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(Investigative Ophthalmology and Visual Science. 2005;46:3095-3101.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-1365
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Potentiation of Lipopolysaccharide-Induced Chemokine and Adhesion Molecule Expression in Corneal Fibroblasts by Soluble CD14 or LPS-Binding Protein

Ken Fukuda,1 Naoki Kumagai,2 Kazutaka Yamamoto,2 Youichiro Fujitsu,2 Nobuhiko Chikamoto,2 and Teruo Nishida2

1From the Departments of Ocular Pathophysiology and 2Biomolecular Recognition and Ophthalmology, Yamaguchi University School of Medicine, Ube City, Yamaguchi, Japan.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. The detection of bacterial lipopolysaccharide (LPS) by human cells is facilitated by LPS-binding protein (LBP) and soluble (s)CD14. The effects of these proteins on chemokine release and adhesion molecule expression in cultured human corneal fibroblasts were examined.

METHODS. The release of chemokines into culture supernatants and the expression of the intercellular adhesion molecule (ICAM)-1 on the cell surface were determined by enzyme-linked immunosorbent assays. The intracellular abundance of chemokine and ICAM-1 mRNAs was quantitated by reverse transcription and real-time polymerase chain reaction analyses. The phosphorylation and degradation of I{kappa}B-{alpha} and the subcellular localization of NF-{kappa}B were examined by immunoblot and immunofluorescence analyses, respectively.

RESULTS. Neither sCD14 nor LBP alone affected the expression of chemokines or ICAM-1 in cultured human corneal fibroblasts. However, sCD14 or LBP enhanced the LPS-induced upregulation of ICAM-1 and the chemokines interleukin-8 and monocyte chemoattractant protein (MCP)-1 in these cells at the protein and mRNA levels. Combined stimulation with LPS and either sCD14 or LBP also induced the phosphorylation and degradation of I{kappa}B-{alpha} and the translocation of NF-{kappa}B from the cytoplasm to the nucleus of corneal fibroblasts.

CONCLUSIONS. LBP and sCD14 may play important roles in the defense of the cornea against bacterial infection, by facilitating the detection of LPS by corneal fibroblasts.

Bacterial corneal ulcer is a major cause of loss of vision in developed and developing countries. This condition results from the destruction of collagen fibrils in the corneal stroma by collagenolytic enzymes produced as a consequence of bacterial infection.1 Pathologic examination has revealed the presence of many infiltrating leukocytes, including neutrophils and macrophages, in or surrounding corneal ulcers.1 We have shown that the interaction of resident corneal fibroblasts with infiltrating leukocytes very likely promotes collagen degradation.2 Whereas corneal fibroblasts alone in culture degrades collagen fibrils to a small extent, neutrophils alone do not. However, the addition of either neutrophils or neutrophil-conditioned medium to corneal fibroblasts results in a marked increase in the amount of collagen degraded by the fibroblasts. We have also demonstrated an interaction between bacteria and corneal fibroblasts in culture, in that elastase produced by Pseudomonas aeruginosa both degrades collagen directly and activates the collagen-degrading matrix metalloproteinases produced by corneal fibroblasts.3 In addition, we found that corneal fibroblasts are able to detect the presence of lipopolysaccharide (LPS), a component of the cell membrane of Gram-negative bacteria, and that they upregulate the expression of the chemokines interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 and that of intercellular adhesion molecule (ICAM)-1 in response.4 Such a response in vivo would be expected to trigger the local infiltration of leukocytes. Corneal fibroblasts may thus function as sentinel and effector cells in the defense of the cornea against bacterial infection.

