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*
Department of Pathology, University of Geneva, Geneva, Switzerland; and
Division of Rheumatology/Allergy and Clinical Immunology, Department of Internal Medicine, University of California, Davis, CA 95616
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain
is very effective in protection against murine lupus. To investigate
the specific contribution of select H-2 haplotypes on the
Ead transgene-mediated disease-suppressing
effect, we generated H-2 congenic (NZB x BXSB)F1
hybrid mice bearing either H-2b/b, H-2d/b, or
H-2d/d haplotype, and compared the transgene-mediated
protective effect on the clinical development (autoantibody production
and glomerulonephritis) of lupus in these F1 hybrids. The
level of protection was most remarkable in mice bearing the
I-E- H-2b/b haplotype but was only minimal in
I-E+ H-2d/d F1 hybrids. Additional
analysis demonstrated a marked suppression of lupus in I-E+
H-2k/k (MRL x BXSB)F1 hybrid mice,
indicating that the transgene is able to suppress autoimmune responses
even in mice already expressing I-E molecules at a homozygous level.
Our results indicate that the level of the transgene-mediated
protection is dependent on the host H-2 haplotype. This suggests that
the autoimmune suppressive activity of the
Ead transgene is likely to be determined
through the interaction of the transgene product with the host MHC
class II molecules, providing new insight into the role of MHC in
lupus-like autoimmunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain, which restores the I-E expression
at the homozygous level (8, 10). This strongly suggests
that the reduced susceptibility associated with the
I-E+ H-2d and
H-2k haplotypes (vs the
I-E- H-2b) can be related
to the expression of the Ea gene, which is absent in the
H-2b mice because of the deletion of the promoter
region of the Ea gene (11).
Studies on lupus-prone mice expressing different levels of the
Ead transgene revealed that the
protection conferred by the transgene is dependent on its level of
expression, but not on that of whole I-E molecules on the surface of B
cells (12, 13). In addition, it has been observed that the
level of protection markedly differs among lupus-prone mice studied:
the highest in BXSB, intermediate in (MRL x
BXSB)F1, and the lowest in (NZB x
BXSB)F1 and (NZW x
BXSB)F1 mice (10, 12, 13). This
indicates that the disease-suppressing effect of the
Ead transgene is likely influenced by
the presence or absence of specific disease-associated alleles present
in different lupus-prone strains. Although the precise mechanisms
responsible for the Ead
transgene-mediated protection of SLE have not yet been defined, the
demonstration that autoantibodies were selectively produced by
nontransgenic B cells in transgenic and nontransgenic double bone
marrow chimeras indicated that B cells, and not T cells, are the major
site of the transgene effect on the suppression of autoimmune responses
(10, 14). Thus, we postulated that
Ead transgene expression may lead to
interference with an efficient interaction between autoreactive T and B
cells by modulating the presentation of pathogenic self-peptides by MHC
class II molecules; this can result from increased formation of I-E
-chain-derived peptides (E peptides) displaying a high affinity
to the I-A molecules or from the induction or enhanced expression of
mixed-haplotype I-E molecules (10, 12, 13). If this is
indeed the case, the protective ability of the
Ead transgene can be markedly
influenced by the host H-2 haplotype because of variabilities in the
binding affinity of individual MHC class II molecules to E peptides
and in the expression level of potential autoimmune-inhibitory
mixed-haplotype I-E molecules in the transgenic mice bearing different
H-2 haplotypes.
