65 66 whereas TLR2 may respond to certain Gram-positive bacterial products such as Staphylococcus aureus, Streptococcus pneumoniae, or Bacillus subtilis. 67 68 Studies by Yang et al. 28 indicate that LPS may not only bind to CD14 but also to TLR proteins, either independently or in conjunction with a CD14 complex. However, it was also reported that LPS binding to TLR4 alone is not as effective in generating a functional cellular response as binding to the CD14-TLR4 complex. 30 66 69 70 Results in our studies indicate, for the first time, expression of both functional CD14 and TLR4 in human cornea cells. 
Significant gaps still remain in our understanding of the mechanisms by which CD14 and TLR4 mediate cellular LPS responses. It has been proposed that sCD14 and TLR mediate LPS responses in nonleukocytes. 34 35 36 37 71 However, our data indicate that corneal cells themselves are capable of expressing endogenous membrane CD14 and thus are not dependent on sCD14 for LPS activation. It is believed that in innate immunologic responses, different combinations of TLRs in conjunction with CD14 may mediate differential inflammatory responses after encountering different groups of pathogens. Thus, it is likely, as has been proposed for other cell types, that TLR4 is the functional signaling component of CD14 in the cornea to Gram-negative bacterial infections. 
Our studies also indicate that LPS binding to the human cornea CD14 receptors initiated a rapid innate immune response by the production of proinflammatory cytokines and chemokines such as IL-6 and -8, which can initiate an efficient host response to Gram-negative corneal infections. The cornea is a unique tissue that is significantly different from CD14-expressing leukocytes in regard to function and biological response to various stimuli and thus offers a novel opportunity to define the innate immunologic responses in this critical nonvascular ocular structure. The activation of corneal CD14 may have both beneficial and detrimental inflammatory effects on the cornea, depending on the effectiveness and duration of the host inflammatory response. There is a fragile balance between generating a successful inflammatory response to eliminate the offending microorganism and an excessive inflammatory response that can result in corneal scarring and blindness. For example, it has been reported that C3H/HeJ mice that do not have a functioning TLR4 receptor are susceptible to Pseudomonas keratitis, which may be in part because of the deficiency in TLR4-mediated corneal innate immune responses that results in poor clearing of this pathogen by corneal host mechanisms. 72 73 74 75 Understanding the molecular pathogenesis of LPS interactions with the cornea and the inflammatory consequences of corneal CD14-TLR4 complex activation may permit the development of novel, specific therapies that can be delivered topically to prevent some of the destructive consequences of ocular Gram-negative infections. 
 
Figure 1.
 
Human corneal cell expression of CD14 mRNA. RT-PCR amplification products of 777 bp were generated from cDNAs, which were reverse transcribed from mRNA of human corneal epithelial cells (A; EP), stromal cells (B; S), or endothelial cells (C; EN), by using primers specific for human CD14. The U937 human monocyte cell line and B-lymphoblastoid JY-1 cells served as positive (right lane) and negative (left lane) controls, respectively, with size markers in the center lane (D). The amplified gene products were subcloned into a plasmid vector and partially sequenced by automated DNA sequencer. The nucleotide sequence of corneal CD14 is presented in (E).
Figure 1.
 
Human corneal cell expression of CD14 mRNA. RT-PCR amplification products of 777 bp were generated from cDNAs, which were reverse transcribed from mRNA of human corneal epithelial cells (A; EP), stromal cells (B; S), or endothelial cells (C; EN), by using primers specific for human CD14. The U937 human monocyte cell line and B-lymphoblastoid JY-1 cells served as positive (right lane) and negative (left lane) controls, respectively, with size markers in the center lane (D). The amplified gene products were subcloned into a plasmid vector and partially sequenced by automated DNA sequencer. The nucleotide sequence of corneal CD14 is presented in (E).
Figure 2.
 
Cell surface expression of CD14 on human corneal cells. Surface expression of CD14 on human corneal epithelial, stromal, and endothelial cells was assessed by flow cytometry. B-lymphoblastoid JY-1 cells, which express no CD14, were used as a negative control and the CD14-expressing U937 human monocyte cell line was used as a positive control (A). CD14 expression was detected on the surface of human corneal epithelial cells (B), human corneal stromal cells (C), and human corneal endothelial cells (D) by using a CD14-specific monoclonal antibody RMO52. Cells treated with an isotype-matched irrelevant antibody served as an additional control (open histogram). The data shown are representative of studies conducted in triplicate.
Figure 2.
 
