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Increases the Severity and Accelerates the Onset of Experimental Autoimmune Uveitis in Transgenic Rats
Laboratories of
*
Immunology and
Molecular and Developmental Biology, and
Veterinary Research and Resources, 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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in the activation of inflammatory cells that mediate autoimmune
pathology, recent studies in IFN--deficient mice paradoxically show
that IFN- confers protection from EAU. Because of the implications
of these findings for therapeutic use of IFN-, we sought to
reexamine these results in the rat, another species that shares
essential immunopathologic features with human uveitis and is the
commonly used animal model of uveitis. We generated transgenic rats
(TR) with targeted expression of IFN- in the eye and examined
whether constitutive ocular expression of IFN- would influence the
course of EAU. We show here that the onset of rat EAU is markedly
accelerated and is severely exacerbated by IFN-. In both wild-type
and TR rats, we found that the disease onset is preceded by induction
of ICAM-1 gene expression and is characterized by selective recruitment
of T cells expressing a restricted TCR repertoire in the retina. In
addition, these events occur 2 days earlier in TR rats. Thus, in
contrast to the protective effects of IFN- in mouse EAU, our data
clearly show that intraocular secretion of IFN- does not confer
protection against EAU in the rat and suggest that IFN- may activate
distinct immunomodulatory pathways in mice and rats during
uveitis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EAU is a predominantly T cell-mediated intraocular inflammatory disease induced in susceptible species by active immunization with ocular-specific proteins (or peptides derived from them) (4, 5) and is transferable to naive syngeneic animals by injection of in vitro-activated CD4+, MHC class II-restricted T cell lines specific to retinal Ags (6, 7). The two major uveitogenic retinal proteins are S-Ag (also termed "arrestin") and interphotoreceptor retinoid-binding protein (IRBP) (4, 5). The experimental animal used in the majority of early studies on EAU has been the Lewis rat, an inbred strain that is highly susceptible to EAU induced by all known uveitogenic Ags (7, 8, 9, 10). Other rat strains show various levels of susceptibility to EAU, but even "resistant" strains develop this disease when pertussis toxin is injected as an additional adjuvant (9, 10, 11). In contrast to rats, most strains of mice were found to be resistant to EAU, and the small number of mouse strains that are susceptible develop disease only when immunized with the retinal Ags at doses much higher than those causing disease in Lewis rats (12). EAU can also be readily induced in primates and severe ocular inflammation develops in monkeys of different species following immunization with S-Ag, IRBP, or peptide determinants of their sequence (13, 14, 15). Taken together with the unique anatomic sequestration of the vertebrate eye, EAU is a useful paradigm of organ-specific autoimmunity mediated by T lymphocytes.
IFN- is a potent transcriptional regulator and a major inducer of
MHC class II gene expression (16). An early feature of a
number of autoimmune diseases is the overexpression of MHC class II
proteins on target cells (17, 18), and aberrant expression
of MHC class II proteins in ocular tissues has also been observed in
ocular disorders such as uveitis, retinoblastoma, proliferative
diabetic retinopathy, and retinitis pigmentosa (19, 20, 21).
As the autoreactive T cells that mediate autoimmune pathology are
predominantly Th1 lymphocytes and produce copious amounts of IFN-,
it was proposed in the early 1980s that induction of ectopic expression
of MHC class II molecules on target cells by IFN- elaborated by
these cells may be a risk factor for developing organ-specific
autoimmune diseases (18, 22). In EAU and in uveitis
patients, IFN- mRNA and protein are detected in the inflamed eye,
and the expression of IFN- mRNA is temporally correlated with the
onset of uveitis, suggesting involvement of IFN- in the induction
and pathogenesis of uveitis (19, 20). However, in the
mouse model of uveitis, depletion of systemic IFN- has been shown to
result in exacerbation of EAU and mouse strains that are resistant to
EAU induction were converted to a susceptible phenotype by peripheral
administration of anti-IFN- Ab (23). The results
from the mouse model of EAU therefore suggest a protective effect of
IFN- in EAU and imply potential benefits of IFN- therapy in the
treatment of uveitis.
