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The Journal of Immunology, 2003, 171: 3010-3018.
Copyright © 2003 by The American Association of Immunologists

Generation and Function of Reactive Oxygen Species in Dendritic Cells During Antigen Presentation 1

Hiroyuki Matsue*, Dale Edelbaum*, David Shalhevet*, Norikatsu Mizumoto*, Chendong Yang*, Mark E. Mummert*, Junichi Oeda{dagger}, Hiroyuki Masayasu{dagger} and Akira Takashima2,*

* Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, TX 75390; and {dagger} Daiichi Pharmaceutical Co., Ltd, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although reactive oxygen species (ROS) have long been considered to play pathogenic roles in various disorders, this classic view is now being challenged by the recent discovery of their physiological roles in cellular signaling. To determine the immunological consequence of pharmacological disruption of endogenous redox regulation, we used a selenium-containing antioxidant compound ebselen known to modulate both thioredoxin and glutaredoxin pathways. Ebselen at 5–20 µM inhibited Con A-induced proliferation and cytokine production by the HDK-1 T cell line as well as the LPS-triggered cytokine production by XS52 dendritic cell (DC) line. Working with the in vitro-reconstituted Ag presentation system composed of bone marrow-derived DC, CD4+ T cells purified from DO11.10 TCR-transgenic mice and OVA peptide (serving as Ag), we observed that 1) both T cells and DC elevate intracellular oxidation states upon Ag-specific interaction; 2) ebselen significantly inhibits ROS production in both populations; and 3) ebselen at 5–20 µM inhibits DC-induced proliferation and cytokine production by T cells as well as T cell-induced cytokine production by DC. Thus, Ag-specific, bidirectional DC-T cell communication can be blocked by interfering with the redox regulation pathways. Allergic contact hypersensitivity responses in BALB/c mice to oxazolone, but not irritant contact hypersensitivity responses to croton oil, were suppressed significantly by postchallenge treatment with oral administrations of ebselen (100 mg/kg per day). These results provide both conceptual and technical frameworks for studying ROS-dependent regulation of DC-T cell communication during Ag presentation and for testing the potential utility of antioxidants for the treatment of immunological disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Reactive oxygen species (ROS) 3 have long been considered as harmful by-products of intrinsic oxygen metabolism or cellular responses to hazardous stimuli. In fact, oxidative stress has been implicated in the pathogenesis of various disorders, including cancers, aging, diabetes, atherosclerosis, chronic inflammation, HIV infection, ischemia-perfusion injury (reviewed in Ref. 1). This classic view, however, appears to require substantial revision. It is now known that ROS at relatively low concentrations serve as essential second messengers mediating cellular responses to many physiological stimuli (reviewed in Ref. 2). For example, T cells generate hydrogen peroxide and/or superoxide anion in response to mitogenic stimuli, such as Con A, superantigens, anti-CD3 mAb, anti-CD28 mAb, and TCR coupling (3, 4, 5, 6, 7, 8). Moreover, treatment of T cells with antioxidant agents or scavenging enzymes results in their unresponsiveness to cytokine receptor signaling and TCR signaling, thereby regulating growth, apoptosis, and gene expression (2, 8, 9, 10). With respect to underlying mechanisms, the thiol/disulfide status of cysteine residues of cytoplasmic proteins is counterregulated by newly generated ROS and by endogenous disulfide reductase systems, and this redox regulation tightly governs transcriptional activities of AP-1 and NF-{kappa}B (11, 12, 13, 14), as well as catalytic potentials of protein tyrosine phosphatases (15, 16, 17, 18, 19).

Dendritic cells (DC) are strategically localized in the environmental interfaces (e.g., skin) and equipped with extremely potent capacities to incorporate and process complex Ag and activate immunologically naive T cells (20). Very recently, Angelini et al. (21) reported that DC generate cysteine after stimulation with LPS or TNF-{alpha} and secrete thioredoxin upon interaction with T cells and that DC-dependent activation of T cells can be inhibited by glutamate (a competitive inhibitor of cystine uptake) or antagonistic mAb against thioredoxin. They interpreted these observations to imply that DC may facilitate the activation of T cells by creating a reducing milieu in the immunological synapse. Ag presentation is not a unidirectional event in which DC simply deliver activation signals to T cells; DC also receive signals back from the responding T cells, thereby undergoing terminal maturation (22, 23, 24). We have reported previously that this bidirectional DC-T cell communication can be intercepted by immunosuppressive reagents, such as dexamethasone (DEX), cyclosporin A (CyA), tacrolimus (FK506), and rapamycin (RAP), unveiling a mechanism by which these drugs suppress adaptive immune responses (25, 26). Thus, it is conceivable that pharmacological disruption of intrinsic redox regulatory pathways in DC and/or T cells may inhibit the DC-T cell communication, thereby suppressing the adaptive immunity.

Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (also called PZ51), is a seleno-organic compound that exhibits multifunctional antioxidant activities (see Fig. 1A for the chemical structure). Although identified originally as a glutathione peroxidase-like compound (27), ebselen is now known to compete with the thioredoxin system far more efficiently by acting as a direct substrate of thioredoxin reductase and a superfast oxidant of thioredoxin (28, 29). Ebselen also inhibits 5- and 15-lipoxygenases, inducible NO synthase, and NADPH oxidase (30, 31, 32). The purposes of this study were to determine the pharmacological impact of this compound on bidirectional DC-T cell interaction and to assess its preclinical efficacy to prevent an adaptive immune response vs an innate inflammatory reaction in skin.



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  		FIGURE 1. Direct impact of ebselen on T cells. A, Chemical structure of ebselen. B, HDK-1 T cells were cultured for 24 h with (•) or without Con A ({circ}) in the presence of ebselen at the indicated concentrations. Culture supernatants were examined for IFN-{gamma} by ELISA. Data are representative of three independent experiments showing the means ± SD from triplicate cultures. Statistically significant differences compared with the Con A-activated samples in the absence of ebselen are indicated with asterisks (*, p < 0.05; **, p < 0.01). C, HDK-1 T cells were cultured for 24 h with IL-2 in the presence of ebselen at the indicated concentrations. Cell viabilities were then measured by trypan blue exclusion. Data are representative of two independent experiments showing the means ± SD from triplicate cultures. Statistically significant differences compared with the control cultures without ebselen are indicated with asterisks (**, p < 0.01). D, HDK-1 T cells were cultured for 24 h with (•) or without IL-2 ({circ}) in the presence of ebselen at the indicated concentrations. Cells were pulsed for the last 16 h with [3H]thymidine. Data are representative of two independent experiments showing the means ± SD from triplicate cultures. Statistically significant differences compared with the control cultures without ebselen are indicated with asterisks (**, p < 0.01). E and F, After a 24-h preincubation with or without 30 µM ebselen, HDK-1 T cells were washed extensively and then examined for IFN-{gamma} production in the presence or absence of Con A (E) and for [3H]thymidine incorporation in the presence or absence of IL-2 (F). Data are representative of two independent experiments showing the means ± SD from triplicate cultures. Statistically significant differences are indicated with asterisks (**, p < 0.01).

