February 2004
Volume 45, Issue 2
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Cornea  |   February 2004
Promotion of Corneal Allograft Survival by the Induction of Oxidative Macrophages
Jun Yamada; Kazuichi Maruyama; Yoichiro Sano; Shigeru Kinoshita; Yukie Murata; Junji Hamuro
Author Affiliations
  • Jun Yamada
    From the Department of Ophthalmology, Meiji University of Oriental Medicine, Kyoto, Japan;
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; and
  • Kazuichi Maruyama
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; and
  • Yoichiro Sano
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; and
  • Shigeru Kinoshita
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; and
  • Yukie Murata
    Basic Research Laboratories, Ajinomoto Co. Inc., Kawasaki, Japan.
  • Junji Hamuro
    Basic Research Laboratories, Ajinomoto Co. Inc., Kawasaki, Japan.
Investigative Ophthalmology & Visual Science February 2004, Vol.45, 448-454. doi:https://doi.org/10.1167/iovs.03-0939
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      Jun Yamada, Kazuichi Maruyama, Yoichiro Sano, Shigeru Kinoshita, Yukie Murata, Junji Hamuro; Promotion of Corneal Allograft Survival by the Induction of Oxidative Macrophages. Invest. Ophthalmol. Vis. Sci. 2004;45(2):448-454. https://doi.org/10.1167/iovs.03-0939.

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Abstract

purpose.A Th1-type immune response was detected in allotransplanted, rejected corneas. Because the intracellular thiol redox status of antigen-presenting cells (APCs) reportedly regulates the Th1/Th2 balance through distinctive cytokine production by APCs, this study was conducted to investigate the effect of the intracellular thiol redox status of macrophages (Mps) on corneal allograft survival.

methods. N,N′-diacetyl-l-cystine dimethylester (NACOMe)2 was injected intraperitoneally into BALB/c (H-2d) mice to induce Mps with a low intracellular glutathione content (icGSH). Corneal grafts from C57BL/10 (H-2b), B10.D2 (H-2d), and DBA/2 (H-2d) donor mice were placed on neovascularized BALB/c graft beds for assessment. B10.D2-grafted recipients were evaluated for donor-specific delayed-type hypersensitivity (DTH), and the cytokines produced by their lymphocytes were examined (IFN-γ, IL-4, and IL-10). In other experiments, naïve BALB/c mice, injected intravenously with Mps of low icGSH content, received B10.D2 corneal grafts.

results.In (NACOMe)2-treated mice, 13 of 20 B10.D2 grafts and 6 of 10 DBA/2 grafts survived indefinitely. No grafts survived in the control mice (P < 0.0001). (NACOMe)2 treatment did not enhance C57BL/10 graft survival. At 2 weeks after B10.D2 grafting, control mice exhibited DTH, but (NACOMe)2-treated mice did not (P < 0.01). Lymphocytes from (NACOMe)2-treated mice did not respond to donor splenocytes. Those of control mice showed Th1-type cytokine secretion. The intravenous transfer of peritoneal Mps from (NACOMe)2-treated mice prolonged corneal allograft survival (P < 0.003).

conclusions. The observed enhanced graft acceptance may be due to the suppression of alloantigen-induced Th1 polarization through the induction of Mps with reduced icGSH levels.

