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Simmons Arthritis Research Center and Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75235; 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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Susceptibility to lupus in murine models has been mapped to several loci, as recently reviewed (4, 5, 6). Our studies have focused on the NZM2410 strain, derived from a cross between New Zealand Black (NZB) and New Zealand White (NZW) mice (7). This lupus-prone strain demonstrates splenomegaly, a significant expansion of activated CD4+ T cells and B cells, high-titered ANAs, and early-onset immune-complex glomerulonephritis (GN) (7, 8). To map these immunological traits, a (NZM2410 x C57BL/6 (B6)) x NZM2410 backcross analysis was undertaken several years ago (9). That mapping study revealed that genomic intervals on chromosomes 1 (Sle1), 4 (Sle2), 7 (Sle3), and 17 (Sle4) were strongly linked to lupus nephritis. Of relevance to this study, the locus on chromosome 7 (Sle3) showed significant linkage to GN, with peak linkage at the pink eye-dilution (p) locus (X2 = 16.7; p < 10-4; logarithm of the odds score = 4). Other independent mapping studies have also mapped lupus susceptibility to murine chromosome 7, close to Sle3. Using an (NZB x NZW)F2 mapping panel, Kono et al. (10) mapped a lupus susceptibility locus, Lbw5, in the vicinity of Sle3. Subsequently, the same workers mapped susceptibility to lymphadenopathy and anti-dsDNA production in another disease model, using an (MRL.Faslpr x B6.Faslpr)F2 analysis, to Lmb3, again in the vicinity of Sle3 (11). Using an (MRL/lpr x CAST/Ei) backcross panel, Watson et al. (12) mapped susceptibility to lupus nephritis to a more centromeric locus on murine chromosome 7, close to ckmm. Thus, this genomic interval on murine chromosome 7 appears to harbor a gene(s) that potentially dictates some critical pathogenic mechanisms leading to lupus in several different disease-prone strains.
Deciphering the relative immunopathological contributions of these loci
to disease has been greatly facilitated by the generation and
characterization of congenic mice bearing these individual lupus
susceptibility intervals (4, 13, 14, 15, 16, 17). Adopting this powerful approach,
we have recently shown that Sle1 triggers loss of tolerance
to chromatin, apparently without any generalized abnormalities in
lymphocyte activation, differentiation, or apoptosis (15, 17). In
contrast, Sle2 leads to generalized B cell hyperactivity,
elevated levels of splenic and peritoneal B1a cells, and increased
serum polyclonal/polyspecific IgM, but with no evidence of IgG ANAs, T
cell defects, or GN (15, 16). This study focuses on B6.NZMc7 mice,
which are B6 mice rendered congenic for the NZM2410-derived
Sle3 interval, as diagrammed in Fig. 1
. Our initial studies with this congenic
strain had revealed increased serum levels of polyclonal/polyspecific
IgM and IgG Abs, accompanied by an 18% incidence of severe (i.e.,
>50% of glomeruli affected) immune complex-mediated GN (15). By
comparing the cellular phenotypes seen in the B6.NZMc7 strain with
those of age-matched B6 controls, the present study aims to understand
the pathogenic mechanisms through which Sle3 might
contribute to disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B6 mice were obtained from The Jackson Laboratory (Bar Harbor,
ME) and subsequently bred in our animal colony. The derivation of B6
congenic mice bearing NZM2410-derived lupus susceptibility intervals
has been detailed previously (14). As depicted in Fig. 1
, B6.NZMc7 mice
are B6 mice congenic (homozygotes) for a 40-cM interval on murine
chromosome 7, encompassing the 95% confidence interval flanking
Sle3, with termini at D7 MIT56 and D7
MIT62. The entire congenic interval is derived from the NZW parent
of the NZM2410 strain. All mice used for this study were bred and
housed in a conventional colony, under identical conditions, at the
University of Florida Department of Animal Resources. As no sex
differences were noted in the expression of any of the phenotypes
described, both male and female mice were used in this study.
Nevertheless, for the in vivo antigenic challenge experiments, female
mice were used.
