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,,,
*
Department of Medicine and Division of Rheumatology and Clinical Immunology,
Center for Mammalian Genetics, and
Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL 32610
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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 development of genome-wide scans has provided new tools for dissecting the pathogenesis of multigenic diseases 17, 18, 19, 20 . Several groups have utilized this approach to identify the positions of loci contributing to lupus susceptibility and currently the positions of at least nine different lupus susceptibility loci have been identified 21, 22 . Our group has concentrated on the NZB/NZW-derived NZM2410 strain 23 in which we have identified three major genomic intervals linked to early onset glomerulonephritis 20 . Each of these SLE susceptibility intervals was subsequently transferred individually onto the nonautoimmune C57BL/6 genome using marker-assisted selection to produce "speed congenics" 24, 25 . Initial characterization of the new congenic strain carrying Sle1, termed B6.NZMc1, detected a loss of tolerance to chromatin at both the T and B cell levels 26, 27 . B6.NZMc1 mice spontaneously produce IgG autoantibodies specific for subnucleosome components of chromatin and develop spontaneous autoreactive T cells responding to histone epitopes 26, 27 . This limited disease, despite a clear break in tolerance, lends support to the threshold liability model for disease susceptibility 18, 20 .
In this report, we use adoptive transfer experiments to demonstrate that Sle1 is functionally expressed in B cells, and that expression in B cells is essential to break tolerance to nuclear autoantigens and develop humoral autoimmunity. These findings provide important new insights into the cell lineages affected by Sle1 and suggest that a loss of tolerance in the B cell compartment can potentiate the activation of T cells to provide signals required for the development of IgG humoral autoimmunity. These results also illustrate the efficacy of congenic strain construction for the dissection of a complex genetic. This work, then, is a crucial first step that establishes the feasibility of using congenics and partial phenotypes in elucidating the complex cellular interactions leading to lupus nephritis in the NZM2410 mouse.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6) and C57BL/6.Thy-1a Igha Gpia (B6.TC) mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and bred in our animal colony. The NZM2410 strain was originally obtained from U. H. Rudofsky (New York State Health Department) and also maintained in our facility. The development of the B6.NZMc1 congenic strain, having a 37-cM interval derived from chromosome 1 of NZM2410, has been described previously 24 . This interval, defined by the markers D1 MIT101 and D1 MIT155, contains the 95% confidence limits for inclusion of Sle1, as determined previously and is of NZW origin 20, 24 .
Preparation of chimeras
A week before cell transfer, mice were placed on sterilized tap
water supplemented with trimethoprim-sulfamethoxazole (1% v/v; Lemmon,
Sellerveill, PA). On the day before cell transfer, host mice were
lethally irradiated with two doses of 525 rad 
-irradiation (3 h
apart) in a Gamma-cell 40 137Cs apparatus (Atomic Energy of
Canada, Ottawa, Canada). A trial experiment with Ig allotype-congenic
B6.TC mice showed that with this split-dose regimen, all circulating B
lymphocytes were of donor origin when examined 2 mo after transfer. B
cells were prepared as described previously, including the use of mAb
and complement to eliminate mature T cells 2 . Both donor and
recipient mice were 2 to 3 mo of age and were sex matched. Each mouse
was given 107 cells in 0.5 ml of HBSS by tail vein
injection.
Flow cytometry
Single-cell suspensions of spleen cells were prepared, followed by lysis of RBC in 0.83% NH4Cl. Cells were first blocked with staining media (PBS with 5% horse serum and 0.05% NaN3) supplemented with 10% normal rabbit serum. Cells were then incubated on ice with preoptimized amounts of labeled mAbs. For the first set of experiments, directly fluoresceinated anti-B220, directly phycoerythrin (PE)-conjugated anti-CD4, and biotinylated anti-CD69, all purchased from PharMingen (San Diego, CA), were used. After washing, cells were incubated in streptavidin (Av)-Quantum Red (QR) (Sigma Chemical, St. Louis, MO), also on ice. For the second group of experiments, in which equal numbers of B cells were cotransferred into recipient mice, alternative combinations of Abs were used to allow the donor origin of the T and B cells to be ascertained. For T cell phenotypic determination, directly fluoresceinated anti-CD90.2 (anti-Thy-1b) was used along with Cychrome C (CyC)-conjugated anti-CD4 or anti-CD8 (PharMingen). For the third color, biotinylated anti-CD69 or anti-CD25 were used, followed by AvPE. This combination was found to give lower background than with AvQR. The T cells of B6.TC origin were identified as CD4+CD90.2- or CD8+CD90.2-, as appropriate. To verify the donor origin of these cells, a combination of anti-CD4-CyC (or anti-CD8-CyC), biotinylated CD90.1(anti-Thy 1a), and fluoresceinated anti-CD90.2 was also used. For phenotypic analysis of B cells, the combination of either directly fluoresceinated anti-IgMa or anti-IgMb and CyC-conjugated anti-B220 was used. As the third color, either biotinylated anti-CD69, -CD25, -B7.1, or -B7.2 was added, again followed by AvPE. Cells were analyzed using a FACScan (Becton Dickinson, San Jose, CA). Dead cells were excluded by forward angle and side scatter profiles. At least 20,000 cells were collected per sample.
