IL-10 has a protective role in experimental autoimmune uveoretinitis

International Immunology, Vol. 10, No. 6, pp. 807–814

© 1998 Oxford University Press

IL-10 has a protective role in experimental autoimmune uveoretinitis Luiz V. Rizzo, Hui Xu, Chi-Chao Chan, Barbara Wiggert1 and Rachel R. Caspi Laboratory of Immunology and 1Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, 10 Center Drive MSC 1857, Bethesda, MD 20892-5897, USA

Keywords: experimental autoimmune uveoretinitis, IL-10

Abstract The role of IL-10 in the regulation of ocular autoimmune disease was studied in experimental autoimmune uveoretinitis (EAU) elicited in mice by immunization with the retinal antigen interphotoreceptor retinoid binding protein. IL-10-deficient mice were susceptible to EAU, indicating that pathogenesis can occur without presence of IL-10. Treatment of normal mice with IL-10 for 5 days after uveitogenic immunization ameliorated subsequent EAU scores, and downregulated antigen-specific production of tumor necrosis factor-α and IFN-γ. A concomitant treatment with IL-4 further reduced disease, and resulted in emergence of antigen-specific IL-4 and IL-10 production, as well as in enhancement of the IgG1 antibody isotype. IL-4 by itself was not protective. Only IL-10, but not IL-4, was able to inhibit the function of differentiated uveitogenic T cells in culture. Expression of mRNA for Th1 and Th2 cytokines in the eye during the course of EAU showed that while a Th1 pattern predominated early, IL-10 mRNA expression coincided with down-regulation of the Th1 response and resolution of EAU. Systemic neutralization of IL-10 during the expression phase of EAU resulted in elevated disease scores. Our results suggest that endogenous IL-10 limits expression of EAU and may play a role in the natural resolution of disease. The data further suggest that exogenous IL-10 may be useful in therapeutic control of autoimmune uveitis. While IL-10 by itself is sufficient to suppress Th1 effector development and function, a concomitant administration of IL-4 is required to shift the autoimmune response towards a non-pathogenic Th2 pathway. Introduction Experimental autoimmune uveoretinitis (EAU) is a prototypic tissue-specific autoimmune disease whose target is the neural retina. EAU can be induced in susceptible rodent strains by immunization with retinal proteins such as the interphotoreceptor retinoid binding protein (IRBP) (1,2). EAU in animals is considered to represent a number of sight-threatening human inflammatory eye diseases of a presumed autoimmune nature, that exhibit strikingly similar pathology to EAU and can be accompanied by detectable immunological responses to ocular antigens (1,3). Our previous studies have shown that a Th1 response underlies the pathogenesis as well as the genetic susceptibility to EAU: susceptible mouse and rat strains are dominant Th1 responders to the uveitogenic retinal antigen, and uveitogenic effector T cells display a Th1-like cytokine profile (4–6). We and others have shown that certain immunological manipulations that resulted in up-regulation of anti-inflammatory and Th2 cytokine responses, such as induction of oral tolerance or

anterior chamber-associated immune deviation, have afforded protection from EAU (7–9). IL-10 is an anti-inflammatory cytokine that was shown to suppress IFN-γ production, inhibit the Th1 response and promote the Th2 response (10). A number of studies described beneficial effects of IL-10 administration in different models of cell-mediated autoimmunity (11–16). Others, however, reported harmful effects of IL-10 in the same models (17–20), suggesting that regulation of cellular responses by IL-10 is complex and may be influenced by many factors. In the present study, we investigated the effect of IL-10 on natural and therapeutic regulation of EAU in the mouse model. Our data show that administration of exogenous IL-10 can ameliorate EAU and that IL-10 synergizes with IL-4 in this effect. IL-10 is able to inhibit proliferation and IFN-γ production by mature uveitogenic effector T cells, and neutralization of endogenous IL-10 in vivo during expression of EAU has an exacerbating effect on disease. Furthermore, spontaneous

Correspondence to: R. R. Caspi Transmitting editor: R. L. Coffman

Received 5 December 1997, accepted 20 February 1998

808 IL-10 protects from EAU resolution of EAU is accompanied by up-regulation of IL-10 message in the eye and by down-regulation of message for Th1-type cytokines. These results support the interpretation that endogenous IL-10 has a role in the natural control and recovery from EAU, and that administration of exogenous IL-10 may have therapeutic potential in inflammatory and autoimmune eye disease.

received three doses of 10,000 U of IL-4 per day and/or three doses of 1000 U (or 330.1 µg, depending on the preparation) of IL-10 per day. Anti-IL-10 treatment was with the neutralizing rat anti-mouse IL-10 mAbs (SX1, SX2 and 2JA) at 250 µg i.p, every other day, starting on day 14 and continued through day 20 after immunization. Controls were treated with an equivalent dose of purified rat Ig (Sigma). Eyes were collected on day 21.