Cell preparation
Splenocytes were depleted of RBCs using 0.83% NH4Cl, and single-cell suspensions were prepared. Total splenic B cells were prepared as described before (17, 18), using pretitrated amounts of anti-Thy-1 (Accurate Chemicals, New York, NY) and rabbit complement (Accurate Chemicals) to lyse T cells, and were typically 85–95% pure. Splenic T cells were prepared as described (17, 18). Briefly, red cell-depleted splenocytes were loaded onto nylon wool columns (Robbins Scientific, Sunnyvale, CA) and incubated at 37°C for 45 min. Nonadherent cells were washed through and then incubated with pretitrated amounts of anti-I-Ab (clone K25–8.7, Accurate Chemicals) and anti-CD24 (PharMingen, San Diego, CA) on ice for 45 min. Ab-bound cells were lysed with rabbit complement (Accurate Chemicals), yielding T cells with >90% purity. Bone marrow cells were obtained by flushing femurs and tibia with medium. Peritoneal cavity cells were obtained by injecting medium into the peritoneal cavity and extracting the cells with transfer pipettes. Prepared single-cell suspensions were counted and used for culture or flow cytometric analysis, as described below.
Flow cytometric analysis, sorting, and Abs
Flow cytometric analysis (FACS) was performed as described previously (16, 19). Briefly, cells were first blocked with staining medium (PBS, 5% horse serum, and 0.05% azide) containing 10% normal rabbit serum. Cells were then stained on ice with optimal amounts of FITC, PE, or biotin-conjugated primary mAbs diluted in staining medium for 30 min. Following two washes, biotin-conjugated Abs were revealed using streptavidin-PE (Life Technologies, Grand Island, NY) or streptavidin-Quantum Red (Sigma, St. Louis, MO). Cell staining was analyzed using a FACScan (Becton Dickinson, San Jose, CA). Dead cells were excluded on the basis of scatter characteristics and propidium iodide (PI) uptake, and 10,000 events were acquired per sample. The following dye- or biotin-coupled Abs were obtained from PharMingen and used at pretitrated dilutions: CD4 (RM4-5), CD5 (53-7.3), CD8 (Ly-2), CD23 (B3B4), CD24 (M1/69), CD25 (7D4), CD43 (S7), CD44 (IM7), CD45R/B220 (RA3-6B2), CD62L (MEL14), CD69 (H1.2F3), CD80/B7-1 (16-10A1), and CD86/B7-2 (GL1).
Cell stimulation assays
F(ab')2 goat anti-mouse IgM (Cappel, Durham, NC)
and LPS (Sigma) were used for B cell stimulation at concentrations from
0.1 to 100 µg/ml. Likewise, graded doses of anti-CD3
(PharMingen), rIL-2 (Chiron Therapeutics, Emeryville, CA) or keyhole
limpet hemocyanin (KLH; Calbiochem, La Jolla, CA) were added to assess
T cell responses to stimuli. For these assays total splenocytes or
splenic T cells (5 x 105/well) were cultured for
72 h in 200-µl cultures, in serum-free HL-1 medium (HyCor
Biomedicals, Irvine, CA), with or without added stimuli. Lymphocyte
response was assessed either by assaying the extent of proliferation,
using [3H]TdR incorporation (ICN Biomedicals, Costa Mesa,
CA) over the last 18 h of culture or by measuring by ELISA the
amount of IL-2, IL-4, or IFN-
secreted at the indicated time points
postculture.
For the experiments depicted in Fig. 4, 5 x 105
splenocytes per well were cultured with anti-CD3 and anti-CD28
(1 µg/ml each, PharMingen). At 24, 48, and 72 h postculture, the
absolute numbers of CD4+ and CD8+ T cells in
culture were determined by counting live (i.e., PI-excluding) cells and
by FACS analysis. For assaying activation-induced cell death (AICD),
total splenocytes were stimulated with anti-CD3 and anti-CD28
for 48 h. Then, blasts (consisting of >90% T cells) were
purified over Ficoll, washed, and recultured with plate-bound
anti-CD3, with or without anti-Fas (2 µg/ml, clone Jo2,
PharMingen) or control hamster IgG (PharMingen). After the indicated
culture periods, the percentage of apoptosis was determined, as
described below.