Ig assay by ELISA
The anti-dsDNA ELISA has been previously described 28 .
Briefly, methylated BSA precoated 96-well microtiter plates (Immulon
II, Dynatech, Chantilly, VA) were coated overnight with 50 µg/ml calf
thymus dsDNA (Sigma). The plates were then blocked with PBS
supplemented with 3% BSA, 0.1% gelatin, and 3 mM EDTA. Sera were
diluted 1/100 and incubated in duplicate for 2 h at room
temperature. For total IgG titers, development was with an alkaline
phosphatase-conjugated anti-mouse IgG (-chain-specific, Jackson
Immunoresearch, West Grove, PA). Final step was with
p-nitrophenyl phosphate (pNPP) substrate (Sigma), in 0.01 M
diethanolamine, pH 9.8. Results have been displayed in arbitrary
units/ml (a.u./ml) based on serial dilution of a standard positive
serum.
Anti-H2A/H2B/dsDNA autoantibodies were assayed on subnucleosome particles which were reconstituted by the addition of 10 µg/ml of a 1:1 mixture of histones H2A and H2B (Boehringer Mannheim, Indianapolis, IN) to plates coated with dsDNA, as above. Sera were diluted 1/100 to 1/400, and development was as outlined above for anti-dsDNA.
For total IgM determination, the plates were coated with affinity-purified monoclonal rat anti-mouse IgM (Bet 2, American Type Culture Collection, Rockville, MD) at a concentration of 3 µg/ml. Following blocking, sera were added at a dilution of 1/1000 and development was with rat Ig-absorbed biotinylated donkey anti-mouse IgM (Jackson ImmunoResearch), followed by avidin-alkaline phosphatase (Sigma) and pNPP substrate. For total IgG determination, the first-step coating was with F(ab')2 donkey anti-mouse IgG (Jackson ImmunoResearch) at 2 µg/ml, followed by blocking. After the addition of sera and washing, development was with polyclonal rabbit anti-IgG2a (Nordic Immunological Labs, San Clemente, CA) followed by alkaline phosphatase-coupled donkey anti-rabbit IgG (Jackson Immunoresearch) absorbed against mouse Ig and pNPP substrate (Sigma).
When necessary, allotype-specific IgM and IgG2a determinations were conducted for the above specificities. In all cases, the coating and blocking steps were conducted as outlined above. However, plates were prepared in triplicate, and an aliquot of diluted sera was added in duplicate to each plate. For IgM allotype-specific assays, the three plates were developed in parallel with biotinylated anti-IgMa (DS1, PharMingen), anti-IgMb (AF6-78.25, PharMingen), or allotype nonspecific donkey anti-mouse IgM. For IgG2a allotype-specific assays, the three plates were developed in parallel with polyclonal rabbit anti-IgG2aa or anti-IgG2ab, or allotype nonspecific anti-IgG2a, (Nordic Immunological Labs). While already highly allotype-specific, the small amount of residual cross-reactivity in the anti-IgG2aa reagent was removed by adsorption to Sepharose 4B-coupled B6 Ig immediately before use. For all subsequent steps, development was as given for equivalent allotype-nonspecific determinations. The a and b allotype titers were compared with each other by using serial dilutions of standard sera from a B6/lpr-Igha (allotype a) and a B6/lpr mouse adjusted in dilution to give equivalent optical density readings when developed by the allotype nonspecific reagent.