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

BALB/c mice (6- to 8-wk-old females) and DO11.10-transgenic mice (33) (6–10 wk old) were bred and maintained in the Animal Research Center facilities at the University of Texas Southwestern Medical Center (Dallas, TX). All of the animal experiments were approved by the Institutional Review Board and conducted according to guidelines of the National Institutes of Health (Bethesda, MD).

Reagents and Abs

The OVA peptide (OVA323–339) was synthesized at the Biopolymers Facility at the University of Texas Southwestern Medical Center. Ebselen was synthesized in-house in Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan) (28). We purchased LPS (Escherichia coli 026:B6) and Con A from Amersham Pharmacia Biotech (Piscataway, NJ), oxazolone (OX) and croton oil (CrO) from Sigma-Aldrich (St. Louis, MO), and polyethylene glycol (PEG; PEG 1500) from Roche (Indianapolis, IN). Recombinant mouse IL-2, IL-4, IL-12, and GM-CSF were purchased from R&D Systems (Minneapolis, MN). The mAb KJ1.26, which recognizes the transgenic TCR complex specific for OVA323–339 peptide, and its isotype-matched control IgG were purchased from Caltag Laboratories (Burlingame, CA). Other Abs were obtained from BD PharMingen (San Diego, CA). None of the reagents, except for LPS, contained detectable amounts of endotoxin as tested by the OCL-100 system (BioWhittaker, Walkersville, MD).

Cell preparations

The XS52 DC line and the HDK-1 T cell clone were maintained as before (25), and their phenotypic and functional features are described elsewhere (23, 24, 34, 35). CD4+ T cells were purified from spleens of DO11.10 mice by two sequential magnetic bead separations as before (26, 36). The resulting T cell preparations containing 95–99% CD4+ cells were used as DO11.10 T cells without further purification. Short-term Th1 or Th2 cultures were generated by stimulating DO11.10 T cells by DC and OVA peptide for 7 days in the presence of IL-12 plus anti-IL-4 mAb or IL-4 plus anti-IL-12 mAb, respectively (37). Bone marrow-derived DC cultures were generated from BALB/c mice in complete RPMI 1640 (38) in the presence of 10 ng/ml GM-CSF as described previously (26, 39, 40). The resulting DC preparations harvested on day 6 or 7 contained >80% CD11c+ cells showing a characteristic phenotype of immature DC (26).

In vitro Ag presentation assays

Freshly isolated DO11.10 T cells (2.5 x 105 cells/ml) were cocultured with gamma-irradiated (1500 rad) bone marrow-derived DC (2.5 x 104 cells/ml) and 2 µg/ml OVA peptide. To test the secondary activation, Th1 and Th2 T cells (5 x 104 cells/ml) were cocultured with gamma-irradiated DC (5 x 103 cells/ml). Cells were pulsed for 8 h with [3H]thymidine (1 µCi/well) and harvested on day 3 for the primary response or on day 2 for the secondary response (26, 38). To test cytokine production, bone marrow DC (4 x 105 cells/ml) and DO11.10 T cells (2 x 106 cells/ml) were cocultured with OVA peptide in the absence of gamma-irradiation. Viabilities of T cells and DC in the cocultures were assessed by measuring propidium iodide (PI) uptake by the KJ1.26+ populations and CD11c+ populations, respectively (26). Cell viability of pure populations, such as XS52 and HDK-1 cell lines, was measured by a trypan blue exclusion assay (24). In some experiments, XS52 DC and HDK-1 T cells were examined for their mitotic potentials by measuring [3H]thymidine uptake in the presence of 10 ng/ml GM-CSF and 200 U/ml IL-2, respectively. These cell lines were also examined for cytokine production after stimulation with 100 ng/ml LPS or 3 µg/ml Con A.

Measurement of the oxidation states in DC and T cells

XS52 DC were stimulated with 100 ng/ml LPS for various periods, and an oxidation-sensitive dye, 5-(and 6-)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA; Molecular Probes, Eugene, OR) was added at 2 µM during the last 15 min of incubation (8). Samples were washed and analyzed by FACS for fluorescence signals within the PI- populations. To study ROS generation during Ag presentation, CM-H2DCFDA was added to the cocultures of bone marrow DC (2.5 x 105 cells/ml) and DO11.10 T cells (5 x 105 cells/ml), and these samples were analyzed by FACS after labeling with PE-conjugated anti-CD11c or KJ1.26 mAb. In some experiments, bone marrow-derived DC preparations were stimulated with LPS as above and examined for CM-H2DCFDA fluorescence signals within the PI-/CD11c+ populations.

Contact hypersensitivity (CH) assays

To test allergic CH responses, BALB/c mice were sensitized by topical application of 1.25% OX on the shaved abdominal skin (day -4) and challenged with 0.5% OX on the right ear or vehicle alone on the left ear (day 0). The swelling responses (the right ear thickness minus the left ear thickness) were examined on days 1 and 2 by a third blinded experimenter using an engineer’s micrometer (38, 41, 42). To test irritant CH, mice received topical application of 1% CrO on the right ear and vehicle alone on the left ear (day 0) and were examined for swelling responses (the right ear thickness minus the left ear thickness) (42).

Ebselen treatments

For in vitro experiments, ebselen was dissolved in DMSO to prepare a stock solution at 6 or 32 mM and added to the cultures at 0–80 µM with the constant DMSO concentration of 0.5%. For in vivo experiments, a 10 mg/ml ebselen solution freshly prepared in 50% DMSO/50% PEG was administered orally at the dose of 100 mg/kg per treatment. Control animals in the placebo group received oral administrations of the vehicle (DMSO/PEG) alone. Ebselen and placebo were administered on 1) days -4 and -3 (postsensitization protocol), 2) days 0 and 1 (postchallenge protocol), or 3) days -4, -3, 0, and 1 (combination protocol).

Statistical analyses

Comparisons between two groups were performed with a two-tailed student’s t test, and more than two groups were compared by ANOVA. Each experiment was repeated at least once to assess reproducibility.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Direct impact of ebselen on mitogen-triggered T cell activation

We first examined the impact of ebselen on mitogen-induced activation of the CD4+ Th1 clone HDK-1. Con A-induced production of IFN-{gamma} was inhibited by ebselen in a dose-dependent fashion, with significant (p < 0.05) effect at 5 µM and almost complete (>95%) inhibition at 20 µM (Fig. 1B). Ebselen affected the viability of HDK-1 T cells, causing 30% reduction at 20–30 µM (Fig. 1C). Moreover, their proliferative response to exogenous IL-2 was abrogated completely by ebselen at 30 µM (Fig. 1D). Importantly, 24-h preincubation of HDK-1 T cells with ebselen at 30 µM did not completely abrogate their responsiveness to Con A (measured by IFN-{gamma} production; Fig. 1E) or to IL-2 (measured by [3H]thymidine uptake; Fig. 1F). Thus, the observed impact of ebselen on T cells did not appear to simply reflect its direct cytotoxicity. In summary, ebselen in a relatively narrow range (5–20 µM) inhibited all tested functional properties of mitogen-activated T cells (i.e., cytokine production, growth, and survival) in a partially reversible manner, corroborating the current notion that ROS act as a second messenger in T cell activation (2).

