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Suppression of the TNF-Induced Increase in IL-1 Expression by Hypochlorite in Human Corneal Epithelial Cells

¹ From the Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan; the ² Department of Biological Sciences, College of Optometry, State University of New York, New York, New York; the ³ Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan; and the ⁴ Laboratory of Biochemistry, National Heart Lung and Blood Institute, Bethesda, Maryland.

	Abstract

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PURPOSE. In response to injury, activated neutrophils release tumor necrosis factor (TNF)- and myeloperoxidase (MPO). TNF in turn causes human corneal epithelial cells to secrete interleukin (IL)-1, whereas MPO results in formation of HClO/OCl^-. The effect of HClO/OCl^- on the expression of the IL-1 gene and protein is unknown. The current study was undertaken to examine in immortalized human corneal epithelial cells whether NaOCl alters TNF-induced increases in expression of IL-1 gene and protein.

METHODS. Semiquantitative RT-PCR and ELISA characterized IL-1 gene and protein expression, respectively. TNF-induced nuclear transfer of nuclear factor (NF)-B was measured by electrophoretic mobility shift assay (EMSA). The isoform of inhibitory protein B (IB) was identified by Western blot analysis.

RESULTS. Exposure to NaOCl (0.75 mM) for 10 minutes caused suppression of TNF-induced increases in IL-1 mRNA and protein, declines in NFB nuclear transfer, and a modification of IB, based on a bandshift detected by Western blot analysis. Modified IB became resistant to TNF-induced proteolysis. Methionine sulfoxide reductase A (MsrA, 10 µM) eliminated the NaOCl-induced IB bandshift.

CONCLUSIONS. NaOCl oxidizes IB at methionine residues and thereby suppresses dissociation of IB from NFB. Decreased dissociation could in turn suppress TNF-induced activation of NFB, resulting in declines in expression of IL-1 gene and protein. These effects suggest that release of HClO/OCl^- in vivo by activated neutrophils may counterbalance TNF-induced NFB-dependent secretion if IL-1 and suppress an excessive inflammatory reaction.

	Introduction

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The corneal epithelium provides a barrier function against infection through the maintenance of tight junctional integrity. If the tight junctions are disrupted as a result of wounding, stromal infection may occur during the healing process if tight junctional re-formation is delayed or inadequate. For the barrier function to be reestablished, the tight junctions must have adequate resistance through the formation of close apposition between neighboring cells in the most superficial cell layer. Formation of tight junctional complexes depends on a myriad of interactions among different cytokine receptor–linked second-messenger cascades that mediate control of corneal epithelial growth, differentiation, and cell survival. In addition, timely closure of epithelial wounds is dependent on the interplay between the release of cytokines, not only from epithelial cells, but also from the stromal keratocytes and immune system cells and on cytokines secreted from corneal nerves and the lacrimal and accessory lacrimal glands. Should the tight junctional integrity remain compromised, tear fluid containing cytokines may travel through the junctions in excessive amounts into the stroma. Some of them may set into play events leading to the breakdown of the stromal matrix and inflammation followed by immunologic responses. One outcome could be infiltration of neutrophils resulting in ulceration of the diseased or damaged tissues.^{1 2 3} , These considerations have prompted an intense effort to gain further understanding of how to control cytokine-mediated responses resulting from infection.

Activated neutrophils infiltrate and aggregate at a corneal inflammatory site. They secrete large amounts of myeloperoxidase (MPO) and produce hydrogen peroxide in phagolysosomes and the extracellular compartment.⁵ MPO catalyzes the production of hypochlorous acid (HClO/OCl^-) from hydrogen peroxide and the chloride ion.^{6 7 8} HClO/OCl^- is a potent antimicrobial agent that affects both the emitting and surrounding cells at an inflammatory site as a result of oxidizing a wide range of biomolecules, particularly thiols, methionine, and amines.⁹ In some other tissues, formation of taurine chloramine (TauCl) has been detected. TauCl is a major chlorination product and is a more stable but less toxic oxidant than HClO/OCl^-.^{7 9 10}

Recent studies have suggested that TauCl has a significant role as an immunomodulator. It can inhibit (1) the production in macrophages of nitric oxide and tumor necrosis factor (TNF)-¹¹ ; (2) the production in bone marrow–derived dendritic cells of TNF, interleukin (IL)-6, -10, and -12, prostaglandin (PG)E₂, and nitrite¹² ; (3) the production in glioma cells of monocyte chemoattractant protein-1 and macrophage inflammatory protein-2¹³ ; and (4) the production in rheumatoid arthritis fibroblast-like synoviocytes of IL-6 and 8.¹⁴ Because the transcription of the genes of these inflammatory proteins is controlled by the transcription factor nuclear factor (NF)-B,^{14 15 16 17} TauCl and its source HClO/OCl elicit their effects through NFB-associated cell-signaling pathways.