Statistics
Comparisons between two groups were performed by Student’s t test. Comparisons among groups were by one-way ANOVA with pairwise testing of all combinations performed by the Student-Newman-Keuls test. Results were calculated with SigmaStat for Windows V1.02 (Jandel Scientific, San Rafael, CA). p values less than 0.05 were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that Sle1 in B6.NZMc1 causes a
break in tolerance to H2A/H2B/dsDNA subnucleosome particles and that
CD69 expression on CD4+ T cells is increased 26 . More
recently, we have also detected a statistically significant increase in
B7.2 expression in B cells of aged B6.NZMc1 mice relative to their B6
congenic partner. A comparison of the expression of these phenotypes in
B6.NZMc1 and B6 mice is presented in Table I
. These results indicate that
Sle1 is capable of mediating autoimmune phenotypes in both T
and B cell compartments.
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The first set of experiments were designed to determine whether
Sle1 was functionally expressed on cells of bone marrow
origin or on a radioresistant host population (or both). To accomplish
this, B6.NZMc1 and B6 host mice were lethally irradiated and given
either B6.NZMc1 or B6 bone marrow. Syngeneic reconstitution of B6 and
B6.NZMc1 were performed to provide negative and positive controls,
respectively, for the possible modulating effects of radiation. Sera
were collected from all mice and assayed for IgG anti-dsDNA and
anti-H2A/H2B/DNA by ELISA as described previously 26 . Results
from the analysis of mice at 12 mo of age are shown in Fig. 1. As expected, syngeneic reconstitution
of normal B6 mice resulted in low titers of autoantibodies, and
syngeneically reconstituted B6.NZMc1 mice had elevated titers of
anti-subnucleosome Abs. As shown by the comparison of the remaining
two groups, bone marrow derived from B6.NZMc1 mice was both necessary
and sufficient to mediate ANA production in B6 host mice, while B6 bone
marrow failed to produce autoantibodies in B6.NZMc1 host. Comparison of
the two groups receiving B6.NZMc1 bone marrow showed no statistically
significant host effect. Interestingly, the titers of IgG
anti-subnucleosome Abs tended to be higher in female mice. Although
the age of onset of renal disease in the parent NZM2410 strain trended
toward a female bias and anti-dsDNA autoantibody titers tend to be
higher in female mice 23 , these trends are not statistically
significant with our current sample size. It is possible that this
gender bias is enhanced in the chimeras, possibly via a differential
effect of radiation allowing a subtle difference to be amplified
between the sexes. In support of this hypothesis, sex-specific
differences in immune response in irradiated, autoimmune mice have been
reported 29 . The IgG anti-dsDNA response at 1 year of age
followed the same trend, although the differences were not as great
(data not shown). This was not surprising, given the low penetrance
(30%) for this specificity in unmanipulated B6.NZMc1 mice 26 .
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Characteristically, B6.NZMc1 mice have shown an age-dependent
increase in the number of activated CD4+ T cells such that
by 12 mo of age there is approximately a 50% increase over age-matched
congenic B6 controls 26 . The chimeric mice were similarly tested for
CD69 expression at 12 mo. As shown in Fig. 2, the total percentage of
CD69+ CD4+ T cells in mice receiving B6.NZMc1
bone marrow was significantly increased in comparison with mice
receiving B6 bone marrow (p < 0.02). These
results are consistent with the autoantibody production data presented
above and indicate that increases in the frequencies of activated
CD4+ T cells are mediated by the expression of
Sle1 in B cells.
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The above experiments demonstrated that bone marrow from B6.NZMc1 mice could successfully transfer the anti-subnucleosome autoantibody phenotype but shed no light on the lineage of hemopoietic cells that must express Sle1 in order for autoimmunity to develop. To address this issue, cotransfer experiments were conducted using the nonautoimmune C57BL/6.Thy-1a Igha Gpia (B6.TC) congenic strain in combination with B6.NZMc1. B6.TC is a B6 congenic strain carrying three introgressed genes which express allelic variants of B6: Thy-1 (chromosome 9); Igh (chromosome 12); and Gpi (chromosome 7). B6.TC mice do not produce autoantibodies and have been successfully used in chimera experiments with gld mice 8 .