Generation and function of ROS in LPS-induced DC activation

To gain new insights into the redox regulation during DC activation, we initially used the stable DC line XS52 derived from the mouse epidermis (34). As reported previously (25), this DC line secreted IL-1{beta}, IL-6, IL-12 p40, and TNF-{alpha} in response to LPS stimulation. Ebselen inhibited the production of all cytokines in dose-dependent manners, with significant (p < 0.05) inhibition detectable at 5–20 µM (Fig. 2A). Ebselen showed no detectable cytotoxicity to XS52 DC, except at the highest concentration of 30 µM (Fig. 2B). By contrast, ebselen inhibited their proliferative responses to exogenous GM-CSF at much lower concentrations (1–10 µM; Fig. 2C). Once again, 24-h preincubation with ebselen even at 30 µM did not completely abrogate the potential of XS52 DC to secrete IL-1{beta} upon LPS stimulation (Fig. 2D) or to proliferate in responses to GM-CSF (Fig. 2E). Thus, ebselen at relatively low concentrations (5–20 µM) inhibits LPS-induced cytokine production by XS52 DC without affecting cell viability, with the implication that this DC line may produce ROS in response to LPS stimulation.



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  		FIGURE 2. Direct impact of ebselen on DC. A, XS52 DC were cultured for 24 h with (•) or without LPS ({circ}) in the presence of ebselen at the indicated concentrations. Culture supernatants were examined for the indicated cytokines by ELISA. Data are representative of three independent experiments showing the means ± SD from triplicate cultures. Statistically significant differences compared with the LPS-activated samples in the absence of ebselen are indicated with asterisks (*, p < 0.05; **, p < 0.01). B and C, XS52 DC were cultured for 24 h with GM-CSF in the presence of ebselen at the indicated concentrations and examined for cell viabilities by trypan blue exclusion (B). XS52 DC were cultured for 24 h with (•) or without GM-CSF ({circ}) in the presence of ebselen at the indicated concentrations and examined for [3H]thymidine uptake (C). Data are representative of two independent experiments showing the means ± SD from triplicate cultures. Statistically significant differences compared with the control cultures without ebselen are indicated with asterisks (*, p < 0.05; **, p < 0.01). D and E, After a 24-h preincubation with or without 30 µM ebselen, XS52 DC were washed extensively and then examined for IL-1{beta} production in the presence or absence of LPS (D) and for [3H]thymidine incorporation in the presence or absence of GM-CSF (E). Data are representative of two independent experiments showing the means ± SD from triplicate cultures. Statistically significant differences are indicated with asterisks (**, p < 0.01).

 
To test the above possibility more directly, we measured ROS generation in XS52 DC by using a cell permeant, oxidation-sensitive dye CM-H2DCFDA, which remains nonfluorescent until oxidation by peroxides, peroxynitrite, or hydroxy radical (8). A robust increase in the fluorescence intensity was observed 15 min after exposure of XS52 DC to hydrogen peroxide (serving as a positive control), validating our assay system (Fig. 3A, left panel). Significant, albeit modest, elevation in the fluorescence intensity became detectable at 4 h after LPS stimulation (Fig. 3A, right panel, and B). Importantly, LPS-induced ROS generation in XS52 DC was inhibited by ebselen in a dose-dependent manner, with significant (p < 0.05) suppression at 10 µM and almost complete inhibition at 40 µM (Fig. 3C, •). XS52 DC remained fully viable after a 4-h incubation with ebselen even at 80 µM, again documenting their relative resistance to ebselen-induced cell death. Importantly, LPS stimulation also elevated CM-H2DCFDA fluorescence signals in the CD11c+ populations in bone marrow-derived DC preparations and ebselen at 20 µM inhibited LPS-induced ROS generation substantially (Fig. 3D). Taken together, our observations document a previously unrecognized potential of DC to generate ROS in response to LPS stimulation.



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  		FIGURE 3. LPS-induced ROS generation in DC. A, XS52 DC were incubated for 15 min with CM-H2DCFDA in the presence (filled histograms) or absence (open histograms) of 0.025% H2O2 (left panel). Alternatively, XS52 DC were cultured for 4 h in the presence (filled histograms) or absence of LPS (open histograms), and CM-H2DCFDA was added during the last 15-min incubation period (right panel). The samples were then examined for fluorescence profiles within the PI- populations. B, XS52 DC were cultured for the indicated periods with (•) or without LPS ({circ}), and CM-H2DCFDA was added during the last 15-min incubation periods. ROS production was examined at the indicated time points by measuring the fluorescence intensities within the PI- populations. Data are representative of two independent experiments showing the means ± SD (n = 3) of the mean fluorescence intensities. Statistically significant differences compared with the control cultures without LPS are indicated with asterisks (**, p < 0.01). C, XS52 DC were cultured for 4 h with or without 100 ng/ml LPS in the presence of ebselen at the indicated concentrations. CM-H2DCFDA was added during the last 15-min incubation period. Data are representative of two independent experiments showing the means ± SD (n = 3) of the percent inhibition of LPS-induced ROS production (•) and the percent cell viability measured by PI uptake ({blacktriangleup}). {triangleup}, The baseline cell viability in the absence of LPS and ebselen. Statistically significant differences compared with LPS-activated samples in the absence of ebselen are indicated with asterisks (*, p < 0.05; **, p < 0.01). D, Bone marrow DC were treated for 4 h in the presence (filled histograms) or absence of LPS and/or 20 µM ebselen (open histograms), and CM-H2DCFDA was added during the last 15-min incubation period. The samples were then examined for fluorescence profiles within the CD11c+/PI- populations. E, Bone marrow DC were incubated for 24 h in the presence or absence of LPS (100 ng/ml) and/or ebselen (20 µM) and then examined for surface expression of CD86 and CD40. Data shown are the means ± SD (n = 3) of mean fluorescent intensity in the CD11c+/PI- populations. Statistically significant differences are indicated with asterisks (*, p < 0.05; **, p < 0.01).

 
As an initial step toward understanding the biological consequence of elevated ROS production in DC, we next examined whether ebselen would affect phenotypic changes in DC after LPS stimulation. As expected, LPS treatment caused marked up-regulation of the surface expression of both CD86 and CD40 by the CD11c+ populations in bone marrow DC preparations. Interestingly, ebselen (20 µM) significantly suppressed LPS-dependent CD86 up-regulation, without inhibiting CD40 up-regulation (Fig. 3E). Likewise, in the absence of LPS stimulation, ebselen modestly down-regulated steady-state expression levels of CD86, but not CD40. These observations suggest, although indirectly, that a modest increase in intracellular ROS concentrations may serve as one mechanism for regulating surface expression of CD86.