Cell surface expression of CD14 on human corneal cells. Surface expression of CD14 on human corneal epithelial, stromal, and endothelial cells was assessed by flow cytometry. B-lymphoblastoid JY-1 cells, which express no CD14, were used as a negative control and the CD14-expressing U937 human monocyte cell line was used as a positive control (A). CD14 expression was detected on the surface of human corneal epithelial cells (B), human corneal stromal cells (C), and human corneal endothelial cells (D) by using a CD14-specific monoclonal antibody RMO52. Cells treated with an isotype-matched irrelevant antibody served as an additional control (open histogram). The data shown are representative of studies conducted in triplicate.
Figure 3.
 
LPS-induced CD14-dependent human corneal epithelial cell intracellular Ca2+ response. Cultured HCE-T cells were grown on glass coverslip culture dishes to approximately 50% to 70% confluence and 2μ M of the fluorescent calcium probe fura-2/AM was incorporated. (A) Cells treated with 0.1% human serum alone; (B) treated with 50 ng/ml LPS plus 0.1% human serum; (C) pretreated with 0.5 μg/ml anti-CD14 monoclonal antibody Leu-M3 30 minutes before 50 ng/ml LPS plus 0.1% human serum; (D) treated simultaneously with 50 ng/ml LPS plus 0.1% human serum and 300 ng/ml polymyxin B. Intracellular free calcium concentration was determined by measuring the ratio of fluorescence at excitation wavelengths of 340 and 380 nm.
Figure 3.
 
LPS-induced CD14-dependent human corneal epithelial cell intracellular Ca2+ response. Cultured HCE-T cells were grown on glass coverslip culture dishes to approximately 50% to 70% confluence and 2μ M of the fluorescent calcium probe fura-2/AM was incorporated. (A) Cells treated with 0.1% human serum alone; (B) treated with 50 ng/ml LPS plus 0.1% human serum; (C) pretreated with 0.5 μg/ml anti-CD14 monoclonal antibody Leu-M3 30 minutes before 50 ng/ml LPS plus 0.1% human serum; (D) treated simultaneously with 50 ng/ml LPS plus 0.1% human serum and 300 ng/ml polymyxin B. Intracellular free calcium concentration was determined by measuring the ratio of fluorescence at excitation wavelengths of 340 and 380 nm.
Figure 4.
 
LPS-augmented CD14 mRNA expression in human corneal epithelial cells. The effect of LPS on CD14 mRNA expression was measured by quantitative RT-PCR in primary human corneal epithelial cells and HCE-T cells. (A) CD14 mRNA expression in freshly isolated human corneal epithelial cells was examined 3 hours after the addition of LPS (50 ng/ml). (B) In an mRNA kinetic study, CD14 mRNA expression in HCE-T cells was examined 3, 6, 12, 24, and 48 hours after the addition of LPS (50 ng/ml). (C) In an LPS dose–response study, the effect of increasing concentrations of LPS (0, 10, 50, and 100 ng/ml and 1 and 10 μg/ml) on CD14 mRNA expression was determined in HCE-T cells 12 hours after LPS exposure. The relative intensity of CD14 mRNA expression was normalized with the mRNA expression of 18S rRNA for each experimental condition. The data shown are representative of triplicate experiments. All values are expressed as mean ± SD. Statistically significant differences in CD14 mRNA expression were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (*P < 0.001).
Figure 4.
 
LPS-augmented CD14 mRNA expression in human corneal epithelial cells. The effect of LPS on CD14 mRNA expression was measured by quantitative RT-PCR in primary human corneal epithelial cells and HCE-T cells. (A) CD14 mRNA expression in freshly isolated human corneal epithelial cells was examined 3 hours after the addition of LPS (50 ng/ml). (B) In an mRNA kinetic study, CD14 mRNA expression in HCE-T cells was examined 3, 6, 12, 24, and 48 hours after the addition of LPS (50 ng/ml). (C) In an LPS dose–response study, the effect of increasing concentrations of LPS (0, 10, 50, and 100 ng/ml and 1 and 10 μg/ml) on CD14 mRNA expression was determined in HCE-T cells 12 hours after LPS exposure. The relative intensity of CD14 mRNA expression was normalized with the mRNA expression of 18S rRNA for each experimental condition. The data shown are representative of triplicate experiments. All values are expressed as mean ± SD. Statistically significant differences in CD14 mRNA expression were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (*P < 0.001).
Figure 5.
 
