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*
Laboratory of Immunology, National Eye Institute, and
Department of Immunology Instituto de Ciêancias Biomédicas, Sao Paulo, Brazil; and
Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, and
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Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The use of oral tolerization in the clinic would only be possible under either one of two conditions: the eliciting Ag is known (in this case treatment may need to be customized for each patient), or feeding of an Ag present in the target organ and likely to be exposed during the inflammatory process can generate suppressor cells secreting Ag-nonspecific anti-inflammatory cytokines that, acting locally, would decrease inflammation. This phenomenon (bystander suppression) is of utmost importance from the clinical point of view, since in most cases the triggering Ag in human autoimmune diseases is not known. Investigators have reported that Lewis rats fed OVA and immunized with MBP and OVA given separately s.c. were protected from EAE 12 . The protective effect could be adoptively transferred by CD8+ cells from OVA-fed animals into MBP- plus OVA-injected rats 12 . Furthermore, it was shown that oral tolerization with mouse MBP suppressed EAE in the SJL mouse, in which autoimmunity to proteolipid protein appears to play a primary role, suggesting that Ag-driven bystander suppression following oral tolerization with autoantigens may play some role in suppression of EAE following oral tolerization with MBP in this model 13 .
Oral tolerance has been used successfully as an immunotherapeutic approach to many experimental models of autoimmune diseases. Although clinical trials of oral tolerance in multiple sclerosis and arthritis did not show positive results, a clinical trial using S-Ag as the oral tolerogen in the treatment of autoimmune uveitis rendered very encouraging results 14 . To improve on the efficacy of this alternative approach to the treatment of autoimmunity it becomes imperative to understand the mechanisms involved in the development and maintenance of tolerance by oral administration of Ag. It has been suggested that anergy or deletion of Ag-specific cells and active suppression are responsible for the development of oral tolerance. It is important for us to determine parameters are involved in defining the dominant mechanism and to establish the participation of cytokines in the process. It is therefore important to understand the basic mechanisms involved in this phenomenon by taking advantage of the animal models available. It has been suggested that feeding of small amounts of protein would result in tolerance by active suppression, whereas a high dose would result in anergy or T cell deletion in a similar fashion to tolerance induced by i.v. injection of Ag 15, 16 . It is likely that these mechanisms are overlapping and will occur concomitantly, with one or the other predominating. It was also proposed that inhibition of systemic delayed-type hypersensitivity after feeding proteins is due to active suppression, whereas inhibition of systemic humoral immunity may result from T cell anergy 17, 18, 19 . Using the murine model of EAU induced by immunization with interphotoreceptor retinoid binding protein (IRBP), we have shown that oral tolerance elicited by three feedings of 0.2 mg of IRBP followed by one injection of IL-2 at priming (3x + IL-2) correlates with the production of TGF-ß, IL-4, and IL-10 by Peyer’s patch (PP) cells. Suppression could also be elicited by 5x feeding, with minimal production of cytokines 20 . This has led us to propose that tolerance induced by the 3x + IL-2 regimen involves mainly regulatory cells, whereas protection induced by the 5x regimen involves mainly anergy or deletion of the uveitogenic effector T cells 20 .
To examine the importance of IL-4 and IL-10 in oral tolerance induction, knockout mice (KO) deficient for IL-4 and/or IL-10 and their normal littermates were fed IRBP using either the 3x + IL-2 or the 5x regimen and were challenged with a uveitogenic dose of IRBP. We report here that IL-4 and IL-10 KO mice were not protected against EAU by the 3x + IL-2 feeding regimen that protects their normal littermates against the development of disease. However, these same IL-deficient mice were protected by the 5x regimen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice of either sex were obtained from The Jackson Laboratory (Bar Harbor, ME). IL-4-deficient mice (IL-4 KO) and IL-10-deficient mice (IL-10 KO) were gifts from Drs. Klaus Rajewsky, Werner Müller, and Ralf Kühn (Institute of Genetics, University of Kohn, Kohn, Germany) and were bred to the C57BL/6 background by one of us (R.A.M.). Mice deficient in both IL-4 and IL-10 (DKO) were bred by R.A.M. from the single KO mice. Animals were kept in microisolator cages under specific pathogen-free conditions and were handled in compliance with National Eye Institute, National Institutes of Health, and Instituto de Ciêancias Biomédicas-University of Sao Paulo guidelines for animal use.