Following lethal irradiation, B6.NZMc1 mice were given T cell-depleted
bone marrow in the following three combinations: 1 B6.TC and B6; 2
B6.TC and B6.NZMc1; and 3 B6.NZMc1. Chimerism was assessed
periodically by two-color allotype-specific flow cytometry of PBL and,
at the end of the experiment, on splenocytes. In addition, serum Ig
levels were quantitated by IgM and IgG2a allotype-specific ELISA. Two
separate experiments were conducted, one in which the host mice were
all B6.NZMc1 and the second in which host mice were (B6.NZMc1 x
B6.TC)F1. Because the results were comparable, they have
been combined. Mice given a combination of bone marrow from normal
B6.TC (Igha; Thy-1a) and either normal B6
(Ighb; Thy-1b) or autoantibody-prone B6.NZMc1
(Ighb; Thy-1b) congenic strains had cellular
(Table II) and serologic (Table III) evidence of repopulation by both
donors. The peripheral blood results are shown for mice 3 mo after
reconstitution; results were similar when examined at 5 mo (data not
shown). Interestingly, the relative proportion of T and B cells derived
from the normal B6.TC donor appeared to be greater when coinfused with
B6.NZMc1 than with normal B6 bone marrow. This trend was seen across
two separate experiments in both males and females and therefore
represents four independent determinations. Better balance was seen in
IgD staining, a pattern we have also seen in analogous combinations
using the single congenic B6.C20 (Igha; Thy-1b)
strain. Studies performed on splenic lymphocytes 1 year after
reconstitution showed similar results, although the composition was
more strongly tilted toward the non-B6.TC donor. There were no
consistent differences in the way the T and B cell compartments were
repopulated between the donors, suggesting that there was no selective
expansion or elimination due to cell type-specific expression of the
allelic differences. In B6.NZMc1 host mice receiving only B6.NZMc1 bone
marrow, no evidence of B6.TC-derived cells could be found, confirming
the allele-specific properties of the assays. However, in
(B6.NZMc1 x B6.TC)F1 host mice, a small percentage of
Thy-1a/b T (i.e., host-derived) cells could be found in the
spleen. Typically, these amounted to 1% of the total lymphocyte
population and were no more than 3% (data not shown). In these same
mice, no a allotype B cells could be found, as would have been expected
if host-derived B cells survived radiation.
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). Compared with IgM, serum total
IgG2a was more disproportionately of the b allotype, a finding that we
have also seen before 1, 2 . Again, relatively more IgM and IgG2a
derived from the B6.TC donor were present when the bone marrow was
paired with B6.NZMc1. However, in all cases, evidence of double
chimerism was present in mice coinfused with B6.TC bone marrow. Sle1 was functionally expressed on B cells
To determine whether B cells derived from B6.TC and B6.NZMc1 mice
would produce anti-H2A/H2B/dsDNA autoantibodies in autoimmune
double-chimeric mice, IgG2a anti-H2A/H2B/dsDNA titers were
determined using an allotype-specific ELISA. Results are shown for mice
1 year after reconstitution (Fig. 3).
Mice receiving only B6.NZMc1 bone marrow had high titers of IgG2a
autoantibodies, consistent with earlier results for total IgG, while
mice receiving a combination of normal B6.TC and normal B6 bone marrow
were essentially negative. In mice receiving a combination of normal
B6.TC and autoantibody-prone B6.NZMc1 bone marrow, the autoantibody
response was exclusively of the b allotype, strongly suggesting that
Sle1 must be expressed in a B cell to mediate a loss of
tolerance to nuclear Ags. Similar to the results of the first set of
experiments, penetrance of the B6.NZMc1 phenotype was 
50% in these
chimeras, somewhat decreased from the 80% typically seen in
unmanipulated mice. This penetrance was not decreased by dilution by
B6.TC-derived B cells (6 of 14 vs 7 of 14 for B6.NZMc1 alone;
p > 0.9).
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By allotype-specific three-color flow cytometry, B cells were
phenotyped for expression of a number of activation markers, including
CD80 (B7.1), CD86 (B7.2), CD25, and CD69. No consistent differences
were seen in any of these parameters with the exception of B7.2, which
is inducibly expressed on B cells. Compared with the other activation
markers, B7.2 staining did not result in the delineation of two
populations. Instead, differences were measured in median expression,
and an example is shown in Fig. 4. Here,
the profile of B220+ cells is displayed and the quadrants
are drawn to find the median level of expression of B7.2 for B6.TC- and
B6-derived B cells, as identified by allele-specific IgM expression.
Only modest differences in median level were seen based on allotype
(Fig. 4A, left). In mice receiving only B6.NZMc1
bone marrow, nearly three-fourths of the B cells were above this median
level (Fig. 4A, right). In mice receiving a
combination of B6.TC and B6.NZMc1, two-thirds of the B6.NZMc1-derived B
cells were above the median seen for B cells of B6.TC origin (Fig. 4A, middle). A compilation of the levels of
expression of B7.2 in mixed chimeras strongly suggest that this
up-regulation of B7.2 is due to an intrinsic property of B cells
expressing Sle1 (Fig. 4B).