Penetrating keratoplasty in normal eyes has emerged worldwide as the most common and successful form of solid tissue transplantation. 1 2 However, corneal grafts in high-risk (i.e., immune-privilege–abrogated) eyes continue to carry a poor prognosis. Even intensive systemic immunosuppressive therapy is often of no avail. 2 3 4 Therefore, a better understanding of rejection mechanism is necessary for the development of a new immunomodulation strategy in patients who undergo keratoplasty. 
Immunologic studies in rodents have revealed that donor-specific DTH is chiefly responsible for the destructive alloimmunity that follows orthotopic corneal grafting. 5 6 DTH is typically mediated by CD4+ T cells of the Th1 phenotype. They are the polar opposites of CD4+ Th2 cells. 7 The Th1–Th2 balance during their induction, maturation, and maintenance is regulated by cytokines secreted by APCs. Ocular APCs, including Langerhans cells (LCs) and infiltrating macrophages (Mps), mediate the DTH response at both the afferent and efferent phases of corneal graft rejection. The corneal allograft acceptance achieved by Slegers et al. 8 in rats on depletion of conjunctival Mps supports the hypothesis that these cells play an important role in graft rejection, probably through the local activation of the Th1-type response. Sano et al., 9 who found that the intravenous (IV) adoptive transfer of TGF-β–treated Mps promotes graft survival, indicated that modulated Mps can regulate the recipients’ immune response. These observations suggest that modulating the functional phenotypes of Mps may be useful for preventing corneal allograft rejection. 
Tissue stromal reaction is frequently accompanied by the generation of reactive oxygen species (ROS), which in turn regulate the cytokine milieu at inflamed tissue sites. Glutathione (GSH) constitutes the first line of the cellular defense against oxidative injury. 10 Intracellular GSH (icGSH) in Mps is essential for the secretion of IL-12 by regulating MAPK p38 activity, 11 12 13 14 15 which indicates that icGSH is critical for determining whether the Th1 or Th2 response predominates. 12 14 Furthermore, exposure of Mps to IFN-γ increased, whereas exposure to IL-4 decreased, the icGSH/oxidized icGSH (icGSSG) ratio. 14 16 Mps with decreased icGSH are known as oxidative Mps (OMps) and those with increased amounts as reductive Mps (RMps). 11 13 14 17 Their balance dictates the cytokine networks of the immune system through the distinctive production of nitric oxide (NO), IL-12, IL-18, and IFN-γ by RMps, and of IL-6, IL-10, and prostaglandin (PG)E2 by OMps. 14 Therefore, it is reasonable to expect that RMps contribute to the induction of DTH responses governed by Th1 cells. 
We hypothesized that OMp induction in a graft recipient would lead to prolonged corneal allograft survival by abrogating the Th1-mediated DTH response in the absence of Th1-stimulating cytokines. Our results confirm that OMp induction is of great benefit at both the clinical and pathologic levels. 
Materials and Methods
Animals
Male BALB/c (H-2d), C57BL/10 (H-2b), and DBA/2 (H-2d) mice were purchased from SLC, (Osaka, Japan) at 7 to 10 weeks of age. Same-age B10.D2 (H-2d) mice were purchased from The Jackson Laboratory, (Bar Harbor, ME). All were treated in accordance with the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research. All experiments were approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine. 
Reagents
N,N′-diacetyl-l-cystine dimethylester ((NACOMe)2) was purchased from Bachem (Bubendorf, Switzerland; E-1770) and diethyl maleate (DEM, D9-770-3) from Sigma-Aldrich (St. Louis, MO). Glutathione diethylester ((GSH-(OEt)2) was synthesized at our request at the Peptide Research Institute (Minoo, Japan) and its purity of more than 98.3% confirmed by HPLC. The former two compounds induce OMps with a reduced amount of icGSH, the latter induces RMps with an elevated amount of icGSH. They are used widely as modulators of the thiol redox status of Mps and at the doses used, they are without cellular toxicity in vivo. 11 13 14 15 16 18 19 To induce OMps and RMps, we delivered three intraperitoneal (IP) injections in the following order: 200 μg/500 μL (NACOMe)2, 250 μg/500 μL DEM, and 2 mg/500 μL ((GSH-(OEt)2). Monochlorobimane (MCB, M-1381; Molecular Probes, Eugene, OR) was used to stain for icGSH. 11 13 14 15 16 18 19  
Qualitative Determination of icGSH
Peritoneal cell suspensions (300 μL), adjusted to 3 × 105 cells/mL in phenol-red–free RPMI-1640 medium, were placed on Chamber Slides (catalog no. 136439, Laboratory-Tek; Nunc, Roskilde, Denmark), and incubated at 37°C for 3 hours in a CO2 incubator. Nonadherent cells were removed, the culture was washed three times with the same medium, and 300 μL MCB adjusted to 10 mM in the same medium was added and allowed to react for 30 minutes. Fluorescence intensity, reflective of the amount of icGSH in adherent Mps, was monitored by argon-ion laser cytometry on a workstation (ACAS 570; Meridian Instruments, Okemos, MI). To detect the icGSH levels, we used a fluorescent MCB probe with excitation and emission wavelengths of 350 and 460 nm, respectively. The cell-permeant MCB probe is nonfluorescent but forms a fluorescent adduct with GSH in a reaction catalyzed by glutathione S-transferase. 
Preparation of Peritoneal Mps for Adoptive Transfer
After three IP injections of 200 μg (NACOMe)2 or saline alone, peritoneal cells harvested from five mice were inoculated into a microplate (catalog no. 167008; Nunc) containing phenol-red–free RPMI-1640 without antibiotics and incubated for 3 hours. Adherent cells were used as resident peritoneal Mps. After three washes and collection by vigorous pipetting on ice, 1 × 106 peritoneal Mps in PBS were transferred intravenously into naive BALB/c mice. 
Orthotopic Corneal Transplantation to High-Risk Eyes
Before all surgical procedures, each recipient was deeply anesthetized with an IP injection of 3 mg ketamine and 0.0075 mg xylazine. Corneal neovascularization (referred to as high-risk graft beds) was induced by placing three interrupted 11-0 sutures (Sharpoint; Vanguard, Houston, TX) in the central cornea 2 weeks earlier. 20 The corneal transplantation technique has been described. 21 Briefly, the central 2 mm of the donor cornea was excised and secured in recipient graft beds with eight interrupted 11-0 nylon sutures. In this series of experiments, examination of all grafted eyes after 72 hours confirmed no complications such as hyphema, infection, or loss of the anterior chamber. Transplant sutures were removed in all cases on day 7. Grafts were examined by slit lamp biomicroscopy twice a week. At each time point, grafts were scored for opacification from 0 to 5+, according to a scoring system described elsewhere. 21 Grafts exhibiting an opacity score of 3+ or greater (moderate stromal opacity with only pupil margin visible) at 2 weeks or 2+ or greater (mild deep stromal opacity with pupil margin and iris vessels visible) after 3 weeks were considered rejected (immunologic failure). 
Preparation of Lymphocytes from Graft Recipients
Ipsilateral draining lymph nodes (LNs) and spleens were harvested from graft recipients and pressed through nylon mesh to produce a single-cell suspension. Lymphocytes from each recipient were separated in the subsequent experiments. Red blood cells were lysed by TE buffer (10 mM Tris-HCl and 0.1 mM EDTA) and washed twice. In adoptive transfer experiments, pooled cells from the LNs and spleen of each mouse (5 × 106) were injected IV into naive BALB/c recipients. In cell culture experiments, T cells were purified to more than 95% by Thy1.2 microbeads according to the manufacturer’s instructions (MACS; Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany) and cultured. 
Cell Culture for Evaluating Donor-Specific Responses
T cells (4 × 105) were resuspended in 96-well plates and stimulated with irradiated (2000 R) B10.D2 splenocytes in serum-free medium at 37°C in a 5% CO2 atmosphere. The serum-free medium 22 consisted of RPMI-1640 medium, 10 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin (all from BioWhittaker, Walkersville, MD), and 1 × 10−5 M 2-mercaptoethanol (Sigma-Aldrich) supplemented with 0.1% bovine serum albumin (Sigma-Aldrich), ITS+ culture supplement (1 μg/mL iron-free transferrin, 10 ng/mL linoleic acid, 0.3 ng/mL Na2Se, and 0.2 μg/mL Fe(NO3)3; Collaborative Biomedical Products, Bedford, MA). 
ELISA and Proliferation Assays
Cultures were grown for 24, 48, or 72 hours. At each time point, supernatants were collected and analyzed for their IFN-γ-, IL-4-, and IL-10 content with ELISA kits used according to the manufacturer’s instructions (BD Pharmingen, San Diego, CA). Similar cultures were incubated for 120 hours, pulsed with tritiated thymidine (0.5 μCi/well) during the final 8 hours, and harvested with a cell harvester (MicroMate 196; Perkin-Elmer, Meriden, CT). Radioactivity was measured on a beta counter (Matrix 9600; Perkin Elmer). 
Assessment of Delayed Hypersensitivity
Mice treated with (NACOMe)2 or saline received B10.D2 corneal allografts. Two weeks later, they received injections in the right ear pinnae of 1 × 106 irradiated (2000 R) B10.D2 or DBA/2 spleen cells in 10 μL Hanks’ balanced salt solution. 23 At 24 and 48 hours after ear challenge, ear thickness was measured with a low-pressure micrometer (Mitutoyo; MTI Corp., Paramus, NJ). 
Statistical Methods
We constructed Kaplan-Meier survival curves and used the Breslow-Gehan-Wilcoxon test to compare the probabilities of allograft survival. To compare proliferation responses, secreting cytokines, and DTH responses, we used Student’s t-test. P < 0.05 was deemed significant. 
Results
Fate of Orthotopic Corneal Allografts on Modulation of icGSH
In the first set of experiments, B10.D2 donor grafts were placed on high-risk (neovascularized) graft beds of BALB/c mice. This combination shares the same major histocompatibility complex (MHC) molecule, but displays different minor histocompatibility (minor H) antigens. To modulate the icGSH of APCs, BALB/c mice were treated with the following reagents at 0, 4, and 7 days before corneal grafting. In BALB/c hosts injected with (NACOMe)2 to induce OMps, the survival of grafted B10.D2 corneas was prolonged significantly (65% acceptance at 8 weeks, n = 20, P < 0.0001), compared with the saline-treated group (n = 15). Treatment with the OMp-inducer DEM also promoted B10.D2 cornea graft survival (30% acceptance at 8 weeks, n = 10, P < 0.0001), compared with the saline-treated group. In contrast, treatment with the RMp-inducer GSH-(OEt)2 (n = 10) produced results similar to those in the saline-treated controls. Because our scoring system did not unequivocally detect graft rejection earlier than 2 weeks and because peritoneal Mps of grafted mice tended to polarize spontaneously to RMps, further accelerated rejections were not detected (Fig. 1A) . 
On day 14, saline- or (NACOMe)2-treated mice (n = 5 each) and, on day 42, (NACOMe)2-treated mice with either rejected or healthy corneas (n = 2 each) were killed and their enucleated eyes were examined histologically. Representative results are shown in Figures 1B 1C 1D 1E . Rejected corneas from saline-treated donors manifested copious amounts of infiltrating cells and edematous stroma (Fig. 1B) , as did rejected corneas from (NACOMe)2-treated donors (Fig. 1D) . In contrast, accepted corneas from (NACOMe)2-treated donors exhibited no infiltrating cells and appeared healthy at 14 (Fig. 1C) and 42 days (Fig. 1E) after transplantation. 
To confirm the effect of (NACOMe)2 on the survival of corneal allografts with only minor H incompatibility, BALB/c recipients were given DBA/2 corneal allografts that shared the same MHC molecule as the recipients, but displayed minor H different from both BALB/c and B10.D2 mice. As shown in Figure 1F , (NACOMe)2 treatment significantly promoted graft survival (40% acceptance, n = 10, P < 0.0002) compared with saline treatment (n = 10). 