Due to the potential importance of this thesis, we generated (NZB x BXSB)F1 hybrids bearing the H-2b/b, H-2d/b, or H-2d/d haplotype and (MRL x BXSB)F1 hybrids bearing the H-2k/b or H-2k/k haplotype, and compared the level of the Ead transgene-mediated protection from autoimmune manifestations (autoantibody production and glomerulonephritis) in relation to the host H-2 haplotype. We report in this paper that the autoimmune inhibitory effect of the Ead transgene is dependent on the H-2 haplotype, suggesting that the action of the Ead transgene is mediated through interaction with the host MHC class II molecules.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A BXSB Ead transgenic line, BXSB-E-1, which expresses the transgene at a high level, was established as previously described (10). BXSB.H-2d, BXSB.H-2k, and NZB.H-2b congenic mice, created by backcross procedures at the 12th, 12th, and 10th generations, respectively, were described (6, 8, 15). NZB and MRL mice were purchased from Bomholtgard (Ry, Denmark). (NZB x BXSB)F1 and (MRL x BXSB)F1 mice were generated by local breeding. The presence of the transgene in F1 offsprings was screened by Southern blot analysis, as described previously (10, 12). A total of 15–30 mice were serially followed for serology, and when animals were moribund, complete autopsy was performed to establish the mortality rate due to glomerulonephritis. Mice were bled from the retroorbital plexus and resulting sera were kept at -20°C until use.
Serological assays
Serum levels of IgG anti-DNA autoantibodies were determined by ELISA, and results are expressed in titration units, as described previously (16). Serum levels of gp70-anti-gp70 immune complexes (gp70 IC) were quantified by an ELISA combined with the precipitation of sera with polyethylene glycol (average m.w. 6000), and results are expressed as µg/ml of gp70 complexed with anti-gp70 Abs, as described previously (6).
Histopathology
Samples of all major organs were obtained at autopsy, and histological sections were stained with either the periodic acid-Schiff reagent or with hematoxylin and eosin. Glomerulonephritis was scored on a 0-to-4 scale, in blind, based on the intensity and extent of histological changes, as described (17), according to Pirani and Salinas-Madrigal (18). Grades 3 and 4 glomerulonephritis were considered significant contributors to clinical disease and/or death.
Cytofluorometric analysis
The expression of I-E molecules in peripheral blood B cells was
analyzed by first staining them with FITC-conjugated anti-mouse µ
chain (LO-MM-9) mAb (19), and then by incubation
with biotinylated antiE (H81.98.21.1) (20) or
anti-EdEßb (Y-17)
(21) mAb, and then with PE-conjugated streptavidin (Caltag
Laboratories, San Francisco, CA). Lastly, they were analyzed with
FACScan (Becton Dickinson, Mountain View, CA).
Statistical analysis
Survival curves were estimated with BMDP statistical software (22) and compared using the Breslow statistic (23). Statistical analysis for serological parameters was performed with the Wilcoxon two-sample test. Probability values >5% were considered insignificant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the role of the H-2 haplotype on the
Ead transgene-mediated protection of
SLE, (NZB x BXSB)F1 hybrids bearing
different H-2 haplotypes (H-2b/b,
H-2d/b, or H-2d/d) with or
without the transgene were generated, and the effect of the transgene
on the clinical development of SLE was assessed in these
F1 female mice. (NZB x
BXSB)F1 nontransgenic females of
H-2b/b, H-2d/b, and
H-2d/d haplotypes developed typical SLE; 50% of
them died of glomerulonephritis by 7, 9, and 15 mo, respectively (Fig. 1
). Although the high-level expression of
the transgene was very effective in the protection from SLE occurring
in both H-2b/b and H-2d/b
F1 females, the protective effect of the
Ead transgene was far stronger in the
H-2b/b F1 females (Fig. 1).
In fact, two-thirds of the H-2b/b
F1 females bearing the transgene were still alive
at 18 mo of age, whereas 50% of the H-2d/b
F1 female transgenics died of glomerulonephritis
by 17 mo of age (p < 0.001). Most strikingly,
the expression of the transgene barely altered the survival rate of the
H-2d/d F1 females
(p > 0.1; Fig. 1).
