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*
Department of Ophthalmology, University of Minnesota, Minneapolis, MN 55455;
Center for Ophthalmic Research, Brigham and Women’s Hospital, Boston, MA 02115; and
Department of Ophthalmology, Johns Hopkins Medical School, Baltimore, MD 21287
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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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Experimental evidence for active tolerance has been reported in the experimental autoimmune encephalomyelitis (EAE)3 model as well. For example, autologous myelin basic protein (MBP) is less encephalitogenic than heterologous MBP (7), suggesting that there is active tolerance to MBP, and that it is broken by cross-reactive heterologous Ags. Conversely, evidence of immunologic ignorance in the CNS was revealed by studies of mice carrying a transgenic (Tg) TCR specific for MBP (8). Another mechanism of tolerance was revealed by reports of thymic mRNA expression of various autoantigens, including retinal rod photoreceptor cell arrestin (i.e., S-Ag) and MBP (9, 10). Arrestin, an immunopathogenic autoantigen that mediates experimental autoimmune uveoretinitis (EAU) (11), is a member of a large family of proteins with significant sequence homology (12). Tolerance to widely expressed arrestin family members could contribute to tolerance of rod photoreceptor cell arrestin.
Consequently, there is clear need to examine a neural Ag in a less complicated system (13). Tg mice expressing foreign Ags are frequently used to study tolerance and autoimmunity (reviewed in Refs. 14, 15, 16). Testing the influence of the retinal microenvironment on tolerance is possible using Tg mice whose expression of a neo-self-Ag is directed to the retina and elsewhere. We have chosen Tg mice that express ß-galactosidase (ß-gal) in retinal photoreceptor cells, and asked whether EAU could be mediated by an immune response to the ß-gal transgene. An inflammatory, tissue-destructive autoimmune response was found in the Tg mice with high level retinal expression, and there was no evidence for tolerance resulting from that level of retinal expression. Control Tg mice that express ß-gal systemically and normal non-Tg mice were included in the studies for comparison. Our results extend previous studies by demonstrating immunopathology mediated by CD4 T cells specific for a foreign transgene expressed in a neural tissue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The rho-ß-gal mice were derived from C57BL/6 2174-31 founders
expressing ß-gal under the control of a rhodopsin promoter
(17). Expression is first seen 7–8 days postnatally and
increases linearly with age. The lo-arr-ß-gal mice (low level retinal
expression) and hi-arr-ß-gal mice (high level retinal expression)
were derived from CD-1 mice expressing ß-gal using two different
forms of the arrestin promoter (18, 19, 20). Col-ß-gal mice
were provided by Dr. B. deCrombrugghe (M. D. Anderson Cancer
Center, Houston, TX) as a B6D2F1 mouse expressing ß-gal using the
-2000 murine 
2(I) collagen promoter (21). Col-ß-gal
mice express modest levels of ß-gal in several tissues, including the
retina. All Tg mice were backcrossed onto the B10.A background,
providing the MHC type (I-Ak) known for immune
reactivity to ß-gal (22), and the EAU-permissive B10
background (23). The rho-ß-gal mice were used after 5–6
backcrosses, the hi-arr-ß-gal mice were used after 5–10 backcrosses,
and the lo-arr-ß-gal and the col-ß-gal mice were used after 9–11
backcrosses. B10.A mice purchased from Charles River (Wilmington, MA)
and transgene-negative littermates were also used. Mice were maintained
under specific pathogen-free conditions on lactose-free chow.