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Purified splenic B cells or T cells, or T cell blasts, prepared as detailed above, were cultured, with or without added stimuli. Aliquots of this culture were assayed at 0, 24, 48, and 72 h for the percentage of apoptotic cells in culture, using hypotonic lysis and PI incorporation, as described before (20). Briefly, the cell pellets were resuspended in hypotonic PI solution (3.4 mM sodium citrate, 50 µg/ml PI, 0.1% Triton X-100, 1 mM Tris, and 0.1 mM EDTA) and analyzed by FACS, using logarithmic scales. Apoptotic nuclei were distinguished by their hypodiploid DNA content and scatter characteristics, after excluding the debri. Cell death was also assayed by monitoring annexin binding, as described (21). Annexin is known to bind to phosphatidylserine exposed on the outer leaflets of cells undergoing apoptosis. For assaying this, lymphocytes or T cell blasts were stained with PE- or biotin-labeled anti-CD4, anti-CD8, or anti-B220, as described above. Following a washing step, the cells were incubated with annexin-FITC (PharMingen) in HEPES-containing buffer and then analyzed on a flow cytometer.
IL ELISAs
IL produced in culture was assayed by ELISA, as described
previously (17). The following reagents were purchased from PharMingen:
anti-IL-2; anti-IL-4; anti-IFN-; biotinylated
anti-IL-2; biotinylated anti-IL-4; biotinylated
anti-IFN-; and rIL-2, rIL-4, and IFN- standards. Briefly,
Immulon I plates (Dynatech Laboratories, Chantilly, VA) precoated with
the "capture" Ab (anti-IL-2, anti-IL-4, or
anti-IFN-) were blocked and then incubated for 2 h with
culture supernatants (diluted 1:2) or serial dilutions of the rIL
standard. Captured IL was detected using biotin-coupled anti-IL,
avidin-alkaline phosphatase (1:10,000, Sigma), and
p-nitrophenylphosphate substrate (Sigma). ODs were converted to pg/ml
using the derived standard curve.
In vivo immunization
B6 and B6.NZMc7 mice (four or five mice per group, age 2.5 mo)
were injected i.p. with DNP-KLH (100 µg/mouse; Calbiochem) in CFA and
boosted 2 weeks later with 100 µg/mouse DNP-KLH in IFA. Immunized
mice were seromonitored for anti-hapten Abs for several weeks
thereafter, as illustrated in Results. At sacrifice, the
spleens were stained with peanut agglutinin to assess the size and
numbers of germinal centers, as described below. In addition, the T
cell response to the immunogen was gauged by measuring IL production
(as detailed above) upon rechallenge with KLH in vitro. To assess
response to thymus-independent Ags, independent groups of mice (four
per group) were challenged i.p. with trinitrophenol (TNP)-LPS (20
µg/mouse, Sigma) and seromonitored for anti-hapten response for 2
mo postchallenge. Briefly, serum anti-DNP and anti-TNP Abs were
determined by assaying serial dilutions of the test sera on
DNP-BSA-coated (Calbiochem) or TNP-BSA-coated (Accurate Chemicals)
Immulon-II plates (Dynatech), respectively. Bound Abs were revealed
with alkaline phosphatase-conjugated anti-mouse IgM or
anti-mouse IgG (Boehringer Mannheim, Indianapolis, IN), utilizing
p-nitrophenylphosphate as a substrate. The isotypes of anti-DNP Abs
were determined by using isotype-specific enzyme conjugates. The
avidity of these serum Abs was determined by gauging the amount of
competitor (
-DNP-lysine, Sigma) that was required for reducing the
ELISA reactivity to DNP-BSA by 50% (ID50).