During inflammation, activated neutrophils infiltrate the cornea and can induce the epithelium to express IL-1,^{18 19} -1ß,^{18 20} -6, and -8²⁰ and TNF.^{20 21} Expression of their genes is ultimately dependent on dissociation of the inhibitory protein B (IB) from NFB, which results in activation of NFB.^{14 22 23} In the present study, we investigated the effect of NaOCl on expression in the epithelium of (1) gene and protein of the proinflammatory cytokine IL-1, (2) nuclear transfer of NFB, and (3) oxidation status of the isoform of IB (IB).

	Methods

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Reagents
Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 mixture and fetal bovine serum (FBS) were from Iwaki (Tokyo, Japan). Cholera toxin, insulin, gentamicin, RNA extraction buffer (TRIzol), oligo dT primer, and RNase H^- reverse transcriptase (SuperScript II) were from Invitrogen (Groningen, The Netherlands). Human epidermal growth factor (EGF) was obtained from BD Biosciences (Bedford, MA), recombinant human TNF and an immunoassay system (An’Alyza) from Genzyme Techne (Minneapolis, MN), recombinant RNasin RNase inhibitor from Promega (Madison, WI), and NaOCl from Wako (Tokyo, Japan). Alkaline phosphatase (AP) and calpain inhibitor I were purchased from Roche Diagnostics Corp. (Mannheim, Germany), a protein assay kit from Bio-Rad Laboratories (Hercules, CA), and polyvinylidene difluoride membranes from Millipore (Bedford, MA). The affinity-purified rabbit polyclonal antibody for IB was from Santa Cruz Biotechnology (Santa Cruz, CA). Donkey anti-rabbit IgG linked to horseradish peroxidase, an enhanced chemiluminescence (ECL) Western blot detection system, and [-³²P] adenosine triphosphate (ATP) were from Amersham Pharmacia Biotech (Buckinghamshire, UK). T4 oligonucleotide kinase was from Takara Biomedical (Shiga, Japan), and DNA (HotStartTaq) polymerase was from Qiagen (Chatsworth, CA).

Culture of Human Corneal Epithelial Cells
Santen Pharmaceutical Co., Ltd. (Osaka, Japan) generously provided simian virus (SV)40-immortalized human corneal epithelial (HCE) cells. They were cultured in DMEM/Ham’s F-12 mixture medium, supplemented with 10% FBS, 5 µg/mL cholera toxin, 10 ng/mL human EGF, and 40 ng/mL gentamicin. Cultures were incubated at 37°C under fully humidified 95% air and 5% CO₂. All experiments were performed with HCE cells seeded at a density of 8 x 10⁵ cells per 35-mm dish and cultured overnight.

Cell Treatment with OCl^- and Stimulation with TNF
NaOCl was diluted to 40 mM with 60 mM sodium phosphate buffer (pH 8.3) consisting of 60 mM Na₂HPO₄ and 60 mM NaH₂PO₄. Cells were exposed to various concentrations of NaOCl for 10 minutes in a 37°C incubator. After the treatment, cells were washed with phosphate-buffered saline (PBS). To activate NFB, HCE cells were exposed to 30 ng/mL TNF for various periods after treatment with NaOCl.