Ags and immunizations
Mice were immunized s.c. with 50–100 µg of ß-gal in CFA
supplemented with 2.5 mg/ml Mycobacterium tuberculosis
H37Ra. One microgram of pertussis toxin (Sigma, St. Louis, MO) was
administered i.p. on the same day. Purified ß-gal was purchased from
Prozyme (San Leandro, CA). A set of 169 synthetic peptides
corresponding to the entire length of ß-gal, each 17 residues long
and offset by seven residues, was purchased from Chiron Technologies
(San Diego, CA). To reduce the number of samples required to screen
lymph node (LN) cells for proliferative responses, 34 pools containing
five peptides each were made (see example in Table III
). A synthetic
peptide corresponding to residues 461–480 (LRHNPGGPSSAMPLVLSYFQ) of
murine interphotoreceptor retinoid-binding protein (IRBP) was used in
some experiments (24). IRBP is currently the most
well-characterized immunopathogenic retinal autoantigen used in murine
EAU studies (25).
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Nine days after priming, draining LNs from several mice were pooled and stimulated with 75 µg/ml ß-gal in T-25 flasks in RPMI 1640 with 10% FCS. The T cells were restimulated at 9- to 10-day intervals with irradiated splenocytes and Ag to make T cell lines. No exogenous IL-2 was used to develop the MT-1 line. Clones were derived from the MT-1 line by limiting dilution. A line specific for the 461–480 peptide of IRBP was similarly made in B10 mice primed with the 461–480 peptide. Peptides of ß-gal or IRBP were used at 2 µM to stimulate some T cell cultures as specified. Adoptive transfer of EAU was performed with either ß-gal-specific or IRBP461–480-specific, T cells collected 2 days postactivation with Ag and splenic APC, and transferred i.p. in 0.5 ml of saline. For proliferation assays, draining LN cells were prepared and dispensed into 96-well plates at 5 x 105 cells/well in RPMI 1640 with 10% FCS. Ag was added as indicated. After 3 days of culture, 1 µCi of [3H]TdR was added, and the cultures were harvested 18 h later. Assays of line cells and clones were performed with 6 x 105 irradiated splenocytes and 4–6 x 104 T cells/well. [3H]TdR was added after 48 h.
Cytokine assays
The cytokine content of culture supernatants taken from assays as described above was determined by ELISA according to the manufacturer’s suggestions (PharMingen, San Diego, CA). The Ab pairs and the murine cytokine standards were purchased from PharMingen.
ELISA
Serial dilutions of sera were added to ELISA plates coated with 500 ng/well of ß-gal, washed, and then incubated with alkaline phosphatase-conjugated anti-mouse IgG (Sigma). After washing, the wells were incubated with p-nitrophenyl phosphate substrate. Absorbance was measured at 405 nm.
Evaluation of EAU
Eyes were fixed overnight in 10% buffered formalin, paraffin embedded, sectioned (5 microns), and stained with hematoxylin and eosin. The slides were examined in a masked fashion according to previously reported criteria (26).
X-Gal staining
Minor modifications were made to a published protocol (27). Cryostat sections of OCT-embedded tissue were fixed in PBS containing 2% paraformaldehyde and 0.25% glutaraldehyde for 7 min. The sections were washed, covered in X-Gal substrate and incubated at 37°C for 1 h. The sections were counterstained with Nuclear Fast Red for 3 min.
o-Nitrophenyl ß-D-galactopyranoside (ONPG) assay
Quantitation of ß-gal in the eyes was performed as previously described (17), using purified Escherichia coli ß-gal (Prozyme) as the standard, and ONPG (Sigma) as the colorimetric substrate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using Tg mice, retinal expression of a new Ag, ß-gal, was
achieved without the consequences of mechanical inoculation used in
previous studies. Expression in tissues from Tg mice was examined by
X-Gal staining (Fig. 1). High level
expression was found in the photoreceptor cell layer of the retina in
the rho-ß-gal mice (Fig. 1C) and hi-arr-ß-gal mice (Fig. 1B); no expression was found in ß-gal-negative littermates
(Fig. 1A). A slight stain was found in the lo-arr-ß-gal
mice only after prolonged incubation in the X-Gal substrate.
Col-ß-gal mice expressed a small amount of ß-gal in the peripheral
retina (Fig. 1D), skin (Fig. 1E), kidney (Fig. 1F), and elsewhere. Thymuses from the Tg mice were also
examined by X-Gal staining and RT-PCR. No evidence of ß-gal or
ß-gal mRNA was found (data not shown).