Immunochemistry
Immunochemistry on splenic sections was performed, as described previously (8). Briefly, fragments of spleens were snap frozen in liquid nitrogen and stored at -70°C, until time of sectioning. Cryosections 0.4–0.6 µm were fixed in acetone and air dried. Slides were rehydrated with TBS-0.1% BSA and then blocked with 5% normal rabbit serum and 0.3% H202/100% MeOH. T cells were tagged by sequential incubations with biotin-coupled anti-CD3 (1 µg/ml, PharMingen) and avidin-alkaline phosphatase. B cells were identified using FITC-coupled anti-B220 (5 µg/ml, PharMingen) and peroxidase-coupled anti-FITC (Boehringer Mannheim). In addition, the size and numbers of germinal centers in the spleens of the immunized mice were assessed using biotin-coupled peanut agglutinin. All incubations were performed for 30 min at room temperature in a moist chamber. Sections were developed using the peroxidase substrate diaminobenzidine (Sigma) and the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium (Sigma) and then washed and mounted using Permount (Fisher, Pittsburgh, PA).
Statistics
Data obtained for the B6.NZMc7 mice were compared with those from the control B6 mice using the Student’s t test. For all experiments, the mean (±SEM) values observed are shown.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sle3 did not seem to impact the development or cellular
composition of the primary lymphoid organs. Thus, the marrow and thymus
from B6.NZMc7 mice exhibited the appropriate distributions of B cells
and T cells, respectively, at the different developmental stages (data
not shown). All differences observed were confined to the secondary
lymphoid organs. Although B6.NZMc7 mice did not differ from B6 controls
in the size or cellularity of their spleens, the T cell and B cell
zones in these mice were poorly organized, with frequent intermingling
of T cell and B cell foci, compared with B6 spleens, as illustrated in
Fig. 2. However, this immunohistological
phenotype was a relatively late event, being apparent only in the older
age group (9- to 12-mo-old mice). B6.NZMc7 spleens did not differ from
B6 spleens in the numbers or size of germinal centers after immune
challenge (data not shown).
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illustrates the distribution of T
cell and B cell subsets in the peripheral lymphoid organs of B6.NZMc7
mice. Because the total splenocyte numbers did not differ between the 2
strains, any observed differences in lymphocyte percentages also
reflect differences in absolute numbers. The B6.NZMc7 spleens did not
differ significantly from B6 controls in the numbers of B1, B2, or
total T cells, at any age. However, B6.NZMc7 spleen and lymph node (LN)
had significantly greater percentages and numbers of CD4+ T
cells relative to CD8+ T cells, leading to elevated CD4:CD8
ratios compared with age-matched B6 controls (Fig. 3A; Table I). This pattern was
noted at all ages tested. Thus, 1- to 3-mo-old and 4- to 6-mo-old
B6.NZMc7 spleens exhibited mean splenic CD4:CD8 ratios of 2.0, and
1.77, respectively, compared with the corresponding B6 ratios of 1.34
and 1.22 (p < 0.05 and p <
0.001, respectively). Likewise, B6.NZMc7 LN also exhibited significant
increases in the absolute numbers of CD4+ T cells, but not
CD8+ T cells, compared with B6 controls. Thus, for example,
4- to 6-mo-old B6.NZMc7 nodes exhibited 31.7% CD4+ T cells
and mean CD4:CD8 ratios of 1.07, compared with the corresponding B6
values of 23.9% and 0.82, respectively (p <
0.01). Interestingly, the T cell:B cell ratios and the absolute numbers
of T cells were significantly elevated in B6.NZMc7 LN compared with B6
controls, at all ages, as illustrated in Table I.
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B6.NZMc7 splenic T cells also appeared to be spontaneously
activated, having increased expression of CD69, compared with B6 T
cells (Table I and Fig. 3A). This phenotype became
progressively stronger with age. Thus, in 9- to 12-mo-old B6.NZMc7
spleens, on the average, 33.4% of CD4+ T cells expressed
CD69, compared with 21.9% of B6 CD4+ T cells
(p < 0.001). A similar trend was seen with the
expression of two other activation markers, CD25 and CD44 (data not
shown). Although splenic CD8+ T cells from B6.NZMc7 mice
also appeared to be more activated than their B6 counterparts, these
differences were not as pronounced as those seen with CD4+
T cells. In contrast, no significant differences were seen in the
activation status of B6 and B6.NZMc7 LN T cells (Table I).