Methods

Assay of antigen-specific responses of a uveitogenic effector T cell line

Animals B10.A female mice (6–8 weeks old) were purchased from the National Cancer Institute Research Facility (Frederick, MD). C57BL/6 mice (6–12 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME) or bred in-house at the NIH 10A animal facility. Mice were kept in microisolator cages under specific pathogen-free conditions, and were handled in compliance with NIH and NEI guidelines for animal use.

Reagents and cytokines IRBP was isolated from bovine retinas as described previously, by concanavalin A (Con A)–Sepharose affinity chromatography and fast performance liquid chromatography (21). IRBP preparations were aliquoted and stored at –70°C. BSA, α-methyl-mannoside, Con A, pertussis toxin (PTX) and complete Freund’s adjuvant (CFA) were purchased from Sigma (St Louis, MO), horseradish peroxidase (HRP)–streptavidin was from Southern Biotechnologies Associates (Birmingham, AL). Mycobacterium tuberculosis strain H37RA were from Difco (Detroit, MI). Recombinant murine IL-4, tumor necrosis factor (TNF)-α and IFN-γ were obtained from PharMingen (San Diego, CA) or from R & D Systems (Minneapolis, MN). Recombinant murine IL-10 and IL-4 were a generous gift from Dr Paulo Vieira and Dr Kevin Moore from DNAX Research Institute (Palo Alto, CA) and from Dr Satwant Narula, Shering Plough (Nutley, NJ). Anti-IL-10 mAb treatment was with a 1:1:1 mixture of the monoclonals SX1, SX2 and 2JA (DNAX) that had been affinity purified from ascites fluid as previously described (22).

Immunization B10.A mice were immunized s.c. with 50–100 µg of IRBP in 0.2 ml emulsion 1/1 v/v with CFA that had been supplemented with M. tuberculosis to the final concentration of 2.5 mg/ml and were given 1 µg PTX in 0.1 ml i.p. as an additional adjuvant. IL-10-deficient animals on the C57BL/6 background and their wild-type littermates were immunized with a modified protocol of 100–150 µg IRBP in CFA that contained only 1.0 mg/ml mycobacteria and received 2 µg of PTX i.p. This modified protocol, with reduced mycobacterial component and increased PTX and antigen, was used to prevent the very severe inflammatory response at the site of immunization in IL-10-deficient mice, on the one hand, and to compensate for the relatively lower susceptibility to EAU of B6 mice compared to B10.A, on the other.

Treatment B10.A mice were treated i.p. with recombinant murine IL-10 or recombinant murine IL-4 in a volume of 0.1 ml. Mice

A Th1-like long-term T cell line specific to the IRBP-derived peptide 161–180 was derived from B10.RIII mice, and was characterized and propagated as described previously (6). For measurement of antigen-specific responses, rested line cells (2.53105/0.2 ml well) were stimulated with peptide 161– 180 (2 µg/ml) in the presence irradiated splenic antigenpresenting cells (APC; 2.53105/well). IL-4 or IL-10 were added to the wells at the beginning of culture. Supernatants for cytokine assays were removed from the wells after 48 h. Parallel cultures to be assayed for proliferation were pulsed with 1 µCi [3H]thymidine for an additional 16–18 h. Thymidine uptake was determined by standard liquid scintillation counting

Cytokine assay by ELISA IL-2, IL-4 and IFN-γ in culture supernatants were analyzed by ELISA as described elsewhere (9) Briefly, Corning ELISA microtiter plates were coated with capture antibody for each cytokine (cytokine ELISA antibody pairs from PharMingen) in 0.5 M sodium bicarbonate/carbonate buffer, pH 9.6, overnight at 4°C. After washing unbound antibodies and blocking the free sites in the plate samples were added and incubated overnight at 4°C. A biotinylated secondary antibody was added (cytokine ELISA antibody pairs from PharMingen) for 1 h under shaking at room temperature; after washing unbound antibodies streptavidin–HRP was added and the color reaction was developed using o-phenylenediamine. Concentrations of each cytokine were calculated by extrapolation from a standard curve created by adding recombinant cytokine to each plate. TNF-α was measured by ELISA, using a kit from Endogen (Woburn, MA). ELISA plates were read at 490 nm with an ELISA reader (Molecular Devices, Sunnyvale, CA).