Reagents
IRBP was isolated from bovine retinas as described previously
using Con A-Sepharose affinity chromatography and fast performance
liquid chromatography 21, 22 . IRBP preparations were aliquoted and
stored at -70°C. BSA, 
-methyl-mannoside, Con A, pertussis toxin
(PTX), CFA, and conalbumin were purchased from Sigma (St. Louis, MO),
horseradish peroxidase-streptavidin was obtained from Southern
Biotechnologies Associates (Birmingham, AL). PHA-p and
Mycobacterium tuberculosis strain H37RA were purchased from
Difco (Detroit, MI). Cyanogen bromide-activated Sepharose 4B was
obtained from Pharmacia (Piscataway, NJ).
Antibodies
The anti-IL-10 mAb-producing hybridomas (SX1, SX2, and 2S5) were provided by Drs. Kevin Moore and Robert Coffman (DNAX Research Institute, Palo Alto, CA). The J4-1 (rat anti-4-hydroxy-3-nitro-phenyl acetyl) was used as a control as described previously 23 . Polyclonal rat IgG1 isolated from naive male rats by affinity chromatography was also used as a control in some experiments. Ab against TGF-ß was obtained by immunizing chickens with recombinant TGF-ß in alum. The Abs were recovered from sera by immunoaffinity chromatography using an anti-chicken Ig Ab coupled to Sepharose. TGF-ß-neutralizing activity of the Ab obtained from the column was tested in a CCL-64 assay 20 using rTGF-ß in different concentrations. One microgram of Ab was shown to neutralize 100 pg of TGF-ß in vitro.
Cell culture medium
T cells were cultured in DMEM supplemented with 10% FCS (HyClone, Logan, UT) or 0.5% normal mouse serum obtained from syngeneic naive mice (as indicated), 10-5 M 2-ME, (Sigma), 2 mM L-glutamine, 0.1 mM nonessential amino acids, and vitamins (Life Technologies, Grand Island, NY) 23 .
Recombinant cytokines
Recombinant human IL-2 was purchased from Boehringer Mannheim
(Mannheim, Germany), recombinant murine IL-5, and IFN-
were obtained
from Genzyme (Boston, MA) or from R&D Systems (Minneapolis, MN).
Recombinant murine IL-4 and IL-10 were gifts from Dr. Satwat Narula,
Schering Plough (Nutley, NJ). Recombinant TGF-ß was a gift from Dr.
Chi-Chao Chan (National Eye Institute, National Institutes of Health)
or was purchased from R&D Systems, Promega (Madison, WI), or Life
Technologies.
Immunization
Mice were immunized s.c. with 100 µg of IRBP in 0.2 ml of emulsion (1/1, v/v) with CFA that had been supplemented with Mycobacterium tuberculosis to a final concentration of 1 mg/ml and were given 2 µg of PTX in 0.1 ml of RPMI 1640 i.p. as an additional adjuvant.
Anti-cytokine treatment of mice
Mice fed IRBP were given i.p. eight equal doses of the anti-IL-10 Ab mixture, J4-1 mAb (rat IgG1 anti-4-hydroxy-3-nitrophenylacetyl), or polyclonal rat IgG starting 1 day before initiation of the feeding and spread twice a week until the termination of the experiment. Mice fed or not fed IRBP (3x + IL-2 regimen) were given i.p. two equal doses of 8 mg of chicken anti-TGF-ß Ab or chicken control Ab 1 day before the first feeding and 7 days after immunization with a uveitogenic dose of IRBP. Mice were given three doses of 0.5 µg of the anti-IL-4 Ab 11B.11 starting 1 day before the feeding of IRBP began and then every 3 days. Alternatively, mice were given three doses of 0.5 µg of 11B.11 every 3 days starting on day 7 after immunization with a uveitogenic dose of IRBP.