Generation and function of ROS in DC and T cells during Ag-specific interaction

In the next set of experiments, we sought to determine whether DC (and T cells) would produce ROS during Ag presentation. For this aim, we cocultured bone marrow-derived DC with CD4+ T cells freshly purified from the DO11.10 TCR-transgenic mice in the presence of OVA peptide (serving as a relevant Ag). This system provides an exceptionally powerful tool to study Ag-specific, bidirectional DC-T cell communication because DO11.10 T cells proliferate vigorously and produce various cytokines (IFN-{gamma}, IL-2, and IL-4) only upon stimulation with both DC and Ag, whereas bone marrow DC produce a different set of cytokines (IL-6 and IL-12) only in the presence of both T cells and Ag (26). When CM-H2DCFDA was added in combination with hydrogen peroxide (serving as a positive control) to DC-T cell cocultures in the absence of Ag, strong fluorescence signals were detected at 15 min in both CD11c+ populations (i.e., DC) and KJ1.26+ populations (DO11.10 T cells), validating our ROS detection system (Fig. 4A, left panels). It should be noted that the hydrogen peroxide treatment caused modest apoptosis of DO11.10 T cells, but not of bone marrow DC, as assessed by FITC-annexin V binding (data not shown).



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  		FIGURE 4. ROS production in DC and T cells during Ag presentation. A, Bone marrow DC and DO11.10 T cells were cultured for 2 h in the absence of OVA peptide, and 0.025% H2O2 (closed histograms) or PBS alone (open histograms) was added along with CM-H2DCFDA during the last 15-min culture period (left panels). Alternatively, bone marrow DC and DO11.10 T cells were cultured for 4 or 6 h in the presence (closed histograms) or absence of OVA peptide (open histograms), and CM-H2DCFDA was added during the last 15-min culture period (right panels). The samples were then examined for ROS generation by measuring fluorescence profiles within the CD11c+/PI- populations (DC) and the KJ1.26+/PI- populations (DO11.10 T cells). B, Bone marrow DC and DO11.10 T cells were cultured for the indicated periods in the presence (•) or absence of OVA peptide ({circ}), and CM-H2DCFDA was added during the last 15-min culture periods. The samples were then examined for ROS generation in DC (left) and DO11.10 T cells (right). Data are representative of two independent experiments showing the means ± SD (n = 3) of mean fluorescent intensity. Statistically significant differences compared with the control cultures without OVA peptide are indicated with asterisks (*, p < 0.05; **, p < 0.01). C, Bone marrow DC and DO11.10 T cells were cocultured with OVA peptide at the indicated concentrations, and CM-H2DCFDA was added during the last 15-min culture periods. The samples were then examined for ROS generation in DC at 6 h and DO11.10 T cells at 4 h. Data shown are the means ± SD (n = 3) of mean fluorescent intensity. Statistically significant differences compared with the control cultures without OVA peptide are indicated with asterisks (*, p < 0.05; **, p < 0.01). D, Bone marrow DC and DO11.10 T cells were cocultured with or without OVA peptide in the presence of ebselen at the indicated concentrations of ebselen, and CM-H2DCFDA was added during the last 15-min culture periods. The samples were then examined for ROS generation in DC at 6 h and DO11.10 T cells at 4 h. Data are representative of two independent experiments showing the means ± SD (n = 3) of the percent inhibition of Ag-dependent ROS generation. Statistically significant differences compared with the control cultures with OVA peptide in the absence of ebselen are indicated with asterisks (**, p < 0.01).

 
When CM-H2DCFDA was added to the "complete" cocultures containing all three components, significant fluorescence signals became detectable in both DC and DO11.10 T cells (Fig. 4A, right panels). Interestingly, ROS generation appears to occur with somewhat different time kinetics in DC (4–6 h) vs T cells (2–4 h) during Ag-specific interaction (Fig. 4B, •). CM-H2DCFDA remained mostly nonfluorescent in the "incomplete" cocultures without OVA peptide, indicating Ag specificity (Fig. 4B, {circ}). OVA peptide dose dependency experiments revealed that CM-H2DCFDA fluorescence signals increased in both DC and DO11.10 T cells in an Ag concentration-dependent fashion, further supporting our conclusion that DC and T cells generate ROS during Ag presentation (Fig. 4C). When added to the complete cocultures, ebselen dose-dependently inhibited CM-H2DCFDA oxidation in both DC and T cells, with significant (p < 0.01) inhibition observed at 20 µM for DC and 10 µM for T cells (Fig. 4D).

With respect to the functional outcome, ebselen suppressed Ag-specific, DC-dependent proliferation by DO11.10 T cells in a dose-dependent manner, with significant (p < 0.01) inhibition at 10 µM and almost complete (>95%) inhibition at 15 µM (Fig. 5A). Consistent with our observations with the HDK-1 and XS52 lines, ebselen at these concentrations partially reduced the viability of DO11.10 T cells, but not of bone marrow DC (Fig. 5B). Ebselen also suppressed IFN-{gamma}, IL-2, and IL-4 production by T cells as well as IL-6 and IL-12 production by DC, with significant (p < 0.01) inhibition at 5–10 µM and >80% inhibition at 15–20 µM (Fig. 5C). We interpreted these results to suggest a potent pharmacological activity of ebselen to disrupt bidirectional signaling between DC and T cells during Ag presentation.



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  		FIGURE 5. In vitro effects of ebselen on the primary Ag presentation. A, DO11.10 T cells were cocultured with {gamma}-irradiated bone marrow DC and OVA peptide in the continuous presence of ebselen at the indicated concentrations and examined for [3H]thymidine uptake on day 3. Data are representative of three independent experiments showing the means ± SD from triplicate cultures. Statistically significant differences compared with the control cultures without ebselen are indicated with asterisks (**, p < 0.01). B and C, DO11.10 T cells were cocultured for 24 h with bone marrow DC and OVA peptide in the presence of ebselen at the indicated concentrations. The samples were examined for cell viabilities (PI uptake) within the KJ1.26+ populations (left) and the CD11c+ populations (right) by FACS (B). Culture supernatants were examined for the indicated cytokines by ELISA (C). Data are representative of two independent experiments showing the means ± SD from triplicate cultures. Statistically significant differences compared with the control cultures without ebselen are indicated with asterisks (*, p < 0.05; **, p < 0.01).

 
To study the impact of ebselen on the secondary T cell activation, we generated Th1 cells and Th2 cells from DO11.10 T cells. The resulting T cell populations both proliferated vigorously when restimulated with both bone marrow DC and OVA peptide (Fig. 6A). Ebselen inhibited the proliferation of both Th1 and Th2 cells in dose-dependent manners, with almost complete (>95%) inhibition achieved at 10 µM. Th1 cells secreted relatively large amounts of IFN-{gamma} and IL-2 upon restimulation with DC and OVA peptide, whereas Th2 cells secreted IL-4 (Fig. 6B). Again, ebselen at 10 µM inhibited the secretion of all three cytokines significantly (p < 0.01) and substantially (70–80%). Thus, ebselen prevents not only the primary T cell activation, but also the secondary activation of both Th1 and Th2 cells.