Antibody isotype determination Anti-IRBP-specific antibodies were measured as described elsewhere (5) Briefly, ELISA plates were coated overnight with 2 µg/ml of dialyzed IRBP using the buffer described above. After blocking and washing, serum samples were plated overnight at 4°C. The next day unbound material was washed and plates were developed using HRP-conjugated goat anti-IgG subclass-specific antibodies (Southern Biotechnology Associates). Concentrations were extrapolated from a standard curve from hyperimmunized mice. In these assays, the concentration of anti-IRBP antibody was estimated using standard curves constructed by coating wells with antiIg antibody against the appropriate isotype and adding polyclonal Ig standards of the pertinent isotype.

IL-10 protects from EAU 809 Histopathology and EAU grading Eyes were obtained 7–28 days after immunization or after adoptive transfer of IRBP-responsive T cells (as specified in the text). Freshly enucleated eyes were fixed for 1 h in 4% phosphate-buffered glutaraldehyde and transferred into 10% phosphate-buffered formaldehyde until processing. Fixed and dehydrated tissue was embedded in methacrylate and 4– 6 µm sections, cut through the pupillary–optic nerve plane, were stained by standard hematoxylin & eosin. Six sections cut at different levels were examined for each eye in a masked fashion by one of us (C. C. C.), and the presence and extent of lesions was determined. Incidence and severity of EAU were scored on a scale of 0–4 in half point increments, according to a semiquantitative system described previously (23). Briefly, the minimal criterion to score an animal as positive by histopathology was inflammatory cell infiltration of the ciliary body, choroid or retina. Progressively higher grades were assigned for presence of discrete lesions in the tissue such as vasculitis, granuloma formation, retinal folding and/ or detachment, photoreceptor damage, etc. The grading system takes into account lesion type, size and number. RT-PCR analysis of lymphokine gene expression in the retina Mice were perfused under rompun/ketamine anesthesia with 10 ml of cold PBS plus heparin through an aortic cannulation to clear the retinal vessels and decrease the background from blood-borne lymphocytes. Total RNA was extracted from the enucleated eyes at different time points after immunization using the RNAzol B method, and RNA was reverse transcribed and amplified using primers for IL-2, IL-4, IL-5, IL-10, TNF-α and IFN-γ, as described elsewhere (24,25). Then 1 µg RNA was transcribed using the MMLV-H reverse transcriptase. After this reaction, the cDNA-containing solution was used for specific sequence amplification using 1 U Taq DNA polymerase and 80 ng each of sense and antisense primers. The number of cycles was chosen after preliminary optimization experiments for each gene product: IL-2, 35 cycles; IFNγ, 28; IL-4, 30; IL-10, 23; TNF, 24; and HPRT, 23 cycles. Finally, PCR products were separated on 1% agarose gels and were analyzed by Southern blot hybridization with fluoresceinlabeled internal probes using the ECL-39 oligo labeling and detection system and Hyperfilm-ECL (Amersham, Amersham, UK). Results were normalized using HPRT as a housekeeping gene and naive controls in each experiment for a measure of baseline gene expression of the cytokines measured. Reproducibility and data presentation Experiments were repeated at least twice. Response patterns were highly reproducible. Unless stated otherwise, graphs show representative experiments. Statistical analysis of EAU scores was performed by Snedecor and Cochran’s test for linear trend proportions (non-parametric, frequency based) (26). Each mouse (average of both eyes) was treated as one statistical event. Statistical analysis of proliferation and cytokine production was by independent t-test. P , 0.05 was considered significant. Results

IL-10-deficient mice develop EAU We first wished to examine whether expression of EAU would be affected by absence of endogenous IL-10. IL-10-deficient

Fig. 1. IL-10-deficient mice are susceptible to EAU. IL-10-deficient mice on C57BL/6 background and their wild-type littermates were immunized with 100–150 mg IRBP (in CFA containing 1 µg/ml MT) and were given 2 µg PTX. EAU was scored by histopathology on a scale of 0–4, 19 days after immunization. The incidence of EAU as positive/total mice in the group is shown within the columns. Results are a composite of five separate experiments. The difference between EAU scores of the IL-10 knockout and wild-type mice was not statistically significant.