Next, we examined the capacity to promote corneal graft survival with MHC+, minor H-disparate combinations. Despite B10.D2 donor graft survival, (NACOMe)2 treatment failed to enhance C57BL/10 allograft survival (0% acceptance, n = 10), as in saline-treated recipients (n = 10; data not shown). These results clearly show that modulation of the intracellular thiol redox status has suppressive effects on the rejection of minor H incompatible-, but not of MHC-incompatible grafts. 
Alloresponse of T Cells from (NACOMe)2-Treated Mice
T cells from ipsilateral cervical LNs and spleens of (NACOMe)2- or saline-treated mice that had received B10.D2 corneal allografts were purified 1 or 2 weeks after transplantation. Purified T cells from BALB/c mice injected subcutaneously in the neck with 1 × 107 B10.D2 splenocytes 1 week earlier served as the positive control. T cells (2.5 × 105) were cocultured for 24 to 72 hours with 2.5 × 105 x-irradiated B10.D2 or DBA/2 splenocytes in 96-well plates, and supernatants were collected to measure cytokine secretion. T-cell proliferation was analyzed after 5 days. At 1 week after transplantation, T cells from mice injected with (NACOMe)2 or saline manifested neither positive proliferation nor cytokine secretion (data not shown), but at 2 weeks after transplantation, T cells from these mice exhibited increased proliferative responses (Fig. 2A) and cytokine production (Fig. 2B) . DBA/2 splenocyte-stimulation of T cells from saline-treated, (NACOMe)2-treated, and positive control mice did not induce proliferation and cytokine secretion. On exposure to B10.D2 splenocytes, T cells from saline-treated grafted (n = 6) and positive control (n = 4) mice proliferated and secreted significant amounts of IFN-γ, but not IL-4 and IL-10. In contrast, T cells from (NACOMe)2-treated graft-recipient mice (n = 10) showed less proliferation and less secretion of IFN-γ. Our results indicate that (NACOMe)2 treatment suppressed the Th1 alloresponse in corneal graft recipients. 
Effect of Adoptively Transferred Lymphocytes on Graft Acceptance
To evaluate the presence of regulatory T cells in (NACOMe)2-treated mice that showed corneal allograft (B10.D2) acceptance at 2 weeks, lymphocytes from their cervical LNs and spleens were pooled and adoptively transferred by IV injection into naive BALB/c mice (one donor equivalent per recipient). Immediately thereafter, lymphocyte recipients (n = 10) received B10.D2 corneal allografts onto high-risk graft beds. The adoptive transfer of lymphocytes did not enhance corneal graft survival (data not shown), indicating that graft acceptance in (NACOMe)2-treated mice may not be due to the induction of regulatory T cells. 
Effect of Adoptive Transfer of OMps on Corneal Allograft Survival
We assessed the capacity of OMps to promote corneal graft survival. The IV transfer of OMps is capable of converting Mps of recipient mice to OMp. 18 19 Peritoneal Mps from (NACOMe)2- or saline-treated BALB/c mice were collected and their icGSH content was determined by MCB staining. As shown in Figure 3A , Mps from (NACOMe)2-treated mice were OMps with low icGSH content, whereas Mps from saline-treated mice were RMps with high icGSH content (Fig. 3B) . Peritoneal Mps (1 × 106) were injected IV into naive BALB/c mice at the time of B10.D2 corneal grafting onto high-risk eyes. The adoptive transfer of OMps (n = 10, P < 0.003) significantly promoted corneal graft survival (n = 9; Fig. 3C ), whereas that of RMps did not, suggesting that OMps can suppress corneal allograft rejection in vivo. 
Effect of (NACOMe)2 Treatment on Donor-Specific DTH
We evaluated the acquisition of donor-specific DTH, which plays a central role in mediating corneal graft rejection. After (NACOMe)2 or saline treatment, one panel of mice received B10.D2 corneal allografts in high-risk graft beds as well as an intradermal inoculation of x-irradiated B10.D2 or DBA/2 splenocytes into the right ear pinnae for a DTH assay (n = 5 in each group). All groups of mice did not show any DBA/2-specific response (data not shown). As shown in Table 1 , whereas saline-treated mice manifested positive DTH responses 2 weeks after grafting, in (NACOMe)2-treated mice, the donor-specific DTH response was significantly lower (P < 0.005). These results indicate that (NACOMe)2 treatment can enhance graft survival by suppressing allosensitization by the corneal allograft. 
Discussion
Ours is the first report to show the relevance of the intracellular thiol redox status of Mps (or APCs) in the regulation of corneal allograft rejection. OMps with low icGSH secrete the Th2-type cytokines IL-6, IL-10, and PGE2, and prevent the differentiation of naive T cells (Th0) into Th1 cells. 12 14 In contrast, RMps with high icGSH, but not OMps, secrete the Th1-type cytokines IFN-γ and IL-12 that are essential for Th1 differentiation. 11 12 14 16 Because corneal graft rejection can be suppressed by preventing the Th1 response or by inducing the Th2 response, 24 we posited that (NACOMe)2 may exert an indirect effect by inducing OMps that can skew the Th1/Th2 balance to Th2. 11 12 13 14 15 16 18 19 We found that in most (NACOMe)2-treated graft recipients a reduced Th1 response was elicited, whereas the donor-specific Th2-type response was not elevated (Fig. 2) . This suggests that the Th1 response may be crucial for the rejection of corneal allografts introduced into vascularized high-risk beds. This hypothesis is supported by the observation that the adoptive transfer of lymphocytes did not promote graft acceptance, whereas that of OMps did (Fig. 3C) . These results indicate that graft survival in (NACOMe)2-treated mice was not solely ascribed to the induction of Th2 cells. The role of Th2 cells in allograft rejection is controversial, and it remains uncertain whether Th2 cells or Th2 cytokines play a preventive role in allograft rejection. Th2 cells may be innocuous, they may actively inhibit Th1 activity, and they may even be pathogenic under certain conditions. Consequently, it is plausible that the abrogation of Th1 induction due to skewing toward OMps participates in the observed prolongation of corneal allografts. 
In murine transplantation models, a high amount of IFN-γ was detected in rejected corneas. 25 Considering that IFN-γ efficiently converts Mps to RMps, 14 15 it is conceivable that IFN-γ activation renders the converted cells capable of producing IL-12. Consequently, skewing toward Th1 and elevation of an alloantigen-specific DTH response occur. In this context, it can be concluded that OMp induction may function as a blockade against the amplification loop of a RMp/Th1/DTH circuit triggered by corneal allografting. 
(NACOMe)2 injected IP may reach the eye through the blood circulation and affect the thiol redox status of infiltrating Mps. Resident tissue Mps are present in the iris, ciliary body, uvea, retina, conjunctiva, and corneal limbus. 20 21 22 23 24 Normal murine corneal stroma does not express MHC class II, but contains small numbers of CD11b+ and CD11c+ cells thought to be immature dendritic cells (DCs) and immature Mps. 26 27 Possibly, the redox status of Mps dictates their function as APCs even in eyes. Because the cytokine profile produced by DCs is also regulated by their intracellular redox status, as is the case in Mps, 19 ocular LCs may also participate in the observed graft survival in (NACOMe)2-treated mice. The relevance of Mps and LCs in corneal graft rejection will be determined by extensive studies examining the role of their intracellular redox status in the local microenvironment. The effect of local induction of OMps by the subconjunctival injection of (NACOMe)2 is under investigation in our laboratory. 
Mps play diverse and relevant roles in the host defense against invasive and noxious insult. 17 28 The importance of the innate immune response has been reevaluated based on findings concerning the essential role played by Mp-produced IFN-γ and the Toll-like receptor (TLR) family–MyD88–NF-κB system. 29 30 31 A pro- or anti-inflammatory paradigm has been proposed for the functional heterogeneity of Mps. 32 33 34 35 36 We consider RMps proinflammatory and OMps anti-inflammatory. 14 According to the Mps depletion study in vivo, Torres et al. 37 showed that conjunctival Mps depletion downregulates both the Th1 and Th2 response after corneal allografts. Brewer et al. 38 reported that systemic Mps depletion shifts the expected Th1 response to a Th2 response—a finding that coincides with the observation of Slegers et al. 8 who showed corneal allograft acceptance in rats depleted of conjunctival Mps. Desmedt et al. 39 argued that Mps efficiently elicit cellular immunity by selectively suppressing an already generated Th2-dependent response and thereby act as Th1-dedicated APCs. 
RMps produced an elevated IFN-γ/IL-10 ratio, a functional index of RMps, whereas OMps produced a reduced ratio. IL-10 produced by OMps is an anti-inflammatory cytokine that suppresses MHC class II expression. This may at least partially contribute to graft acceptance after OMp induction. TGF-β is a potent inducer of OMp 19 and Sano et al. 9 reported that IV adoptive transfer of TGF-β–treated Mps promotes corneal graft survival. 
Because the redox status of nonmacrophage APC (DCs, LCs) may also be affected by pharmacological agents, it is certain that they contribute to the graft survival reported. 
Minor H antigens, rather than antigens encoded within the MHC, are the most important initiators of alloimmunity after orthotopic corneal transplantation. 21 40 These antigens are loaded onto self-MHC molecules and presented to recipient T cells by the so-called indirect pathway of allorecognition. Thus, APC modulation by (NACOMe)2 may be effective only in minor-H–incompatible grafts (Figs. 1A 1F) , because APCs, including donor- and host-derived APCs, are the only cells that are able to activate allospecific T cells in both afferent and efferent phases. In contrast, not only MHC-encoded alloantigens are recognized through the direct pathway of allorecognition that is irrelevant to recipient APCs, but many of the direct alloreactive T cells have the phenotype of memory Th1-type cells. Because cornea contains bone-marrow–derived cells that may express MHC after grafting and serve as APCs for activating recipient T cells directly, 26 27 and because their secreted IFN-γ strongly induces RMp locally, (NACOMe)2 treatment may be unable to promote C57BL/10 graft survival. This type of dissociation between B10.D2 and C57BL/10 has already been observed. 24  
Strategies have been devised to suppress donor-specific corneal allograft rejection in rodent models, including induction of donor-specific tolerance 9 41 42 and inhibition of the induction of donor-specific Th1 cells. 24 43 Donor-specific regulatory cells induced by those strategies may protect corneal allografts indefinitely. From this perspective, by facilitating the long-term acceptance of donor corneal grafts, our strategy for inhibiting the Th1-mediated response is a significant step toward achieving the survival and acceptance of corneal grafts in a clinical setting. 
 Supported in part by Research Grant 15791012 from the Japanese Ministry of Education, Culture and Science and research funds from the Kyoto Foundation for the Promotion of Medical Science.
 Submitted for publication August 27, 2003; revised October 19, 2003; accepted November 7, 2003.
 Disclosure: J. Yamada, None; K. Maruyama, None; Y. Sano, None; S. Kinoshita, None; Y. Murata, Ajinomoto Co., Inc. (F); J. Hamuro, Ajinomoto Co., Inc. (F)
 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: Jun Yamada, Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajiicho, Hirokoji-agaru Kawaramachi-dori, Kamigyo-ku, Kyoto, Japan 602-0841; [email protected].
 