LPS is an amphipathic molecule with a large hydrophobic component and a small hydrophilic domain.5 The lipophilic portion of LPS, known as lipid A, is the active component.6 In an aqueous environment, LPS exists as polymeric aggregates, with the hydrophilic polysaccharide component facing outward, and the lipid A region facing inward.5 Polymeric LPS binds poorly to leukocytes and at low concentrations fails to provoke responses in these cells.7 Several serum proteins and lipids are capable of binding to LPS,8 9 and among such proteins soluble (s)CD1410 and LPS-binding protein (LBP)11 play key roles in its recognition. LBP functions as a carrier for LPS and promotes its biological effects by rendering it monomeric and thereby exposing lipid A.12 LBP then transfers LPS monomers to the binding site either of CD14 expressed on the cell surface or of sCD14.13 The addition of LBP to serum-free cell cultures thus enhances LPS-induced cellular responses by a factor of 100 to 1000.14 sCD14 is able to substitute for membrane-bound CD14 in promotion of the binding of LPS to its receptor, toll-like receptor (TLR)-4. We have shown that low concentrations of human serum increase both the potency and efficacy of LPS with regard to its actions in corneal fibroblasts,4 suggesting the presence in normal serum of a soluble factor (or factors) that may be necessary for the activation of these cells through their LPS receptors.

We and others have shown that LPS is recognized by and activates structural cells in the cornea.4 15 We have now examined the effects of sCD14 and LBP on chemokine release and the expression of ICAM-1 by LPS-stimulated human corneal fibroblasts.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Materials
Eagle’s minimum essential medium (MEM), fetal bovine serum, and phosphate-buffered saline (PBS) were obtained from Invitrogen-Gibco (Grand Island, NY). All materials used for cell culture were endotoxin-minimized. LPS derived from Escherichia coli was obtained from Sigma-Aldrich (St. Louis, MO); tissue culture dishes from Greiner Bio-One (Kemsmuenster, Austria); eight-well chamber slides from Nalge Nunc (Naperville, IL); a mouse monoclonal antibody (mAb) to ICAM-1 from BD-Pharmingen (San Diego, CA); rabbit polyclonal antibodies to the p65 subunit of NF-{kappa}B and normal mouse immunoglobulin (Ig)G from Santa Cruz Biotechnology (Santa Cruz, CA); horseradish peroxidase–conjugated goat antibodies to mouse IgG from Chemicon (Temecula, CA); Alexa Fluor 488–conjugated goat antibodies to rabbit IgG from Molecular Probes (Eugene, OR); human recombinant sCD14 and LBP and paired antibodies for human IL-8 and MCP-1 enzyme-linked immunosorbent assays (ELISAs) from R&D Systems (Minneapolis, MN); rabbit polyclonal antibodies to I{kappa}B-{alpha} and a mouse mAb to phosphorylated I{kappa}B from Cell Signaling Technologies (Beverly, MA); a one-step substrate system (TMB) from Dako (Carpinteria, CA); PCR reagents (QuantiTect SYBR Green) from Qiagen (Hilden, Germany); micro-BCA assay reagents from Pierce (Rockford, IL); and enhanced chemiluminescence reagents from GE Healthcare (Piscataway, NJ).

Isolation and Culture of Human Corneal Fibroblasts
Human corneas were obtained from Mid-America Transplant Service (St. Louis, MO), Northwest Lions Eye Bank (Seattle, WA), and The Eye Bank of Wisconsin (Madison, WI). The donors were white men and women ranging in age from 4 to 65 years. After the center of each donor cornea was punched out for corneal transplantation surgery, the remaining rim of tissue was used for the present experiments. The human material was used in strict accordance with the basic principles of the Declaration of Helsinki. Corneal fibroblasts were prepared and cultured as described previously.16 Each cornea was digested separately with collagenase to provide a suspension of corneal fibroblasts. The cells from each cornea were cultured independently in MEM supplemented with 10% fetal bovine serum in 60-mm dishes until they had achieved ~90% confluence. They were used for the present experiments after four to six passages. The purity of the cell cultures was judged on the basis of both the distinctive morphology of corneal fibroblasts and their reactivity with antibodies to vimentin in immunofluorescence analysis.16 No contamination with corneal epithelial cells was detected.

ELISA for IL-8 and MCP-1
The concentrations of IL-8 and MCP-1 in culture supernatants were determined by ELISA, as previously described,16 with measurement of absorbance at 450 nm. The limit of detection for each assay was 5 pg/mL. The concentrations of chemokines in culture supernatants were normalized by expression as nanograms of chemokine per 1 x 106 cells.