Lymphokine assays
IL-2 was measured in supernatants collected 24 h after
antigenic stimulation by the HT-2 bioassay as previously described
23 . Briefly, cell culture supernatants were obtained from the cell
lines and clones at different time points after Ag stimulation. The
supernatant dilutions were distributed in 96-well plates. HT-2 cells
were added to the wells at 104 cells/well. Cultures were
pulsed 16–18 h later with 1 µCi/well of [3H]thymidine
for 6 h. To confirm the presence of IL-2, anti-IL-2 Ab (S4B6,
American Type Culture Collection, Manassas, VA) and/or anti-IL-4 Ab
11B11 (provided by Dr. William Paul, National Institutes of Health,
Bethesda, MD) were added to some wells. The IL-2 concentration was
extrapolated from a standard curve obtained by culturing HT-2 cells in
rIL-2. IL-2 was also measured by ELISA using the Ab pairs from
PharMingen (La Jolla, CA) and using recombinant murine IL-2
(PharMingen) as a standard. Briefly, 96-well microtiter plates (Costar,
Cambridge, MA) were coated with the appropriate anti-cytokine Ab (1
µg/ml in 0.5 M sodium carbonate/bicarbonate, pH 9.6, buffer). After
blocking the plates with BSA and overnight incubation with
supernatants, the plates were developed using biotin-conjugated
anti-cytokine Abs, followed by a 45-min incubation at 37°C. After
additional washes, horseradish peroxidase-conjugated streptavidin
(Southern Biotechnology Associates, Birmingham, AL) was added before
development with o-phenylene diamine. IL-4 was measured by
ELISA using the Ab pairs from PharMingen. Supernatants for IL-4
measurement were collected 48 h after antigenic stimulation.
IFN- was measured by ELISA using Ab pairs from PharMingen in
supernatants collected after 48 h of antigenic stimulation. IL-10
was measured using an ELISA kit from Endogen (Boston, MA) on
supernatants collected 48 h after antigenic stimulation. TGF-ß
was measured using a minikit from Promega on supernatants collected
after 48 h of antigenic stimulation.
Magnetic cell sorting
CD4 and CD8 cells were isolated by magnetic cells sorting using a magnetic cell-sorting magnet and microbeads coated with anti-mouse CD4 or anti-mouse CD8 Abs according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). The isolation was performed using positive and negative selection columns. Briefly, PP were obtained and teased to a single cell suspension as described above. Magnetic labeling was performed with the appropriate Ab-coated microbeads for negative selection in the refrigerator for 20 min. After negatively selecting for either CD4 or CD8, the resulting population was put through a positive selection column with the respective Ab-coated microbeads. The effectiveness of the cell selection process was evaluated by FACS. The CD4+ cell separation contained <5% CD4- cells and <0.2% CD8+ cells. The CD8+ cell separation had <7.5% CD8- cells and <0.2% CD4+ cells.
Histopathology and EAU grading
Eyes were obtained 17–22 days after immunization. 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- to 6-µm sections, cut through the pupillary-optic nerve plane, were stained with standard hematoxylin and eosin. Six sections cut at different levels were examined for each eye in a masked fashion by one of us (C.C.C. or L.V.R.), and the presence and extent of lesions were determined. The incidence and severity of EAU were scored on a scale of 0–4 in half-point increments according to a semiquantitative system described previously 24 . 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 the 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.
Statistical analysis of the data was performed using the Snedcor and
Cochran linear trend in proportions where appropriate;
p 
0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that protection against the development
of EAU induced by the 3x + IL-2 feeding regimen is likely to involve
anti-inflammatory cytokines (IL-4, IL-10, and TGF-ß), whereas
protection induced by the 5x regimen might involve anergy or deletion
of the uveitogenic T cells 20 . To investigate the importance of IL-4
and IL-10 on the development of EAU as well as on the ability to induce
oral tolerance, we fed IRBP to IL-10- or IL-4-deficient (KO) mice
(either the 3x + IL-2 regimen or the 5x regimen). Unfed KO mice
developed EAU that did not differ significantly in incidence from that
of normal littermates and had similar histological features, indicating
that IL-4 and/or IL-10 are not necessary for the induction of EAU (Fig. 1
). However, disease severity was
slightly lower in the KO mice than in their normal littermates. Their
ability to develop protective oral tolerance also differed from that of
their normal siblings. Both the 3x + IL-2 and the 5x regimens were
protective in wild-type (wt) mice (C57BL/6). In contrast, IL-4 and
IL-10 KO mice were protected only by the 5x regimen, but not by the
3x + IL-2 regimen (Figs. 1 and 2). Mice
deficient in both cytokines (double KO) were also insensitive to the
3x + IL-2 regimen, but were protected by the 5x regimen (Fig. 3). These results reinforce the
hypothesis that at least two pathways are involved in tolerance induced
by mucosal presentation of Ag.
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). This result supports the
interpretation that the absence of protection in IL-10 KO mice was due
to their lack of IL-10 and was not a secondary effect of enterocolitis.