Figure 1.
Fate of corneal allografts in BALB/c high-risk graft beds on modulation of the thiol redox state. (A) BALB/c mice treated with (NACOMe)2 (n = 20; ○), DEM (n = 10; □), GSH-(OEt)2 (n = 10; •), or saline (n = 15; ▪) received B10.D2 corneal allografts. (B– E) Histologic appearance of B10.D2 allografts after 14 days in saline- (B) or (NACOMe)2-treated (C) recipients. Rejected (D) and healthy (E) B10.D2 allografts at 42 days in (NACOMe)2-treated recipients. Arrows: endothelial layer. (F) DBA/2 corneal allografts in BALB/c mice treated with (NACOMe)2 (n = 10; ○) or saline (n = 10; ▪).
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Fate of corneal allografts in BALB/c high-risk graft beds on modulation of the thiol redox state. (A) BALB/c mice treated with (NACOMe)2 (n = 20; ○), DEM (n = 10; □), GSH-(OEt)2 (n = 10; •), or saline (n = 15; ▪) received B10.D2 corneal allografts. (B– E) Histologic appearance of B10.D2 allografts after 14 days in saline- (B) or (NACOMe)2-treated (C) recipients. Rejected (D) and healthy (E) B10.D2 allografts at 42 days in (NACOMe)2-treated recipients. Arrows: endothelial layer. (F) DBA/2 corneal allografts in BALB/c mice treated with (NACOMe)2 (n = 10; ○) or saline (n = 10; ▪).
Figure 1.
 