Whole-Cell ELISA for ICAM-1
For detection of ICAM-1, a whole-cell ELISA was performed as described.4 17 18 Corneal fibroblasts (1 x 103 cells per well) were cultured in 96-well flat-bottomed microtiter plates for 72 hours. The culture medium was then changed to serum-free MEM, and the cells were incubated for an additional 24 hours. After replacement of the medium with MEM supplemented with various concentrations of sCD14 or LBP, in the absence or presence of LPS (10 ng/mL), the cells were incubated for 24 hours, washed twice with PBS, and fixed for 15 minutes at room temperature with PBS containing 1% paraformaldehyde. The cells were then washed with PBS containing 0.1% bovine serum albumin (BSA), incubated for 1 hour at 37°C with a mAb to ICAM-1 (1:10,000 dilution) in PBS containing 1% BSA (PBS-BSA), washed three times with PBS-BSA, and incubated for 1 hour at 37°C with horseradish peroxidase–conjugated goat antibodies to mouse IgG in PBS-BSA. After they were washed three times with PBS-BSA, the cells were incubated for 15 minutes in the dark with 100 µL of TMB solution. The reaction was then terminated by the addition of 50 µL of 1 M H2SO4, and the absorbance of each well was determined at a wavelength of 450 nm with a microplate reader (PowerWave XS; Bio-Tek Instruments, Winooski, VT).

Quantitative RT-PCR Analysis
The medium of corneal fibroblasts was changed to serum-free MEM, and the cells were cultured for an additional 24 hours. After incubation for a further 6 hours in MEM with sCD14 or LBP, in the absence or presence of LPS (10 ng/mL), the fibroblasts were washed with PBS, and total RNA was extracted (MagNa Pure system; Roche Molecular Biochemicals, Mannheim, Germany). The RNA was subjected to reverse transcription (RT) with a kit (Promega, Madison, WI), and the resultant cDNA was subjected to real-time polymerase chain reaction (PCR) analysis by rapid cycling in glass capillaries with a thermocycler (LightCycler; Roche Molecular Biochemicals), as described previously.19 20 21 The reaction was performed in a final volume of 20 µL with PCR reagents (QuantiTect SYBR Green; Qiagen) and specific primers. After an initial denaturation step at 95°C for 10 minutes, 40 cycles of amplification were performed. Each cycle comprised denaturation at 94°C for 15 seconds, primer annealing at 55°C for 20 seconds, and elongation at 72°C for 10 seconds (IL-8 and MCP-1) or 14 seconds (ICAM-1 and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). SYBR Green I fluorescence was monitored at the end of each cycle to obtain a measure of the amount of PCR product formed. After completion of the amplification protocol, melting curve analysis was performed to confirm the specificity of amplification by cooling the sample to 65°C at a rate of 20°C/s, maintaining a temperature of 65°C for 10 seconds, and then heating at a rate of 0.1°C/s to 97°C, with continuous measurement of fluorescence. The fluorescence signal (F) was plotted against temperature (T) to generate a melting curve for each sample. The melting curve was then converted to a melting peak by plotting the negative derivative of fluorescence with respect to temperature against temperature (–dF/dT versus T). Each PCR product gives rise to a specific melting temperature. The sequences of the PCR primers for IL-8, MCP-1, ICAM-1, and GAPDH were as described previously.4 The thermocycler PCR data were analyzed with the dedicated software (LightCycler; Roche Molecular Biochemicals), which first adjusts the values for each sample by subtraction of the background fluorescence generated during the initial cycles. A fluorescence threshold is then set at 5% of full scale, and the software determines the cycle number at which each sample reached this threshold. The cycle number corresponding to the fluorescence threshold is inversely related to the log of the initial template concentration. Given that amplification occurs in an exponential manner, a difference in cycle threshold of 1 corresponds to an approximately twofold difference in relative transcript abundance. The amount of IL-8, MCP-1, or ICAM-1 mRNA was normalized by that of GAPDH mRNA and is presented in arbitrary units (1 unit corresponds to the value for cells incubated in the absence of LPS, sCD14, and LBP).