In addition to the T cells, B6.NZMc7 B cells also appeared to be
spontaneously activated. As illustrated in Fig. 3B, B6.NZMc7
splenic B cells from 9- to 12-mo-old mice exhibited significantly
higher levels of surface I-Ab, CD44, and B7-2. This
phenotype was late in onset, as B cells from younger B6 and B6.NZMc7
spleens did not differ from each other, by surface phenotype. At all
ages tested, B6.NZMc7 spleens did not differ from B6 spleens with
respect to the numbers or percentages of B1a or B1b cells. Likewise,
there were no significant differences in the numbers or percentages of
B1a or B1b cells in their peritoneal cavities (data not shown).
We next assessed the functional status of B6.NZMc7 lymphocytes.
B6.NZMc7 and B6 splenic B cells showed a similar proliferative response
to anti-IgM and LPS (data not shown). However, when stimulated with
anti-CD3 and anti-CD28, B6.NZMc7 T cells behaved differently.
As illustrated in Fig. 4, B6.NZMc7
CD4+ T cells demonstrated significantly increased expansion
in culture, compared with B6 CD4+ T cells. Thus, by 72
h postculture, B6.NZMc7 CD4+ T cells exhibited more than a
3-fold increase in absolute numbers (p <
0.004). A similar trend was not noted for CD8+ T cells.
Indeed, in all B6 cultures, there were always more CD8+ T
cells than CD4+ T cells 72 h poststimulation, but the
reverse was true with B6.NZMc7 T cells, as can be seen in Fig. 4. In
both the B6 and B6.NZMc7 cultures, 72 h poststimulation, the T
cells were uniformly large in size and exhibited similarly high levels
of CD69 and Fas and reduced levels of L-selectin, typical of activated
T cell blasts (data not shown).
The increase in absolute numbers of B6.NZMc7 CD4+ T cells
in culture could reflect an increased production of nascent T cells
and/or reduced apoptosis of activated T cells. Indeed, both appear to
be contributing, as illustrated in Figs. 5 and 6.
Compared with B6 T cells, B6.NZMc7 T cells showed significantly
increased proliferation, as assessed by [3H]TdR
incorporation, in response to stimulation with anti-CD3 (Fig. 5A), and IL-2 (Fig. 5B). These experiments were
performed with 2- to 4-mo-old mice, and their splenic T cells did not
differ in the surface levels of CD3 or IL-2R (data not shown). However,
B6.NZMc7 T cells produced similar quantities of IL-2, IL-4, and IFN-
upon stimulation (data not shown).
Sle3 impacts T cell apoptosis
Apoptosis was studied by two complementary methods, hypotonic lysis with PI incorporation (20) and annexin binding (21), as detailed in Materials and Methods. In the former method, apoptotic cells were identified by their hypodiploid DNA content and light scatter characteristics. The latter method is based on the principle that dying cells expose phosphatidylserine on the outer membrane leaflets, which can be tagged by annexin binding. This method has the added advantage of allowing double or triple staining with other surface markers. This permits one to gauge cell death in selected lymphocyte populations.
By both methods, B6 and B6.NZMc7 splenic B cells showed similar rates
of apoptosis, with or without anti-IgM stimulation, as illustrated
in Fig. 6A. Likewise, splenic T cells from both strains
showed similar rates of spontaneous apoptosis in culture, with no
stimuli added. In contrast, B6.NZMc7 T cells exhibited reduced AICD.
For these experiments, splenocytes were first stimulated for 48 h
with anti-CD3 and anti-CD28. Then, the lymphoblasts were Ficoll
purified from the cultures, washed, and recultured on
anti-CD3-coated wells for the indicated durations. Importantly, the
6 h data points should accurately reflect the apoptotic rates of
the plated T cell blasts, as this time lapse is too short for any cell
divisions to have occurred. Importantly, the B6.NZMc7 T cell blasts
consistently showed reduced apoptotic rates compared with the B6 T cell
blasts, at the 6 h and 40 h time points.