In this study, we have addressed the possibility that the paradoxical
nature of the effects of IFN- in uveitis is an epiphenomenon of the
mouse species that is relatively resistant to EAU (24). We
have therefore generated transgenic (TR) rats with constitutive
expression of IFN- in the eye and examined the effects of IFN- on
the induction, progression and susceptibility to experimental
uveitis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stud males, vasectomized males, and female SPF Hsd Sprague Dawley SD rats were purchased from Harlan Sprague Dawley (Frederick, MD). LEW rats, 8–12 wk old, were purchased from Charles River Breeding Laboratories (Raleigh, NC). All animal procedures conformed to Institutional Guidelines and the Association for Research in Vision and Ophthalmology (ARVO) Resolution on Use of Animals in Research.
Generation of 
A-crystallin/IFN- transgenic
rats
Sixty-day-old female Sprague-Dawley SD rats were injected (i.p.)
with 40 µg luteinizing hormone-releasing hormone antagonist (LH-RHa)
((des, gly10, D-ala6,
pro9), LH-RHa, ethyl amide) (Sigma, St. Louis, MO) on day 4
(day 0 is mating day). Superovulation was subsequently induced by i.p.
injection of 10 IU pregnant mare serum chorionic gonadotrophin (PMSG)
(Sigma) on day -2. On day 0, animals received i.p. 10 IU human
chorionic gonadotrophin (HCG) (Sigma) and were individually mated with
stud males. On the morning of day 1, embryos were collected by tearing
the ampullae in CZB medium (25) under a dissecting
microscope. Disruption of the cumulus cells was by incubation of the
zygotes at room temperature in medium containing 1% hyaluronidase
(type IV-S, Sigma). Embryos were then washed and maintained in CZB
medium under 5% CO2 at 37°C. Twenty hours after HCG
injection, zygotes at the pronuclear stage were injected with
linearized 1267-bp A-crystallin/IFN- cDNA fragment (26, 27) and cultured for 1 h in CZB medium at 37°C, 5%
CO2 before transfer. For embryo transfer, 3-mo-old female
rats were synchronized with a single ip injection of 80 µg LH-RHa on
day -4. On day 0, females were individually mated with vasectomized
males. Ten embryos were transferred into the infundibula of
anesthetized pseudopregnant females. Tail DNA from the live-born pups
was screened for the presence of the transgene by PCR.
Induction of EAU
For EAU experiments, TR Sprague-Dawley rats were crossed with
wild-type (WT) Lewis rats for 13 generations to derive an IFN- TR
Lewis rat line. Eight-week-old TR and WT litter mates were used for EAU
studies. The animals were immunized by a single hind footpad injection
with bovine S-Ag (50 µg) emulsified in Hunter’s adjuvant (TiterMax;
CytRx, Norcross, GA) and sacrificed at 24-h time intervals (two rats
per time point) starting on day 5 thru day 14 after immunization. One
eye from each rat was used for RNA isolation and the other for
histology.
Histological analysis
Eyes were carefully dissected out, fixed in 4% glutaraldehyde for 30 min and transferred to 10% buffered formalin. Specimens were dehydrated through graded alcohols and embedded in methacrylate. Serial transverse sections through the pupillary optic nerve plane were cut and stained with hematoxylin and eosin. Photographs of representative sections were taken on a Zeiss photomicroscope.
Reverse transcription (RT) PCR analysis
Eight-week-old TR or WT littermate rats were perfused
extensively before the eyes were enucleated. The retina and lens were
carefully removed under a dissecting microscope and total RNA was
isolated using TriZol reagent according to the procedures recommended
by the manufacturer (Life Technologies, Gaithersburg, MD). All RNA
samples were digested with RNase-free DNase 1 (Life Technologies) for
30 min, purified by phenol/chloroform extractions and precipitated in
0.4 M LiCl. RNA (10 µg) was annealed to oligo(dT)12–16
coupled to magnetic beads (Dynal Corporation, Lake Success, NY) for 15
min. The immobilized mRNA was washed and suspended in reaction mix (10
mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.75 mM
MnSO4, 4 mM dNTP, 2.5 mM DTT, 4 U RNasin) containing 2 U
Retrotherm reverse transcriptase (Epicentre Technologies, Madison, WI)
and 100 U SuperScript II reverse transcriptase (Life Technologies).
cDNA synthesis was performed by incubation at 40°C for 10 min,
followed by a gradual increase in temperature to 65°C over a 10-min
period and continued incubation at 65°C for an additional 50 min.