Immunoblot Analysis of I{kappa}B-{alpha}
The phosphorylation and degradation of I{kappa}B-{alpha} in corneal fibroblasts were examined by immunoblot analysis, as described previously.22 In brief, after incubation for 30 minutes at 37°C with MEM containing LPS and either sCD14 or LBP, the cells were washed twice with PBS, lysed with radioimmunoprecipitation buffer, and assayed for protein concentration. Cell lysates (20 µg of protein) were subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel under reducing conditions, and the separated proteins were transferred electrophoretically to a polyvinylidene difluoride membrane. After blocking of nonspecific sites, the membrane was incubated with antibodies to I{kappa}B-{alpha} or to phosphorylated I{kappa}B-{alpha}, and immune complexes were detected with the use of enhanced chemiluminescence reagents.

Immunofluorescence Staining for NF-{kappa}B
Immunostaining for NF-{kappa}B in corneal fibroblasts was performed as described previously.22 In brief, cell monolayers grown on eight-well chamber slides were incubated at 37°C first for 24 hours in serum-free MEM and then for 30 minutes with MEM containing sCD14, LBP, or LPS. The cells were then washed twice with PBS, fixed with 4% paraformaldehyde in PBS, and washed an additional three times with PBS before permeabilization with 100% methanol for 6 minutes at –20°C. Nonspecific adsorption of antibodies was blocked by incubation for 30 minutes with PBS containing 3% BSA, and the cells were then incubated for 1 hour at room temperature with antibodies to the p65 subunit of NF-{kappa}B (1:100 dilution in PBS-BSA), washed, and incubated for 30 minutes at room temperature with Alexa Fluor 488–conjugated secondary antibodies (1:500 dilution in PBS-BSA). The cells were finally washed, mounted in mounting medium (Vectashield; Vector Laboratories, Inc., Burlingame, CA), and examined with a fluorescence microscope (Axiovert; Carl Zeiss Meditec, München-Hallbergmoos, Germany).

Statistical Analysis
Data are expressed as means ± SEM for assays performed in quadruplicate unless indicated otherwise. Differences were evaluated by the unpaired Student’s t-test or by analysis of variance (ANOVA) followed by the Dunnett or Scheffé test. P < 0.05 was considered statistically significant.


   Results
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 Abstract
 Methods
 Results
Discussion
 References
 
Effects of sCD14 and LBP on Chemokine Release by LPS-Stimulated Human Corneal Fibroblasts
We first examined the effects of sCD14 and LBP on the release of both the CXC chemokine IL-8 and the CC chemokine MCP-1 from human corneal fibroblasts in the absence or presence of LPS. The cells were cultured for 24 hours with various concentrations of sCD14 (0.1–500 ng/mL) or LBP (0.01–1000 ng/mL) in the absence or presence of LPS (10 ng/mL), and the amount of IL-8 or MCP-1 in the culture supernatant was then determined by ELISA. Corneal fibroblasts constitutively released small amounts of IL-8 into the culture medium, and this process was stimulated by LPS, consistent with our previous observations.4 In the absence of LPS, sCD14 did not stimulate IL-8 release by these cells. In the presence of LPS, however, sCD14 induced a marked concentration-dependent increase in the amount of IL-8 released into the culture medium (Fig. 1A) . LBP alone also had no effect on the release of IL-8 from corneal fibroblasts, but in the presence of LPS it stimulated IL-8 release in a concentration-dependent manner (Fig. 1B) . Similarly, corneal fibroblasts constitutively released small amounts of MCP-1 into the culture medium, and this process was stimulated by LPS, as shown previously.4 Whereas sCD14 or LBP alone did not significantly affect this process, in the presence of LPS each of these proteins induced a marked concentration-dependent increase in the amount of MCP-1 released into the culture medium (Fig. 2) .