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Retinas from the Tg mice were assayed with ONPG at 4 wk of age. Rho-ß-gal and hi-arr-ß-gal retinas contained approximately 58 and 66 ng of ß-gal, respectively. These amounts correspond to concentrations of approximately 19 and 22 µg/ml in the retina based on a calculated retinal volume of 3 µl. These are clearly immunologically active levels, well above the level of 0.5 ng/ml that is known to be immunologically significant in hen egg lysozyme-expressing Tg mice, for example (28). No activity was detected in the retinas of lo-arr-ß-gal mice by ONPG. Approximately 4 ng of ß-gal was found per col-ß-gal retina. No ß-gal activity was found in thymuses or sera from these Tg mice by ONPG assay.
Adoptive transfer of EAU
It is well known that adoptive transfer of T cells specific for
Ags associated with retinal photoreceptor cells can induce EAU in
rodents (26, 29). Accordingly, the adoptive transfer of
EAU by activated, ß-gal-specific T cells would demonstrate that the
ß-gal expressed in the Tg mice can be processed and presented by
local APC at levels capable of recognition by T cells. The MT-1 T cell
line was made from normal B10.A mice immunized s.c. with ß-gal in
CFA. Several clones were isolated from the MT-1 line and were found to
be responsive to ß-gal in vitro in a dose-dependent manner (Fig. 2). EAU was induced in both the
rho-ß-gal and hi-arr-ß-gal mice following adoptive transfer with
the MT-1 line (Fig. 3, A and
B, and Table I). Three of
eight clones examined to date, 3E9, 1G8, and 1H9, also transferred EAU
to the hi-arr-ß-gal mice (Fig. 3C and Table I). No EAU was
found in the lo-arr-ß-gal mice. These results show the dose-dependent
efferent immune recognition of ß-gal and immunopathogenesis of
autoimmune disease in the retina. The histopathology of EAU mediated by
ß-gal-specific T cells was concentrated on the photoreceptor cells,
especially in the later stages of the inflammation, similar to that
observed following the adoptive transfer of uveitogenic T cells
specific for the 461–480 peptide of IRBP (Fig. 3, G and
H), a known uveitogenic retinal protein (23).
The col-ß-gal mice were not susceptible to adoptive transfer of EAU
(Table I), nor did they express evidence of autoimmunity in any of
several other tissues using this transfer protocol.
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The MT-1 line and the clones were CD4+ and
/ß TCR+; there was no particular association
with any TCR Vß usage (data not shown). Cytokine analysis of MT-1
line cells and the pathogenic 3E9 clone shows that TNF- and
substantial levels of IFN-
are produced, but little or no IL-2 or
IL-4 (Table II). Assays of other T cells
were used to confirm assay reliability for detection of IL-2, IL-4, and
IL-6 in the culture supernatants (data not shown).
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Immunizations were performed using a procedure adapted from a
protocol designed to induce EAU with IRBP in mice (30).
The hi-arr-ß-gal mice developed EAU (Fig. 3
, E and
F), but the col-ß-gal and lo-arr-ß-gal mice did not
(Table I). Clearly, tolerance capable of protecting the mice from
actively induced, ß-gal-mediated EAU was not present in mice
expressing ß-gal only in the retina. The lack of disease in the
col-ß-gal mice has several possible explanations; the amount of
ß-gal in the col-ß-gal retinas may be too low to support a
pathogenic response, or there may be tolerance to ß-gal resulting
from the low, systemic level of expression. Evidence for partial
tolerance is shown below.
