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* Laboratory of Immunology,
Laboratory of Retinal Cell and Molecular Biology; NEI, NIH, Bethesda, Maryland and
Department of Biomedical Sciences, Ohio University School of Medicine, Athens, Ohio; and
Epigen Consulting, Inc., Arlington, Massachusetts
Correspondence: Rachel R. Caspi, Laboratory of Immunology, NEI, NIH, Bldg. 10, Rm. 10N222, 10 Center Dr., Bethesda, MD 20892. E-mail: rcaspi{at}helix.nih.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-)-induced expression of major histocompatibility complex class I and class II molecules. Here, we investigate the effect of MMI on experimental autoimmune uveoretinitis (EAU) and study its mechanism(s). Treatment of EAU with MMI administered in drinking water inhibited induction of the disease and associated antigen (Ag)-specific proliferation and cytokine production by draining lymph node cells (LNCs). The treatment was protective only if administered during the first but not during the second week after immunization, suggesting an effect on the induction phase of EAU. It is interesting that MMI inhibited disease in IFN- knockout mice, indicating that the in vivo protective effect is IFN--independent. Flow cytometric analysis of draining LNCs extracted 5 days after immunization showed that MMI partly to completely reversed the increase in Mac-1+/class I+/class II+ cells induced by immunization and reduced the proportion of B7-1 and CD40-positive cells, suggesting a deficit in the Ag-presenting cell (APC) population. APC from untreated mice largely restored antigen-specific proliferation of MMI-treated LNCs. We suggest that MMI inhibits EAU at least in part by preventing the recruitment and/or maturation of APC, resulting in reduced generation of Ag-specific T cells.
Key Words: Autoimmune disease • T lymphocytes • immunoregulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mechanism by which MMI inhibits the development of autoimmune diseases is not fully understood. It has been suggested that MMI inhibits antigen (Ag) presentation by inhibiting the expression of major histocompatibility complex (MHC) molecules on the target tissue [3
, 5
, 6
]. In cultured rat FRTL-5 thyrocytes, MMI inhibits transcription of MHC class I and class II genes through inhibition of interferon- (IFN-)-induced class II transactivator [6
7
8
]. However, the exact mechanism underlying immunosuppressive activity of MMI in vivo still remains unclear, as there are multiple factors that influence the expression of MHC class I and class II molecules.
In the present study, we tested the in vivo effects of MMI on the induction of experimental autoimmune uveoretinitis (EAU) in mice. EAU is a CD4+ T helper cell type 1 (Th1)-mediated autoimmune disease model induced by immunization with retinal Ags such as interphotoreceptor retinoid-binding protein (IRBP) [9
]. The effector Th1-like cells produce high IFN- and low interleukin (IL)-4 and IL-5 in response to the Ag and transfer the disease to genetically compatible, naive recipients. Disease pathogenesis involves recognition by these T cells of Ag presented by MHC class II-positive Ag-presenting cells (APC) and a subsequent inflammatory cascade [10
]. We report that MMI suppressed the induction of disease when administered during the afferent stage of EAU but not if given during the expression phase. Protected animals had reduced T cell proliferation and cytokine responses to Ag. The protective effect of MMI was also seen in IFN- knockout (GKO) mice, indicating that it is IFN--independent. We present evidence that inhibition of Ag-specific responses is secondary to reversal of the immunization-induced maturation and/or accumulation of MHC class I+/class II+/Mac-1+ APC in draining lymph nodes (LNs) and can be corrected by addition of APC from untreated animals.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene (GKO) were developed, screened, and back-crossed for eight generations onto the C57BL/6 background by Dalton et al. [11
] and were obtained from Genentech (Nutley, NJ). All animals were housed under conventional conditions, were given water and chow ad libitum, and were used at 2–6 months of age. The use of the animals conformed to institutional guidelines.