Residual RNA was removed by hydrolysis with 2 M NaOH for 30 s and
samples were washed three times in TE (10 mM Tris (pH 7.5), 0.1 mM
EDTA) and suspended in 50 µl TE/glycerol solution (1:1). One
microliter cDNA/magnetic beads, 0.4 µM primer, 0.2 µM dNTP were
subjected to hot start PCR with 1.5 U AmpliTaq Gold DNA polymerase
(Perkin-Elmer, Foster City, CA) in a total volume of 50 µl. Samples
were incubated at 95°C for 10 min to activate the AmpliTaq Gold and
amplification was carried out for 30 cycles of 30 s each at
95°C, 60°C, and 72°C. This was followed by a final 10-min
extension at 72°C. The primers used for PCR amplifications were: for
rat ß-actin, 5'-TTGTAACCAACTGGGACGATATGG-3' and
5'-GATCTTGATCTTCATGGTGCTAGG-3' (28); for ACry-IFN-
transgene, 5'-CAGAGGCTCCTGTCTGACTCACTGC-3' and
5'-CTGGATTCCGGCAACAGCTGGTGGAC-3' (26, 27); for ICAM-1,
5'-ATCCGTGCAGGTGAACTGCTCTTC-3' and 5'-CTCTGCTGTTTGTGCTCTCCAG-3'
(29); for RT1-B 5'-ATGCCGCTCAGCAGAGCTCTGAT-3' and
5'-CTCGACTGTCTCTGACACCAGACATGT-3' (30). Each of the
Vß primers used corresponds to a unique sequence located upstream of
the 5' coding region of 1 of the 20 LEW rat Vß TCR cDNAs and these
PCR primers have previously been used to analyze rat Vß TCR gene
usage (31). The antisense Cß primer
(5'-CAATGGATCCCGAGGGTAGCCTTTTGTTTGTCTGCAATCT-3') containing a
BamHI site is specific to LEW rat TCR C region segment and
because this sequence is common to both LEW rat Cß1 and Cß2, no
bias toward amplification of TCR cDNAs encoding either C region Cß
gene element was expected. To control for possible DNA contamination of
mRNAs used as target for PCR amplification, first-strand synthesis
containing each mRNA sample without reverse transcriptase was
performed; failure to obtain RT-PCR products with any of the PCR
amplimers confirmed the absence of contaminating DNA templates. All
cDNA preparations used in this study were found to be suitable
substrates for PCR amplification on the basis of efficient
amplification of a ß-actin sequence.
Southern blot analysis
The amplification reaction (0.7 vol) was electrophoresed on a 1.5% agarose gel, transferred and fixed onto Hybond N+ nylon membranes as recommended by the manufacturer (Amersham, Arlington Heights, IL.). Filters were prehybridized for 2 h at 50°C in 6x SSPE, 5x Denhardt’s solution, 0.5% SDS, and hybridization was performed in the same solution containing fluorescein-dUTP 3'-end-labeled oligonucleotide probes for 12 h at 50°C. The oligonucleotide probes were labeled using the ECL 3'-oligolabeling system (Amersham) and each probe used is internal to the sense and antisense primers employed for PCR amplification. The sequences of the oligonucleotides used as hybridization probes are 5'-TCGGAAGATCGAAAGTCCGGAGCT-3' and 5'CAAACAAGGAGACCTTGGGTGGAGTCACCGT-3, complementary to murine ICAM-1 and rat TCR Cß, respectively. After hybridization, filters were washed two times at room temperature in 5x SSC, 0.1% SDS, followed by a 30-min high stringency wash in 0.1x SSC, 0.1% SDS at 50°C. Filters were then incubated with an anti-fluorescein Ab conjugated to horseradish peroxidase for 30 min and signal detection was based on the enzymatic reduction of peroxide as recommended for the ECL 3'-oligolabeling and detection system (Amersham).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A-crystallin-IFN- Sprague Dawley
transgenic rats
Transgenic animals are valuable tools for the study of in vivo
functions of cytokines, growth factors and other bioactive
polypeptides. The mouse has become the species of choice for transgenic
studies because its genetics is well understood and the methods used
for manipulating the mouse embryo to generate TR mice are relatively
easy. On the contrary, to obtain transgenic rat, derivation of
competent embryos at the pronuclear stage after superovulation is
problematic and the success rate depends on the age and rat strain. In
this study, we experimented with several rat strains, at ages varying
from 30 to 60 days, and superovulation was induced with varying
concentrations of PMSG (data not shown). We had most success with
superovulation of 60-day-old Sprague-Dawley strain with 10 IU PMSG.