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Similar phenotypic studies were conducted on T cells using the
Thy-1 allelic marker. Our earlier results had indicated that mice
receiving B6.NZMc1 bone marrow had an increased percentage of
CD4+ T cells expressing the very early activation Ag CD69
(Fig. 2). To determine whether this was an intrinsic property of T
cells expressing Sle1, spleen cells were subjected to
three-color flow cytometric analysis. After first gating on
CD4+ cells, the two-color profile of CD69 and
allele-specific CD90 expression was displayed. A compilation of these
results is shown in Fig. 5. Compared with
our earlier data, only two mice showed clearly elevated levels of CD69
expression on CD4+ T cells, and in both cases the
B6.TC-derived T cells showed equivalent or greater expression. Thus,
these data suggest that the activation of CD4+ T cells to
elevate CD69 expression is a secondary consequence rather than an
intrinsic property.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our initial experiments, we transferred bone marrow reciprocally between the two congenic strains B6 and B6.NZMc1. To rule out the possibility of radiation-induced modifications of phenotype, we also included syngeneic reconstitution. These studies established that Sle1 was functionally expressed in cells of bone marrow origin. Moreover, we were able to recapitulate the phenotypes in radiation chimeras that had been found in unmanipulated mice. However, these experiments did not determine which lineages were functionally expressing the locus. To ascertain this, additional experiments were conducted in which congenic normal and B6.NZMc1 bone marrow were coinfused into lethally irradiated young B6.NZMc1 and (B6.NZMc1 x B6.TC)F1 mice. The congenic normal B6.TC strain was chosen because there were detectable allelic differences in a T cell (CD90) and a B cell (Igh) marker. Over time, the T and B cells derived from each donor would all be positively and negatively selected under identical conditions, reconstituting an intact immune system. Any differences in phenotype between the donor T and B cells should be attributable as an intrinsic property of that cell population conferred by expression of the NZMc1 interval. In fact, our experiments established that B cells of B6.NZMc1 origin behaved differently than B6.TC B cells developing in the same environment. In the absence of Sle1, B6.TC- and B6-derived B cells were much more comparable in their behavior. These results strongly imply that Sle1 is functionally expressed on B cells and contributes to a loss of humoral tolerance to nuclear Ags, an important component of SLE.
There are some issues that must be addressed regarding interpretation of our results. First, despite coinfusion of an equal number of B cells from the two donor strains, reconstitution was unbalanced and favored the b allotype strains. We considered the possibility of allotype suppression 30 or problems of minor histocompatibility differences and therefore repeated the experiment with (B6.NZMc1 x B6.TC)F1 host mice. Surprisingly, reconstitution ratios were unchanged. The imbalance raised the concern that lack of detectable anti-subnucleosome autoantibodies of B6.TC origin was merely the result of a numerical disadvantage in the number of precursor B cells. Fortunately, the imbalance was less marked in mice receiving a combination of B6.TC and B6.NZMc1 bone marrow. Despite the increased percentage of B6.TC-derived B cells in mice receiving a combination of B6.TC and B6.NZMc1 bone marrow, these B6.TC-derived cells produced, if anything, less autoantibody than when paired with normal B6 B cells. This clearly shows that the B6.NZMc1 B cells behave differently, indicating that Sle1 changes the intrinsic properties of B cells.
A second issue to consider is one of allotype skewing of the autoantibody response. That is, it is possible that the b allotype has an increased propensity to produce anti-chromatin Ab. This has been reported for allotype heterozygous (B6/lpr x B6/lpr-Igha)F1 mice 31 . Although seen for a few different autoantibody specificities, the amount of skewing was modest for IgG2a anti-chromatin and would not account for the extent of differences seen in the present experiments. Moreover, skewing in the lpr mice was not completely one-sided; a significant minority of mice were predominantly of the a allotype 31 . Finally, inasmuch as we have previously found that Sle1 is expressed in an allele-dose fashion 27 , we tested a cohort of 17 (B6.TC x B6.NZMc1)F1 mice for IgG2a anti-H2A/H2B/dsdDNA. Although of low titer and with incomplete penetrance, the autoantibody response was skewed only about 60:40 toward the b allotype (data not shown).
The observed results cannot be easily explained by transfer of B6.NZMc1
B cells already committed to producing autoantibodies. Donors were
young mice, and autoantibodies did not become evident until many months
after transfer, making it unlikely that a significant number of
activated B cells with anti-subnucleosome specificity were present.
The time course of autoantibody development in the chimeras
recapitulated that which were seen in unmanipulated B6.NZMc1 mice 26
and duplicates previous experience with B6/lpr chimeras 1 .