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  		FIGURE 1. Concentration-dependent effects of sCD14 and LBP on IL-8 release by human corneal fibroblasts. Cells deprived of serum for 24 hours were incubated for 24 hours in the absence ({circ}) or presence (•) of LPS (10 ng/mL) and with the indicated concentrations of sCD14 (A) or LBP (B). The amount of IL-8 released into the culture medium was then determined by ELISA. Data are expressed as nanograms of IL-8 per 1 x 106 cells and are the mean ± SEM of results of three experiments. *P < 0.01 (Scheffé test) versus the corresponding value for cells incubated with LPS in the absence of sCD14 or LBP.

 


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  		FIGURE 2. Concentration-dependent effects of sCD14 and LBP on MCP-1 release by human corneal fibroblasts. Serum-deprived cells were incubated for 24 hours in the absence ({circ}) or presence (•) of LPS (10 ng/mL) and with the indicated concentrations of sCD14 (A) or LBP (B). The amount of MCP-1 released into the culture medium was then determined by ELISA. Data are expressed as nanograms of MCP-1 per 1 x 106 cells and are the mean ± SEM of results of three experiments. *P < 0.05, **P < 0.01 (Scheffé’s test) versus the corresponding value for cells incubated with LPS in the absence of sCD14 or LBP.

 
Effects of sCD14 and LBP on Chemokine mRNA Abundance in LPS-Stimulated Human Corneal Fibroblasts
The effects of sCD14 and LBP on the abundance of chemokine mRNAs in corneal fibroblasts were examined by quantitative RT-PCR analysis after incubation of the cells with each agent (100 ng/mL) in the absence or presence of LPS (10 ng/mL). Control fibroblasts contained a small amount of IL-8 mRNA, the abundance of which was not significantly increased by incubation with sCD14 or LBP alone. Whereas stimulation with LPS alone induced an ~67-fold increase in the abundance of IL-8 mRNA in these cells, consistent with our previous observations,4 stimulation with LPS in the presence of sCD14 or LBP resulted in ~520- and ~255-fold increases, respectively, in the amount of these transcripts compared with control values (Fig. 3A) . Similarly, control fibroblasts contained a small amount of MCP-1 mRNA, the abundance of which was not significantly increased by sCD14 or LBP alone. Stimulation with LPS alone induced an ~8-fold increase in the abundance of MCP-1 mRNA in these cells, consistent with our previous results,4 but stimulation with LPS in the presence of sCD14 or LBP resulted in ~30- and ~22-fold increases, respectively, in the amount of MCP-1 mRNA (Fig. 3B) .



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  		FIGURE 3. Effects of sCD14 and LBP on the abundance of chemokine mRNAs in LPS-stimulated human corneal fibroblasts. Serum-deprived cells were incubated for 6 hours with or without sCD14 or LBP (each at 100 ng/mL) in the absence or presence of LPS (10 ng/mL), after which the amounts of IL-8 (A) and MCP-1 (B) mRNAs were determined by quantitative RT-PCR analysis. The amounts of IL-8 and MCP-1 mRNAs were normalized by that of GAPDH mRNA and are presented in arbitrary units (1 unit corresponds to the value for cells incubated in the absence of sCD14, LBP, and LPS). Data are means ± SEM of results of four experiments. *P < 0.01 (Scheffé test) versus the corresponding value for cells incubated with LPS in the absence of sCD14 or LBP.

 
Effects of sCD14 and LBP on Surface Expression of ICAM-1 by LPS-Stimulated Human Corneal Fibroblasts
The effects of sCD14 and LBP on the expression of ICAM-1 at the surface of human corneal fibroblasts were examined by whole-cell ELISA after culture of the cells for 24 hours with various concentrations of sCD14 or LBP in the absence or presence of LPS (10 ng/mL). As we showed previously,4 corneal fibroblasts expressed ICAM-1 at the cell surface constitutively and this expression was increased by LPS. Although sCD14 alone had no effect on the surface expression of ICAM-1, it increased the expression of this adhesion molecule in LPS-stimulated cells in a concentration-dependent manner (Fig. 4A) . Similarly, LBP induced a concentration-dependent increase in the surface expression of ICAM-1 by these cells only in the presence of LPS (Fig. 4B) .