mice on the B6 background and their wild-type littermates were immunized with a uveitogenic regimen of IRBP (see Methods) and eyes were collected for histopathological analysis after 19 days. IL-10-deficient mice developed EAU that was histologically indistinguishable from that developed by wild-type littermates, except that their EAU scores tended to be lower, although the difference between the groups was not statistically significant (P , 0.26) (Fig. 1). These data indicate that pathogenesis of EAU can develop in the complete absence of IL-10 production.

Early treatment with IL-10 protects from EAU and synergizes with IL-4 Mice were treated with three daily injections of 1000 U IL-10, or IL-10 plus 10,000 U IL-4, during the first 5 days after immunization for induction of EAU (Fig. 2). The results showed that IL-10 treatment alone has a partial protective effect from disease, and is able to down-regulate antigen-specific production of IFN-γ and TNF by primed lymph node cells collected from the treated mice 21 days after immunization (Fig. 2a and b). IL-4 alone was not protective at this dose (not shown). However, when the IL-10 treatment was combined with IL-4, protection from EAU was more pronounced than with IL-10 alone. Furthermore, in addition to suppression of antigen-specific IFN-γ and TNF responses, there was also an up-regulation of antigen-specific IL-4 and IL-10 production by primed lymph node cells (Fig. 2c and d). Thus, while IL-10 alone appears to be sufficient to down-regulate the Th1 response, IL-4 is needed to achieve a shift towards a Th2 cytokine profile. To further address the issue of response shift, we analyzed the anti-IRBP serum antibody isotypes. Because IFN-γ and IL-4 promote antibody isotype switching to IgG2a and IgG1 respectively, the amounts of these antibody isotypes are

810 IL-10 protects from EAU

Fig. 2. Early treatment with IL-10 or with IL-10 plus IL-4 protects from EAU and shifts the cytokine response. B10.A mice were immunized with 100 µg IRBP in CFA plus PTX. The treated groups received 33/day 10,000 U of recombinant IL-4 and/or 33/day 1000 U of recombinant IL-10 on days: 0, 1, 2, 3 and 4. The control group received no treatment. Eyes and lymph node cells were collected 21 days after immunization. EAU grading was by histopathology. The incidence of EAU as positive/total is shown within the columns. Statistical analysis of difference from control is shown above the columns. Cytokine production to IRBP in culture was measured by ELISA. For statistical analysis of antigen-specific cytokine production, parallel groups from both experiments were combined (since the supernatants were pooled, yielding one sample per group, analysis of a single experiment is not valid). The difference between IL-10 treated and untreated groups in IFN-γ production was significant at P , 0.05, but was not significant for TNF.

Table 1. IgG2a and IgG1 antibody isotype responses in mice treated with IL-10 or with a combination of IL-10 and IL-4 Experiment

Treatment group

IgG2aa (ng/ml) IgG1a (ng/ml)

1

PBS IL-10

13,830 9680

23,800 6568

2

PBS IL-10 IL-10 1 IL-4

14,380 3379 7876

18,910 7378 23,040

aThe

sera originated from the same mice whose EAU scores and cytokine profiles are shown in Fig 2.

considered to be reflective of the type of response (Th1 or Th2) generated in vivo. While IL-10 treatment alone was able to down-regulate both isotypes (P , 0.05 compared to control), consistent with a general suppression of the helper response, IL-10 plus IL-4 down-regulated the IgG2a response, but restored the IgG1 response, consistent with enhancement of Th2 help (Table 1).

Sustained treatment with IL-10, but not with IL-4, protects from EAU In another series of experiments the efficacy of treatment with IL-4 alone or IL-10 alone was tested during the afferent phase

(days 1–14), the efferent phase (days 13–19) or throughout the entire 3 weeks after uveitogenic immunization. While IL-4 alone had no protective effect at any stage of EAU, IL-10 alone could completely protect from disease, but only if it was administered throughout the entire course of EAU from day 0 through day 19 (data not shown).