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  		FIGURE 6. In vitro effects of ebselen on the secondary T cell activation. A, Th1-polarized (left panel) or Th2-polarized T cells (right panel) derived from DO11.10 T cells were cultured for 2 days with {gamma}-irradiated bone marrow DC and OVA peptide in the presence of ebselen at the indicated concentrations. Data are representative of three independent experiments showing the means ± SD (n = 3) of [3H]thymidine uptake on day 2. Statistically significant differences compared with the control cultures without ebselen are indicated with asterisks (**, p < 0.01). B, Th1-polarized (top) or Th2-polarized T cells (bottom) derived from DO11.10 T cells were cultured for 24 h with bone marrow DC and OVA peptide in the presence or absence of 10 µM ebselen. Culture supernatants were then examined for the indicated cytokines by ELISA. Data are representative of two independent experiments showing the means ± SD from triplicate cultures. Statistically significant differences are indicated with asterisks (**, p < 0.01).

 
In vivo impact of ebselen on allergic vs irritant CH responses

Ag presentation is the initial and key event for the induction of adaptive immune responses. Thus, our in vitro observations may imply a potential utility of ebselen in the treatment of immunological disease. We tested this concept in CH responses to reactive haptens, a standard animal model of allergic contact dermatitis (38, 41, 42). Mice were sensitized with OX on day -4, challenged with the same hapten on day 0, and examined for ear swelling responses on days 1 and 2. In the first set of experiments, we administered ebselen (100 mg/kg per day) or vehicle alone (placebo group) orally in both the postsensitization phase (days -4 and -3) and the postchallenge phase (days 0 and 1). Ebselen given in this combination protocol significantly (p < 0.001) diminished the extent of ear swelling responses as compared with the placebo control (Fig. 7A). When administered only during the postsensitization phase (days -4 and -3), ebselen showed a significant (p < 0.05), albeit rather limited efficacy (Fig. 7B). The postelicitation administration protocol (days 0 and 1) appeared to be as effective as the combination protocol, producing significant (p < 0.001) and substantial (50–70%) inhibition.



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  		FIGURE 7. In vivo effects of ebselen on the onset of CH responses. A and B, BALB/c mice were sensitized on day -4 by topical application of 1.25% OX onto shaved abdominal skin and challenged on day 0 on the ears. Ebselen ({blacksquare}) or vehicle alone ({square}) were administered orally on days -4, -3, 0, and 1 (A) or during the sensitization phase (days -4 and -3) and/or the elicitation phase (days 0 and 1) (B). Data shown are the ear swelling responses (means ± SEM, n = 10) on days 1 and 2. Statistically significant differences are indicated with asterisks (*, p < 0.05; **, p < 0.01; and ***, p < 0.001). C, BALB/c mice were painted with 1% CrO on the ears on day 0 and examined for ear swelling responses. Ebselen ({blacksquare}) or vehicle alone ({square}) was administered orally on days 0 and 1. Data shown are the ear swelling responses (means ± SEM, n = 10) on days 1 and 2. All data shown are representative of two independent experiments.

 
Because ebselen is known to exert anti-inflammatory activities (43, 44, 45), we next examined its impact on the onset of irritant CH responses, which represent innate inflammatory reactions to chemically induced tissue injury (42). Mice were painted with CrO on day 0 and examined for ear swelling responses on days 1 and 2. Ebselen treatment given during the postelicitation phase (days 0 and 1) failed to significantly suppress irritant CH responses (Fig. 7C). In our pharmacokinetics study in mice, ebselen concentrations in the serum samples reached >15 µM in 8 h after a single oral administration of 100 mg/kg and remained >5 µM for the next 16 h (data not shown). Thus, it seems reasonable to conclude that ebselen in an "immunosuppressive" dose range inhibits T cell-mediated adaptive immune responses in the skin, without significantly affecting innate inflammation responses.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have made two major findings in the present study. First, we have observed that DC generate ROS in response to LPS stimulation and during Ag-specific interaction with T cells. T cells have been reported to generate ROS in response to artificial mitogens, and DC are known to express inducible NO synthase after treatment with LPS or cytokines (e.g., IFN-{gamma}) (3, 4, 5, 6, 7, 8, 46). To the best of our knowledge, however, this is the first report documenting ROS generation in DC during Ag presentation. Second, we have identified previously unrecognized potentials of ebselen to prevent LPS-induced DC activation, disrupt bidirectional DC-T cell activation processes during Ag presentation, and suppress the expression of adaptive immune responses in living animals. Although ebselen is known to inhibit anti-CD3 mAb-induced ROS production in T cells (8) and suppress inflammatory reactions (43, 44, 45), its pharmacological impact on DC or its preclinical efficacy against adaptive immune responses has not been reported in the literature. Both findings imply that endogenously produced ROS in DC and T cells act as second messengers in altering their functions during Ag presentation (2).

Our primary purpose was to determine the immunological consequence of pharmacological disruption of the intrinsic redox regulatory pathways. In this regard, ebselen at 10–20 µM efficiently inhibited 1) Con A-triggered cytokine production and IL-2-dependent proliferation by the HDK1 T cell line, 2) LPS-induced cytokine production and GM-CSF-dependent proliferation by the XS52 DC line, 3) LPS-induced CD86 up-regulation in bone marrow-derived DC, 4) DC-dependent proliferation and cytokine production by freshly isolated DO11.10 T cells and by primed Th1 and Th2 populations, and 5) T cell-dependent cytokine production by bone marrow DC. Ebselen in the same concentration range also inhibited ROS generation in 6) XS52 DC line and bone marrow DC after LPS stimulation and 7) bone marrow DC and HDK-1 T cells during Ag-specific interaction. Thus, our observations support the current concept that ROS at low concentrations play physiological roles in cellular signaling via regulating the redox states of transcription factors (e.g., NF-{kappa}B and AP-1) and various enzymes involved in signal transduction (e.g., protein tyrosine phosphatases, receptor tyrosine kinases, mitogen-activated protein kinases, protein kinase C) (1, 11, 12, 13, 14, 15, 16, 17, 18, 19). With respect to pharmacological mechanisms of action, ebselen has been reported to inhibit multiple redox regulatory pathways (27, 30, 31, 32), presumably via altering its redox states by forming ebselen selenol and ebselen diselenide (28, 29). Some of our observations, especially those with LPS-stimulated DC, may be explained by the known activity of ebselen to inhibit LPS-dependent nuclear translocation of NF-{kappa}B p50 (47). It is also conceivable that ebselen may inhibit Ag-specific DC-T cell interaction by interrupting with cysteine/thioredoxin-mediated, unidirectional communication from DC to T cells, an intriguing mechanism recently proposed by Angelini et al. (21). Our data along with recent reports by others form the framework for our understanding of functions of redox regulation in the immune system.