To otherwise account for these results, it would have to be conjectured
that the generation of autoreactive memory B cells was limited to a
short period early in the life of the B6.NZMc1 mouse, that these cells
arose and homed to the bone marrow without ever secreting detectable
autoantibody in the donor, and that only after many months of latency
in the host did they became Ab-secreting cells. This would appear to be
a very unlikely series of events. Furthermore, in the lpr
system, where the possibility for carryover is likely to be higher due
to a failure in apoptosis, no differences in kinetics, specificity, or
outcome with or without specific depletion of bone marrow B cells was
seen 32 . The finding that B7.2, an important inducible costimulatory
molecule 33 , was differentially expressed on B6.NZMc1 B cells in
B6TC/B6.NZMc1 mixed chimeras further raises our confidence that
Sle1 is functionally expressed on B cells and is an
interesting finding in of itself. The differences in the mixed chimeras
were actually more striking than in the unmanipulated mice (Table I).
The mixed chimeras have the advantage of providing internal controls
for unknown environmental factors, thus allowing subtle differences to
be detected. It has been suggested that B7.2 may positively influence
the development of a Th2 response 34, 35 . Moreover, selective
blockade of CD86 (B7.2) but not CD80 (B7.1) by chronic mAb treatment
resulted in a marked reduction in serum IgG anti-dsDNA titers in
(NZB x NZW)F1 mice 36 . Taken together, it is
unlikely that lack of anti-subnucleosome autoantibody of B6.TC
origin was due to an artifact of the experimental system.
In contrast to our results for B cells, the data on T cells suggest that the increased activation of CD4+ T cells is a secondary event. If so, this would imply that loss of tolerance in the B cell compartment can provide sufficient stimulatory signals to cause activation of the T cell compartment. The data, however, are less conclusive. First, only two mice receiving a combination of B6.TC and B6.NZMc1 bone marrow had substantially elevated CD69 expression. Second, in these older chimeras (13–15 mo total age), we found that in the syngeneically reconstituted B6.NZMc1 mice, a variable subset (ranging from 1 to 5%) of CD4+ T cells had lost CD90 expression and that these cells tended to be CD69+. Therefore, in the mixed chimeras, the CD90.2-CD4+ T cells could have been contaminated with a small number of CD90.2- T cells that were actually of CD90.2+ origin. Unfortunately, although a small population, this contamination was theoretically sufficient to affect our results. Finally, although the abnormal Sle1-expressing B cells may have stimulated both normal- and Sle1-derived T cells, it is uncertain whether nucleosome-specific T cells were derived from both sources. We plan to address the issue of an intrinsic T cell defect more definitively using B6.NZMc1 mice with targeted deletions of T and B cells.
In addition to identifying the functional lineage expression of Sle1, these results have other important implications. First, they demonstrate the feasibility of our approach to dissecting a complex genetic disease. It was feared that mice with only a single susceptibility locus would have such a subtle perturbation of the immune system that a reproducible phenotype would not be identifiable. This has been a problem in similar approaches taken in the analysis of type I diabetes mellitus 37 . Even with a reproducible phenotype, penetrance might have been too low for practical exploitation in small-group adoptive transfer experiments. Additionally, phenotypic expression might have been so delayed in onset as to preclude analysis due to survival problems. Finally, radiation effects might have adversely affected expression of the phenotype. It was therefore important that we could demonstrate that the phenotype could be transferred with high fidelity.
Second, adoptive transfer experiments such as these are important in more narrowly focusing candidate gene searches. For example, the acute phase reactant serum amyloid protein (SAP) is in the middle of the chromosome 1 interval, and differences in SAP titers have been described in some strains of autoimmune mice 38, 39 . Moreover, SAP has been shown to be important in the clearance of chromatin 40, 41 , and the B6.NZMc1 phenotype of increased titers of anti-subnucleosome Abs makes this an intriguing gene. However, this gene is expressed on hepatocytes and would therefore be an unlikely candidate. In fact, this conclusion has been confirmed by direct sequencing 42 and comparable SAP mRNA expression between B6 and B6.NZMc1 (L. Morel et al., unpublished observations).
Finally, we expect that some combinations of susceptibility-loci bi- and tricongenic mice will develop overt SLE. By combining individual susceptibility-loci congenic donors in mixed chimera experiments, we now have the tools to dissect the cellular interactions causing autoimmunity in these very important murine models.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eric S. Sobel, University of Florida Department of Medicine, Box 100221 JHMHC, Gainesville, FL 32610-0221. E-mail address:
3 Current address: Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75235.
4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; B6, C57BL/6; B6.TC, C57BL/6.Thy-1a Igha Gpia, CyC, Cychrome C; a.u., arbitrary units; PE, phycoerythrin; Av, streptavidin; dsDNA, double-stranded DNA. pNPP, p-nitrophenyl phosphate.