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). Both autoantibody responses were
nearly completely suppressed in the H-2b/b
F1 female transgenics at any age tested (4, 6,
and 8 mo of age; p < 0.001), but the level of the
suppression was significantly less in the H-2d/b
female transgenics, despite comparable development of these
autoantibody responses in both H-2d/b and
H-2d/b nontransgenics. Consistent with a marginal
effect of the transgene on the survival rate in the
H-2d/d F1 females, these
mice exhibited small decreases in serum levels of gp70 IC at 6 and 8 mo
of age (p < 0.001) and of IgG anti-DNA
autoantibodies at 8 mo of age (p < 0.05).
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), and significantly
prolonged their survival rate (50% mortality rate due to
glomerulonephritis: 8 mo in transgenics and 5 mo in nontransgenics)
(Fig. 2). In contrast, significant but
relatively small decreases in serum levels of gp70 IC, but not of IgG
anti-DNA autoantibodies, were noted in F1
males bearing either the H-2d/b or
H-2d/d haplotype, whose life-spans were barely
extended by the presence of the transgene (50% mortality rate in
H-2d/b mice: 7 mo in transgenics and 6 mo in
nontransgenics; 50% mortality rate in H-2d/d
mice: 8 mo in transgenics and 7 mo in nontransgenics).
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(H81.98.21.1) mAb, at levels
comparable to those found in nontransgenic H-2d/d
F1 hybrid mice (Fig. 3A). However, as expected, the
surface density of a mixed-haplotype I-E molecule,
EdEßb, as recognized
by the Y-17 mAb, was higher in the H-2b/b
transgenic F1 hybrids than that of the
H-2d/b F1 transgenics,
although the latter mice expressed a higher level of the
EdEßb heterodimer than
their nontransgenic H-2d/b littermates did (Fig. 3B).
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The data obtained with (NZB x BXSB)F1 hybrid mice bearing the three different H-2 haplotypes have shown that the protective ability of the Ead transgene is strongly linked to the host H-2 haplotype. Because the disease-suppressing activity of the transgene was the lowest in the H-2d/d (NZB x BXSB)F1 hybrids already expressing I-E molecules at a homozygous level, we studied the protective ability of the transgene in lupus-prone mice bearing another I-E+ haplotype, H-2k. Because NZB mice bearing the H-2k haplotype are not available, we generated (MRL x BXSB)F1 hybrids bearing either H-2k/b or H-2k/k haplotype, and the effect of the transgene on the clinical development of SLE was assessed in both H-2k/b and H-2k/k F1 male mice (only male hybrids carrying the Yaa gene are able to develop a lethal form of SLE) (24).
The transgene-induced suppression of a lupus-like syndrome was highly
significant in both H-2k/b and
H-2k/k (MRL x BXSB)F1
transgenic males (Fig. 4). The 50%
cumulative mortality rates of both transgenic F1
male hybrids were 14 and 15.5 mo, respectively, compared with their
nontransgenic male littermates (H-2k/b, 5.5 mo;
H-2k/k, 8 mo). The greatly prolonged survival in
the transgenic F1 males was reflected most in
serum levels of gp70 IC (Table III). At 4
mo of age, serum gp70 IC concentrations in both
H-2k/b and H-2k/k
F1 transgenic males were markedly reduced
compared with those of nontransgenic F1 male mice
(p < 0.001). A partial reduction in IgG
anti-DNA levels was seen only in the H-2k/b
F1 males (p = 0.01) and
not in the H-2k/k F1 males
(p > 0.1). These results suggest that the
protective capacity of the Ead
transgene may be more effective in the H-2k/b
F1 males than in the H-2k/k
F1 males. This was further supported by the
analysis of their F1 female hybrids, which
develop a mild, but not lethal, form of lupus. The expression of the
transgene reduced the production of IgG anti-DNA autoantibodies
only in H-2k/b (MRL x
BXSB)F1 female hybrids (p
< 0.001), and not in H-2k/k
F1 females (p > 0.1)
(Table III). Although the H-2k/k
F1 females developed low levels of gp70 IC
detectable at 8 mo of age, its production was completely inhibited by
the presence of the transgene (p < 0.01), as
is the case for the H-2k/b
F1 females (p < 0.001).