Anti-ß-gal immune responses of Tg mice
Evidence for tolerance to ß-gal as a self-Ag was also sought
from in vitro assays. Draining LN cells from Tg and control mice primed
with ß-gal were tested in proliferation assays. Based on the dose
responses of normal, immunized mice to ß-gal (Fig. 4A), 0.63 µM (75 µg/ml) Ag
was used to survey the responses of the various Tg and non-Tg control
mice. Both lo- and hi-arr-ß-gal mice were indistinguishable from
B10.A controls, but col-ß-gal mice exhibited a modestly reduced
response (p = 0.05 compared with
ß-gal-negative littermates; Fig. 5).
The reduced response of the col-ß-gal mice was the result of an
upward shift in the dose-response curve, so that the use of high doses
of ß-gal in the proliferation assays significantly restored their
responsiveness, which was lost at low doses (Fig. 4B). The
dose-response curve of the lo-arr-ß-gal mice was unaffected (Fig. 4C). Unprimed mice had no response to ß-gal.
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B), were found in all ß-gal-primed mice,
revealing little evidence for tolerance in B cell responsiveness. The
titers of unprimed control mice, both Tg and normal, were below
detection. Even though the hi-arr-ß-gal mice expressed ß-gal in the
retina sufficient to be susceptible to EAU, they did not appear to
develop tolerance to ß-gal based on that expression. Peptide specificity of the response to ß-gal
It has been proposed that tolerance to self-Ags need not be
complete if tolerance to the immunodominant epitopes of the self-Ag is
induced and maintained, since biologically significant responses are
unlikely to be generated to the cryptic sites (31). Using
Tg mice with widespread expression of hen egg lysozyme (HEL), it was
shown that tolerance was preferentially induced to immunodominant
determinants (32). The modest decrease in the responses of
the col-ß-gal mice described above raised the possibility that T
cells specific for subdominant or cryptic sites in ß-gal were raised
by the adjuvant-promoted response and were responsible for the ß-gal
responses. The Tg mice described here allow this hypothesis to be
easily tested, since the reference response of normal, ß-gal-negative
mice to ß-gal can be determined. The 34 peptide pools described in
Materials and Methods were used to screen mice for responses
to ß-gal. Based on preliminary screening assays showing that the peak
response to the immunodominant group 14 peptides was achieved at
approximately 1 µM, 2 µM peptide was used in subsequent
experiments. A highly reproducible pattern of responses was found (Fig. 6). The patterns of responses of the hi-
and lo-arr-ß-gal mice were indistinguishable from each other and from
those of the non-Tg B10.A control mice, further demonstrating
immunological ignorance of the retinal ß-gal.
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(data not shown). Cytokine production by draining LN cells
The differences in the responses of ß-gal-immunized col-ß-gal
mice compared with those of the other mice were potentially due to
differences in cytokine production. Ag-stimulated production of
cytokines that directly promote T cell proliferation, IL-2 and IL-4,
was very low in all ß-gal-primed mice and was not substantially
different between Tg and non-Tg mice (Table III). Levels of TNF- and IL-6, which
are associated with the pathogenesis of EAU (33) and EAE
(34), were not different between any groups of mice.
Although some difference in IFN- production, also associated with
EAU (35), was observed between Tg and non-Tg mice,
col-ß-gal mice were not different from hi-arr-ß-gal mice. Since all
mice produced substantial amounts of IFN-, the biological
significance of the approximately 3- to 4-fold difference between B10.A
and col-ß-gal mice is uncertain.
Specificity of the MT-1 line and the 3E9 clone
The MT-1 line was screened with the peptide groups after 15–20
cycles of ß-gal stimulation and propagation. Even though the line was
highly selected in vitro, it responded to multiple dominant sites
similar to those identified in the previous screening assays of primed
LN cells (Fig. 7). Screening the
pathogenic T cell clone 3E9 that was derived from the MT-1 line
revealed that it responded well to group 14, as did MT-1 cells (Fig. 7). When tested against the individual peptides of this group, peptides
70 and 71 were found to stimulate the proliferation of 3E9 T cells,
identifying the overlapping region of these peptides as the site
of recognition (Table IV).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of the Tg mouse strategy described in this report support the interpretation that sequestration plays an important role in the immune privilege of the retina. The ability to express a new self Ag in the desired tissues and at different levels also provides a model that will allow more critical testing of immune deviation phenomena. Importantly, the use of transgenes allows the critical control of testing the immune response in the absence of the Ag, a condition that can be difficult to achieve with many self-Ags.