Received for publication June 8, 1998. Accepted for publication November 4, 1998.
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 K. R. Kumar, L. Li, M. Yan, M. Bhaskarabhatla, A. B. Mobley, C. Nguyen, J. M. Mooney, J. D. Schatzle, E. K. Wakeland, and C. Mohan Regulation of B cell tolerance by the lupus susceptibility gene ly108. Science, June 16, 2006; 312(5780): 1665 - 1669. [Abstract] [Full Text] [PDF]
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 A. K. Stanic, C. M. Stein, A. C. Morgan, S. Fazio, M. F. Linton, E. K. Wakeland, N. J. Olsen, and A. S. Major Immune dysregulation accelerates atherosclerosis and modulates plaque composition in systemic lupus erythematosus PNAS, May 2, 2006; 103(18): 7018 - 7023. [Abstract] [Full Text] [PDF]
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Z. Xu, B. Duan, B. P. Croker, E. K. Wakeland, and L. Morel Genetic Dissection of the Murine Lupus Susceptibility Locus Sle2: Contributions to Increased Peritoneal B-1a Cells and Lupus Nephritis Map to Different Loci J. Immunol., July 15, 2005; 175(2): 936 - 943. [Abstract] [Full Text] [PDF]
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Y. Chen, D. Perry, S. A. Boackle, E. S. Sobel, H. Molina, B. P. Croker, and L. Morel Several Genes Contribute to the Production of Autoreactive B and T Cells in the Murine Lupus Susceptibility Locus Sle1c J. Immunol., July 15, 2005; 175(2): 1080 - 1089. [Abstract] [Full Text] [PDF]
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M. Wakui, L. Morel, E. J. Butfiloski, C. Kim, and E. S. Sobel Genetic Dissection of Systemic Lupus Erythematosus Pathogenesis: Partial Functional Complementation between Sle1 and Sle3/5 Demonstrates Requirement for Intracellular Coexpression for Full Phenotypic Expression of Lupus J. Immunol., July 15, 2005; 175(2): 1337 - 1345. [Abstract] [Full Text] [PDF]
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K. Liu, C. Liang, Z. Liang, K. Tus, and E. K. Wakeland Sle1ab Mediates the Aberrant Activation of STAT3 and Ras-ERK Signaling Pathways in B Lymphocytes J. Immunol., February 1, 2005; 174(3): 1630 - 1637. [Abstract] [Full Text] [PDF]
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B R Lauwerys and E K Wakeland Genetics of lupus nephritis Lupus, January 1, 2005; 14(1): 2 - 12. [Abstract] [PDF]
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M. Wakui, J. Kim, E. J. Butfiloski, L. Morel, and E. S. Sobel Genetic Dissection of Lupus Pathogenesis: Sle3/5 Impacts IgH CDR3 Sequences, Somatic Mutations, and Receptor Editing J. Immunol., December 15, 2004; 173(12): 7368 - 7376. [Abstract] [Full Text] [PDF]
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Z. Xu, E. J. Butfiloski, E. S. Sobel, and L. Morel Mechanisms of Peritoneal B-1a Cells Accumulation Induced by Murine Lupus Susceptibility Locus Sle2 J. Immunol., November 15, 2004; 173(10): 6050 - 6058. [Abstract] [Full Text] [PDF]
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S. Atencio, H. Amano, S. Izui, and B. L. Kotzin Separation of the New Zealand Black Genetic Contribution to Lupus from New Zealand Black Determined Expansions of Marginal Zone B and B1a Cells J. Immunol., April 1, 2004; 172(7): 4159 - 4166. [Abstract] [Full Text] [PDF]
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 H. S. Lee, Y. H. Chung, T. G. Kim, T. H. Kim, J. B. Jun, S. Jung, S. C. Bae, and D. H. Yoo Independent association of HLA-DR and FC{gamma} receptor polymorphisms in Korean patients with systemic lupus erythematosus Rheumatology, December 1, 2003; 42(12): 1501 - 1507. [Abstract] [Full Text] [PDF]
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J. E. Wither, G. Lajoie, S. Heinrichs, Y.-C. Cai, N. Chang, A. Ciofani, Y.-H. Cheung, and R. MacLeod Functional Dissection of Lupus Susceptibility Loci on the New Zealand Black Mouse Chromosome 1: Evidence for Independent Genetic Loci Affecting T and B Cell Activation J. Immunol., August 15, 2003; 171(4): 1697 - 1706. [Abstract] [Full Text] [PDF]