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), mostly because the
anti-IL-4 treatment decreased disease severity by itself.
Inhibition of disease development by 11B.11 may be more pronounced at
higher doses (B. Sun, S. H. Sun, P. B. Silver, L.V.R., and
R.R.C., manuscript in preparation).
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Neither IL-4 nor IL-10 is required for the decrease in lymphocyte proliferative responses induced by oral administration of Ag
It was recently reported by others that neither IL-4 nor IL-10 is
essential to the development of oral tolerance under conditions known
to the authors to induce active suppression 28, 29 . These studies
were conducted using nominal Ag as the tolerogen, and lymphocyte
proliferation or Ab production as a readout. The immunological
peculiarities inherent to autopathogenic Ags such as IRBP (here of
bovine origin) make the evaluation of tolerance more complex, since it
is assumed that some form of tolerance to autologous IRBP already
exists, and that the induction of autoimmunity occurs by breaking the
tolerant state. Our data show that one cannot protect IL-4 and IL-10 KO
mice from EAU using the 3x + IL-2 regimen. However, when we looked at
the lymphoproliferative response against IRBP in the draining lymph
node (LN), we were surprised to observe that proliferation to IRBP was
indeed diminished in the fed animals even though the disease scores
were similar in the fed and nonfed groups (Fig. 6). A functional dissociation between in
vitro proliferative responses and disease in the EAU model has been
reported previously 30, 31 . Several nonmutually exclusive mechanisms
can be invoked to explain this dissociation: 1) lymphocyte
proliferation is not a sensitive assay to detect pathogenic cells; 2)
different antigenic epitopes or cell populations are involved in these
two responses; while pathology by definition reflects a response to
autologous epitopes, proliferation and IL-2 production are likely to
reflect largely a response to nonself (bovine) epitopes of IRBP; and 3)
timing differences exist, i.e., proliferation is assayed at the time
that pathology is already fully developed, and most of the cells
involved in the pathogenic response have migrated out of the draining
LN. Regardless of which explanation is correct, it is apparent from the
proliferation data that some form of tolerance was induced in the KO
mice by the 3x + IL-2 regimen, which did not translate into protection
from disease. It is also interesting that LN cell proliferation in
response to IRBP was reduced in the unfed IL-10 KO compared with wt
mice, suggesting that although the development of EAU is not altered by
the absence of IL-10, this cytokine is required for the full expression
of a normal proliferative response.
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The balance between different types of cytokines plays an
important role in the development of EAU and oral tolerance 32, 33 .
Therefore, we decided to measure cytokines expressed in the draining
LN, which is the site of immunization; in PP, which are the putative
sites for the development of tolerance; and in spleen, which is
required for the development of oral tolerance 34 (Fig. 7). We have postulated that protection
against EAU induced by the 3x + IL-2 regimen is cytokine dependent,
whereas the 5x regimen induces cytokine-independent protection. When
we evaluated the cytokine production in response to IRBP in the
draining LN, spleen, and PP of wt and IL-4 or IL-10 KO mice, we
observed significant differences other than, of course, the lack of the
respective cytokines in the KO mice.
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A). We also did not observe any
changes in IL-4 production in the LN or spleen of IL-10 KO mice, as we
had previously reported in another system 35 . PP were not evaluated
in the IL-10 KO animals because the inflammatory bowel disease they
develop impedes correct interpretation of the results. This finding
suggests that IL-4, although crucial to the development of protection
by the 3x + IL-2 regimen, is not associated with the lack of
protection observed in the IL-10-deficient animals.
IL-10 synthesis was not detected in the IL-10 KO animals, and as shown
previously, neither of the oral tolerance regimens was able to induce
IRBP-specific IL-10 synthesis in the LN of treated animals (Fig. 7B). There were no differences in IL-10 production in PP
between wt mice and IL-4 KO, confirming our previous results.
Interestingly, IL-10 production was significantly decreased in the
spleen of IL-4 KO mice compared with that in wt animals. The
significance of this finding is hard to gauge, since in all other
organs evaluated there appears to be no difference between the IL-4 KO
mice and wt mice regarding IRBP-specific IL-10 production.
Nevertheless, it is possible that the lack of IL-4 results in a
decrease in a specific IL-10-producing cell population that, at the
time we evaluated cytokine production, is predominately located in the
spleen, and this population may be important for the development of
oral tolerance. Adoptive transfer experiments are under way to clarify
this question.