Notably, the surface density of I-E molecules, as determined by
staining with anti-E mAb, on circulating B cells from these two
different transgenic mice was comparable to that found in nontransgenic
H-2k/k F1 hybrid mice (data
not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The comparative analysis of (NZB x BXSB)F1
hybrid mice bearing three different H-2 haplotypes has demonstrated
that the inhibitory effect of the Ead
transgene on clinical development of SLE (autoantibody production and
glomerulonephritis) is the strongest with the
H-2b/b haplotype, intermediate with the
H-2d/b haplotype, and the weakest with the
H-2d/d haplotype (Table IV). This strongly suggests that the
disease-suppressing effect conferred by the transgene is determined by
specific mechanism(s) linked to the host H-2 haplotype. This is
consistent with the observation that the autoimmune-inhibitory effect
mediated by the transgene is somehow more selective for anti-gp70
autoantibody than for anti-DNA Ab, as best shown in
H-2k/k (MRL x BXSB)F1
hybrid mice. All these data strongly argue against the idea that the
prevention of SLE results from a nonspecific functional defect in B
cells (and/or other APCs) secondary to overexpression of the transgene,
as was the case in mice bearing a high-copy number of the
Abk transgene (25).
Instead, as discussed below, the action of the
Ead transgene may be more
specifically involved in the process of autoimmune responses.
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).
Our demonstration, a remarkable association of the
Ead transgene-mediated protective
effect with the host H-2 haplotype, favors a model that the transgene
expression in B cells may modulate the presentation of pathogenic
self-peptides by MHC class II molecules (I-A and/or I-E), thereby
interfering with an excessive activation of potential autoreactive T
and B cells. We propose two possible mechanisms of modulation for
autoantigen presentation as a result of either excessive generation of
E peptides with a high affinity to the I-A molecules, thereby
decreasing the use of I-A molecules for presentation of pathogenic
self-peptides, or the induction or increased production of unique
mixed-haplotype I-E molecules, such as
EdEßb, which interfere
with the presentation of pathogenic determinants by other MHC class II
molecules as a consequence of capturing self-peptides (10, 12, 13, 26). In this regard, it is important to note that the
transgenic H-2d mice express only a single type
of I-Ed molecule and not additional novel I-E
molecules with different peptide-binding specificities (Table IV). This
likely is also the case in mice bearing the H-2k
haplotype. The transgenic H-2k mice can produce a
novel mixed-haplotype
EdEßk heterodimer
(Table IV); however, its peptide-binding specificity should be
identical with that of the conventional I-Ek
(EkEßk) molecule
because a single amino acid difference localized at the junctional
region between the 2 domain and the transmembrane domain between the
extracellular region of the Ed and
Ek chains cannot affect the peptide-binding
specificity of these two I-E heterodimers (27). Thus, a
marked suppression observed in the H-2k/k
(MRL x BXSB)F1 transgenic mice supports the
importance of the E peptide-dependent mechanism for the inhibition
of autoantibody production. In addition, the protection of the disease
in the H-2k/k transgenic mice, whose expression
of I-E molecules does not quantitatively and qualitatively differ from
nontransgenic littermates, further argues against the proposal that the
transgene effect is a consequence of thymic selection of a harmful
autoreactive T cell repertoire.