Thus this strain was used for generation of ACry-IFN-
rats.
The chimeric ACry-IFN- (A-crystallin promoter fused to murine
IFN- coding sequence) construct used for microinjection has
previously been used to direct expression of IFN- into the lens
(26, 27). One out of forty live-born pups carried the
transgene and manifested bilateral cataract. This rat was used to
establish a line of ACry-IFN- TR Sprague-Dawley rats by
backcrossing to WT rats. Because the genetics of the Lewis rat strain
is better understood and this strain is commonly utilized for uveitis
studies, homozygous Sprague-Dawley rats were crossed with WT Lewis rats
and after 13 backcrosses an IFN- TR Lewis rat strain was
established. The TR progeny exhibited microphthalmia and microphakia
and stably transmitted the transgene through the germline in a normal
Mendelian fashion. Because of the lens specificity of the
A-crystallin promoter element, transgene expression occurs
preferentially in the lens and its effects are initially manifested and
confined to the lens. The lens architecture in the adult TR eyes is
cataractous and has lost its normal spherical appearance. Normal lens
fiber cells, the anterior subcapsular monolayer epithelia and the
equatorial nuclear bow region are not identifiable. In contrast to
adults, these effects are not seen in newborn rats (Fig.
1B). The effects of IFN- on
the lens is only apparent after the first week of life (Fig.
1C) and becomes more pronounced with time. After the first
month of postnatal life, the lens capsule begins to disintegrate and
this is accompanied by the release of lens material into the anterior
chamber and vitreous cavity (Fig. 1D). By histologic
examination, the adult ACry-IFN- TR rats have a normal cornea,
iris, and ciliary body. However, the choroid is significantly thickened
compared with that of the WT rat, and the adult TR rats eventually
develop retinal infoldings, a morphological feature also seen in a TR
mouse expressing IFN- in the retina under direction of the rhodopsin
promoter (32). The essential histopathological features
observed in the TR rat lens have previously been observed in BALB/c and
FVB/N TR mice expressing the ACry-IFN- transgene, suggesting that
these effects are a direct consequence of expression of IFN- and
cannot be attributed to insertional mutation of a critical gene at the
site of transgene integration.
Transcriptional activation of ACry-IFN- and
IFN--inducible genes
To examine whether the morphological changes seen in the eye
correlated with expression of the transgene, we isolated mRNAs from TR
and WT eyes and transgene expression was determined by reverse
transcriptase-PCR (RT-PCR). The PCR primers were designed to
specifically amplify cDNA fragments coding for the ACry-IFN-
transgene but not the endogenous IFN-. All poly(A) RNA used for
first-strand synthesis were found to be suitable substrates for
comparative RT-PCR analysis as revealed by comparable levels of
amplification of ß-actin transcripts (see Fig.
2A). As shown, ACry-IFN-
expression was found only in TR but not in WT eyes. To determine
whether the biological activity of the secreted IFN- extends to the
retina, we examined whether transcription of IFN--inducible genes is
activated in the retina of TR rats. As shown in Fig. 2B, the
RT1-B gene (equivalent to mouse MHC class II), which is normally not
expressed in the retina, is transcriptionally activated in the TR but
not in the WT rat retina. As the biological activities of IFN- are
primarily mediated by the interferon regulatory factor (IRF) family of
transcription factors (16), we also examined whether
expression of IRF genes is up-regulated in the TR retina. Indeed,
transcription of the genes coding for two IRF members, IRF-1 and
interferon consensus sequence-binding protein (ICSBP), is significantly
activated in the TR compared with the WT retina (Fig. 2B).