Although we found no evidence for ß-gal-specific tolerance in the Tg mice that expressed ß-gal via the retinal photoreceptor cell promoters, expression of ß-gal in other sites can clearly be tolerogenic. Thymic expression of a ß-gal transgene induced tolerance in both CD4 and CD8 T cells if ß-gal was expressed in the medullary epithelial cells (38, 39). Recognition of ß-gal in skin grafts by CD8 cytotoxic T cells has shown that ß-gal can be recognized as a minor histocompatibility Ag in the context of class I MHC (40). Elsewhere, expression of a ß-gal transgene in mouse brain parenchyma was found using a glial fibrillary acidic protein promoter (41). Immunization of these mice with ß-gal led to a mononuclear infiltrate in the areas of expression, but no clinical signs of disease were observed. Interestingly, these mice were not tolerant to ß-gal, although their expression level is lower than we have found in the rho- or hi-arr-ß-gal mice (unpublished observations). Partial tolerance to ß-gal was found in Tg mice, where expression was directed to lymphoid tissues at a concentration of 20 ng/spleen using an Ig heavy chain promoter and µ transmembrane sequence (42). A moderate reduction in Ab response and affinity to ß-gal was found following immunization of those Tg mice, revealing some tolerance induction. There was no indication of immunopathology resulting from autoimmunity.
In the current study, the hi-arr-ß-gal mice expressed at a much higher concentration in the retina, but did not induce tolerance by the assays we used. Since the expression of a substantial level of ß-gal in the retina was not tolerogenic, we conclude that the environment of the retina sequestered the Ag, maintaining tolerance and ocular immune privilege until challenged with a bolus of activated T cells resulting from adoptive transfer or immunization in the presence of CFA and pertussis toxin.
The consequences of Tg expression in the CNS have been examined in a few other reports (43). In these cases the foreign transgenes have been targets for CD8 CTL activity, unlike our CD4-mediated pathology. There have been a number of reports of the activity of CD8 T cells to transgenic Ags leading to autoimmune disease; most of these demonstrate CTL responses to lymphocytic choriomeningitis virus (LCMV) or influenza hemagglutinin Ags expressed in islets in diabetes models. CD4 T cell-mediated pathology was demonstrated by Lo et al. (44), using the hemagglutinin Ag in islet cells. Forster et al. (45) showed that expression of Tag using a rat insulin promoter has a limited ability to induce tolerance in CD4 T cells. However, it is not clear whether it is the thymic expression associated with islet cell promoters or pancreatic expression that is responsible for the observed tolerance in that study. The thymic expression commonly found in islet Ag systems complicates the interpretations of peripheral tolerance. Also, the level of expression in the islet cell models is generally extremely low compared with the high level of retinal expression in our system, which is not further complicated by thymic expression.
In a different model, Goodnow et al. (46) used a double transgenic mouse with the anti-hen egg lysozyme (HEL) TCR Tg and HEL expression directed to the thyroid or islet cells, or systemically. They found extensive infiltrates in the thyroid and pancreas, but not pathology. There have also been numerous demonstrations of tolerance induction by transgenic protein Ags. In contrast, what we have shown is a lack of tolerance to a very well-expressed transgene in the retina and the CD4-mediated immunopathology directed to it.