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  		FIGURE 4. Concentration-dependent effects of sCD14 and LBP on ICAM-1 expression at the surface of LPS-stimulated human corneal fibroblasts. Serum-deprived cells were incubated for 24 hours in the absence ({circ}) or presence (•) of LPS (10 ng/mL) and with the indicated concentrations of sCD14 (A) or LBP (B). The surface expression of ICAM-1 was then assessed by whole-cell ELISA. Data are expressed as absorbance at 450 nm and are the mean ± SEM of results of three experiments. *P < 0.05, **P < 0.01 (Dunnett test) versus the corresponding value for cells incubated with LPS in the absence of sCD14 or LBP.

 
Effects of sCD14 and LBP on ICAM-1 mRNA Abundance in LPS-Stimulated Human Corneal Fibroblasts
The effects of sCD14 and LBP on the abundance of ICAM-1 mRNA in corneal fibroblasts were examined by RT and real-time PCR analysis. The abundance of ICAM-1 mRNA was not affected by incubation of cells with sCD14 or LBP alone at 100 ng/mL. Whereas stimulation with LPS (10 ng/mL) alone induced an ~11-fold increase in the abundance of ICAM-1 mRNA in these cells, consistent with our previous observations,4 exposure of the cells to sCD14 or LBP in the presence of LPS resulted in ~57- and ~38-fold increases, respectively, in the amount of these transcripts (Fig. 5) .



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  		FIGURE 5. Effects of sCD14 and LBP on the abundance of ICAM-1 mRNA in LPS-stimulated human corneal fibroblasts. Serum-deprived cells were incubated for 6 hours, with or without sCD14 or LBP (each at 100 ng/mL) in the absence or presence of LPS (10 ng/mL), after which the amount of ICAM-1 mRNA was determined by quantitative RT-PCR analysis. The amount of ICAM-1 mRNA was normalized by that of GAPDH mRNA and is presented in arbitrary units (1 unit corresponds to the value for cells incubated in the absence of sCD14, LBP, and LPS). Data are means ± SEM of values from three experiments. *P < 0.05, **P < 0.01 (Scheffé test) versus the value for cells incubated with LPS in the absence of sCD14 or LBP.

 
Effects of sCD14 and LBP on the Activation of NF-{kappa}B in LPS-Stimulated Human Corneal Fibroblasts
Signaling by NF-{kappa}B and its inhibitor I{kappa}B-{alpha} has been implicated in induction of the genes for chemokines such as IL-8 and MCP-1 and for adhesion molecules such as ICAM-1 in several cell types.23 We therefore examined the possible role of this signaling pathway in the effects of sCD14 and LBP on chemokine and ICAM-1 expression in LPS-stimulated corneal fibroblasts. Cells were incubated for 30 minutes with sCD14 or LBP (each at 100 ng/mL) in the absence or presence of LPS (10 ng/mL), after which the phosphorylation and degradation of I{kappa}B-{alpha} were examined by immunoblot analysis. Whereas sCD14, LBP, and LPS alone were each without effect, exposure of the cells to sCD14 or LBP in the presence of LPS resulted in both the phosphorylation and degradation of I{kappa}B-{alpha} (Fig. 6) .



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  		FIGURE 6. Effects of sCD14 and LBP on the phosphorylation and degradation of I{kappa}B-{alpha} in LPS-stimulated corneal fibroblasts. Serum-deprived cells were incubated for 30 minutes with sCD14 or LBP (each at 100 ng/mL) in the absence or presence of LPS (10 ng/mL) and were then lysed and subjected to immunoblot analysis with antibodies to I{kappa}B-{alpha} and to phosphorylated I{kappa}B-{alpha} (pI{kappa}B-{alpha}). Data are representative of results in three independent experiments.