Total RNA Extraction and Semiquantitative Reverse Transcription-Polymerase Chain Reaction
In preparation for RT-PCR, the cellular RNA was isolated with RNA extraction reagent (TRIzol). Single-stranded cDNA was prepared by RT with isolated total RNA used as a template with oligo dT primer, dNTP mix, recombinant RNasin RNase inhibitor and RNase H^- reverse transcriptase. PCR was performed with DNA polymerase (HotStartTaq) with primers based on published sequences as follows: for IL-1: sense, 5'-caaggagagcatggtggtagtagcaaccaacg-3', and antisense, 5'-tagtgccgtgagtttcccagaagaagaggagg-3'¹⁸ ; for ß-actin: sense, 5'-accaactgggacgacatggag-3' and antisense, 5'-cgtgaggatcctcatgaggtagtc-3'.²⁴ PCR was initiated in a thermal cycler programmed at 95°C for 15 minutes, and then at 94°C for 1 minute, 65°C for 1 minute, and 72°C for 2 minutes (30 cycles). The sizes of the PCR products for IL-1 and ß-actin were 407 and 354 bp, respectively. After PCR, the content of each tube was analyzed by 1.5% agarose gel electrophoresis. The bands were visualized with ethidium bromide. The density of bands was determined using an image-analysis program provided by Scion Corp. (Frederick, MD).

Enzyme-Linked Immunosorbent Assay
After treatment, cells were collected in PBS and centrifuged at 1000g for 5 minutes at 4°C. Cell lysis solution, containing 50 mM Tris(hydroxymethyl)aminomethane (Tris)/Cl (pH 7.6), 300 mM NaCl, and 0.5% Triton X-100, was added to pelleted cells and kept for 2.5 hours on ice with frequent vortexing. The lysates were centrifuged at 16,000g for 5 minutes, and the resultant supernatant was diluted with PBS. The concentration of IL-1 was colorimetrically measured at 450 nm by an ELISA system (An’Alyza). The cellular protein concentration in cell lysates was determined with a protein assay kit with bovine serum albumin used as a standard.

Electrophoretic Mobility Shift Assay
For the purification of nuclei, pelleted cells were resuspended in buffer A, consisting of 1.5 mM MgCl₂, 10 mM KCl, and 0.5 mM dithiothreitol (DTT), buffered with 10 mM HEPES-KOH (pH 7.9) for 10 minutes on ice. Lysis was completed by vortexing for 30 seconds. The lysates were centrifuged at 16,000g for 30 seconds at 4°C. The purified nuclear pellet was resuspended in buffer B (25% glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 M EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM DTT, and 20 mM HEPES-KOH (pH 7.9), with frequent vortexing for 20 minutes on ice. After centrifugation at 16,000g for 2 minutes, the supernatant was saved to obtain a nuclear extract. The protein amount was determined with the protein assay kit. An oligonucleotide probe containing the sequence of the NFB binding site (sense, 5'-agcttCAGAGGGGACTTTCCGAGAGG-3' antisense, 5'-tcgaCCTCTCGGAAAGTCCCCTCTGa-3') was labeled with [-³²P]-ATP, using T4 oligonucleotide kinase, which was incubated with 5 µg nuclear extract for 20 minutes at room temperature. The NFB that bound to the labeled oligonucleotide probe was fractionated on 4% polyacrylamide gel and visualized by autoradiography.

Western Blot Analysis
Pelleted cells were resuspended in TNE buffer (10 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1 mM EDTA, and 1% Nonidet P-40) containing 20 µM PMSF and 10 mg/mL calpain inhibitor I. The suspension was vortexed for 30 seconds and centrifuged at 16,000g for 5 minutes at 4°C. The supernatant was then added to an equal volume of 2x gel loading buffer (100 mM Tris-Cl [pH 6.8], 4% SDS, 20% glycerol, 0.5 mg/mL bromophenol blue, and 10% ß-mercaptoethanol) and boiled for 5 minutes. The protein assay kit was used to determine cellular protein content. A constant amount of extract (60 µg) was separated on a 12% polyacrylamide gel and electrotransferred to PVDF membranes. To be able to detect a small change in apparent molecular weight of IB, for example, an increase due to phosphorylation, the assay was continued until IB reached close to the bottom of an elongated separating gel (approximately 10 cm long).²⁵ Membranes were blocked in 5% nonfat milk in wash buffer (10 mM Tris-Cl [pH 7.5], containing 100 mM NaCl and 0.1% Tween 20) and immunoblotted with the affinity-purified rabbit polyclonal antibody for IB at a dilution of 1:400. Immunocomplexes were detected by binding to donkey anti-rabbit IgG linked to horseradish peroxidase at a dilution of 1:500, followed by use of the ECL Western blot detection assay kit.