We have shown that the administration of IL-2 can potentiate the
tolerogenic effect of oral administration of Ag 20 . IL-2 was also
shown to be pivotal to the maintenance of tolerance in naive animals,
since IL-2-deficient mice develop spontaneous autoimmunity 36 .
Consequently, we decided to measure IL-2 synthesis in response to IRBP
stimulation in the KO mice and their normal littermates. Interestingly,
IL-10 KO mice synthesized lower levels of IL-2 than their normal
littermates. IL-2 production in the IL-10 KO was undetectable in the
spleen and LN of those mice that received either one of the tolerogenic
regimens. As was the case for TGF-ß, the different lymphoid organs
showed different cytokine patterns; this lack of a consistent cytokine
profile among spleen, LN, and PP suggests that different populations of
Ag-specific T cell are present in these organs at the time we analyzed
them for cytokine production. The decreased IL-2 production by IL-10 KO
mice may be linked to the diminished proliferative response to IRBP
seen in the LN of these mice. The pattern of IFN- production
followed the same observed for IL-2 (Fig. 7E). We observed
no difference between the groups regarding their production of IL-5,
and TNF profiles were unremarkable among the different strains of mice
(data not shown).
Many reports in the literature have suggested that TGF-ß plays a
pivotal role in the development of oral tolerance mediated by cytokines
37, 38, 39 . As we had previously shown 20 , TGF-ß synthesis is
enhanced in wt animals after feeding, and the 3x + IL-2 regimen
induces higher levels of TGF-ß than the 5x regimen (Fig. 7C). Interestingly, TGF-ß expression was absent in the LN
of IL-4 or IL-10 KO mice. Nevertheless, IRBP-specific TGF-ß synthesis
in cytokine-deficient mice was equivalent to that in wt animals in both
spleen and PP. One could speculate that the decrease in TGF-ß
production is associated with the lack of induction of oral tolerance
to IRBP in the cytokine-deficient mice.
Because some concern was raised regarding the source of the cytokines
measured in response to IRBP, we isolated CD4+ cells and
CD8+ cells from the PP of mice fed the 3x + IL-2 regimen.
The results show that when CD4+ PP cells are stimulated
with IRBP in vitro, the synthesis of IL-4, IL-10, and TGF-ß is
similar to that observed when the whole cell population from PP is
used. In contrast, when CD8+ cells are stimulated with
IRBP, there is a significant decrease in the synthesis of all cytokines
analyzed (Fig. 8). These data suggest
that CD4+ cells are responsible for most of the IL-4, IL-10
and TGF-ß synthesis observed after feeding the 3x + IL-2 regimen to
mice.
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The huge costs involved in obtaining the amounts of TGF-ß necessary
to perform these experiments forced us to limit the number of mice to
three in some of the TGF-ß-treated groups. Reconstitution was
ineffective in restoring the effectiveness of the 3x + IL-2 feeding
regimen in these animals (Fig. 9A). Furthermore, unlike the
IL-10-treated mice, wild-type unfed mice treated with TGF-ß were not
protected against the development of EAU, confirming our previously
published data 40 and data from a manuscript in preparation (H. Xu,
L.V.R., P. B. Silver, and R.R.C.). Because of the small number
of animals used in these experiments, we decide to take another
approach to study the role of TGF-ß in the deficient induction of
tolerance in the KO mice. We treated these animals with a polyclonal Ab
raised against TGF-ß in chickens, using doses previously described as
effective in neutralizing TGF-ß in vivo. Our results suggest that
Ab-treated wt animals are still capable of responding to the 3x + IL-2
regimen with induction of tolerance to (Fig. 9B).