Fate of corneal allografts in BALB/c high-risk graft beds on modulation of the thiol redox state. (A) BALB/c mice treated with (NACOMe)2 (n = 20; ○), DEM (n = 10; □), GSH-(OEt)2 (n = 10; •), or saline (n = 15; ▪) received B10.D2 corneal allografts. (B– E) Histologic appearance of B10.D2 allografts after 14 days in saline- (B) or (NACOMe)2-treated (C) recipients. Rejected (D) and healthy (E) B10.D2 allografts at 42 days in (NACOMe)2-treated recipients. Arrows: endothelial layer. (F) DBA/2 corneal allografts in BALB/c mice treated with (NACOMe)2 (n = 10; ○) or saline (n = 10; ▪).
Fate of corneal allografts in BALB/c high-risk graft beds on modulation of the thiol redox state. (A) BALB/c mice treated with (NACOMe)2 (n = 20; ○), DEM (n = 10; □), GSH-(OEt)2 (n = 10; •), or saline (n = 15; ▪) received B10.D2 corneal allografts. (B– E) Histologic appearance of B10.D2 allografts after 14 days in saline- (B) or (NACOMe)2-treated (C) recipients. Rejected (D) and healthy (E) B10.D2 allografts at 42 days in (NACOMe)2-treated recipients. Arrows: endothelial layer. (F) DBA/2 corneal allografts in BALB/c mice treated with (NACOMe)2 (n = 10; ○) or saline (n = 10; ▪).
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Figure 2.
Proliferation response (A) and cytokine secretion (B) by T cells from corneal transplant recipients. (A) (NACOMe)2- or saline-treated BALB/c mice received B10.D2 corneal allografts. After 14 days, draining LNs were removed, and purified T cells were cultured with B10.D2 or DBA/2 stimulator cells. BALB/c mice, subcutaneously injected with 1 × 107 B10.D2 splenocytes were the positive control. (B) IFN-γ, IL-4, and IL-10 secretion in those culture supernatants were assayed. Positive controls (♦, ⋄). (NACOMe)2- (▪, □) and saline-treated mice (•, ○) were shown (filled and open symbols showed B10.D2 and DBA/2 stimulation, respectively). *P < 0.001. Error bars, SE.
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Proliferation response (A) and cytokine secretion (B) by T cells from corneal transplant recipients. (A) (NACOMe)2- or saline-treated BALB/c mice received B10.D2 corneal allografts. After 14 days, draining LNs were removed, and purified T cells were cultured with B10.D2 or DBA/2 stimulator cells. BALB/c mice, subcutaneously injected with 1 × 107 B10.D2 splenocytes were the positive control. (B) IFN-γ, IL-4, and IL-10 secretion in those culture supernatants were assayed. Positive controls (♦, ⋄). (NACOMe)2- (▪, □) and saline-treated mice (•, ○) were shown (filled and open symbols showed B10.D2 and DBA/2 stimulation, respectively). *P < 0.001. Error bars, SE.
Figure 2.
 