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, B and C). Thus, both CD4+ and
CD8+ T cell blasts showed impaired AICD, even in the
presence of anti-Fas Abs. B6 and B6.NZMc7 T cells showed no
consistent difference in the expression levels of Fas or FasL (data not
shown). Although T cell blasts from the lupus-prone NZM2410 strain
exhibited a similar phenotype (Fig. 6, B and C),
they were very sensitive to Fas-mediated apoptosis. The latter
observation correlates well with the heightened expression of Fas on
NZM2410 blasts, compared with B6 or B6.NZMc7 T cell blasts (data not
shown). Sle3 augments the immune response to antigenic challenge
The immune responsiveness of these mice was assessed by
challenging them with a T-dependent (DNP-KLH), or a T-independent
(TNP-LPS) Ag. As illustrated in Fig. 7,
upon secondary challenge with DNP-KLH, B6.NZMc7 mice showed
significantly elevated levels of IgM (about 3–5 fold) and IgG (nearly
two-fold) anti-DNP Abs, for several months postchallenge. The
anti-DNP Abs in B6 and B6.NZMc7 mice did not differ in their
avidity for the hapten (Fig. 7E) or the IgG subclass
distribution (data not shown). Importantly, B6.NZMc7 T cells responded
more vigorously to rechallenge with KLH by IL production (IL-2
production is depicted in Fig. 7F). However, these two
strains did not differ in the relative quantities of IL-4 vs IFN-
produced upon rechallenge, suggesting that Sle3 does not
impact the Th1/Th2 balance (data not shown). B6 and B6.NZMc7
immune-challenged mice did not differ in the numbers or sizes of
germinal centers, as revealed by staining with peanut agglutinin.
Finally, B6 and B6.NZMc7 mice showed a similar humoral response when
challenged with the T-independent Ag, TNP-LPS (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several investigators have examined the features of T cells in the various murine models of lupus, as well as in human SLE. Of relevance to this work, an expansion of activated CD4+ T cells has been well documented in several lupus-prone (or genetically related) strains, including (NZB x NZW) F1, (NZB x SWR) F1, NZB.H2bm12, NZM2410, NZW, and Faslpr/lpr mice, as well as in human lupus (Refs. 22–25 and C. Mohan, unpublished observations). Although several of the mouse models are also characterized by elevated CD4:CD8 ratios, this has not been a characteristic feature of human SLE (reviewed in 25). Experiments involving knockouts, Ab-mediated blocking, and in vitro cultures have clearly demonstrated the critical role played by Th cells in driving B cell autoantibody production in murine and human lupus (26, 27, 28, 29, 30, 31, 32). Importantly, parallel studies have documented generalized abnormalities in T cell signal transduction (33, 34, 35, 36, 37) and apoptosis (38, 39, 40, 41, 42, 43) in murine and human lupus.
Superimposed on this generalized (i.e., polyclonal) T cell activation, a select few potentially pathogenic Th clones are likely to be further expanded in an Ag-driven fashion. This concept is supported by TCR clonotype analyses (44, 45, 46) and the demonstration of T cell antigenic specificities to a variety of nuclear, DNA-binding proteins, including histones, nucleosomes, ribosomal P2, and DNase I, as well as ANA-derived idiopeptides (18, 47, 48, 49, 50, 51, 52, 53). It is likely that the combined events of generalized, polyclonal T cell activation and the focused expansion of a few Ag-specific T cell clones are together responsible for driving ANA production by pathogenic B cell clones and/or end-organ disease.
The present study advances an interval on murine chromosome 7 as
harboring one such gene that may be responsible for the intrinsic,
generalized T cell abnormality that characterizes lupus. As discussed
earlier, mapping studies indicate that this genomic interval confers
susceptibility to ANA and/or GN in several murine models of lupus,
including the NZM2410 strain (9, 10, 11, 12). To elucidate the specific
immunological contributions of Sle3 independent of other
lupus susceptibility loci, this genomic interval has been selectively
bred onto the B6 background. Analysis of these B6.NZMc7 congenic mice
reveals that Sle3 leads to an increase in the absolute
numbers of splenic and LN CD4+ T cells and a reduction in
CD8+ T cells, leading to elevated CD4:CD8 ratios (Table I).