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V. A. Adarichev, J. C. Valdez, T. Bardos, A. Finnegan, K. Mikecz, and T. T. Glant Combined Autoimmune Models of Arthritis Reveal Shared and Independent Qualitative (Binary) and Quantitative Trait Loci J. Immunol., March 1, 2003; 170(5): 2283 - 2292. [Abstract] [Full Text] [PDF]
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 P. L. Cohen Autoimmunity and Lymphoproliferation: Two Genes are Worse than One Mol. Interv., November 1, 2002; 2(7): 427 - 430. [Abstract] [Full Text]
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E. S. Sobel, L. Morel, R. Baert, C. Mohan, J. Schiffenbauer, and E. K. Wakeland Genetic Dissection of Systemic Lupus Erythematosus Pathogenesis: Evidence for Functional Expression of Sle3/5 by Non-T Cells J. Immunol., October 1, 2002; 169(7): 4025 - 4032. [Abstract] [Full Text] [PDF]
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E. S. Sobel, M. Satoh, Y. Chen, E. K. Wakeland, and L. Morel The Major Murine Systemic Lupus Erythematosus Susceptibility Locus Sle1 Results in Abnormal Functions of Both B and T Cells J. Immunol., September 1, 2002; 169(5): 2694 - 2700. [Abstract] [Full Text] [PDF]
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 X. Shi, C. Xie, D. Kreska, J. A. Richardson, and C. Mohan Genetic Dissection of SLE: SLE1 and FAS Impact Alternate Pathways Leading to Lymphoproliferative Autoimmunity J. Exp. Med., August 5, 2002; 196(3): 281 - 292. [Abstract] [Full Text] [PDF]
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P. P. L. Chiu, A. M. Jevnikar, and J. S. Danska Genetic Control of T and B Lymphocyte Activation in Nonobese Diabetic Mice J. Immunol., December 15, 2001; 167(12): 7169 - 7179. [Abstract] [Full Text] [PDF]
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 U. Wellmann, M. Letz, A. Schneider, K. Amann, and T. H. Winkler An Ig {micro}-heavy chain transgene inhibits systemic lupus erythematosus immunopathology in autoimmune (NZB x NZW)F1 mice Int. Immunol., December 1, 2001; 13(12): 1461 - 1469. [Abstract] [Full Text] [PDF]
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L. Morel, K. R. Blenman, B. P. Croker, and E. K. Wakeland The major murine systemic lupus erythematosus susceptibility locus, Sle1, is a cluster of functionally related genes PNAS, January 23, 2001; (2001) 31336098. [Abstract] [Full Text]
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D. D. Desai and T. N. Marion Induction of anti-DNA antibody with DNA-peptide complexes Int. Immunol., November 1, 2000; 12(11): 1569 - 1578. [Abstract] [Full Text] [PDF]
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J. M. Otto, R. Chandrasekeran, C. Vermes, K. Mikecz, A. Finnegan, S. E. Rickert, J. T. Enders, and T. T. Glant A Genome Scan Using a Novel Genetic Cross Identifies New Susceptibility Loci and Traits in a Mouse Model of Rheumatoid Arthritis J. Immunol., November 1, 2000; 165(9): 5278 - 5286. [Abstract] [Full Text] [PDF]
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L. Morel, B. P. Croker, K. R. Blenman, C. Mohan, G. Huang, G. Gilkeson, and E. K. Wakeland Genetic reconstitution of systemic lupus erythematosus immunopathology with polycongenic murine strains PNAS, June 6, 2000; 97(12): 6670 - 6675. [Abstract] [Full Text] [PDF]
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O. T. M. Chan, M. P. Madaio, and M. J. Shlomchik B Cells Are Required for Lupus Nephritis in the Polygenic, Fas-Intact MRL Model of Systemic Autoimmunity J. Immunol., October 1, 1999; 163(7): 3592 - 3596. [Abstract] [Full Text] [PDF]
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L. Morel, K. R. Blenman, B. P. Croker, and E. K. Wakeland The major murine systemic lupus erythematosus susceptibility locus, Sle1, is a cluster of functionally related genes PNAS, February 13, 2001; 98(4): 1787 - 1792. [Abstract] [Full Text] [PDF]
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