However, transcription of the gene coding for the housekeeping
transcription factor -pal, a key regulator of eukaryotic initiation
factor-2 (33), is not differentially activated,
suggesting that the effects of IFN- in the retina is specific and
restricted to genes that encode gamma-activation sequence (GAS) in
their promoters (34, 35). Enhanced expression of MHC class
II and IFN--responsive transcription factors genes clearly suggests
that the transgene is biologically active in the retina.
The onset of EAU is accelerated and more severe in
IFN- TR rats
In unimmunized rats, there is very little difference between WT
and TR retinas. The only observable difference is in TR rats over 3 mo
of age. These rats manifest retinal folds and a mild choroiditis
without involvement of the photoreceptor layer (compare Fig. 3
, A and B). To
evaluate the effects of constitutive expression of IFN- in the eye
on the induction and severity of uveitis, WT and TR rat littermates
were immunized with S-Ag and the course of EAU was monitored over a
2-wk period. EAU manifested as grossly inflamed uvea and neuroretina,
culminating in blindness. Eyes were enucleated at various time points
and subjected to histological analysis as indicated in Materials
and Methods. As shown in Fig. 3, the onset of disease occurred 3
days earlier in the TR rats (compare Fig. 3, C and
D) with the appearance of inflammatory cells in the
vitreous, uvea, and neuroretina. The number of inflammatory cells
increased with time and paralleled the degree of tissue destruction.
The first signs of EAU in the retina of WT rats was observed on day 10
after immunization. In contrast, by this time, extensive destruction of
the retina has already occurred in the TR eyes and the photoreceptor
cell and outer nuclear layers are completely obliterated.
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TR rat retinas
As shown above, MHC class II expression is up-regulated in the
retina of TR rats compared with their WT littermates. Because enhanced
expression of MHC class II in a tissue or cell promotes Ag presentation
and increases the avidity of the TCR (36), we examined the
retinal T cell repertoire during EAU to determine whether local
expression of IFN- influenced the recruitment of T cells into the
retina. Beginning from day 6 until day 14 after immunization, RNA
derived from the retina at various time points were used for cDNA
synthesis and RT-PCR analysis as described in Materials and
Methods. Thirty cycles of PCR amplification was found to be
optimum for analyses of Vß TCR transcripts, and under this condition
the amplification product reflected the relative abundance of the TCR
mRNAs in the retina (31). It should be emphasized that TCR
transcripts are normally not present in the retina of naive animals
(31). Results of Southern blot hybridization of the PCR
products from WT and TR retinas with a Cß-specific probe are shown in
Fig. 4. In rats immunized with S-Ag, TCR
transcripts are detectable beginning on postimmunization day 7 and day
9 in TR and WT rats, respectively. On the respective days, Vß8.1,
Vß8.2, Vß9, Vß14, and Vß16 TCRs were the most abundant
transcripts detected in TR retina while Vß5, Vß8.1, Vß9, Vß12,
Vß13, Vß14, Vß15, and Vß18 were found in the WT retina.
Twenty-four hours after the initial infiltration of the retinas by
these T cell clonotypes, nonspecific recruitment of all Vß
subfamilies is observed. These results have been obtained in three
independent experiments and are consistent with previous reports
showing that Vß5, Vß8, Vß9, and Vß14 are among the earliest T
cells subtypes recruited into the retina and thus may be involved in
the etiology of EAU (31). Recruitment of similar T cell
repertoires into the site of autoreactive attack (retina) in both WT
and TR rats, suggests that accelerated onset and severity of EAU in the
TR rat is not due to differences in Ag priming.