Reflecting its potent antioxidant activities, preclinical efficacy of ebselen has been evaluated in a variety of disease models. For example, ebselen has been reported to prevent LPS-induced pulmonary inflammation and adjuvant-induced arthritis in rats by inhibiting neutrophil recruitment (44, 45). Ebselen inhibited phorbol ester-induced oxidative and inflammatory changes in mouse skin, suggesting its potential utility as a chemopreventative agent against cancer development (48). Ebselen prevented the onset of Con A-induced liver injury in mice; interestingly, this protection was associated with reduced TNF-{alpha} and elevated IL-10 serum levels (49). Finally, neuroprotective activities of ebselen have been observed in animal models of focal cerebral ischemia (50, 51, 52), as well as in human patients with acute ischemic stroke or aneurysmal subarachnoid hemorrhage (53, 54). These observations illustrate striking diversity of physiological and pathological roles played by ROS and multifunctionality of ebselen. The present study has revealed a new physiological role of ROS (i.e., regulation of Ag presentation) and a new disease target of ebselen (i.e., adaptive immune disorders).

We have previously studied the pharmacological effects of CyA, RAP, and DEX on DC and T cells in the same in vitro-reconstituted Ag presentation systems (26). These conventional immunosuppressants inhibited certain aspects of Ag presentation rather selectively in relatively low- and broad-dose ranges (i.e., 10-8-10-6 M for CyA, 10-10–10-8 M for RAP, and 10-9–10-6 M for DEX). For example, RAP in the above dose range selectively inhibited DC-dependent T cell proliferation without affecting cytokine production by T cells or DC. By contrast, ebselen inhibited all tested changes in a relatively high and extremely narrow concentration range. The observed difference among the tested agents most likely reflects the difference in their mechanisms of action. The CyA-cyclophilin A complex binds to calcineurin, thereby inhibiting TCR-mediated signal transduction pathways (e.g., NFAT-dependent transcription of cytokine genes) (55, 56, 57). The RAP-FKBP12 complex binds to the mammalian target of RAP, thereby inhibiting protein synthesis at the translational level, cytokine receptor signaling, and CD28-mediated NF-{kappa}B activation (57, 58, 59, 60). DEX directly inhibits gene transcription by competing for DNA binding sites in the promoter regions or cross-coupling with many transcription factors (e.g., AP-1 and NF-{kappa}B) (61, 62, 63). As described above, ebselen inhibits various signaling cascades by affecting transcription factors and enzymes involved in signal transduction. Thus, it is tempting to speculate that the combination of ebselen with one or more immunosuppressants may exert synergistic augmentation of immunosuppressive properties. Conversely, it is possible that ebselen (and perhaps other antioxidants) may produce unexpected immunological adverse effects when unconsciously coadministered with an immunosuppressive agent.

In summary, we have demonstrated ROS generation in both DC and T cells during Ag presentation and pharmacological activities of ebselen to inhibit Ag-specific DC-T cell interaction in vitro and allergic CH responses in animals. It is equally important to note several key questions that remain to be clarified. First, one must determine molecular identities and quantities of the ROS being generated in DC and T cells. Second, pharmacological mechanisms by which ebselen modulates the DC-T cell communication must be elucidated at molecular levels. It is also crucial to study underlying mechanisms for the observed difference between DC and T cells in their susceptibility to ebselen-mediated cytotoxicity. Finally, relative safety and preclinical efficacy of ebselen treatment must be tested in different models of immunological disorders. Nevertheless, we believe that the present study provides both conceptual and technical frameworks for studying the roles played by endogenously produced ROS in DC and T cells in their Ag-specific communication and, thus, the initiation of adaptive immunity.


   Acknowledgments
 
We thank Lesa Ellinger for her technical support and Pat Adcock for her secretarial assistance.


   Footnotes
 
1 This work was supported by research grants from the National Institutes of Health (RO1-AI46755, RO1-AR35068, RO1-AR43777, and RO1-AI43232) and from Daiichi Pharmaceutical Co., Ltd. Back

2 Address correspondence and reprint requests to Dr. Akira Takashima, Department of Dermatology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9069. E-mail address: akira.takashima{at}utsouthwestern.edu Back

3 Abbreviations used in this paper: ROS, reactive oxygen species; DC, dendritic cell; DEX, dexamethasone; CyA, cyclosporin A; RAP, rapamycin; OX, oxazolone; CrO, croton oil; PEG, polyethylene glycol; PI, propidium iodide; CM-H2DCFDA, 5-(and 6-)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester; CH, contact hypersensitivity. Back