Proliferation response (A) and cytokine secretion (B) by T cells from corneal transplant recipients. (A) (NACOMe)2- or saline-treated BALB/c mice received B10.D2 corneal allografts. After 14 days, draining LNs were removed, and purified T cells were cultured with B10.D2 or DBA/2 stimulator cells. BALB/c mice, subcutaneously injected with 1 × 107 B10.D2 splenocytes were the positive control. (B) IFN-γ, IL-4, and IL-10 secretion in those culture supernatants were assayed. Positive controls (♦, ⋄). (NACOMe)2- (▪, □) and saline-treated mice (•, ○) were shown (filled and open symbols showed B10.D2 and DBA/2 stimulation, respectively). *P < 0.001. Error bars, SE.
Proliferation response (A) and cytokine secretion (B) by T cells from corneal transplant recipients. (A) (NACOMe)2- or saline-treated BALB/c mice received B10.D2 corneal allografts. After 14 days, draining LNs were removed, and purified T cells were cultured with B10.D2 or DBA/2 stimulator cells. BALB/c mice, subcutaneously injected with 1 × 107 B10.D2 splenocytes were the positive control. (B) IFN-γ, IL-4, and IL-10 secretion in those culture supernatants were assayed. Positive controls (♦, ⋄). (NACOMe)2- (▪, □) and saline-treated mice (•, ○) were shown (filled and open symbols showed B10.D2 and DBA/2 stimulation, respectively). *P < 0.001. Error bars, SE.
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Figure 3.
icGSH content of peritoneal Mps and fate of B10.D2 corneal allografts after adoptive transfer of Mps. Peritoneal Mps from (NACOMe)2-treated (A) and saline-treated (B) mice were stained with 10 μM MCB for 30 minutes at 37°C. The fluorescence intensity revealing the amount of icGSH in adherent Mps was monitored by argon-ion laser cytometry and a computer workstation. Yellowish or red staining indicates the abundant presence of icGSH. (C) After IV injection of either (NACOMe)2- (n = 10; ○) or saline-treated (n = 9; ▪) Mps into naive BALB/c hosts, B10.D2 corneal grafts were placed on high-risk graft beds. *P < 0.003.
View OriginalDownload Slide
 