LPS induced human corneal epithelial cell IL-6. LPS induction of HCE-T cell IL-6 secretion was examined by ELISA. (A) The effect of LPS (50 ng/ml) exposure on secreted IL-6 from 0 to 48 hours was measured in the cell culture supernatants. (B) The CD14 dependence of this response was determined in some studies by pretreating cells with either an anti-CD14 monoclonal antibody Leu-M3 (0.5 μg/ml) or an antibody to a common determinant of human MHC class I HLA (0.5 μg/ml). The data shown are representative of triplicate experiments. All values are expressed as mean ± SD. Statistically significant differences in secreted IL-6 in LPS-treated HCE-T cells were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (*P < 0.0002) in (A). The significance of differences in secreted IL-6 in LPS-treated HCE-T cells in the presence of anti-CD14 monoclonal antibody compared with those in the absence of the anti-CD14 antibody were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (**P < 0.02) in (B).
Figure 5.
 
LPS induced human corneal epithelial cell IL-6. LPS induction of HCE-T cell IL-6 secretion was examined by ELISA. (A) The effect of LPS (50 ng/ml) exposure on secreted IL-6 from 0 to 48 hours was measured in the cell culture supernatants. (B) The CD14 dependence of this response was determined in some studies by pretreating cells with either an anti-CD14 monoclonal antibody Leu-M3 (0.5 μg/ml) or an antibody to a common determinant of human MHC class I HLA (0.5 μg/ml). The data shown are representative of triplicate experiments. All values are expressed as mean ± SD. Statistically significant differences in secreted IL-6 in LPS-treated HCE-T cells were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (*P < 0.0002) in (A). The significance of differences in secreted IL-6 in LPS-treated HCE-T cells in the presence of anti-CD14 monoclonal antibody compared with those in the absence of the anti-CD14 antibody were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (**P < 0.02) in (B).
Figure 6.
 
LPS-induced human corneal epithelial cell IL-8. (A) The effect of LPS (50 ng/ml) on secreted HCE-T cell IL-8 from 0 to 48 hours was measured in the cell culture supernatants by ELISA. (B) The CD14 dependence of this response was determined by pretreating cells with either an anti-CD14 monoclonal antibody Leu-M3 (0.5 μg/ml) or an antibody to a common determinant of human MHC class I HLA (0.5 μg/ml). The data shown are representative of triplicate experiments. All values are expressed as mean ± SD. Statistically significant differences in secreted IL-8 in LPS-treated HCE-T cells were determined by the ANOVA with probabilities shownfor both the overall significance and the pair-wise comparison (*P < 0.001) in (A). The significance of differences in secreted IL-8 in LPS-treated HCE-T cells in the presence of anti-CD14 monoclonal antibody compared with those in the absence of the anti-CD14 antibody were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (**P < 0.005) in (B).
Figure 6.
 
LPS-induced human corneal epithelial cell IL-8. (A) The effect of LPS (50 ng/ml) on secreted HCE-T cell IL-8 from 0 to 48 hours was measured in the cell culture supernatants by ELISA. (B) The CD14 dependence of this response was determined by pretreating cells with either an anti-CD14 monoclonal antibody Leu-M3 (0.5 μg/ml) or an antibody to a common determinant of human MHC class I HLA (0.5 μg/ml). The data shown are representative of triplicate experiments. All values are expressed as mean ± SD. Statistically significant differences in secreted IL-8 in LPS-treated HCE-T cells were determined by the ANOVA with probabilities shownfor both the overall significance and the pair-wise comparison (*P < 0.001) in (A). The significance of differences in secreted IL-8 in LPS-treated HCE-T cells in the presence of anti-CD14 monoclonal antibody compared with those in the absence of the anti-CD14 antibody were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (**P < 0.005) in (B).
Figure 7.
 