Reagents
IRBP was isolated from bovine retinas by concanavalin A (Con A)-Sepharose affinity chromatography and high-performance liquid chromatography as described previously [12
]. MMI, bovine serum albumin (BSA), 
-methyl-D-mannopyranoside (-MMP), Con A, pertussis toxin (PTX), and complete Freund’s adjuvant (CFA) were purchased from Sigma Chemical Co. (St. Louis, MO). Horseradish peroxidase-streptavidin was purchased from Southern Biotechnology Associates (Birmingham, AL). Mycobacterium tuberculosis strain H37RA was purchased from Difco (Detroit, MI). Ab pairs for enzyme-linked immunosorbent assay (ELISA) were purchased from PharMingen (La Jolla, CA) and Southern Biotechnology Associates. Purified murine recombinant IFN- was generously provided by Maurice K. Gately of Hoffman LaRoche (Nutley, NJ).
Treatment with MMI
Starting on the day of immunization, MMI was administered in drinking water (degassed Milli-Q water, 150 ml/bottle, wrapped with aluminum foil) at 2 mg/ml or as indicated. The drug was refreshed every other day, and the water consumption was recorded. In some experiments, the drug was withdrawn on day 8 (afferent treatment, covering the Ag-priming phase) or was started on day 8 (efferent treatment, starting after the priming phase).
Immunization
B10.A mice were immunized subcutaneously in the thighs and base of tail with 50 µg IRBP in 0.2 ml emulsion, 1:1 v/v, with CFA containing 2.5 mg/ml M. tuberculosis and were simultaneously injected intraperitoneally (i.p.) with 0.5 µg PTX in 0.1 ml as an additional adjuvant. In experiments with GKO and C57BL/6 mice, the concentrations of IRBP and PTX were doubled (100 µg and 1 µg, respectively) to achieve the maximum disease-inducing conditions.
Histopathology and EAU grading
Whole eyes were collected and prepared for histopathologic evaluation on day 21. The eyes were immersed for 1 h in 4% phosphate-buffered glutaraldehyde and transferred into 10% phosphate-buffered formaldehyde until processing. Fixed and dehydrated ocular tissue was embedded in methacrylate, and 4- to 6-µm sections were cut through the pupillary-optic nerve plane. Sections were stained by hematoxylin and eosin. An ocular pathologist evaluated the presence or absence of disease in a masked way after examining six sections cut at different levels for each eye. Severity of EAU for each eye was scored on a scale of 0 (no disease)–4 (maximum disease) in half-point increments, according to a semiquantitative system described previously [13
]. Briefly, the minimal criterion to score an eye as positive by histopathology was inflammatory cell infiltration of the ciliary body, choroid, or retina (EAU grade 0.5). Progressively higher grades were assigned for the presence of discrete lesions in the tissue, such as vasculitis, granuloma formation, retinal folding, and/or detachment, photoreceptor damage. The grading system takes into account lesion type, size, and number.
Delayed type hypersensitivity
Two days before the termination of an experiment, mice received 10 µg IRBP in 10 µl intradermally into the pinna of one ear. The other ear was injected similarly but with phosphate-buffered saline (PBS). Ear swelling was measured at the termination of the experiment 48 h later with a spring-loaded micrometer. Delayed type hypersensitivity (DTH) results are expressed as Ag-specific swelling, calculated as the difference between the thickness of the IRBP-injected ear and the PBS-injected ear.
Lymphocyte proliferation
Draining LNs (inguinals and iliacs) were collected at the termination of an experiment and pooled within each group. Triplicate cultures of 5 x 105 cells/0.2 ml/well were stimulated with graded doses in 96-well flat-bottom plates in RPMI 1640 containing 1% naive mouse serum and 20 mg/ml -MMP. The cultures were incubated for 60 h and were pulsed with 3H-thymidine (1.0 µCi/10 µl/well) for the last 18 h. The plates were harvested using a PHD cell harvester (Cambridge Technology, Watertown, MA), and radioisotope incorporation was determined using standard liquid scintillation.
For evaluating the effect on APC function, LN cells (LNCs) of C57BL/6 mice immunized with IRBP (150 µg) + PTX (0.5 µg) and treated with MMI (2 mg/ml) were collected 7 days after immunization. Single-cell suspensions containing 5 x 105 cells/well were made for proliferation assay with or without addition of a normal APC cell (5x105 cells/well) from syngeneic splenocytes.