icGSH content of peritoneal Mps and fate of B10.D2 corneal allografts after adoptive transfer of Mps. Peritoneal Mps from (NACOMe)2-treated (A) and saline-treated (B) mice were stained with 10 μM MCB for 30 minutes at 37°C. The fluorescence intensity revealing the amount of icGSH in adherent Mps was monitored by argon-ion laser cytometry and a computer workstation. Yellowish or red staining indicates the abundant presence of icGSH. (C) After IV injection of either (NACOMe)2- (n = 10; ○) or saline-treated (n = 9; ▪) Mps into naive BALB/c hosts, B10.D2 corneal grafts were placed on high-risk graft beds. *P < 0.003.
Figure 3.
 
icGSH content of peritoneal Mps and fate of B10.D2 corneal allografts after adoptive transfer of Mps. Peritoneal Mps from (NACOMe)2-treated (A) and saline-treated (B) mice were stained with 10 μM MCB for 30 minutes at 37°C. The fluorescence intensity revealing the amount of icGSH in adherent Mps was monitored by argon-ion laser cytometry and a computer workstation. Yellowish or red staining indicates the abundant presence of icGSH. (C) After IV injection of either (NACOMe)2- (n = 10; ○) or saline-treated (n = 9; ▪) Mps into naive BALB/c hosts, B10.D2 corneal grafts were placed on high-risk graft beds. *P < 0.003.
icGSH content of peritoneal Mps and fate of B10.D2 corneal allografts after adoptive transfer of Mps. Peritoneal Mps from (NACOMe)2-treated (A) and saline-treated (B) mice were stained with 10 μM MCB for 30 minutes at 37°C. The fluorescence intensity revealing the amount of icGSH in adherent Mps was monitored by argon-ion laser cytometry and a computer workstation. Yellowish or red staining indicates the abundant presence of icGSH. (C) After IV injection of either (NACOMe)2- (n = 10; ○) or saline-treated (n = 9; ▪) Mps into naive BALB/c hosts, B10.D2 corneal grafts were placed on high-risk graft beds. *P < 0.003.
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Table 1.
 
View Table
 
Donor-Specific Delayed Hypersensitivity 2 Weeks after Grafting
Table 1.
 
Donor-Specific Delayed Hypersensitivity 2 Weeks after Grafting
Corneal Allograft Treatment Ear Challenge Ear Swelling (μm ± SD) n
B10.D2 (NACOMe)2 B10.D2 38 ± 22 5
B10.D2 Saline B10.D2 110 ± 18* 5
None (NACOMe)2 B10.D2 36 ± 20 5
None Saline B10.D2 30 ± 22 5
 After (NACOMe)2 or saline treatment, one panel of mice received a B10.D2 corneal allograft. Either B10.D2 or DBA/2 splenocytes were ear challenged, and swelling responses were assessed at 2 weeks after transplantation.
*  Response significantly higher than in the control (P < 0.005).
The authors thank Takeshi Kezuka for helpful discussions of the experiments. 
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Figure 1.
Fate of corneal allografts in BALB/c high-risk graft beds on modulation of the thiol redox state. (A) BALB/c mice treated with (NACOMe)2 (n = 20; ○), DEM (n = 10; □), GSH-(OEt)2 (n = 10; •), or saline (n = 15; ▪) received B10.D2 corneal allografts. (B– E) Histologic appearance of B10.D2 allografts after 14 days in saline- (B) or (NACOMe)2-treated (C) recipients. Rejected (D) and healthy (E) B10.D2 allografts at 42 days in (NACOMe)2-treated recipients. Arrows: endothelial layer. (F) DBA/2 corneal allografts in BALB/c mice treated with (NACOMe)2 (n = 10; ○) or saline (n = 10; ▪).
View OriginalDownload Slide
 
Fate of corneal allografts in BALB/c high-risk graft beds on modulation of the thiol redox state. (A) BALB/c mice treated with (NACOMe)2 (n = 20; ○), DEM (n = 10; □), GSH-(OEt)2 (n = 10; •), or saline (n = 15; ▪) received B10.D2 corneal allografts. (B– E) Histologic appearance of B10.D2 allografts after 14 days in saline- (B) or (NACOMe)2-treated (C) recipients. Rejected (D) and healthy (E) B10.D2 allografts at 42 days in (NACOMe)2-treated recipients. Arrows: endothelial layer. (F) DBA/2 corneal allografts in BALB/c mice treated with (NACOMe)2 (n = 10; ○) or saline (n = 10; ▪).
Figure 1.
 
Fate of corneal allografts in BALB/c high-risk graft beds on modulation of the thiol redox state. (A) BALB/c mice treated with (NACOMe)2 (n = 20; ○), DEM (n = 10; □), GSH-(OEt)2 (n = 10; •), or saline (n = 15; ▪) received B10.D2 corneal allografts. (B– E) Histologic appearance of B10.D2 allografts after 14 days in saline- (B) or (NACOMe)2-treated (C) recipients. Rejected (D) and healthy (E) B10.D2 allografts at 42 days in (NACOMe)2-treated recipients. Arrows: endothelial layer. (F) DBA/2 corneal allografts in BALB/c mice treated with (NACOMe)2 (n = 10; ○) or saline (n = 10; ▪).
Fate of corneal allografts in BALB/c high-risk graft beds on modulation of the thiol redox state. (A) BALB/c mice treated with (NACOMe)2 (n = 20; ○), DEM (n = 10; □), GSH-(OEt)2 (n = 10; •), or saline (n = 15; ▪) received B10.D2 corneal allografts. (B– E) Histologic appearance of B10.D2 allografts after 14 days in saline- (B) or (NACOMe)2-treated (C) recipients. Rejected (D) and healthy (E) B10.D2 allografts at 42 days in (NACOMe)2-treated recipients. Arrows: endothelial layer. (F) DBA/2 corneal allografts in BALB/c mice treated with (NACOMe)2 (n = 10; ○) or saline (n = 10; ▪).
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Figure 2.
Proliferation response (A) and cytokine secretion (B) by T cells from corneal transplant recipients. (A) (NACOMe)2- or saline-treated BALB/c mice received B10.D2 corneal allografts. After 14 days, draining LNs were removed, and purified T cells were cultured with B10.D2 or DBA/2 stimulator cells. BALB/c mice, subcutaneously injected with 1 × 107 B10.D2 splenocytes were the positive control. (B) IFN-γ, IL-4, and IL-10 secretion in those culture supernatants were assayed. Positive controls (♦, ⋄). (NACOMe)2- (▪, □) and saline-treated mice (•, ○) were shown (filled and open symbols showed B10.D2 and DBA/2 stimulation, respectively). *P < 0.001. Error bars, SE.
View OriginalDownload Slide
 
Proliferation response (A) and cytokine secretion (B) by T cells from corneal transplant recipients. (A) (NACOMe)2- or saline-treated BALB/c mice received B10.D2 corneal allografts. After 14 days, draining LNs were removed, and purified T cells were cultured with B10.D2 or DBA/2 stimulator cells. BALB/c mice, subcutaneously injected with 1 × 107 B10.D2 splenocytes were the positive control. (B) IFN-γ, IL-4, and IL-10 secretion in those culture supernatants were assayed. Positive controls (♦, ⋄). (NACOMe)2- (▪, □) and saline-treated mice (•, ○) were shown (filled and open symbols showed B10.D2 and DBA/2 stimulation, respectively). *P < 0.001. Error bars, SE.
Figure 2.
 