Immunolocalization of CD14 in human cornea. CD14 was immunolocalized in whole human cornea using a specific anti-human CD14 monoclonal antibody (B, D, F, and H) or biotin-conjugated anti-mouse IgG as a negative control (A, C, E, and G). The localizations of CD14-immunoreactive products are shown in cells of the epithelium (B, D, arrows) and stroma and endothelium (F, H, arrowheads). The data are representative of independent experiments conducted in triplicate.
Figure 7.
 
Immunolocalization of CD14 in human cornea. CD14 was immunolocalized in whole human cornea using a specific anti-human CD14 monoclonal antibody (B, D, F, and H) or biotin-conjugated anti-mouse IgG as a negative control (A, C, E, and G). The localizations of CD14-immunoreactive products are shown in cells of the epithelium (B, D, arrows) and stroma and endothelium (F, H, arrowheads). The data are representative of independent experiments conducted in triplicate.
Figure 8.
 
LPS-augmented TLR4 mRNA expression in human corneal epithelial cells. TLR4 RT-PCR amplification product (1139 bp) was generated from cDNAs, which were reverse transcribed from HCE-T cell mRNA, using primers specific for human TLR4 (A). The effect of LPS on the induction of TLR4 mRNA was measured by Northern blot analysis in HCE-T cells (B) and by quantitative RT-PCR, constitutively or with the addition of LPS, in freshly isolated human corneal epithelial cells (C). LPS-induced TLR4 mRNA expression was examined after pretreatment with the anti-CD14 monoclonal antibody, Leu-M3 (0.5μ g/ml) in (B). The relative intensity of TLR4 mRNA expression was determined by densitometry in comparison with β-actin mRNA expression (bar graph). Data are representative of experiments conducted five times. The data obtained by densitometric analysis and by quantitative RT-PCR are expressed as mean ± SD. Statistically significant differences in TLR4 mRNA expression in freshly isolated human corneal epithelial cells and LPS-treated HCE-T cells in the absence or presence of anti-CD14 antibody were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (*P < 0.0001;** P < 0.0002, respectively).
Figure 8.
 
LPS-augmented TLR4 mRNA expression in human corneal epithelial cells. TLR4 RT-PCR amplification product (1139 bp) was generated from cDNAs, which were reverse transcribed from HCE-T cell mRNA, using primers specific for human TLR4 (A). The effect of LPS on the induction of TLR4 mRNA was measured by Northern blot analysis in HCE-T cells (B) and by quantitative RT-PCR, constitutively or with the addition of LPS, in freshly isolated human corneal epithelial cells (C). LPS-induced TLR4 mRNA expression was examined after pretreatment with the anti-CD14 monoclonal antibody, Leu-M3 (0.5μ g/ml) in (B). The relative intensity of TLR4 mRNA expression was determined by densitometry in comparison with β-actin mRNA expression (bar graph). Data are representative of experiments conducted five times. The data obtained by densitometric analysis and by quantitative RT-PCR are expressed as mean ± SD. Statistically significant differences in TLR4 mRNA expression in freshly isolated human corneal epithelial cells and LPS-treated HCE-T cells in the absence or presence of anti-CD14 antibody were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (*P < 0.0001;** P < 0.0002, respectively).
Figure 9.
 
HCE-T cell TLR4 corneal epithelial cell surface expression. Corneal epithelial cell surface expression of TLR4 was assessed by flow cytometry. (A) B-lymphoblastoid JY-1 cells, which express no TLR4, were used as a negative control and the TLR4-expressing human monocyte leukemia THP-1 cells were used as a positive control. (B) Expression was detected with the TLR4-specific monoclonal antibody HTA125. Cells treated with an isotype-matched irrelevant antibody served as an additional control (open histogram). The data shown are representative of studies conducted in triplicate.
Figure 9.
 
HCE-T cell TLR4 corneal epithelial cell surface expression. Corneal epithelial cell surface expression of TLR4 was assessed by flow cytometry. (A) B-lymphoblastoid JY-1 cells, which express no TLR4, were used as a negative control and the TLR4-expressing human monocyte leukemia THP-1 cells were used as a positive control. (B) Expression was detected with the TLR4-specific monoclonal antibody HTA125. Cells treated with an isotype-matched irrelevant antibody served as an additional control (open histogram). The data shown are representative of studies conducted in triplicate.
Figure 10.
 