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ICAM-1 plays an important role in the extravasation of
inflammatory cells into tissues (37). In endotoxin-induced
uveitis (EIU), an animal model of human anterior uveitis, ICAM-1 is
expressed in the eye just before the clinical or histological signs of
ocular inflammation and Ab to the ß2 integrin Mac-1
(CD11b/CD18), which interacts with ICAM-1, was shown to inhibit EIU
(38). Because the ICAM-1 gene contains GAS elements in its
promoter sequence, its expression is inducible by IFN- (16, 34, 35). We therefore examined whether there is a differential
pattern of ICAM-1 gene expression in the WT and TR retinas and if this
is the basis of accelerated recruitment of cells into the TR rat
retina. Results of RT-PCR analysis for the expression of ICAM-1 in the
retina during the course of S-Ag-induced EAU is shown in Fig. 5. ICAM-1 mRNA transcripts are detected
in the retina of TR rats on day 7 postimmunization. Although our RT-PCR
assay is semi-quantitative, the amount of ICAM-1 mRNA in the retina, as
indicated by band intensity, appeared to increase as the disease
progressed. In contrast to the TR rats, detection of ICAM-1 transcripts
in WT rat retinas is not observed until day 9 postimmunization. It is
therefore interesting to note that, onset of EAU and detection of TCR
Vß transcripts in the retina are temporally correlated with
expression of ICAM-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the eye and have used these rats to
investigate the role of IFN- in the induction, progression, and
susceptibility to experimental uveitis. The most notable effects of
IFN- in the rat eye include microphakia, impaired lens fiber
formation, cataract, and formation of retinal infoldings. Our
demonstration that genes coding for two members of the IRF
transcription factor family, IRF-1 and ICSBP, as well as ICAM-1 (data
not shown) and MHC class II, are transcriptionally activated in TR but
not in WT rat retina provides further suggestive evidence that the
secreted IFN- is biologically active in the eye.
It is also of note that TR mice with targeted expression of IFN- in
the retina under direction of the murine rhodopsin promoter had
previously been reported (32). In contrast to the IFN-
TR rats, these mice exhibited intense intraocular inflammation
accompanied by destruction of the photoreceptor layer. However, these
mice share some of the features found in the IFN- TR rats, including
bilateral cataracts, development of retinal infoldings, and
up-regulated expression of MHC class II gene in their retinas. A
possible explanation for the lack of retinal inflammation in IFN- TR
rats may be related to the fact that exposure of the rat retina to
IFN- occurs much later in life than in the TR mice. Whereas the
rhodopsin promoter targeted expression of IFN- to the mouse eye
retinal development, IFN- expression in the rat under direction of
the A-crystallin promoter is restricted to the lens during embryonic
development. The retina, anterior chamber, and vitreous cavity become
exposed to IFN- only after the first month of postnatal life
following the rupture of the lens capsule. Thus, in the TR rat the lens
serves as an endogeneous depot of intraocular IFN-. A major
advantage of the rat model for studying the role of IFN- in EAU is
that it is not complicated by the damage of photoreceptors due to
overexpression of IFN- during development, and confounding effects
of preexisting inflammatory cells in the eye are eliminated.
Results of experiments comparing the kinetics of induction and
pathology of EAU in WT and TR rats reveal that constitutive expression
of IFN- in the eye accelerates the onset and increases the severity
of uveitis. It is of note that local expression of IFN- in the eye
appears to have marginal effects on the uveitogenic T cell population
recruited into the retina as very little or no quantitative differences
were observed between WT and TR rats. It is remarkable that in both the
TR and WT rats, T lymphocytes expressing Vß8, Vß9, Vß14, and
Vß15 were the earliest clonotypes to infiltrate the retina. The
correlation between the temporal expression of disease and detection of
these T cell subtypes in the retina had previously been reported
(31) and is strongly suggestive of a role for these cells
in the initiation of EAU. Recruitment of a similar T cell repertoire
into the retina at the time of disease onset, albeit delayed in the WT
eyes, suggests that the primary impact of IFN- is in the target
tissue and not at the level of Ag priming of the pathogenic T cells.
Interestingly, although a similar T cell repertoire is recruited, the
development of pathology occurs within a shorter time frame in the
IFN- TR eyes, suggesting that IFN- potentiates the pathogenicity
of the recruited cells. These results are in accord with the well
recognized proinflammatory roles of IFN- in the immunopathogenic
mechanisms of several experimental models of autoimmunity
(39). These include autoimmune thyroditis in mice
(40), autoimmune insulin-dependent diabetes in mice
(41), and experimental autoimmune peripheral neuritis in
the rat (42). However, in recent studies of experimental
allergic encephalomyelitis (EAE), an animal model of multiple
sclerosis, IFN- was found to confer protection against autoimmune
pathology (43, 44, 45).