Only IL-10, but not IL-4, is effective in suppressing the mature uveitogenic Th1-like effector cell Uveitogenic T cells from a long-term line specific to peptide 161–180 of IRBP were stimulated in culture with the specific antigen presented on irradiated splenic APC, in the presence of recombinant mouse IL-10 or IL-4. Only IL-10, but not IL-4, was able to suppress proliferation and production of IFN-γ in response to antigen (Fig. 3). This is in keeping with the result that only IL-10, but not IL-4 when administered by itself, was able to protect mice from EAU.

Up-regulation of IL-10 mRNA in eyes of uveitic mice coincides with resolution of EAU Mice were given a uveitogenic regimen of IRBP. Eyes were collected at 7 day intervals, and expression of mRNAs for IFN-γ, TNF-α, IL-2, IL-4 and IL-10 in the eyes was determined by semiquantitative RT-PCR. The disease course in relation to the time points analyzed is as follows: 7 days after immunization is before clinical manifestations are apparent,

IL-10 protects from EAU 811

Fig. 3. IL-10, but not IL-4, suppresses antigen-driven proliferation and IFN-γ production by uveitogenic effector T cells. T line cells (B10.RIII, peptide 161–180 specific) were stimulated with 2 µg/ml of the peptide in the presence of APC. IL-10 (100 U/ml) or IL-4 (1000 U/ml) were added at the onset of culture. Shown is IFN-γ protein as measured in the supernatant after 48 h of by ELISA and proliferation after 60 h as [3H]thymidine uptake. Statistical analysis of proliferation: comparison of the triplicate samples for each replicate experiment showed the difference in each to be highly statistically significant (P , 0.0005). For analysis of antigen-specific cytokine production, parallel groups from both experiments were combined as in Fig. 2. The difference in IFN-γ production between IL-10 treated and untreated cells was significant at P , 0.0005.

day 14 coincides with clinical onset (typically day 12–14), day 21 is during peak expression of disease and day 35 is well into the resolution phase. Figure 4 shows that type 1 and proinflammatory cytokines (IFN-γ, IL-2 and TNF-α) dominated the early and peak phases of disease. IL-4 appeared relatively early, but continued to rise into the resolution phase, whereas IL-10 first became detectable on day 28, coinciding with the early resolution phase and continued to rise through day 35. Thus, local expression of IL-10 message coincides with the natural recovery of mice from EAU.

Neutralization of IL-10 during the expression phase of EAU up-regulates disease In order to address the question whether endogenous IL-10 might be involved in controlling expression of EAU under normal conditions, B10.A mice immunized for EAU induction were treated with neutralizing anti-IL-10 antibodies starting on day 14 (coinciding with clinical onset of disease) through day 20 (Fig. 5). While mice treated with control antibodies developed low EAU scores, animals receiving anti-IL-10 antibodies developed significantly more disease. Discussion A number of sight threatening human uveitic diseases that affect the posterior pole of the eye are thought to be cell

mediated by virtue of responding favorably to anti-T cell therapy. The present study was initiated to explore the regulatory role and possible therapeutic potential of IL-10 in this type of ocular autoimmunity, as represented by the EAU model, which is dependent on a dominant Th1 response. Unexpectedly, IL-10-deficient mice developed reduced EAU scores in comparison to controls. Although the difference in EAU scores between IL-10 knockout mice and controls was not statistically significant at the number of mice analyzed here, we feel that this difference is real. IL-10 knockout mice are subject to inflammatory bowel disease and develop severe inflammatory responses at the site of immunization. We hypothesize that the reduced EAU scores might have resulted from recruitment of the inflammatory leukocytes, that otherwise would have ended up in the eye, to other inflammatory sites. Administration of exogenous IL-10 was able to ameliorate EAU and synergized with IL-4 in this effect. IL-4 alone, however, was ineffective. While mice treated with IL-10 by itself had a depressed Th1 response, as judged by reduced antigen-specific production of IFN-γ, only those mice that received IL-4 in addition to IL-10 exhibited evidence of a shift towards an up-regulated Th2 response to IRBP. These data are also supported by the antibody subclass shift in the treated mice: while the IL-10 treated mice had a depressed IgG1 and IgG2a response, treatment with IL-4 restored the

812 IL-10 protects from EAU

Fig. 4. Kinetics of lymphokine mRNA expression in uveitic eyes of IRBP-immunized B10.A mice. Mice were perfused with PBS before enucleation of eyes. The mRNA extracted from the eye was subjected to RT-PCR amplification followed by Southern blotting. Specific bands were detected with 32P-labeled internal probes. Shown is relative band intensity by densitometry compared to the housekeeping gene HPRT.