A relatively limited protection in the H-2d/d
transgenic mice expressing only the conventional
I-Ed molecules was unexpected because the
I-Ad molecule has been shown to have a high
affinity to one of the E peptides (28). However,
expression of the two endogenous Ead
genes in the H-2d mice may be sufficient to
inhibit the autoimmune responses mediated by I-Ad
molecules because the introduction of two copies of the
Ead transgene is able to almost
completely prevent the development of SLE in BXSB
(I-E-, H-2b) mice
(8), whose I-Ab molecule also has a
high affinity to the E peptide, like the I-Ad
molecule (29). If this is so, it can be speculated that
I-Ed molecules may be responsible for the
development of relatively weak autoimmune responses occurring in the
H-2d/d F1 hybrid mice and
that these I-Ed-dependent autoimmune responses
can only be slightly suppressed, if at all, by the E
peptide-dependent mechanism, because of a possible limited affinity of
the I-Ed molecules to E peptides. In contrast
to the H-2d and H-2k mice,
the H-2b mice express only the mixed-haplotype
EdEßb heterodimer in
the presence of the transgene (Table IV), and its expression level is
much higher than that seen in H-2d/b and
H-2k/b heterozygous mice. If the
EdEßb heterodimer is
indeed implicated in the autoimmune suppressive activity, the best
protection observed in H-2b mice can be explained
by the additive effect of the E peptide-mediated and
EdEßb mixed
haplotype-mediated inhibition of the presentation of pathogenic
self-peptides by MHC class II molecules.
The present results indicate that the protective effect conferred by
the Ead transgene is likely to be much
more complex than we thought initially, but support that the overall
disease-suppressing effect of the Ead
transgene can be determined by several different factors. They include:
1) the affinity of individual MHC class II molecules to peptides
derived from the transgene product, I-E -chains
(28, 29, 30); 2) the expression level of potential autoimmune
inhibitory mixed-haplotype I-E molecules generated in the transgenic
mice; and 3) the relative contribution of individual MHC class II
molecules to the development of autoimmune response characteristics of
SLE. Accordingly, the interpretation for a better protection observed
in H-2d/b heterozygous (NZB x
BXSB)F1 mice than H-2d/d
F1 mice and in H-2k/b
(MRL x BXSB)F1 mice than
H-2k/k F1 mice is complex
because these H-2 heterozygous mice express multiple I-A and I-E
molecules in which the precise contributions of each class II molecule
to the development of SLE and whose affinities to E peptides have
been poorly defined. This would also explain why the introduction of
the Eaz transgene in (C57BL x
NZB) x NZB backcross mice has no effect on the development of
autoantibody production (31).
In conclusion, our data indicate that the H-2 haplotype is an important genetic factor which controls the protective effect of the Ead transgene. Because the expression of two copies of the Ea gene is capable of providing protection from SLE (8, 9, 32), the possible role of the MHC class II Ea gene as a lupus-protective gene in mice is likely to be influenced by the host MHC haplotype. However, it should be stressed that the effect of the Ead transgene can be modulated by genetic factors other than the MHC class II genes present in the genetic background of individual lupus-prone mice. This was best exemplified by the demonstration that the level of the protection conferred by the transgene is complete in H-2b BXSB males (10) but only partial in H-2b/b (NZB x BXSB)F1 male mice, as shown in this study. Furthermore, our present and previous studies also revealed that the protective effect of the transgene is counteracted by the presence of the Yaa gene in mice highly predisposed to SLE such as (NZB x BXSB)F1 and (NZW x BXSB)F1 mice. This observation is consistent with the thesis that the action of the Yaa gene may be implicated in the process of efficient interaction of autoreactive T and B cells (24, 33, 34). Clearly, further understanding of the Ead transgene-mediated protective mechanism and the Yaa gene-mediated accelerating mechanism would help elucidate the molecular and cellular basis central to the development of murine SLE.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Shozo Izui, Department of Pathology, Centre Médical Universitaire, 1211 Geneva 4, Switzerland. E-mail address:
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; E peptides, I-E -chain-derived peptides; gp70 IC, gp70-anti-gp70 immune complexes; Yaa, Y-linked autoimmune acceleration.
Received for publication August 30, 1999. Accepted for publication October 13, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain. J. Exp. Med. 178:1189. chain transgene: protective role of I-E chain-derived peptides with a high affinity to I-Ab molecules. Eur. J. Immunol. 26:307.[Medline]
u and Eßu chain association: where lies the anomaly?. J. Immunol. 137:948.[Abstract]
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