LPS-induced TLR4-dependent HCE-T cell intracellular Ca2+ response. Cultured HCE-T cells were grown on glass coverslip culture dishes to approximately 50% to 70% confluence, and the fluorescent calcium probe fura-2/AM was incorporated at 2 μM. The HCE-T cells were then treated with either 50 ng/ml LPS plus 0.1% human serum (A), or were pretreated with 0.5 μg/ml of the anti-TLR4 monoclonal antibody HTA125 for 30 minutes before 50 ng/ml LPS plus 0.1% human serum (B). Intracellular free calcium concentration was determined by measuring the ratio of fluorescence at excitation wavelengths of 340 and 380 nm.
Figure 10.
 
LPS-induced TLR4-dependent HCE-T cell intracellular Ca2+ response. Cultured HCE-T cells were grown on glass coverslip culture dishes to approximately 50% to 70% confluence, and the fluorescent calcium probe fura-2/AM was incorporated at 2 μM. The HCE-T cells were then treated with either 50 ng/ml LPS plus 0.1% human serum (A), or were pretreated with 0.5 μg/ml of the anti-TLR4 monoclonal antibody HTA125 for 30 minutes before 50 ng/ml LPS plus 0.1% human serum (B). Intracellular free calcium concentration was determined by measuring the ratio of fluorescence at excitation wavelengths of 340 and 380 nm.
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Figure 1.
 
Human corneal cell expression of CD14 mRNA. RT-PCR amplification products of 777 bp were generated from cDNAs, which were reverse transcribed from mRNA of human corneal epithelial cells (A; EP), stromal cells (B; S), or endothelial cells (C; EN), by using primers specific for human CD14. The U937 human monocyte cell line and B-lymphoblastoid JY-1 cells served as positive (right lane) and negative (left lane) controls, respectively, with size markers in the center lane (D). The amplified gene products were subcloned into a plasmid vector and partially sequenced by automated DNA sequencer. The nucleotide sequence of corneal CD14 is presented in (E).
Figure 1.
 
Human corneal cell expression of CD14 mRNA. RT-PCR amplification products of 777 bp were generated from cDNAs, which were reverse transcribed from mRNA of human corneal epithelial cells (A; EP), stromal cells (B; S), or endothelial cells (C; EN), by using primers specific for human CD14. The U937 human monocyte cell line and B-lymphoblastoid JY-1 cells served as positive (right lane) and negative (left lane) controls, respectively, with size markers in the center lane (D). The amplified gene products were subcloned into a plasmid vector and partially sequenced by automated DNA sequencer. The nucleotide sequence of corneal CD14 is presented in (E).
Figure 2.
 
Cell surface expression of CD14 on human corneal cells. Surface expression of CD14 on human corneal epithelial, stromal, and endothelial cells was assessed by flow cytometry. B-lymphoblastoid JY-1 cells, which express no CD14, were used as a negative control and the CD14-expressing U937 human monocyte cell line was used as a positive control (A). CD14 expression was detected on the surface of human corneal epithelial cells (B), human corneal stromal cells (C), and human corneal endothelial cells (D) by using a CD14-specific monoclonal antibody RMO52. Cells treated with an isotype-matched irrelevant antibody served as an additional control (open histogram). The data shown are representative of studies conducted in triplicate.
Figure 2.
 
Cell surface expression of CD14 on human corneal cells. Surface expression of CD14 on human corneal epithelial, stromal, and endothelial cells was assessed by flow cytometry. B-lymphoblastoid JY-1 cells, which express no CD14, were used as a negative control and the CD14-expressing U937 human monocyte cell line was used as a positive control (A). CD14 expression was detected on the surface of human corneal epithelial cells (B), human corneal stromal cells (C), and human corneal endothelial cells (D) by using a CD14-specific monoclonal antibody RMO52. Cells treated with an isotype-matched irrelevant antibody served as an additional control (open histogram). The data shown are representative of studies conducted in triplicate.
Figure 3.
 
LPS-induced CD14-dependent human corneal epithelial cell intracellular Ca2+ response. Cultured HCE-T cells were grown on glass coverslip culture dishes to approximately 50% to 70% confluence and 2μ M of the fluorescent calcium probe fura-2/AM was incorporated. (A) Cells treated with 0.1% human serum alone; (B) treated with 50 ng/ml LPS plus 0.1% human serum; (C) pretreated with 0.5 μg/ml anti-CD14 monoclonal antibody Leu-M3 30 minutes before 50 ng/ml LPS plus 0.1% human serum; (D) treated simultaneously with 50 ng/ml LPS plus 0.1% human serum and 300 ng/ml polymyxin B. Intracellular free calcium concentration was determined by measuring the ratio of fluorescence at excitation wavelengths of 340 and 380 nm.
Figure 3.
 