Treatment of Cell Lysates with AP and MsrA
After exposure to 0.75 mM NaOCl for 10 minutes, cells were collected in PBS, centrifuged at 1000g for 5 minutes, and resuspended in 30 µL cold TNE buffer. The cell suspension was vortexed for 30 seconds and centrifuged at 16,000g for 5 minutes. The pH value of 30 µL of the supernatant was adjusted to 9.7 with 4.5 µL 0.1 N NaOH, then mixed with 10 U AP and incubated at 37°C for 30 minutes. The reaction was terminated by adding 4.5 µL 0.1 N HCl. To treat with methionine sulfoxide reductase (Msr)A, cell lysates were prepared in 30 µL cold TNE buffer, supplemented with 15 mM DTT. Recombinant yeast MsrA was prepared as reported previously.²⁶ Cell lysates were treated with 10 µM MsrA for 15 minutes at 37°C. To evaluate the effects of DTT on the shifted band, cell lysates were incubated for 30 minutes at 37°C without MsrA. After these enzyme treatments, each sample was mixed with 2x gel loading buffer and boiled for 5 minutes for Western blot analysis.

	Results

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Effect on Transcription of IL-1
To examine whether NaOCl affects transcription of IL-1, we determined the amount of IL-1 message by semiquantitative RT-PCR. A 10-minute pretreatment with 0.75 mM NaOCl had no effect on the basal levels of expression of IL-1 mRNA at 0 hours (no stimulation with TNF; data not shown). These basal levels were subtracted, and increased message levels were plotted. Exposure for 1 hour to TNF (30 ng/mL) increased expression of IL-1 mRNA, but it decreased at 3 hours (Fig. 1A) . Even though NaOCl pretreatment did not affect this transient pattern of change, it significantly decreased TNF-induced increase in expression of IL-1 mRNA at 30 minutes and 1 hour (P < 0.05), whereas no difference was observed at 3 hours. TNF stimulation-dependent expression of the IL-1 gene was decreased by approximately 70% at 30 minutes.

View larger version (31K): [in this window] [in a new window]   Figure 1. Pretreatment with NaOCl inhibited transcription of IL-1. (A) Cells that were seeded at a density of 8 x 105 cells per 35-mm dish and cultured overnight were treated with NaOCl (0.75 mM) or vehicle (60 mM sodium phosphate buffer) for 10 minutes at 37°C. Cells were then washed with PBS, fresh medium was added containing TNF (30 ng/mL), and cells were incubated at 37°C. At the indicated times, cells were harvested for total RNA extraction. (B) The concentration of NaOCl was varied up to 1 mM. After incubation with TNF (30 ng/mL) for 1 hour, cells were subjected to total RNA extraction. Detection of IL-1 and ß-actin mRNA was performed by semiquantitative RT-PCR. All the results were normalized in comparison with ß-actin, and basal message levels without stimulation with TNF were subtracted. Data are the mean ± SEM of results in three independent experiments. *Significantly different from cells treated with TNF by Student’s t-test unpaired analysis (P < 0.05).

Effect on Expression of IL-1 Protein
An ELISA was used to determine the effects of a 10-minute pretreatment with 0.75 mM NaOCl on the TNF (30 ng/mL)-induced increases in expression of IL-1 protein. At 1 hour, the TNF-induced increase without NaOCl pretreatment was 2.3-fold larger than with NaOCl pretreatment (Fig. 2A) . After 3 hours, it declined to less than 1.8-fold. Dose-dependent inhibition of expression of IL-1 protein by pretreatment with NaOCl was observed at doses up to 1 mM (Fig. 2B) .

View larger version (20K): [in this window] [in a new window]   Figure 2. Pretreatment with NaOCl inhibited IL-1 protein expression. (A) Cells were treated with NaOCl (0.75 mM) or vehicle (60 mM sodium phosphate buffer) for 10 minutes at 37°C. Cells were then washed with PBS, and fresh medium was added containing TNF (30 ng/mL) and incubated at 37°C. At the indicated times, cells were harvested and lysed. (B) The concentration of NaOCl was varied up to 1 mM. After incubation with TNF (30 ng/mL) for 1 hour, cell lysates were prepared. The amount of IL-1 in cell lysates was measured by ELISA. Protein amounts are expressed in nanograms per milligram of total cellular protein assayed in corresponding cellular lysates. Data are the mean ± SEM of results from three independent experiments. *Significantly different from cells treated with TNF by Student’s t-test unpaired analysis (P < 0.05).