Determination of cytokine production
Draining LNCs were removed at the termination of an experiment and pooled within each group. The cells were cultured in 96-well flat-bottom plates (1x106 cells/0.2 ml/well) with 50 µg/ml IRBP in RPMI-1640 medium containing 1% fresh-frozen syngeneic mouse serum and 20 mg/ml -MMP. Supernatants were collected for cytokine production analysis after 48 h. IFN-, IL-4, IL-5, IL-6, and IL-10 were measured by ELISA using Ab pairs from PharMingen, as described previously [14
].
Adoptive transfer of EAU
B10.RIII mice were infused with 1.5 x 106 cells from a long-term uveitogenic T cell line that was cultured in the laboratory by alternate cycles of Ag stimulation and IL-2-driven expansion. Eyes were collected from the recipients after 14 days and were evaluated for EAU by histopathology.
Cell staining and flow cytometry
The following mAb were purchased from PharMingen: PE-conjugated anti-B7.1 (16-10A1), anti-CD40 (H1.2F3), anti-B220 (RA3-6B2), anti-CD4 (H129.19), fluorescein isothiocyanate (FITC)-conjugated polyclonal anti-MHC class I, anti-MHC class II, and anti-Mac-1 (M1/70). Single-cell suspensions (5x105 cells/well) consisting of total draining LNCs were incubated with Fc block (PharMingen) for 15 min to block Fc receptors. For two-color fluorescence, cells were double-stained with various PtdEtn- and FITC-conjugated Ab. Following washing, stained cells were resuspended in PBS containing 1% BSA, 0.1% sodium azide, and 0.6 mg/ml propidium iodide (PtdIns, Sigma Chemical Co.). Live cells, excluding PtdIns, were gated and analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA).
Reproducibility and statistical analysis
Experiments were repeated at least twice and usually three or more times. Statistical analysis of EAU scores was by Snedecor and Cochran’s test for linear trend proportions (nonparametric, frequency-based) [15
]. Each mouse (average of both eyes) was treated as one statistical event. Delayed hypersensitivity scores and LNC proliferation were analyzed by independent t-test. Probability values of P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and IL-12 production [16
]. To test if MMI specifically inhibit the Th1 pathway of immunopathogenesis of EAU, primed LNCs from MMI-treated and control groups were collected on day 21 and were stimulated in vitro with IRBP. Cytokines were measured by ELISA in supernatants collected 48 h after stimulation (24 h for IL-2 assay). Type 1 cytokines were assessed by measurement of IFN- and IL-2, whereas type 2 cytokines were measured by IL-4 and IL-5. LNC cultures from MMI-treated mice showed a decreased production of IL-2, IL-5, and IFN- compared with those from controls and no major change in IL-4 (low IL-4 response is typical of mice on the C57BL/6 background; Fig. 2
). Thus, in MMI-treated mice, depressed levels of type 1 cytokines were not accompanied by a Th2 shift when tested 21 days after immunization.
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-independent-deficient mice are susceptible to EAU and develop full-blown pathology [17
]. As prior studies on MMI conducted mostly ex vivo suggested that its effects are mediated through suppression of IFN- resulting in inhibition of the expression of MHC class I and class II molecules [7
, 8
], we investigated whether MMI can ameliorate the induction of EAU in the absence of IFN-. GKO mice and wild type (WT) C57BL/6 mice immunized with IRBP were treated with MMI at 2 mg/ml in drinking water from the day of immunization. As shown in Figure 3
, left, histopathology scores of eyes collected 21 days after immunization revealed that MMI inhibited the induction of EAU equally well in GKO and WT mice. DTH responses in GKO mice were typically increased over those of WT, a phenomenon noted previously by us [16
] as well as by others. Nevertheless, MMI treatment reduced the DTH response in GKO mice to the same extent as it did in WT mice (P<0.02; Fig. 3
, right). These data indicate that contrary to what was expected, the in vivo protective effect of MMI is IFN--independent.