Received for publication April 11, 2003. Accepted for publication July 17, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Droge, W.. 2002. Free radicals in the physiological control of cell function. Physiol. Rev. 82:47.[Abstract/Free Full Text]
  2. Reth, M.. 2002. Hydrogen peroxide as second messenger in lymphocyte activation. Nat. Immunol. 3:1129.[Medline]
  3. Sekkat, C., J. Dornand, M. Gerber. 1988. Oxidative phenomena are implicated in human T-cell stimulation. Immunology 63:431.[Medline]
  4. Williams, M. S., P. A. Henkart. 1996. Role of reactive oxygen intermediates in TCR-induced death of T cell blasts and hybridomas. J. Immunol. 157:2395.[Abstract]
  5. Los, M., H. Schenk, K. Hexel, P. A. Baeuerle, W. Droge, K. Schulze-Osthoff. 1995. IL-2 gene expression and NF-{kappa}B activation through CD28 requires reactive oxygen production by 5-lipoxygenase. EMBO J. 14:3731.[Medline]
  6. Weber, G. F., S. Abromson-Leeman, H. Cantor. 1995. A signaling pathway coupled to T cell receptor ligation by MMTV superantigen leading to transient activation and programmed cell death. Immunity 2:363.[Medline]
  7. Hildeman, D. A., T. Mitchell, T. K. Teague, P. Henson, B. J. Day, J. Kappler, P. C. Marrack. 1999. Reactive oxygen species regulate activation-induced T cell apoptosis. Immunity 10:735.[Medline]
  8. Devadas, S., L. Zaritskaya, S. G. Rhee, L. Oberley, M. S. Williams. 2002. Discrete generation of superoxide and hydrogen peroxide by T cell receptor stimulation: selective regulation of mitogen-activated protein kinase activation and Fas ligand expression. J. Exp. Med. 195:59.[Abstract/Free Full Text]
  9. Iwata, S., T. Hori, N. Sato, Y. Ueda-Taniguchi, T. Yamabe, H. Nakamura, H. Masutani, J. Yodoi. 1994. Thiol-mediated redox regulation of lymphocyte proliferation: possible involvement of adult T cell leukemia-derived factor and glutathione in transferrin receptor expression. J. Immunol. 152:5633.[Abstract]
  10. Nakashima, I., H. Suzuki, M. Kato, A. A. Akhand. 2002. Redox control of T-cell death. Antioxid. Redox Signal. 4:353.[Medline]
  11. Abate, C., L. Patel, F. J. Rauscher, III, T. Curran. 1990. Redox regulation of Fos and Jun DNA-binding activity in vitro. Science 249:1157.[Abstract/Free Full Text]
  12. Hirota, K., M. Matsui, S. Iwata, A. Nishiyama, K. Mori, J. Yodoi. 1997. AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. Proc. Natl. Acad. Sci. USA 94:3633.[Abstract/Free Full Text]
  13. Hayashi, T., Y. Ueno, T. Okamoto. 1993. Oxidoreductive regulation of nuclear factor kB: involvement of a cellular reducing catalyst thioredoxin. J. Biol. Chem. 268:11380.[Abstract/Free Full Text]
  14. Schenk, H., M. Klein, W. Erdbrugger, W. Droge, K. Schulze-Osthoff. 1994. Distinct effects of thioredoxin and antioxidants on the activation of transcription factors NF-{kappa}B and AP-1. Proc. Natl. Acad. Sci. USA 91:1672.[Abstract/Free Full Text]
  15. Caselli, A., R. Marzocchini, G. Camici, G. Manao, G. Moneti, G. Pieraccini, G. Ramponi. 1998. The inactivation mechanism of low molecular weight phosphotyrosine-protein phosphatase by H2O2. J. Biol. Chem. 273:32554.[Abstract/Free Full Text]
  16. Xu, D., I. I. Rovira, T. Finkel. 2002. Oxidants painting the cysteine chapel: redox regulation of PTPs. Dev. Cell 2:251.[Medline]
  17. Barford, D., B. G. Neel. 1998. Revealing mechanisms for SH2 domain mediated regulation of the protein tyrosine phosphatase SHP-2. Structure 6:249.[Medline]
  18. Meng, T. C., T. Fukada, N. K. Tonks. 2002. Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. Mol. Cell. 9:387.[Medline]
  19. Schieven, G. L., J. M. Kirihara, D. E. Myers, J. A. Ledbetter, F. M. Uckun. 1993. Reactive oxygen intermediates activate NF-{kappa}B in a tyrosine kinase-dependent mechanism and in combination with vanadate activate the p56lck and p59fyn tyrosine kinases in human lymphocytes. Blood 82:1212.[Abstract/Free Full Text]
  20. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245.[Medline]
  21. Angelini, G., S. Gardella, M. Ardy, M. R. Ciriolo, G. Filomeni, G. Di Trapani, F. Clarke, R. Sitia, A. Rubartelli. 2002. Antigen-presenting dendritic cells provide the reducing extracellular microenvironment required for T lymphocyte activation. Proc. Natl. Acad. Sci. USA 99:1491.[Abstract/Free Full Text]
  22. Kitajima, T., K. Ariizumi, P. R. Bergstresser, A. Takashima. 1995. T cell-dependent loss of proliferative responsiveness to colony-stimulating factor-1 by a murine epidermal-derived dendritic cell line, XS52. J. Immunol. 155:5190.[Abstract]
  23. Kitajima, T., K. Ariizumi, M. Mohamadzadeh, D. Edelbaum, P. R. Bergstresser, A. Takashima. 1995. T cell-dependent secretion of IL-1{beta} by a dendritic cell line (XS52) derived from murine epidermis. J. Immunol. 155:3794.[Abstract]
  24. Kitajima, T., G. Caceres-Dittmar, F. J. Tapia, J. Jester, P. R. Bergstresser, A. Takashima. 1996. T cell-mediated terminal maturation of dendritic cells: loss of adhesive and phagocytotic capacities. J. Immunol. 157:2340.[Abstract]
  25. Kitajima, T., K. Ariizumi, P. R. Bergstresser, A. Takashima. 1996. A novel mechanism of glucocorticoid-induced immune suppression: the inhibition of T cell-mediated terminal maturation of a murine dendritic cell line. J. Clin. Invest. 98:142.[Medline]
  26. Matsue, H., C. Yang, K. Matsue, D. Edelbaum, M. E. Mummert, A. Takashima. 2002. Contrasting impacts of immunosuppressive agents (rapamycin, FK506, cyclosporin A, and dexamethasone) on bidirectional dendritic cell-T cell interaction during antigen presentation. J. Immunol. 169:3555.[Abstract/Free Full Text]
  27. Muller, A., E. Cadenas, P. Graf, H. Sies. 1984. A novel biologically active seleno-organic compound-I: glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ51 (ebselen). Biochem. Pharmacol. 33:3235.[Medline]
  28. Zhao, R., H. Masayasu, A. Holmgren. 2002. Ebselen: a substrate for human thioredoxin reductase strongly stimulating its hydroperoxide reductase activity and a superfast thioredoxin oxidant. Proc. Natl. Acad. Sci. USA 99:8579.[Abstract/Free Full Text]
  29. Zhao, R., A. Holmgren. 2002. A novel antioxidant mechanism of ebselen involving ebselen diselenide, a substrate of mammalian thioredoxin and thioredoxin reductase. J. Biol. Chem. 277:39456.[Abstract/Free Full Text]
  30. Schewe, C., T. Schewe, A. Wendel. 1994. Strong inhibition of mammalian lipoxygenases by the antiinflammatory seleno-organic compound ebselen in the absence of glutathione. Biochem. Pharmacol. 48:65.[Medline]
  31. Hattori, R., R. Inoue, K. Sase, H. Eizawa, K. Kosuga, T. Aoyama, H. Masayasu, C. Kawai, S. Sasayama, Y. Yui. 1994. Preferential inhibition of inducible nitric oxide synthase by ebselen. Eur. J. Pharmacol. 267:R1.[Medline]
  32. Cotgreave, I. A., S. K. Duddy, G. E. Kass, D. Thompson, P. Moldeus. 1989. Studies on the anti-inflammatory activity of ebselen: ebselen interferes with granulocyte oxidative burst by dual inhibition of NADPH oxidase and protein kinase C. Biochem. Pharmacol. 38:649.[Medline]