Although the total numbers of splenic (CD4+ and
CD8+) T cells are relatively similar between the 2 strains,
an age-dependent increase is observed in the absolute numbers of
activated CD4+ T cells in B6.NZMc7 spleens. Indeed, this in
vivo phenotype is recapitulated in vitro, as demonstrated by the
accumulation of activated CD4+ T cells in culture, upon
stimulation. Both increased cell proliferation and reduced AICD appear
to contribute to this accumulation.
This accumulation of activated T cells over time may be causally
related to the B cell surface phenotype (i.e., increased expression of
activation markers; Fig. 3B). Indeed, this perpetual, low
grade stimulation of bystander B cells may be responsible for the low
grade, polyclonal humoral autoimmunity seen in this strain (15).
Finally, as one would predict, Sle3 augments the immune
response to T-dependent, but not T-independent, Ags (Fig. 7). Thus far,
no differences have been noted in the functional status or the subset
distributions (B1a, B1b, and B2) of B cells between B6 and B6.NZMc7
mice. Ongoing experiments, utilizing bone marrow transfers and B cell-
or T cell-deficient mice, are aimed at dissecting out the relative
contributions of B cells, T cells, and non-lymphocytes to the observed
phenotypes in this strain.
It is important to note that these phenotypes are also present in the
original NZM2410 strain, as well as in other lupus-prone strains, as
discussed above. Indeed, in the NZM2410 strain, these phenotypes are
even more pronounced (C. Mohan et al., manuscript in preparation).
Also, in contrast to the lupus-prone strains, the B6.NZMc7 mice show
relatively low levels of ANAs and a lower penetrance of GN (15).
Additional genes that differ between the B6 and NZM2410 strain are
likely to account for these differences. Indeed, it appears that in the
presence of genes that actively breach tolerance to chromatin (e.g.,
Sle1), loci such as Sle3 can have a dramatic
impact on the amplification and diversification of the incipient
autoimmune response. This notion is supported by the robust humoral and
cellular autoimmune phenotypes seen in B6.NZMc1/c7 bicongenic mice
(Fig. 8) (90).
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-chain (c), and
c–associated tyrosine kinase (Jak3)
all result in impaired lymphocytic homeostasis (as exemplified by an
age-dependent accumulation of activated/memory CD4+, but
not CD8+, T cells), often accompanied by features of
autoimmunity (57, 58, 59, 60, 61, 62, 63, 64). Through the use of TCR transgenic mice, this
expansion of activated CD4+ T cells in several of these
models has been attributed to impaired T cell AICD (65, 66, 67, 68, 69). CTLA4 is
yet another molecule that plays a critical role in peripheral T cell
tolerance (70), as CTLA4-/- mice display massive
lymphoproliferation, overt autoimmunity, and early mortality (71, 72).
Additional molecules that impact immune responsiveness, T cell
activation, and CD4:CD8 ratios include members of the
NF-
B/RelB family, ikB, and ZAP-70
(73, 74, 75, 76, 77).
Although the above molecules do not map to the study interval on murine
chromosome 7, they do suggest that Sle3 could be impinging
on the same biochemical pathways underlying T cell expansion and AICD.
Signaling studies with B6.NZMc7 mice are currently in progress, to
explore this further. Equally important, these other molecules have to
be viewed as potential candidate genes for human lupus or for systemic
autoimmunity in general. T cells with low anti-self avidity are
likely to escape thymic censorship and exit to the periphery, where
they are likely to encounter self-Ags. Anti-self T cells with aberrant
thresholds for expansion/cell death (such as those with Sle3
or allelic variants of the other molecules described above), would be
more readily triggered into activation and may not be as readily purged
from the immune system. Such inefficiency in peripheral T cell
tolerance may be expected to manifest differently, depending on the
genetic background. Thus, on the B6 background, this might simply lead
to a low grade polyclonal T cell activation, leading to serological and
cellular phenotypes similar to those observed in B6.NZMc7 mice. On the
other hand, the presence of additional susceptibility genes could
transform this phenotype into a more pathological one, as discussed
above (Fig. 8). Importantly, similar epistatic relationships with
background genes have also been described for other molecules that
impact T cell homeostasis. For instance, the
Faslpr/lpr allele leads to low grade humoral
autoimmunity on the B6 background, but to high grade, pathological
autoimmunity on the MRL background (39, 55, 78).