 
We next examined the effects of sCD14 and LBP on the subcellular localization of the p65 subunit of NF-{kappa}B in LPS-stimulated corneal fibroblasts by immunofluorescence analysis. Under basal conditions, NF-{kappa}B immunofluorescence was localized predominantly to the cytoplasm of corneal fibroblasts (Fig. 7A) . No immunofluorescence was apparent in cells stained with normal rabbit IgG as a negative control (data not shown). Whereas treatment of cells with sCD14, LBP, or LPS alone had no effect on the subcellular localization of NF-{kappa}B (Figs. 7B 7C 7D) , stimulation with sCD14 or LBP in the presence of LPS resulted in translocation of NF-{kappa}B from the cytoplasm to the nucleus (Figs. 7E 7F) . These results thus suggest that the NF-{kappa}B–I{kappa}B-{alpha} pathway is activated by the combined stimulation of corneal fibroblasts with LPS and either sCD14 or LBP.



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  		FIGURE 7. Effects of sCD14 and LBP on the subcellular localization of NF-{kappa}B in LPS-stimulated corneal fibroblasts. Serum-deprived cells were incubated for 30 minutes with sCD14 or LBP (each at 100 ng/mL) in the absence or presence of LPS (10 ng/mL) and were then fixed and subjected to immunofluorescence analysis with antibodies to the p65 subunit of NF-{kappa}B. Scale bar, 50 µm. Data are representative of results in three independent experiments.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
We have shown that sCD14 and LBP each enhanced the LPS-induced release of IL-8 and MCP-1 from human corneal fibroblasts, the LPS-induced expression of ICAM-1 at the surface of these cells, and the LPS-induced increase in the abundance of IL-8, MCP-1, and ICAM-1 mRNAs. The transcription factor NF-{kappa}B was activated in response to simultaneous stimulation of the cells with LPS and either sCD14 or LBP. These results suggest that sCD14 and LBP may facilitate the response of human corneal fibroblasts to LPS in vivo.

We previously showed that the various effects of LPS on corneal fibroblasts were potentiated by the presence of low concentrations of human serum,4 suggesting that a factor (or factors) in serum is essential for the activation of these cells via their LPS receptors. Several serum proteins and lipids are able to bind LPS.8 9 In the present study, we have shown that two serum-derived soluble factors, sCD14 and LBP, potentiate the innate immune response of corneal fibroblasts to LPS. LBP derived from serum renders LPS monomeric and thereby exposes the lipid A moiety and facilitates its detection by LPS receptors expressed on corneal fibroblasts. Although LBP is considered an acute phase protein, it is present at a substantial concentration (~10 µg/mL) in the serum of healthy individuals.24 25 CD14 is a glycophosphatidylinositol-anchored protein that is constitutively expressed on the surface of various cell types, including monocytes and neutrophils, as well as human corneal fibroblasts.4 15 In addition to its membrane-bound form (mCD14), CD14 is present in a soluble form in serum at a concentration of ~2 µg/mL.26 27 28 Increased concentrations of sCD14 in serum have been associated with inflammatory conditions, such as Kawasaki disease,28 atopic dermatitis,27 liver disease,26 rheumatoid arthritis,29 systemic lupus erythematosus,30 and Sjögren’s syndrome,30 and sCD14 has also been categorized as an acute-phase protein.31

Although the central cornea lacks blood vessels, soluble serum factors such as serum albumin and antibodies are present in the normal cornea, albeit at reduced levels (~20% and 50%, respectively) compared with those in serum. The concentrations of such factors are lower in the central region than in the periphery of the cornea.32 Although the concentrations of sCD14 and LBP in the cornea remain to be determined, our in vitro experiments indicate that the levels of sCD14 and LBP in serum of healthy individuals are much higher than are those necessary for maximum effects on the expression of chemokines and ICAM-1 in corneal fibroblasts in the presence of LPS. Our present study was performed with corneal fibroblasts derived from the peripheral cornea, and so we cannot exclude the possibility that such fibroblasts differ from those in the central cornea in their LPS responsiveness. We have also found that the tear fluid of healthy adults contains sCD14 and LBP at concentrations that are markedly lower than those apparent in serum from the same individuals but that are sufficiently high to exert maximum effects on the innate immune response of corneal fibroblasts in the presence of LPS (unpublished data). In addition, the concentrations of serum proteins in the tear fluid of individuals with bacterial keratitis are increased as a result of secretion from the lacrimal gland and conjunctival exudate, and the serumlike ocular discharge bathes the de-epithelialized corneal stroma. The serum factors sCD14 and LBP thus may promote the response of corneal fibroblasts to LPS in situ.