  33. Murphy, K. M., A. B. Heimberger, D. Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+CD8+TRClow thymocytes in vivo. Science 250:1720.[Abstract/Free Full Text]
  34. Xu, S., K. Ariizumi, G. Caceres-Dittmar, D. Edelbaum, K. Hashimoto, P. R. Bergstresser, A. Takashima. 1995. Successive generation of antigen-presenting, dendritic cell lines from murine epidermis. J. Immunol. 154:2697.[Abstract]
  35. Takashima, A., D. Edelbaum, T. Kitajima, R. K. Shadduck, G. L. Gilmore, S. Xu, R. S. Taylor, P. R. Bergstresser, K. Ariizumi. 1995. Colony-stimulating factor-1 secreted by fibroblasts promotes the growth of dendritic cell lines (XS series) derived from murine epidermis. J. Immunol. 154:5128.[Abstract]
  36. Mummert, M. E., D. Mummert, D. Edelbaum, F. Hui, H. Matsue, A. Takashima. 2002. Synthesis and surface expression of hyaluronan by dendritic cells and its potential role in antigen presentation. J. Immunol. 169:4322.[Abstract/Free Full Text]
  37. Randolph, D. A., C. J. L. Carruthers, S. J. Szabo, K. M. Murphy, D. D. Chaplin. 1999. Modulation of airway inflammation by passive transfer of allergen-specific Th1 and Th2 cells in a mouse model of asthma. J. Immunol. 162:2375.[Abstract/Free Full Text]
  38. Matsue, H., K. Matsue, M. Walters, K. Okumura, H. Yagita, A. Takashima. 1999. Induction of antigen-specific immunosuppression by CD95L cDNA-transfected "killer" dendritic cells. Nat. Med. 5:930.[Medline]
  39. Kumamoto, T., E. K. Huang, H. J. Paek, A. Morita, H. Matsue, R. F. Valentini, A. Takashima. 2002. Induction of tumor-specific protective immunity by in situ Langerhans cell vaccine. Nat. Biotechnol. 20:64.[Medline]
  40. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, R. M. Steinman. 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176:1693.[Abstract/Free Full Text]
  41. Mummert, M. E., M. Mohamadzadeh, D. I. Mummert, N. Mizumoto, A. Takashima. 2000. Development of a peptide inhibitor of hyaluronan-mediated leukocyte trafficking. J. Exp. Med. 192:769.[Abstract/Free Full Text]
  42. Mizumoto, N., T. Kumamoto, S. C. Robson, J. Sévigny, H. Matsue, K. Enjyoji, A. Takashima. 2002. CD39 is the dominant Langerhans cell-associated ecto-NTPDase: modulatory roles in inflammation and immune responsiveness. Nat. Med. 8:358.[Medline]
  43. Gao, J. X., A. C. Issekutz. 1993. The effect of ebselen on polymorphonuclear leukocyte and lymphocyte migration to inflammatory reactions in rats. Immunopharmacology 25:239.[Medline]
  44. Gao, J. X., A. C. Issekutz. 1993. The effect of ebselen on polymorphonuclear leukocyte migration to joints in rats with adjuvant arthritis. Int. J. Immunopharmacol. 15:793.[Medline]
  45. Haddad, E., K. McCluskie, M. A. Birrell, D. Dabrowski, M. Pecoraro, S. Underwood, B. Chen, G. T. De Sanctis, S. E. Webber, M. L. Foster, M. G. Belvisi. 2002. Differential effects of ebselen on neutrophil recruitment, chemokine, and inflammatory mediator expression in a rat model of lipopolysaccharide-induced pulmonary inflammation. J. Immunol. 169:974.[Abstract/Free Full Text]
  46. Bogdan, C.. 2001. Nitric oxide and the immune response. Nat. Immunol. 2:907.[Medline]
  47. Shimohashi, N., M. Nakamuta, K. Uchimura, R. Sugimoto, H. Iwamoto, M. Enjoji, H. Nawata. 2000. Selenoorganic compound, ebselen, inhibits nitric oxide and tumor necrosis factor-alpha production by the modulation of Jun-N-terminal kinase and the NF-{kappa}B signaling pathway in rat Kupffer cells. J. Cell. Biochem. 78:595.[Medline]
  48. Nakamura, Y., Q. Feng, T. Kumagai, K. Torikai, H. Ohigashi, T. Osawa, N. Noguchi, E. Niki, K. Uchida. 2002. Ebselen, a glutathione peroxidase mimetic seleno-organic compound, as a multifunctional antioxidant. Implication for inflammation-associated carcinogenesis. J. Biol. Chem. 277:2687.[Abstract/Free Full Text]
  49. Tiegs, G., S. Kusters, G. Kunstle, H. Hentze, A. K. Kiemer, A. Wendel. 1998. Ebselen protects mice against T cell-dependent, TNF-mediated apoptotic liver injury. J. Pharmacol. Exp. Ther. 287:1098.[Abstract/Free Full Text]
  50. Takasago, T., E. E. Peters, D. I. Graham, H. Masayasu, I. M. Macrae. 1997. Neuroprotective efficacy of ebselen, an anti-oxidant with anti-inflammatory actions, in a rodent model of permanent middle cerebral artery occlusion. Br. J. Pharmacol. 122:1251.[Medline]
  51. Namura, S., I. Nagata, S. Takami, H. Masayasu, H. Kikuchi. 2001. Ebselen reduces cytochrome c release from mitochondria and subsequent DNA fragmentation after transient focal cerebral ischemia in mice. Stroke 32:1906.[Abstract/Free Full Text]
  52. Imai, H., H. Masayasu, D. Dewar, D. I. Graham, I. M. Macrae. 2001. Ebselen protects both gray and white matter in a rodent model of focal cerebral ischemia. Stroke 32:2149.[Abstract/Free Full Text]
  53. Yamaguchi, T., K. Sano, K. Takakura, I. Saito, Y. Shinohara, T. Asano, H. Yasuhara. 1998. Ebselen in acute ischemic stroke: a placebo-controlled, double-blind clinical trial. Ebselen Study Group. Stroke 29:12.[Abstract/Free Full Text]
  54. Saito, I., T. Asano, K. Sano, K. Takakura, H. Abe, T. Yoshimoto, H. Kikuchi, T. Ohta, S. Ishibashi. 1998. Neuroprotective effect of an antioxidant, ebselen, in patients with delayed neurological deficits after aneurysmal subarachnoid hemorrhage. Neurosurgery 42:269.[Medline]
  55. Kiani, A., A. Rao, J. Aramburu. 2000. Manipulating immune responses with immunosuppressive agents that target NFAT. Immunity 12:359.[Medline]
  56. Crabtree, G. R.. 1999. Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT. Cell 96:611.[Medline]
  57. Brazelton, T. R., R. E. Morris. 1996. Molecular mechanisms of action of new xenobiotic immunosuppressive drugs: tacrolimus (FK506), sirolimus (rapamycin), mycophenolate mofetil and leflunomide. Curr. Opin. Immunol. 8:710.[Medline]
  58. Sehgal, S. N.. 1998. Rapamune (RAPA, rapamycin, sirolimus): mechanism of action immunosuppressive effect results from blockade of signal transduction and inhibition of cell cycle progression. Clin. Biochem. 31:335.[Medline]
  59. Abraham, R. T., G. J. Wiederrecht. 1996. Immunopharmacology of rapamycin. Annu. Rev. Immunol. 14:483.[Medline]
  60. Abraham, R. T.. 1998. Mammalian target of rapamycin: immunosuppressive drugs uncover a novel pathway of cytokine receptor signaling. Curr. Opin. Immunol. 10:330.[Medline]
  61. Akerblom, I. E., E. P. Slater, M. Beato, J. D. Baxter, P. L. Mellon. 1988. Negative regulation by glucocorticoids through interference with a cAMP responsive enhancer. Science 241:350.[Abstract/Free Full Text]
  62. Auphan, N., J. A. DiDonato, C. Rosette, A. Helmberg, M. Karin. 1995. Immunosuppression by glucocorticoids: inhibition of NF-{kappa}B activity through induction of I{kappa}B synthesis. Science 270:286.[Abstract/Free Full Text]
  63. Scheinman, R. I., A. Gualberto, C. M. Jewell, J. A. Cidlowski, A. S. Baldwin, Jr. 1995. Characterization of mechanisms involved in transrepression of NF-{kappa}B by activated glucocorticoid receptors. Mol. Cell. Biol. 15:943.[Abstract]

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