TNF-Induced Nuclear Transfer of NFB

View larger version (25K): [in this window] [in a new window]   Figure 3. Pretreatment with NaOCl repressed TNF-induced nuclear transfer of NFB. Cells were treated with vehicle (60 mM sodium phosphate buffer) or with NaOCl (0.75 mM) for 10 minutes at 37°C and washed with PBS. Fresh medium containing TNF (30 ng/mL) was added and cells incubated for up to 30 minutes at 37°C. Nuclear extracts were prepared for EMSA. Similar results were obtained in three independent experiments.

NaOCl-Induced Modification of IB

View larger version (40K): [in this window] [in a new window]   Figure 4. NaOCl induced IB band-shifting. Cells were treated with each concentration of NaOCl for 10 minutes at 37°C. (A) The concentration of NaOCl was varied up to 2 mM. Cell lysates were made without stimulation with TNF. (B) Incubation with TNF (30 ng/mL) was performed for up to 30 minutes at 37°C. At the indicated times, cell lysates were prepared. Each lane was loaded with 60 µg protein. Bands reacting with primary antibody specific for IB were visualized with ECL. Similar results were obtained in three independent experiments.

Effect on TNF-Induced Proteolysis of IB

Effect of AP on Modification of IB
Because the band-shifting and -broadening caused by NaOCl was similar to that observed after TNF-induced phosphorylation of serines 32 and 36 on IB, we examined whether exposure of cell lysates to AP before electrophoresis would cause the effect induced by NaOCl to disappear.^{28 29} The results shown in Figure 5A indicate that the band-shifting and -broadening of IB caused by NaOCl was nearly the same as that obtained with NaOCl and AP. This similarity suggests that IB band-shifting and -broadening is not the result of NaOCl induced phosphorylation.

View larger version (41K): [in this window] [in a new window]   Figure 5. Effects on NaOCl-induced IB band-shifting of treatment of cell lysate with AP and MsrA. Cells were treated with NaOCl (0.75 mM) for 10 minutes at 37°C. (A) Pelleted cells were lysed in TNE buffer. Cell lysates (30 µL each) were adjusted to pH 9.7, 10 U AP was added, and cell lysates incubated for 30 minutes at 37°C. Treatment was terminated by neutralization of lysates. (B) Pelleted cells were lysed in TNE buffer containing 15 mM DTT. Cell lysates were treated with 10 µM MsrA for 15 minutes at 37°C. To evaluate effects of DTT on the shifted band, cell lysates were incubated for 30 minutes at 37°C without MsrA. Each lane was loaded with 60 µg protein. Bands that reacted with primary antibody specific for IB were visualized with ECL. Similar results were obtained in three independent experiments.

Effect of MsrA on NaOCl-Induced Alteration of IB

	Discussion

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During corneal inflammation, numerous pro- and anti-inflammatory cytokines are secreted. Two proinflammatory cytokines released by corneal epithelial cells are TNF and IL-1. Another effect in vivo that one or both of them may have is degradation of the corneal matrix in that they stimulate activity and expression of matrix metalloproteinase (MMP)-9 in cultured HCE cells.^{33 34} Besides release of cytokines in the epithelium, infiltrating neutrophils are activated and release MPO during inflammation. It has been suggested that release of MPO mediates the production of HClO/ClO^- to hinder bacterial growth at the site of infection.⁵ Because it has been shown in other tissues that MPO, through the formation of HClO/ClO^-, inhibits release of cytokines during an inflammatory response, we determined in the present study in HCE cells whether NaOCl (i.e., HClO/ClO^-) affects activation of NFB and expression of the IL-1 gene and protein in TNF-stimulated HCE cells. NaOCl transiently suppressed the responses elicited by TNF. Such declines may counterbalance excessive stromal degradation as well as the heightened inflammatory reaction resulting from release of TNF.