The sCD14-LPS complex is thought to bind to mCD14-negative cells such as endothelial cells and certain epithelial cells and thereby to increase their sensitivity to LPS.33 In addition to mediating LPS-induced activation of mCD14-negative cells, sCD14 is able to antagonize the activation of mCD14-positive cells by the LBP-LPS complex, presumably by competing with mCD14 for LPS binding. However, this inhibitory effect requires concentrations of sCD14 higher than those in blood,34 suggesting that it might not be physiologically relevant. We and others have previously shown that corneal fibroblasts express mCD14 at the cell surface.4 15 We have now shown that exogenous sCD14 at concentrations of up to 500 ng/mL (about half the serum value) enhanced the response of corneal fibroblasts to LPS.

The expression of the chemokines IL-8 and MCP-1 and the adhesion molecule ICAM-1 induced by LPS in human corneal fibroblasts was enhanced by sCD14 or LBP. The expression of these molecules in other cell types is regulated at the transcriptional level by NF-{kappa}B.23 Our current results show that sCD14 or LBP, in the presence of LPS, induced the activation of NF-{kappa}B in corneal fibroblasts, suggesting that this transcription factor also contributes to induction of the expression of chemokine and adhesion molecule genes in these cells in response to such stimulation. The signaling molecules responsible for the activation of NF-{kappa}B by LPS and either sCD14 or LBP in corneal fibroblasts remain to be determined, as do the other possible proinflammatory effects of NF-{kappa}B activation in these cells.

The ability of cultured human corneal fibroblasts to respond to viral infection has also been investigated. Herpes simplex virus infection of corneal fibroblasts, but not that of corneal epithelial cells, was found to induce the synthesis of IL-8.35 Adenovirus infection of corneal fibroblasts was shown to activate intracellular signaling by c-Src and the mitogen-activated protein kinases ERK1 and -2, resulting in the expression of IL-8, MCP-1, and ICAM-1.36 These results thus suggest that corneal fibroblasts are able to contribute to induction of an acute inflammatory response not only in bacterial keratitis but also in herpes stromal keratitis and epidemic keratoconjunctivitis.

In contrast to corneal fibroblasts, human corneal epithelial cells appear unable to elicit an innate immune response to LPS37 or viruses35 and may therefore contribute to an immunologically inactive environment at the ocular mucosa. In vivo experiments have also revealed that LPS enters the cornea only at sites of injury.38 Corneal epithelial cells may thus protect the integrity of the corneal surface by acting as both a physical barrier and an immunologic barrier against invasion by bacteria and viruses.


   Acknowledgements
 
The authors thank Kumiko Hara and the staff of Yamaguchi University Center for Gene Research for technical assistance.


   Footnotes
 
Supported by Grants-in-Aid for Scientific Research (KAKENHI) 14207070 and 14571674 and Grant-in-Aid for Young Scientists (B) 16791050 from the Ministry of Education, Science, Sports, and Culture of Japan.

Submitted for publication November 22, 2004; revised April 15, 2005; accepted June 23, 2005.

Disclosure: K. Fukuda, None; N. Kumagai, None; K. Yamamoto, None; Y. Fujitsu, None; N. Chikamoto, None; T. Nishida, 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: Ken Fukuda, Department of Ocular Pathophysiology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube City, Yamaguchi 755-8505, Japan; k.fukuda{at}yamaguchi-u.ac.jp.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

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