Furthermore, KO mice treated with the anti-TGF-ß Ab were not less
susceptible to tolerance induction than animals treated with the
control Ab. These data indicate that if TGF-ß plays a role in oral
tolerance to EAU, it is probably a minor one.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production of murine
Th1-like uveitogenic effector T cells in vitro 41 . A dissociation between lymphocyte proliferation to IRBP and disease status was found. Although the cytokine-deficient mice were not protected against the development of EAU by the 3x + IL-2 regimen, lymphocyte proliferation in response to IRBP was significantly decreased in the LN of the IRBP-fed mice compared with that in the OVA-fed controls. A dissociation between disease status and Ag-specific proliferation has been described previously 30, 31 . It has been suggested that lymphocyte proliferation is not a good indicator of the presence or the activity of pathogenic cells 30, 31 . Because we induce disease with IRBP of heterologous nature (bovine), different antigenic epitopes and cell populations might be involved in the two responses (proliferation and uveitogenicity); while pathogenic cells will respond to autologous epitopes, lymphocyte proliferation in the draining LN will be mostly due to cells responding to the nonself (bovine) epitopes of IRBP. This possibility would account for the differences between our findings and those reported previously that suggested that neither IL-4 nor IL-10 is required for the induction of oral tolerance 29, 42 . In these studies animals were fed and subsequently immunized with exogenous Ags such as OVA, and proliferation and Ab production against these Ags were used as markers for tolerance induction. Because of the nature of the Ags used in these studies, the responses against their homologous counterparts, for instance murine albumin, could not be measured. Another possible explanation for the dissociation between lymphocyte proliferation and disease scores is a difference in the timing of the responses measured. Proliferation is assayed at the time that pathology is already fully developed, and most of the cells involved in the pathogenic response have migrated out of the draining LN. It is possible that all these factors may act in unison contributing to the dissociation between the proliferation results and EAU grade. Nevertheless, it is clear that even in the IL-deficient mice some degree of tolerance was induced by the 3x + IL-2 regimen, although it did not result in protection from the development of uveitis.
In keeping with our original observations 20 , the 3x + IL-2
regimen induced IL-4, IL-10, and TGF-ß production in the PP of normal
mice. The 5x regimen was conspicuously less effective in inducing
these cytokines. As before, we were unable to detect IL-10 production
in the LN of wild-type or KO mice 20 . In contrast to what was
reported by others 43, 44 , Ag-specific production of
IFN- by PP in our experiments was minimal. IFN- and
IL-2 production in spleen and LN cells followed the pattern shown in
the proliferative responses described above, i.e., were suppressed by
both feeding regimens. Here it is important to notice that IL-2
production in the LN of IL-10 KO mice was diminished, and it may be
either the cause or an effect of the diminished LN cell proliferation
seen in response to IRBP on these mice. In contrast, in the PP, IL-2
was enhanced in response to the 3x + IL-2 regimen in both KO and wt
mice. It is noteworthy that IL-4 KO mice had a decreased production of
TGF-ß. This finding is in line with a recent report that IL-4 acts as
a growth factor for TGF-ß-producing, mucosal-specific, regulatory T
cells 27 . The absence of IL-4 or IL-10 also seemed to result in a
reduction of TGF-ß secretion in the LN of fed animals and could be
secondary to a decrease in the production of these cytokines in that
organ. Thus, the need for IL-4 and IL-10 to elicit protective oral
tolerance might be due in part to their effects on TGF-ß production.
To investigate this possibility and because TGF-ß has been reported
in the literature to be a major player in oral tolerance induction in
other systems, we treated the KO mice and their normal littermates with
recombinant TGF-ß. The results showed that the treatment with the
TGF-ß we performed was ineffective in restoring the ability of the
3x + IL-2 feeding regimen to protect these animals against EAU.
Although the doses and administration scheme we used proved to be
effective in another system 40 , it is possible that these doses are
not adequate for the study of oral tolerance, and higher doses
administered more frequently may be required to obtain any effect.
However, the prohibitive costs of such experiments made us decide on
alternative strategy. We treated animals with anti-TGF-ß Ab.
Several different combinations of doses and administration regimens
were used. In these experiments we were limited by the fact that more
than two inoculations of mice with chicken Ab resulted in serum
sickness. The data we present are from a regimen we found to be most
effective in diminishing IgA Ab synthesis, which we used as a marker of
the effectiveness of the Ab to neutralize TGF-ß. The wt animals
remained sensitive to the induction of tolerance by the 3x + IL-2
regimen when treated with anti-TGF-ß Ab. These data combined with
the results of the TGF-ß treatment of KO mice suggest that TGF-ß
plays a minor role in our system compared with the major role it plays
in EAE, for instance. The reason(s) for such difference is unclear at
this point.