LPS-induced CD14-dependent human corneal epithelial cell intracellular Ca2+ response. Cultured HCE-T cells were grown on glass coverslip culture dishes to approximately 50% to 70% confluence and 2μ M of the fluorescent calcium probe fura-2/AM was incorporated. (A) Cells treated with 0.1% human serum alone; (B) treated with 50 ng/ml LPS plus 0.1% human serum; (C) pretreated with 0.5 μg/ml anti-CD14 monoclonal antibody Leu-M3 30 minutes before 50 ng/ml LPS plus 0.1% human serum; (D) treated simultaneously with 50 ng/ml LPS plus 0.1% human serum and 300 ng/ml polymyxin B. Intracellular free calcium concentration was determined by measuring the ratio of fluorescence at excitation wavelengths of 340 and 380 nm.
Figure 4.
 
LPS-augmented CD14 mRNA expression in human corneal epithelial cells. The effect of LPS on CD14 mRNA expression was measured by quantitative RT-PCR in primary human corneal epithelial cells and HCE-T cells. (A) CD14 mRNA expression in freshly isolated human corneal epithelial cells was examined 3 hours after the addition of LPS (50 ng/ml). (B) In an mRNA kinetic study, CD14 mRNA expression in HCE-T cells was examined 3, 6, 12, 24, and 48 hours after the addition of LPS (50 ng/ml). (C) In an LPS dose–response study, the effect of increasing concentrations of LPS (0, 10, 50, and 100 ng/ml and 1 and 10 μg/ml) on CD14 mRNA expression was determined in HCE-T cells 12 hours after LPS exposure. The relative intensity of CD14 mRNA expression was normalized with the mRNA expression of 18S rRNA for each experimental condition. The data shown are representative of triplicate experiments. All values are expressed as mean ± SD. Statistically significant differences in CD14 mRNA expression were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (*P < 0.001).
Figure 4.
 
LPS-augmented CD14 mRNA expression in human corneal epithelial cells. The effect of LPS on CD14 mRNA expression was measured by quantitative RT-PCR in primary human corneal epithelial cells and HCE-T cells. (A) CD14 mRNA expression in freshly isolated human corneal epithelial cells was examined 3 hours after the addition of LPS (50 ng/ml). (B) In an mRNA kinetic study, CD14 mRNA expression in HCE-T cells was examined 3, 6, 12, 24, and 48 hours after the addition of LPS (50 ng/ml). (C) In an LPS dose–response study, the effect of increasing concentrations of LPS (0, 10, 50, and 100 ng/ml and 1 and 10 μg/ml) on CD14 mRNA expression was determined in HCE-T cells 12 hours after LPS exposure. The relative intensity of CD14 mRNA expression was normalized with the mRNA expression of 18S rRNA for each experimental condition. The data shown are representative of triplicate experiments. All values are expressed as mean ± SD. Statistically significant differences in CD14 mRNA expression were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (*P < 0.001).
Figure 5.
 
LPS induced human corneal epithelial cell IL-6. LPS induction of HCE-T cell IL-6 secretion was examined by ELISA. (A) The effect of LPS (50 ng/ml) exposure on secreted IL-6 from 0 to 48 hours was measured in the cell culture supernatants. (B) The CD14 dependence of this response was determined in some studies by pretreating cells with either an anti-CD14 monoclonal antibody Leu-M3 (0.5 μg/ml) or an antibody to a common determinant of human MHC class I HLA (0.5 μg/ml). The data shown are representative of triplicate experiments. All values are expressed as mean ± SD. Statistically significant differences in secreted IL-6 in LPS-treated HCE-T cells were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (*P < 0.0002) in (A). The significance of differences in secreted IL-6 in LPS-treated HCE-T cells in the presence of anti-CD14 monoclonal antibody compared with those in the absence of the anti-CD14 antibody were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (**P < 0.02) in (B).
Figure 5.
 