Fig. 5. Neutralization of IL-10 during efferent phase of EAU exacerbates disease expression. B10.A mice immunized with 50 µg IRBP were treated with neutralizing anti-IL-10 antibodies starting on day 14 (2 days after the clinical onset of disease) through day 20. Eyes were collected on day 21 and were graded by histopathology on a scale of 0–4. The incidence of EAU as positive/total is shown within the columns.

IgG1 (but not IgG2a) antibody titers even above those of untreated mice. This result suggests that while IL-10 can suppress a pathogenic Th1 response, it does not by itself promote a measurable Th2 shift in this system. Separate experiments showed that only IL-10, but not IL-4, was able to inhibit mature uveitogenic effector T cells in culture. These data are in line with the interpretation that IL-4 affects early stages of effector cell development, whereas IL-10 can regulate mature effector cells in a situation where disease has already been established. A cooperative effect between IL-10 and IL-4 was also noted in some other autoimmune situations (13,14,27–29). If the above interpretation that IL-4 mainly affects differentiation of immature effector cells, whereas IL-10 can inhibit the mature effector, is correct, experimental evidence should show that IL-10 administered in the efferent phase can

limit disease expression. Our attempts at IL-10 treatment of established disease, starting on day 13 after immunization, met with variable success, possibly due to our inability to administer sufficient doses of the recombinant cytokine (because of the limited supply). However, the opposite approach, consisting of neutralization of endogenous IL-10 using mAb, resulted in strongly up-regulated EAU scores. This result indicates that endogenous IL-10 may be involved in natural control of the disease. This interpretation is also supported by the kinetics of IL-10 mRNA expression in uveitic eyes, which coincided with natural resolution of EAU and down-regulation of message for proinflammatory and Th1 cytokines. A similar kinetic of IL-10 compared to the proinflammatory cytokines was observed in the central nervous system of mice and rats with experimental autoimmune encephalomyelitis (EAE) and experimental allergic neuritis (30–32), suggesting that this type of regulation may represent a more general phenomenon in tissue-specific autoimmunity. Our results are at variance with those of Cannella et al., Balasa et al. and others, who reported that IL-10 could not protect, or even caused worsening, of EAE and of autoimmune diabetes in mice (17–20). In contrast, our data are in line with several reports describing beneficial effects of IL-10 in cellmediated inflammatory and autoimmune disease models. Thus, EAE, allergic neuritis, diabetes in the NOD mouse, collagen arthritis and experimental thyroiditis were all prevented or ameliorated by administration of exogenous IL-10 (11–16). The present report expands on these data by adding EAU to the list, and presents evidence in favor of a role for endogenous IL-10 in the regulation of the autoimmune process. In summary, our data show that IL-10 can directly and/or indirectly modulate expression of EAU. The data are compatible with the interpretation that endogenous IL-10 plays a role in the natural control and spontaneous resolution of EAU, and strongly suggest that administration of exogenous IL-10 may have therapeutic potential in inflammatory and autoimmune eye disease. To maximize efficacy of an IL-10 therapy in a

IL-10 protects from EAU 813 clinical setting, a concurrent administration of IL-4 might prove beneficial, since a chronic-relapsing disease involves continuous recruitment and priming of new autopathogenic effector cells.

Acknowledgements The authors thank Dr Ricardo T. Gazzinelli, Leslie Stiff-Jones and Roseanne Y. Choi for their invaluable help with certain parts of this study. We wish to express our gratitude to Dr Satwant Narula of Shering-Plough for donating the recombinant murine IL-4 and IL-10, and to Drs Paulo Vieira and Kevin Moore from DNAX for the anti-IL10 antibodies SX1, SX2 and 2JA. We thank Drs Renate Morawetz and Herbert Morse III for donating the original IL-10 knockout breeding stock and some of the animals used in our experiments.