It is possible that under the conditions we described, cytokines such
as IL-4, IL-10, and TGF-ß are being secreted by cells other than
lymphocytes, thus explaining why tolerance against disease development
is induced but cytokine synthesis remains present in response to
retinal Ags. However, when CD4+ cells were depleted from
the PP, the secretion of IL-4, IL-10, and TGF-ß was significantly
decreased. These data suggest that CD4+ cells are indeed
involved in the production of such cytokines. In contrast, depletion of
CD8+ cells did not alter the expression of the same
cytokines, except for a slight enhancement of IL-10 synthesis, probably
due to a decrease in IFN- production. Although it may be argued that
the 3x + IL-2 regimen does not induce tolerance toward IRBP but,
rather, a shift in the type of cells that respond to that Ag, we and
others 9, 10, 11, 12, 13, 15, 16 believe that the definition of tolerance goes
beyond the inability to respond to an Ag. Particularly for
autoantigens, the definition of tolerance must be more flexible, as
demonstrated by the dissociation among disease scores, lymphocyte
proliferation, and cytokine production we reported here.
Interestingly, both IL-4 and IL-10 KO mice had lower EAU scores than
their wt littermates. Although mice in the C57BL/6 background are only
moderately susceptible to EAU, the disease scores obtained with the KO
animals were even lower. In the case of the IL-10 KO mice, we suspect
that their frail physical condition, caused by the inflammatory bowel
disease they develop over time, may have contributed to the diminished
scores. This prediction is supported by the fact that mice treated with
anti-IL-10 Ab were not less susceptible to disease induction than
animals treated with a control Ab (Fig. 4). The lack of IL-4, however,
does seem to affect disease development, since both the IL-4 KO mice
and mice treated with the anti-IL-4 Ab 11.B11 develop lower EAU
scores (Figs. 2 and 5). We have indications that small amounts of IL-4
may be required for the synthesis of IFN- (B. Sun, S. H. Sun,
P. B. Silver, L.V.R., and R.R.C., manuscript in preparation). Although
the role of IL-4 in the development of oral tolerance is undeniable
since we were unable to tolerize the IL-4 KO mice with the 3x + IL-2
regimen, its precise importance remains to be determined, since the
effect of anti-IL-4 treatment on oral tolerance was not
significant. The discrepancy between the results obtained with the KO
mice, which lack IL-4 from birth, and those obtained with
anti-IL-4-treated animals may be due to the fact that precursor
cells require IL-4 to develop, and the complete absence of this
cytokine will impair their development. Whatever the case may be, it
seems that a certain amount of IL-4 may be needed to induce EAU to its
fullest extent.
In conclusion, the data presented here show that the ability to produce IL-10 is not required for disease induction in EAU. However, both IL-10 and IL-4 are required for the induction of protective oral tolerance through the 3x + IL-2 regimen that elicits anti-inflammatory cytokines, but not by the 5x regimen, which seems to be cytokine independent. The requirement for both IL-4 and IL-10 may result from a synergistic response or a sequential need for the two cytokines in inducing and effecting protection, with IL-4 acting at the induction stage and IL-10 at the effector stage. Ag-specific TGF-ß production in response to feeding is decreased in mice deficient in IL-4 and IL-10 and may constitute a part of the mechanism, although neutralization of TGF-ß or its replacement was unable to alter the course of disease in fed animals. A dissociation between tolerance measured by Ag-specific lymphocyte responses in culture and protection from EAU was observed. It may in part reflect responses to self vs nonself epitopes. The data reported here help to elucidate the mechanisms that drive oral tolerance and provide a rationale for exploring strategies that would enhance its protective effect through augmentation of IL-4 and IL-10 to maximize the clinical benefits of oral Ag therapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Prof. Luiz Vicente Rizzo, Laboratório de Imunologia Clínica, Departamento de Imunologia, Instituto de Ciêancias Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1730, Cidade Universitária, Sao Paulo SP, CEP 05508-900, Brazil. E-mail address:
3 Current address: Centre Hospitalier Universitaire Vaudois, Laboratory of AIDS Immunopathogenesis, Division of Infectious Diseases, Department of Internal Medicine, Hospital de Beaumont, 29 ave. Beaumont, 1011 Lausanne, Switzerland.
4 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; EAU, experimental autoimmune uveitis; MBP, myelin basic protein; IRBP, interphotoreceptor retinoid-binding protein; 3x + IL-2, three feedings of 0.2 mg of IRBP followed by one injection of IL-2 at priming; PP, Peyer’s patch; 5x, three feedings of 0.2 mg of IRBP ; KO, knockout; PTX, pertussis toxin; wt, wild-type; LN, lymph node.
Received for publication February 12, 1998. Accepted for publication November 16, 1998.
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References |
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