Our results are also in stark contrast to findings in mouse EAU
(23, 46). Similar to the EAE studies, studies in mouse EAU
have shown that depletion of endogenous IFN- by mAb treatment can
convert genetically resistant mouse strains to an EAU-susceptible
phenotype (23). An important factor that may explain these
differences may relate to innate differences between the
immunomodulatory pathways activated by IFN- in rat and mouse
species. An important pathway proposed for the pathogenesis and/or
recovery from organ-specific autoimmune diseases involves the
production and regulation of the tissue-damaging molecule, nitric oxide
(NO) (47, 48, 49). A key enzyme important for NO production
during inflammation is the inducible NO synthase whose transcriptional
activation is regulated by IFN- (50). Studies in EAE
have found that iNOS/NO exhibit protective or disease-enhancing
properties depending on the species (rat vs mouse) and the mode of
disease induction (active immunization with the autoantigen vs adoptive
transfer of pathogenic T lymphocytes) (47, 51). In rat
EAU, NO has recently been shown to accelerate the onset and to increase
the severity of uveitis through an IFN--dependent mechanism
(52). Results of this study are in accordance with our
current findings in the IFN- TR rat and suggests that the pathogenic
process in the TR rats may in part derive from activation of
macrophages and other inflammatory cells for production of NO. The
proinflammatory effects of IFN- in the rat, as opposed to the mouse,
further suggest that rats and mice may differ in their innate ability
to produce or respond to NO.
Differences between the immunopathogenic mechanisms activated in the
rat and mouse is further underscored by the differential susceptibility
of the two species to EAU induction. S-Ag is one of the best
characterized uveiotogenic proteins. It induces uveitis in Lewis rats
and nonhuman primates (9, 10, 11, 12, 13) and proliferative responses
to this protein have been demonstrated in lymphocytes derived from
patients with uveitis (53). However, most mouse strains
are resistant to EAU and attempts to induce EAU by immunization with
S-Ag in this species have been unsuccessful (12, 24). Only
a small number of mouse strains have been found to be susceptible to
EAU. The disease can only be reproducibly induced using IRBP and, even
so, the amount of the Ag needed to induce disease is greater than 500
times that used in the rat. Taken together, it is reasonable to
conclude that immunopathogenic mechanisms of EAU in the mouse are
dissimilar to those in the rat and nonhuman primates, and thus the
effects of IFN- in mouse EAU may be less representative of its
effects in human uveitis. Although the relative ease of producing TR
mice has made the mouse the species of choice for studying the
biological functions of cytokines and growth factors, the data
presented here provide a cautionary note on extrapolations to other
species and underscores the need for multiple species analyses of
cytokine functions.
The TR rat model of constitutive expression of IFN- in the eye
provides a unique opportunity to dissect the molecular and
immunopathogenic mechanisms involved in anterior and posterior uveitis.
Although IFN- plays a very important role in activating lymphocytes
and mononuclear cells that mediate autoimmune attack of photoreceptor
cells, our results suggest that its paracrine actions may be equally
important and may have an even more insidious effect on predisposition
to autoimmune diseases. As shown in this study, the induction of ICAM-1
gene expression precedes the recruitment and immigration of T cells
into the retina. Both events occur much earlier in the TR eye and are
strongly correlated with the temporal expression of EAU. This
underscores the role of IFN--mediated lymphoid cell extravasation in
the pathogenesis of EAU and suggests that a consequence of constitutive
exposure of ocular cells to IFN-, as occurs during chronic
infections of the eye, is predisposition to a more rapid onset uveitis
in response to pathogens or exposure to a uveitogen. Our data also
suggest that the exact role of IFN- in an organ-specific autoimmune
disease, either protection or exacerbation of the disease, would depend
on the species.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: EAU, experimental autoimmune uveoretinitis; IRBP, interphotoreceptor retinoid-binding protein; RT, reverse transcription; TR, transgenic rats; PMSG, pregnant mare serum chorionic gonadotropin; WT, wild type; IRF, IFN regulatory factor; NO, nitric oxide; LH-RHa, luteinizing hormone releasing hormone antagonist; HCG, human chorionic gonadotropin; GAS, -activation sequence; EAE, experimental allergic encephalomyelitis; EIU, endotoxin-induced uveitis; ICSBP, IFN consensus sequence-binding protein.
Received for publication June 9, 1998. Accepted for publication September 15, 1998.
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References |
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