Abbreviations APC Con A CFA EAU EAE IRBP HRP PTX TNF

antigen-presenting cell concanavalin A complete Freund’s adjuvant experimental autoimmune uveoretinitis experimental autoimmune encephalomyelitis interphotoreceptor retinoid binding protein horseradish peroxidase pertussis toxin tumor necrosis factor

References 1 Gery, I., Mochizuki, M. and Nussenblatt, R. B. 1986. Retinal specific antigens and immunopathogenic processes they provoke. Prog. Retinal Res. 5:75. 2 Caspi, R. R., Roberge, F. G., Chan, C. C., Wiggert, B., Chader, G. J., Rozenszajn, L. A., Lando, Z. and Nussenblatt, R. B. 1988. A new model of autoimmune disease. Experimental autoimmune uveoretinitis induced in mice with two different retinal antigens. J. Immunol. 140:1490. 3 Faure, J. P. 1980. Autoimmunity and the retina. Curr. Top. Eye Res. 2:215. 4 Caspi, R. R., Silver, P. B., Chan, C. C., Sun, B., Agarwal, R. K., Wells, J., Oddo, S., Fujino, Y., Najafian, F. and Wilder, R. L. 1996. Genetic susceptibility to experimental autoimmune uveoretinitis in the rat is associated with an elevated Th1 response. J. Immunol. 157:2668. 5 Sun, B., Rizzo, L. V., Sun, S.-H., Chan, C.-C., Wiggert, B., Wilder, R. L. and Caspil, R. R. 1997. Genetic susceptibility to experimental autoimmune uveitis involves more than a predisposition to generate a Th1-like or a Th2-like response. J. Immunol. 159:1004. 6 Xu, H., Rizzo, L. V., Silver, P. B. and Caspi, R. R. 1997. Uveitogenicity is associated with a Th1-like lymphokine profile: cytokine-dependent modulation of primary and committed T cells in EAU. Cell. Immunol. 178:69. 7 Hara, Y., Caspi, R. R., Wiggert, B., Chan, C. C., Wilbanks, G. A. and Streilein, J. W. 1992. Suppression of experimental autoimmune uveitis in mice by induction of anterior chamber-associated immune deviation with interphotoreceptor retinoid-binding protein. J. Immunol. 148:1685. 8 Saoudi, A., Kuhn, J., Huygen, K., de Kozak, Y., Velu, T., Goldman, M., Druet, P. and Bellon, B. 1993. TH2 activated cells prevent experimental autoimmune uveoretinitis, a TH1-dependent autoimmune disease. Eur. J. Immunol. 23:3096. 9 Rizzo, L. V., Miller-Rivero, N. E., Chan, C. C., Wiggert, B., Nussenblatt, R. B. and Caspi, R. R. 1994. Interleukin-2 treatment potentiates induction of oral tolerance in a murine model of autoimmunity. J. Clin. Invest. 94:1668. 10 de Vries, J. E, 1995. Immunosuppressive and anti-inflammatory properties of interleukin 10. Ann. Med. 27:537.