Proliferation response (A) and cytokine secretion (B) by T cells from corneal transplant recipients. (A) (NACOMe)2- or saline-treated BALB/c mice received B10.D2 corneal allografts. After 14 days, draining LNs were removed, and purified T cells were cultured with B10.D2 or DBA/2 stimulator cells. BALB/c mice, subcutaneously injected with 1 × 107 B10.D2 splenocytes were the positive control. (B) IFN-γ, IL-4, and IL-10 secretion in those culture supernatants were assayed. Positive controls (♦, ⋄). (NACOMe)2- (▪, □) and saline-treated mice (•, ○) were shown (filled and open symbols showed B10.D2 and DBA/2 stimulation, respectively). *P < 0.001. Error bars, SE.
Proliferation response (A) and cytokine secretion (B) by T cells from corneal transplant recipients. (A) (NACOMe)2- or saline-treated BALB/c mice received B10.D2 corneal allografts. After 14 days, draining LNs were removed, and purified T cells were cultured with B10.D2 or DBA/2 stimulator cells. BALB/c mice, subcutaneously injected with 1 × 107 B10.D2 splenocytes were the positive control. (B) IFN-γ, IL-4, and IL-10 secretion in those culture supernatants were assayed. Positive controls (♦, ⋄). (NACOMe)2- (▪, □) and saline-treated mice (•, ○) were shown (filled and open symbols showed B10.D2 and DBA/2 stimulation, respectively). *P < 0.001. Error bars, SE.
View OriginalDownload Slide
Figure 3.
icGSH content of peritoneal Mps and fate of B10.D2 corneal allografts after adoptive transfer of Mps. Peritoneal Mps from (NACOMe)2-treated (A) and saline-treated (B) mice were stained with 10 μM MCB for 30 minutes at 37°C. The fluorescence intensity revealing the amount of icGSH in adherent Mps was monitored by argon-ion laser cytometry and a computer workstation. Yellowish or red staining indicates the abundant presence of icGSH. (C) After IV injection of either (NACOMe)2- (n = 10; ○) or saline-treated (n = 9; ▪) Mps into naive BALB/c hosts, B10.D2 corneal grafts were placed on high-risk graft beds. *P < 0.003.
View OriginalDownload Slide
 
icGSH content of peritoneal Mps and fate of B10.D2 corneal allografts after adoptive transfer of Mps. Peritoneal Mps from (NACOMe)2-treated (A) and saline-treated (B) mice were stained with 10 μM MCB for 30 minutes at 37°C. The fluorescence intensity revealing the amount of icGSH in adherent Mps was monitored by argon-ion laser cytometry and a computer workstation. Yellowish or red staining indicates the abundant presence of icGSH. (C) After IV injection of either (NACOMe)2- (n = 10; ○) or saline-treated (n = 9; ▪) Mps into naive BALB/c hosts, B10.D2 corneal grafts were placed on high-risk graft beds. *P < 0.003.
Figure 3.
 
icGSH content of peritoneal Mps and fate of B10.D2 corneal allografts after adoptive transfer of Mps. Peritoneal Mps from (NACOMe)2-treated (A) and saline-treated (B) mice were stained with 10 μM MCB for 30 minutes at 37°C. The fluorescence intensity revealing the amount of icGSH in adherent Mps was monitored by argon-ion laser cytometry and a computer workstation. Yellowish or red staining indicates the abundant presence of icGSH. (C) After IV injection of either (NACOMe)2- (n = 10; ○) or saline-treated (n = 9; ▪) Mps into naive BALB/c hosts, B10.D2 corneal grafts were placed on high-risk graft beds. *P < 0.003.
icGSH content of peritoneal Mps and fate of B10.D2 corneal allografts after adoptive transfer of Mps. Peritoneal Mps from (NACOMe)2-treated (A) and saline-treated (B) mice were stained with 10 μM MCB for 30 minutes at 37°C. The fluorescence intensity revealing the amount of icGSH in adherent Mps was monitored by argon-ion laser cytometry and a computer workstation. Yellowish or red staining indicates the abundant presence of icGSH. (C) After IV injection of either (NACOMe)2- (n = 10; ○) or saline-treated (n = 9; ▪) Mps into naive BALB/c hosts, B10.D2 corneal grafts were placed on high-risk graft beds. *P < 0.003.
View OriginalDownload Slide
Table 1.
 
View Table
 
Donor-Specific Delayed Hypersensitivity 2 Weeks after Grafting
Table 1.
 
Donor-Specific Delayed Hypersensitivity 2 Weeks after Grafting
Corneal Allograft Treatment Ear Challenge Ear Swelling (μm ± SD) n
B10.D2 (NACOMe)2 B10.D2 38 ± 22 5
B10.D2 Saline B10.D2 110 ± 18* 5
None (NACOMe)2 B10.D2 36 ± 20 5
None Saline B10.D2 30 ± 22 5
 After (NACOMe)2 or saline treatment, one panel of mice received a B10.D2 corneal allograft. Either B10.D2 or DBA/2 splenocytes were ear challenged, and swelling responses were assessed at 2 weeks after transplantation.
*  Response significantly higher than in the control (P < 0.005).
Copyright 2004 The Association for Research in Vision and Ophthalmology, Inc.
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