LPS induced human corneal epithelial cell IL-6. LPS induction of HCE-T cell IL-6 secretion was examined by ELISA. (A) The effect of LPS (50 ng/ml) exposure on secreted IL-6 from 0 to 48 hours was measured in the cell culture supernatants. (B) The CD14 dependence of this response was determined in some studies by pretreating cells with either an anti-CD14 monoclonal antibody Leu-M3 (0.5 μg/ml) or an antibody to a common determinant of human MHC class I HLA (0.5 μg/ml). The data shown are representative of triplicate experiments. All values are expressed as mean ± SD. Statistically significant differences in secreted IL-6 in LPS-treated HCE-T cells were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (*P < 0.0002) in (A). The significance of differences in secreted IL-6 in LPS-treated HCE-T cells in the presence of anti-CD14 monoclonal antibody compared with those in the absence of the anti-CD14 antibody were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (**P < 0.02) in (B).
Figure 6.
 
LPS-induced human corneal epithelial cell IL-8. (A) The effect of LPS (50 ng/ml) on secreted HCE-T cell IL-8 from 0 to 48 hours was measured in the cell culture supernatants by ELISA. (B) The CD14 dependence of this response was determined by pretreating cells with either an anti-CD14 monoclonal antibody Leu-M3 (0.5 μg/ml) or an antibody to a common determinant of human MHC class I HLA (0.5 μg/ml). The data shown are representative of triplicate experiments. All values are expressed as mean ± SD. Statistically significant differences in secreted IL-8 in LPS-treated HCE-T cells were determined by the ANOVA with probabilities shownfor both the overall significance and the pair-wise comparison (*P < 0.001) in (A). The significance of differences in secreted IL-8 in LPS-treated HCE-T cells in the presence of anti-CD14 monoclonal antibody compared with those in the absence of the anti-CD14 antibody were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (**P < 0.005) in (B).
Figure 6.
 
LPS-induced human corneal epithelial cell IL-8. (A) The effect of LPS (50 ng/ml) on secreted HCE-T cell IL-8 from 0 to 48 hours was measured in the cell culture supernatants by ELISA. (B) The CD14 dependence of this response was determined by pretreating cells with either an anti-CD14 monoclonal antibody Leu-M3 (0.5 μg/ml) or an antibody to a common determinant of human MHC class I HLA (0.5 μg/ml). The data shown are representative of triplicate experiments. All values are expressed as mean ± SD. Statistically significant differences in secreted IL-8 in LPS-treated HCE-T cells were determined by the ANOVA with probabilities shownfor both the overall significance and the pair-wise comparison (*P < 0.001) in (A). The significance of differences in secreted IL-8 in LPS-treated HCE-T cells in the presence of anti-CD14 monoclonal antibody compared with those in the absence of the anti-CD14 antibody were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (**P < 0.005) in (B).
Figure 7.
 
Immunolocalization of CD14 in human cornea. CD14 was immunolocalized in whole human cornea using a specific anti-human CD14 monoclonal antibody (B, D, F, and H) or biotin-conjugated anti-mouse IgG as a negative control (A, C, E, and G). The localizations of CD14-immunoreactive products are shown in cells of the epithelium (B, D, arrows) and stroma and endothelium (F, H, arrowheads). The data are representative of independent experiments conducted in triplicate.
Figure 7.
 
Immunolocalization of CD14 in human cornea. CD14 was immunolocalized in whole human cornea using a specific anti-human CD14 monoclonal antibody (B, D, F, and H) or biotin-conjugated anti-mouse IgG as a negative control (A, C, E, and G). The localizations of CD14-immunoreactive products are shown in cells of the epithelium (B, D, arrows) and stroma and endothelium (F, H, arrowheads). The data are representative of independent experiments conducted in triplicate.
Figure 8.
 