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As shown in Figure 5 , draining LNCs of immunized mice showed up to a 15-fold increase in the total % of cells double-positive for Mac-1 and MHC class I or Mac-1 and MHC class II compared with naïve LN. MMI treatment reduced this immunization-induced increase several-fold in some experiments almost to the level of the naïve LNC population. Combined staining for B cell and T cell markers (B-220 and CD3, respectively) confirmed that the inhibition of MHC class I and class II expression occurred in non-B, non-T cells (data not shown). These data are in agreement with observations of others that MMI inhibits the expression of MHC class I and class II molecules on APC [7 , 8 ].
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MMI inhibits APC function and reduces priming of effector T cells
We next tested whether the inhibition of APC-associated markers in the LN translated to a functional deficit in terms of Ag presentation. To test this hypothesis, we examined whether supplementation of draining LNC from MMI-treated mice with "fresh" APC could reverse the inhibited Ag-specific proliferation. C57BL/6 mice were immunized with a uveitogenic regimen of IRBP and were treated with 2 mg/ml MMI. On day 7, draining LNs were harvested, and single-cell suspensions were made for proliferation assay with or without addition of normal APC (irradiated, naïve splenocytes). Figure 6
shows that addition of exogenous APC was able to largely restore the inhibited proliferation of LNC from MMI-treated mice but did not enhance the response of untreated controls. This restoration of proliferation, however, still does not translate to a similar total number of T cells primed with and without MMI treatment. The LN of MMI-treated mice always yielded half or fewer the number of cells than did LN of controls. Thus, despite a similar proliferation potential on a per-cell basis with the APC defect corrected, MMI-treated mice generate on the whole fewer Ag-specific T cells in the draining LN as a result of immunization.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mechanism(s) by which MMI might exert its immunosuppressive and anti-inflammatory effects in vivo are not completely understood. The therapeutic effect of MMI on Graves’ disease had been presumed to result from its activity as a thyroid peroxidase inhibitor, preventing the iodination of thyroglobulin. However, its observed effect of reducing MHC class I expression has led to the suggestion that this also may contribute to its therapeutic efficacy [18
]. Mozes et al. [19
] demonstrated a role for MHC class I molecules in the generation of experimental SLE by showing that MHC class I-deficient mice immunized with the 16/6Id Ab were resistant to this disease despite generating an anti-16/6Id response. Singer et al. [3
] subsequently reported that MMI reduces class I expression on peripheral blood lymphocytes in vivo and prevented development of clinical manifestations of SLE in 16/6Id-immunized mice. Montani et al. [20
] demonstrated that IFN- induced human leukocyte Ag-DR gene expression in rat FRTL-5 thyroid cells (which have no basal class II gene expression). This was suppressed by MMI in a time- and concentration-dependent manner. On the basis of these and similar studies, it has been proposed that much of the effect of MMI depends on inhibition of IFN--dependent mechanisms.
To directly address the question whether IFN- is necessary for protection in EAU, we used IFN--deficient mice as well as WT mice treated with a neutralizing anti-IFN- Ab. Our previous studies showed IFN- per se is not necessary for development of EAU. Mice genetically deficient in IFN- and WT mice treated with neutralizing anti-IFN- Ab develop EAU of equal or greater severity compared with control mice, and the disease develops within a similar timeframe after immunization [17
, 21
]. MMI inhibited the development of EAU in GKO mice as well as in WT mice treated with anti-IFN- Ab, indicating that protection by MMI uses IFN--independent mechanisms and suggesting that inhibition of IFN- may be an epiphenomenon rather than the cause of protection. This was unexpected in view of the prevailing notion that IFN- and IFN--mediated mechanisms underlie the therapeutic effect of MMI, but it in no way negates a central role for down-regulation of MHC molecule expression as part of the mechanism of action. Flow cytometric analysis of LNC from IFN- KO mice revealed an immunization-induced rise in class II+ cells that was inhibited by MMI, although not quite as dramatically as in the WT (data not shown).