Release of TNF by activated neutrophils at the site of infection initiates an inflammatory response by stimulating surrounding cells to produce and release cytokines.³ In the corneal epithelium, it has been shown that exposure to TNF causes these cells to increase expression of IL-1 gene and protein.⁴ Release of IL-1 from these cells is a key factor in the regulation of inflammation and wound healing on the ocular surface.¹⁸ For IL-1 gene and protein expression to be stimulated, the transcription factor NFB must first be activated.²² Its activation is dependent on the dissociation of IB from NFB. The expression of IL-1 gene and protein occurs after phosphorylation and proteolysis of IB.

In the present study, we assessed the effects of NaOCl on the integrity of IB, the activation of NFB, and expression of the IL-1 gene and protein. The results shown in Figure 4A indicate that NaOCl shifted the position of the IB band in a dose-dependent manner. This band-shifting appeared to result from the oxidation of a methionine residue on IB, because band-shifting was lessened by treatment with MsrA (Fig. 5B) . Phosphorylation of IB does not appear to account for the bandshift, because it occurred despite treatment with AP (Fig. 5A) . The results shown in Figure 3 suggest that TNF-induced proteolysis of IB is less pronounced after transient exposure to NaOCl. This result could mean that methionine oxidation protects IB from undergoing phosphorylation, ubiquitination, and subsequent proteolysis and stabilizes its interaction with NFB. The results shown in Figure 3 are in agreement with this suggestion, because preexposure for 10 minutes to NaOCl suppressed activation of NFB. The results in Figures 1 and 2 show that inhibition of NFB resulting from a 10-minute exposure to NaOCl inhibited the expression of the IL-1 gene and protein. Furthermore, the results shown in Figures 1 and 2 are consistent with the time course of NaOCl-induced inhibition of NFB nuclear transfer shown in Figure 3 .

In other studies, there is evidence to suggest that the biological mediator of increases in HClO/ClO^- is TauCl, which is formed from the reaction of taurine with HClO/ClO^-.⁷ TauCl inhibits the production and release of cytokines from lymphocytes, glioma cells, and rheumatoid arthritis fibroblast-like synoviocytes.^{11 12 13 14} Kontny et al.¹⁴ showed that TauCl reduces the activation of transcription factors such as NFB and activating protein (AP)-1 in rheumatoid arthritis fibroblast-like synoviocytes and agree with our finding that NaOCl suppresses NFB activation (Fig. 3) . Barua et al.²³ recently showed in macrophages that TauCl inhibits inducible (i)NOS and expression of the TNF gene by inhibiting upstream signaling pathways that activate IB kinase. Because taurine is present in the corneal epithelium and TauCl spontaneously forms from taurine and HClO/ClO^-, the final mediator may be TauCl. However, it is not yet known whether TauCl is intracellularly synthesized.

Methionine oxidation of various proteins is reported to alter their characteristics.³⁵ For instance, exposure to oxidative stress leads to methionine oxidation of the HIV-2 protease and its inactivation, which can be partially reversed with MsrA.³⁵ Previously, modification of IB by TauCl was not described, whereas we detected NaOCl-induced alteration of IB. It is possible that our ability to detect such a modification is due to a difference in electrophoresis procedures. Presumably, the band-shifting that we detected was the result of methionine oxidation, and it was lessened by treatment with MsrA. Because we detected expression of the MsrA gene in HCE cells with RT-PCR, it is possible that intracellular methionine oxidation may occur in vivo and be reversible (data not shown).

In summary, in HCE cells TNF induced activation of NFB, which resulted in increases in expression of the IL-1 gene and protein. This response can be transiently suppressed in HCE cells by NaOCl (i.e., HClO/OCl^-), which suggests that the time course and magnitude of an immune response in vivo may depend on a host of feedback interactions between various mediators. This suppression occurs as the result of the oxidation of methionine residues on IB, which in turn inhibits activation of NFB by TNF. These results shed new sight on the regulation of the innate immune response in corneal epithelium.

	Footnotes

 
Supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (YM).

Submitted for publication March 21, 2002; revised May 22, 2002; accepted June 6, 2002.

Commercial relationships policy: N.

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: Yusei Miyamoto, Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bioscience Building 402, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan; yusei74{at}k.u-tokyo.ac.jp.

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