Clear evidence for partial tolerance at the T cell level was found in the col-ß-gal mice following immunization, possibly due to the low level of peripheral ß-gal expression, since the substantial levels of retinal expression in the rho- and hi-arr-ß-gal mice did not lead to tolerance. The col-ß-gal mice gave no sign of expression in the thymus at 4 or more weeks of age. Splenocytes or thymocytes from col-ß-gal mice had no ability, in the absence of exogenous ß-gal, to stimulate MT-1 or 3E9 cells when used as APC in proliferation assays (data not shown). The much lower expression levels in the col-ß-gal mice did not provide convincing targets for autoimmune disease in the retina or at other tissue sites of expression. In preliminary results, a trace level of infiltration of the peripheral retina was found in one of eight col-ß-gal mice that were given 500-rad irradiation before transfer with larger numbers (107) of activated MT-1 cells (data not shown). There was no sign of infiltrates in other X-Gal-positive tissues from these mice.
The lack of susceptibility of the col-ß-gal mice to adoptive transfer disease in the retina or at the other systemic sites of expression has several possible explanations. While their systemic expression level was sufficient to reduce the in vitro lymphoproliferative response after priming with the whole molecule, indicating that the presence of ß-gal was recognized by the immune system, it did not support a pathogenic response. Since it is thought that activated T cells have reduced Ag recognition requirements, it would have been reasonable to predict a response by the transferred, activated T cells. There is the possibility that these mice possess regulatory cells such as the CD4+25+ T cells shown to be potent regulators of autoimmunity in other systems (47, 48). There is also the possibility that the activated T cells used in the adoptive transfer acquired altered migratory routes due to contact with Ag in nonlymphoid tissues, resulting in decreased pathogenicity in the eye and elsewhere. Additional Tg mice are being acquired to further study this question.
While the incidence of EAU in the rho- and hi-arr-ß-gal mice was low, its presence is highly significant. We have observed a very large number of rodent eyes over many years, and have not seen spontaneous uveitis resembling EAU. The expression of the transgene itself has not resulted in the appearance of histologic abnormalities or any inflammatory conditions. As shown, immunization of many normal or transgene-negative littermates with ß-gal or their transfer with ß-gal-specific T cells has also never led to the appearance of any histopathology.
Apparently there is nothing special about the known immunopathogenic autoantigens of the retina, since the E. coli enzyme ß-gal can act as such an Ag when present in the retina as a transgene. A substantial level of expression in a sequestered site may be the most important requirements for immunopathogenic autoantigen status. Furthermore, the results show that CD4 T cells raised to a foreign, nonautoantigen in a normal mouse can be immunopathogenic in recipients expressing that Ag.
Our results are most consistent with the interpretation that sequestration is the primary mechanism of retinal immune privilege, and that active mechanisms of ocular immune privilege augment sequestration. These active mechanisms, which are readily induced and detected, may be required to maintain privilege following traumatic or inflammatory injury. The barriers provided by the retinal vascular endothelium and retinal pigment epithelium prevent central tolerance to retinal proteins expressed even at high levels, and peripheral tolerance is limited due to minimal lymphocytic perusal of normal, uninflamed retina and the virtual absence of class II MHC in the retina.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dale S. Gregerson, Department of Ophthalmology, University of Minnesota, 2001 6th Street SE, Minneapolis, MN 55455-3007. E-mail address:
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; Tg, transgenic; EAU, experimental autoimmune uveoretinitis; ß-gal, ß-galactosidase; IRBP, interphotoreceptor retinoid-binding protein; ONPG, o-nitrophenyl ß-D-galactopyranoside; X-Gal, 5-bromo-4-chloro-3-indolyl ß-D-galactoside; HEL, hen egg lysozyme; LN, lymph node.
Received for publication December 18, 1998. Accepted for publication April 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 (I) collagen promoter in transgenic mice. J. Cell Biol. 119:1361.-chains (CD25). J. Immunol. 155:1151.[Abstract]
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) are shown for each group. B,
ELISA titers of anti-ß-gal serum Ab responses of individual Tg
and control mice were obtained with serum samples collected 28–31 days
after immunization with ß-gal. Titers of unprimed Tg and control mice
were below detection. The geometric mean (•) and SD (