11 Rott, O., Fleischer, B. and Cash, E. 1994. Interleukin-10 prevents experimental allergic encephalomyelitis in rats. Eur. J. Immunol. 24:1434. 12 Pennline, K. J., Roque-Gaffney, E. and Monahan, M. 1994. Recombinant human IL-10 prevents the onset of diabetes in the nonobese diabetic mouse. Clin. Immunol. Immunopathol. 71:169. 13 Rabinovitch, A., Suarez-Pinzon, W. L., Sorensen, O., Bleackley, R. C., Power, R. F. and Rajotte, R. V. 1995. Combined therapy with interleukin-4 and interleukin-10 inhibits autoimmune diabetes recurrence in syngeneic islet-transplanted nonobese diabetic mice. Analysis of cytokine mRNA expression in the graft. Transplantation 60:368. 14 Mignon-Godefroy, K., Rott, O., Brazillet, M. P. and Charreire, J. 1995. Curative and protective effects of IL-10 in experimental autoimmune thyroiditis (EAT). Evidence for IL-10-enhanced cell death in EAT. J. Immunol. 154:6634. 15 Bai, X. F., Zhu, J., Zhang, G. X., Kaponides, G., Hojeberg, B., van der Meide, P. H. and Link, H. 1997. IL-10 suppresses experimental autoimmune neuritis and down-regulates TH1-type immune responses. Clin. Immunol. Immunopathol. 83:117. 16 Persson, S., Mikulowska, A., Narula, S., O’Garra, A. and Holmdahl, R. 1996. Interleukin-10 suppresses the development of collagen type II-induced arthritis and ameliorates sustained arthritis in rats. Scand. J. Immunol. 44:607. 17 Wogensen, L., Lee, M. S. and Sarvetnick, N. 1994. Production of interleukin 10 by islet cells accelerates immune-mediated destruction of beta cells in nonobese diabetic mice. J. Exp. Med. 179:1379. 18 Balasa, B. and Sarvetnick, N. 1996. The paradoxical effects of interleukin 10 in the immunoregulation of autoimmune diabetes. J. Autoimmun. 9:283. 19 Lee, M. S., Mueller, R., Wicker, L. S., Peterson, L. B. and Sarvetnick, N. 1996. IL-10 is necessary and sufficient for autoimmune diabetes in conjunction with NOD MHC homozygosity. J. Exp. Med. 183:2663. 20 Cannella, B., Gao, Y. L., Brosnan, C. and Raine, C. S. 1996. IL10 fails to abrogate experimental autoimmune encephalomyelitis. J. Neurosci. Res. 45:735. 21 Pepperberg, D. R., Okajima, T. L., Ripps, H., Chader, G. J. and Wiggert, B. 1991. Functional properties of interphotoreceptor retinoid-binding protein. Photochem. Photobiol. 54:1057. 22 Mosmann, T. R., Schumacher, J. H., Fiorentino, D. F., Leverah, J., Moore, K. W. and Bond, M. W. 1990. Isolation of monoclonal antibodies specific for IL-4, IL-5, IL-6, and a new Th2-specific cytokine (IL-10), cytokine synthesis inhibitory factor, by using a solid phase radioimmunoadsorbent assay. J. Immunol. 145:2938. 23 Chan, C. C., Caspi, R. R., Ni, M., Leake, W. C., Wiggert, B., Chader, G. J. and Nussenblatt, R. B. 1990. Pathology of experimental autoimmune uveoretinitis in mice. J. Autoimmun. 3:247. 24 Gazzinelli, R. T., Hakim, F. T., Hieny, S., Shearer, G. M. and Sher, A. 1991. Synergistic role of CD41 and CD81 T lymphocytes in IFN-gamma production and protective immunity induced by an attenuated Toxoplasma gondii vaccine. J. Immunol. 146:286. 25 Rizzo, L. V., Silver, P., Wiggert, B., Hakim, F., Gazzinelli, R. T., Chan, C. C. and Caspi, R. R. 1996. Establishment and characterization of a murine CD41 T cell line and clone that induce experimental autoimmune uveoretinitis in B10.A mice. J. Immunol. 156:1654. 26 Snedecor, G. W. and Cochran, W. G. 1967. Statistical Methods, 6th edn, p. 246. Iowa State University Press, Ames, IA. 27 van Roon, J. A., van Roy, J. L., Gmelig-Meyling, F. H., Lafeber, F. P. and Bijlsma, J. W. 1996. Prevention and reversal of cartilage degradation in rheumatoid arthritis by interleukin-10 and interleukin-4. Arthritis Rheum. 39:829. 28 Sugiyama, E., Kuroda, A., Taki, H., Ikemoto, M., Hori, T., Yamashita, N., Maruyama, M. and Kobayashi, M. 1995. Interleukin 10 cooperates with interleukin 4 to suppress inflammatory cytokine production by freshly prepared adherent rheumatoid synovial cells. J. Rheumatol. 22:2020. 29 Faust, A., Rothe, H., Schade, U., Lampeter, E. and Kolb, H. 1996. Primary nonfunction of islet grafts in autoimmune diabetic nonobese diabetic mice is prevented by treatment with interleukin4 and interleukin-10. Transplantation 62:648.

814 IL-10 protects from EAU 30 Kennedy, M. K., Torrance, D. S., Picha, K. S. and Mohler, K. M. 1992. Analysis of cytokine mRNA expression in the central nervous system of mice with experimental autoimmune encephalomyelitis reveals that IL-10 mRNA expression correlates with recovery. J. Immunol. 149:2496. 31 Issazadeh, S., Ljungdahl, A., Hojeberg, B., Mustafa, M. and Olsson, T. 1995. Cytokine production in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis:

dynamics of mRNA expression for interleukin-10, interleukin-12, cytolysin, tumor necrosis factor alpha and tumor necrosis factor beta. J. Neuroimmunol. 61:205. 32 Zhu, J., Bai, X. F., Mix, E. and Link, H. 1997. Cytokine dichotomy in peripheral nervous system influences the outcome of experimental allergic neuritis: dynamics of mRNA expression for IL-1 beta, IL6, IL-10, IL-12, TNF-alpha, TNF-beta, and cytolysin. Clin. Immunol. Immunopathol. 84:85.