The study interval on chromosome 7 harbors several candidate genes of immunological interest, including tristetraprolin (TTP), TGF-ß, CD22, and Bcl3. TTP-/- mutant mice exhibit myeloid hyperplasia, cachexia, dermatitis, erosive arthritis, high titers of anti-nuclear (including anti-dsDNA) Abs, and glomerular mesangial thickening (79). CD22-/- mice exhibit increased B cell hyperresponsiveness, elevated serum IgM, serum ANAs, an expansion of B1 cells, and a heightened immune response (80, 81, 82, 83). Interestingly, TGF-ß-/- mice exhibit multifocal inflammatory disease, activated T cells with elevated CD4:CD8 ratios, a more diffuse splenic architecture, lymphadenopathy, and serum ANAs (84, 85, 86, 87). On the other hand, aberrant Bcl3 expression has been commonly associated with B cell lymphoproliferation and leukemogenesis (88). As some of the phenotypes impacted by these candidate molecules overlap with those observed in B6.NZMc7 mice, it is important to ascertain whether any of these represent Sle3. Thus far, we have failed to detect any expression differences or coding region polymorphisms in the TTP and TGF-ß genes, between B6 and NZM2410/NZW (L. Morel and Y. Yu, unpublished observations). With respect to CD22, it is possible that allelic differences between B6 (CD22.2 allele) and NZB/NZW/NZM2410 strains (CD22.1 allele) may be associated with functional differences in B cell signaling (89). Further work is needed to assess the contributions of these allelic differences to the phenotypes seen in B6.NZMc7 mice and to the pathogenesis of lupus in the NZM2410 strain.
Although Sle3 has been described as a single locus in this paper, it is certainly possible that additional genes within this interval are contributing to the observed phenotypes. Ongoing studies with newer congenics bearing shorter genomic intervals are in progress to address the relative contributions of the different regions within the NZM2410-derived chromosome 7 susceptibility interval. Assigning a robust phenotype to these subinterval congenics would greatly boost our efforts to narrow down the vicinity of the culprit gene(s). The eventual identification of the culprit gene(s) on murine chromosome 7 promises to enrich our understanding of how specific defects in T cell function could impact systemic autoimmunity in general and pathogenic ANA formation in particular.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Drs. Chandra Mohan and Edward K. Wakeland, Simmons Arthritis Research Center and the Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75235-8884. E-mail addresses:
3 Abbreviations used in this paper: ANA, anti-nuclear Ab; NZB, New Zealand Black; NZW, New Zealand White; GN, glomerulonephritis; B6, C57BL/6; PI, propidium iodide; AICD, activation-induced cell death; LN, lymph node; TTP, tristetraprolin.
Received for publication October 22, 1998. Accepted for publication March 9, 1999.
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/ß expressing double-negative (CD4-/CD8-) and CD4+ T helper cells in humans augment the production of pathogenic anti-DNA autoantibodies associated with lupus nephritis. J. Immunol. 143:103.[Abstract]
chain. J. Clin. Invest. 101:1448.[Medline]
chain. Proc. Natl. Acad. Sci. USA 92:377. chain. Immunity 2:223.[Medline]
chain plays an essential role in regulating lymphoid homeostasis. J. Exp. Med. 185:189.-chain. J. Immunol. 159:4737.[Abstract]
chain in intrathymic and peripheral T cell selection. J. Exp. Med. 183:1111.B . J. Immunol. 158:5075.[Abstract]
B. J. Exp. Med. 185:1897. in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. Immunity 4:445.[Medline]
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), and CD8+ T cells from B6 (
)
and B6.NZMc7 mice (
). In the depicted experiment, B6.NZMc7 cultures
showed significantly higher numbers of CD4+ T cells,
compared with B6 cultures, at 48 h (p <
0.001) and 72 h (p < 0.004) postculture. This
experiment is representative of five independent experiments.