LPS-augmented TLR4 mRNA expression in human corneal epithelial cells. TLR4 RT-PCR amplification product (1139 bp) was generated from cDNAs, which were reverse transcribed from HCE-T cell mRNA, using primers specific for human TLR4 (A). The effect of LPS on the induction of TLR4 mRNA was measured by Northern blot analysis in HCE-T cells (B) and by quantitative RT-PCR, constitutively or with the addition of LPS, in freshly isolated human corneal epithelial cells (C). LPS-induced TLR4 mRNA expression was examined after pretreatment with the anti-CD14 monoclonal antibody, Leu-M3 (0.5μ g/ml) in (B). The relative intensity of TLR4 mRNA expression was determined by densitometry in comparison with β-actin mRNA expression (bar graph). Data are representative of experiments conducted five times. The data obtained by densitometric analysis and by quantitative RT-PCR are expressed as mean ± SD. Statistically significant differences in TLR4 mRNA expression in freshly isolated human corneal epithelial cells and LPS-treated HCE-T cells in the absence or presence of anti-CD14 antibody were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (*P < 0.0001;** P < 0.0002, respectively).
Figure 8.
 
LPS-augmented TLR4 mRNA expression in human corneal epithelial cells. TLR4 RT-PCR amplification product (1139 bp) was generated from cDNAs, which were reverse transcribed from HCE-T cell mRNA, using primers specific for human TLR4 (A). The effect of LPS on the induction of TLR4 mRNA was measured by Northern blot analysis in HCE-T cells (B) and by quantitative RT-PCR, constitutively or with the addition of LPS, in freshly isolated human corneal epithelial cells (C). LPS-induced TLR4 mRNA expression was examined after pretreatment with the anti-CD14 monoclonal antibody, Leu-M3 (0.5μ g/ml) in (B). The relative intensity of TLR4 mRNA expression was determined by densitometry in comparison with β-actin mRNA expression (bar graph). Data are representative of experiments conducted five times. The data obtained by densitometric analysis and by quantitative RT-PCR are expressed as mean ± SD. Statistically significant differences in TLR4 mRNA expression in freshly isolated human corneal epithelial cells and LPS-treated HCE-T cells in the absence or presence of anti-CD14 antibody were determined by the ANOVA with probabilities shown for both the overall significance and the pair-wise comparison (*P < 0.0001;** P < 0.0002, respectively).
Figure 9.
 
HCE-T cell TLR4 corneal epithelial cell surface expression. Corneal epithelial cell surface expression of TLR4 was assessed by flow cytometry. (A) B-lymphoblastoid JY-1 cells, which express no TLR4, were used as a negative control and the TLR4-expressing human monocyte leukemia THP-1 cells were used as a positive control. (B) Expression was detected with the TLR4-specific monoclonal antibody HTA125. Cells treated with an isotype-matched irrelevant antibody served as an additional control (open histogram). The data shown are representative of studies conducted in triplicate.
Figure 9.
 
HCE-T cell TLR4 corneal epithelial cell surface expression. Corneal epithelial cell surface expression of TLR4 was assessed by flow cytometry. (A) B-lymphoblastoid JY-1 cells, which express no TLR4, were used as a negative control and the TLR4-expressing human monocyte leukemia THP-1 cells were used as a positive control. (B) Expression was detected with the TLR4-specific monoclonal antibody HTA125. Cells treated with an isotype-matched irrelevant antibody served as an additional control (open histogram). The data shown are representative of studies conducted in triplicate.
Figure 10.
 
LPS-induced TLR4-dependent HCE-T cell intracellular Ca2+ response. Cultured HCE-T cells were grown on glass coverslip culture dishes to approximately 50% to 70% confluence, and the fluorescent calcium probe fura-2/AM was incorporated at 2 μM. The HCE-T cells were then treated with either 50 ng/ml LPS plus 0.1% human serum (A), or were pretreated with 0.5 μg/ml of the anti-TLR4 monoclonal antibody HTA125 for 30 minutes before 50 ng/ml LPS plus 0.1% human serum (B). Intracellular free calcium concentration was determined by measuring the ratio of fluorescence at excitation wavelengths of 340 and 380 nm.
Figure 10.
 
LPS-induced TLR4-dependent HCE-T cell intracellular Ca2+ response. Cultured HCE-T cells were grown on glass coverslip culture dishes to approximately 50% to 70% confluence, and the fluorescent calcium probe fura-2/AM was incorporated at 2 μM. The HCE-T cells were then treated with either 50 ng/ml LPS plus 0.1% human serum (A), or were pretreated with 0.5 μg/ml of the anti-TLR4 monoclonal antibody HTA125 for 30 minutes before 50 ng/ml LPS plus 0.1% human serum (B). Intracellular free calcium concentration was determined by measuring the ratio of fluorescence at excitation wavelengths of 340 and 380 nm.
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