We did not see evidence of skewing the response toward the Th2 pathway as a result of MMI treatment, as there was no elevation of Ag-driven IL-4 and IL-5 production by LNCs in any of the experiments. It is interesting that IL-12p40 production was increased after the treatment of MMI (data not shown). ELISA measurement for IL-12p70 for MMI-treated and control groups, however, was barely detectable (data not shown). IL-12p40 dimers have been shown to compete for the IL-12 receptor binding with the IL-12 heterodimer [22 ]. It is tempting to speculate that some of the effects of MMI might include overproduction of the p40 chain and p40 dimer formation.
Experiments in which the treatment was restricted to only the induction phase or only the expression phase of the disease allowed to dissociate effects on generation of the effector cells from effects on their function. These experiments indicated that most of the effect of MMI is exerted during the early stage. Because of the known inhibitory effects of MMI on class I and class II expression, we hypothesized effects on Ag priming and decided to investigate early events in the draining LN that would be associated with APC maturation and Ag presentation. Flow cytometric analysis confirmed inhibitory effects on class I and class II expression on Mac-1+ APC and in addition, revealed inhibitory effects on the costimulatory molecules B7-1 and CD40, which are directly and indirectly involved in priming of the uveitogenic effector T cells. Thus, it seems likely that MMI prevents homing and/or maturation of Mac-1+ APC into the draining LNs. Our study did not directly address to what extent this reflected inhibition of activation of APC already present in the LN versus inhibition of influx of new APC from the site of immunization. Functional data, showing that supplementation with exogenous APC that did not come in contact with MMI corrected the defect in Ag-specific proliferation of LNC extracted from MMI-treated donors, confirmed the interpretation that APC function in MMI-treated mice is compromised. Priming and differentiation of Ag-specific uveitogenic effector T cells also appeared reduced by other criteria. CD4+ to CD4- T cell ratio in immunized LNs rose from 
1:1 to almost 3:1, compatible with proliferation and/or increased recruitment of CD4+ cells from the circulation, and was brought down almost to naïve LN levels by treatment with MMI (data not shown). Furthermore, LNs of MMI-treated mice were on the whole considerably smaller than parallel LNs of their WT controls. In the aggregate, these data support the interpretation that fewer Ag-specific cells were being generated in the MMI-treated than in the control animal.
Although protection from EAU was best when MMI was given during the Ag-priming stage of EAU pathogenesis, there was a modest but reproducible effect when treatment was instituted late after immunization or when treatment was given in parallel with adoptive transfer of already-generated effectors in the form of a T cell line. As in situ Ag presentation (presumably by APC local to the eye) is important to trigger EAU pathology [23 ], we hypothesize that the effects of MMI in the efferent stage may be attributed to reduced Ag presentation to effector T cells in the target organ. This would then parallel the effect of MMI on established Graves’ disease, where inhibition of aberrant MHC molecule expression was suggested to contribute to its therapeutic effect [18 ]. Although within the relatively short timeframe of our EAU experiments, the effect on the efferent phase was modest, it is conceivable that under conditions of chronic disease, this might translate to a significant, cumulative difference in scores over time.
In summary, the administration of MMI during the afferent phase of EAU induction protects from EAU. The suppressive mechanism appears to involve down-regulation of type 1 and type 2 cellular-immune responses as manifested by the suppression of DTH and the inhibition of Ag-driven T cell proliferation and production of IFN-, IL-4, and IL-5. The suppression of cellular function was modest if the drug was given only during the efferent phase of the disease. The protective action of MMI in vivo is independent of endogenous IFN-. It appears to involve, at least in part, inhibition of APC activation and function and as a consequence, reduced Ag priming. As in chronic uveitis, it is thought that new Ag-specific clones are continuously being primed and added to the effector pool, MMI might be useful in the clinical setting by itself or in combination with other treatment modalities for therapy of uveitis in humans.
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ACKNOWLEDGEMENTS |
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Received January 27, 2002; revised August 20, 2002; accepted August 27, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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