By
From the * National Jewish Medical and Research Center, Division of Basic Sciences, Department of
Pediatrics, Denver, Colorado 80206; and the Department of Immunology, University of Colorado
Health Sciences Center, Denver, Colorado 80206
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Abstract |
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Peripheral B cell tolerance was studied in mice of the autoimmune-prone, Fas-deficient MRL/
lpr.H-2d genetic background by introducing a transgene that directs expression of membrane-bound H-2Kb antigen to liver and kidney (MT-Kb) and a second transgene encoding antibody
reactive with this antigen (3-83µ, anti-Kk,b). Control immunoglobulin transgenic (Ig-Tg)
MRL/lpr.H-2d mice lacking the Kb antigen had large numbers of splenic and lymph node B
cells bearing the transgene-encoded specificity, whereas B cells of the double transgenic (Dbl-Tg) MRL/lpr.H-2d mice were deleted as efficiently as in Dbl-Tg mice of a nonautoimmune B10.D2 genetic background. In spite of the severely restricted peripheral B cell repertoire of
the Ig-Tg MRL/lpr.H-2d mice, and notwithstanding deletion of the autospecific B cell population in the Dbl-Tg MRL/lpr.H-2d mice, both types of mice developed lymphoproliferation
and exhibited elevated levels of IgG anti-chromatin autoantibodies. Interestingly, Dbl-Tg
MRL/lpr.H-2d mice had a shorter lifespan than Ig-Tg MRL/lpr.H-2d mice, apparently as an indirect result of their relative B cell lymphopenia. These data suggest that in MRL/lpr mice peripheral B cell tolerance is not globally defective, but that certain B cells with receptors specific
for nuclear antigens are regulated differently than are cells reactive to membrane autoantigens.
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Introduction |
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The MRL/lpr mouse is an intensively studied, but poorly understood model of SLE. The MRL/lpr strain has a variety of genetic defects (1) in addition to carrying the lpr mutation, which is a loss of function allele of the Fas (CD95) gene. Fas and Fas ligand (FasL)1 are members of gene families that are important in immune regulation and cell death (2). The lpr mutation protects cells from the normal death-inducing effects of FasL (3, 4). Although Fas is functionally expressed on many cell types, lpr mice are relatively normal until young adulthood, when they develop massive lymphadenopathy, hypergammaglobulinemia, and autoantibodies to a variety of intracellular constituents, including chromatin, snRNP, and DNA (1, 5). Disease severity is strongly affected by genetic background, and the MRL background is particularly prone to autoimmunity, even in the absence of the lpr defect (1, 8).
Because intrinsic defects in B and T lymphocytes are required in lpr mice for autoimmune disease to occur (9), and because the lpr defect in the Fas-mediated death pathway promotes autoimmunity, several studies have tested the possibility that clonal elimination of autoreactive lymphocytes might be defective in these mice (5, 17). In some studies, central and peripheral T cell tolerance appears relatively unaffected in lpr mice, at least when tested using antigen receptor Tg systems or with T cell superantigens (22, 24). But there are some notable exceptions to this general conclusion (21, 27), suggesting that at least the tempo of and sensitivity to deletion may be impaired in lpr mice.
Parallel studies using B cell receptor (BCR) Tg lpr mice assessed defects in central and peripheral B cell tolerance. Central tolerance to the non-disease-associated autoantigens H-2k,b or membrane-bound hen egg lysozyme appeared to be normal (20, 30), whereas tolerance to the lupus-associated autoantigen, double-stranded DNA, was impaired (31, 32). Therefore, it is possible that nuclear autoantigens are the focus of the autoimmune disease because of an antigen-specific, rather than a global tolerance defect. Several studies have shown that mature peripheral B cells are subject to Fas-induced death subsequent to activation through CD40 and that this death could be specifically rescued by BCR signaling (33). In BCR Tg mice in which B cell anergy develops as a result of exposure to soluble self-antigen, the Fas mutation has little effect until a cognate B/T interaction is generated, at which point defective BCR signaling in anergic B cells prevents their rescue (35). This sensitivity of anergic or antigen nonbinding B cells to T cell killing is blocked in mice lacking functional Fas or FasL (33). Although these studies could not reveal how the lpr defect leads to lupus autoimmunity, they suggest that a global defect in the ability to eliminate autoreactive peripheral B cells could be involved. Consequently, antigen-specific or global tolerance defect(s), or both, may contribute to autoimmunity in the MRL/lpr mouse.
We have previously described a double transgenic (Dbl-Tg) mouse model of peripheral B cell clonal elimination
(37) in which the anti-H-2Kk,b BCR Ig-Tg mouse called
3-83µ is crossed to the MT-Kb mouse, which expresses
the cognate Kb antigen under the control of the sheep metallothionein promoter (38). In these Dbl-Tg mice, B cells
are efficiently eliminated at some time after export from the
bone marrow, apparently as a result of encounter with
liver-expressed Kb antigen, rendering their lymph nodes
nearly devoid of B cells (37). The death pathway that eliminates the autoreactive B cells in this Dbl-Tg model is not
known, but the cells can only be partially rescued by overexpression of Bcl-2 (39). In this study, we have evaluated
the effect of the MRL/lpr genetic background on the deletion of autoreactive B cells in this model.
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Materials and Methods |
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Mice and Their Genetic Typing.
All mice were bred and maintained under specific pathogen-free conditions at the Biological Resource Center of the National Jewish Medical and Research Center (NJMRC, Denver, CO). B10.D2nSn/J mice (henceforth referred to as B10.D2) were obtained from Jackson Laboratory (Bar Harbor, ME). B10.D2 congenic mice bearing the 3-83µFlow Cytometry.
Flow cytometric analysis of lymphoid tissues was performed as described (20) using a FACScalibur® flow cytometer and PC-LYSIS data analysis software (Becton Dickinson, San Jose, CA).ELISA Analysis of Serum Immunoglobulins.
Sera were taken at 6-8 wk of age (young mice) or 5 mo of age (aged mice). Total IgG and IgM concentrations were measured by a sandwich ELISA using specific goat anti-mouse polyclonal reagents (Southern Biotechnology Associates, Birmingham, AL). Unlabeled goat anti-mouse antibodies (10 µg/ml) in PBS were adhered to polystyrene Immunolon microtiter plates (Dynatech Laboratories, Inc., Chantilly, VA) by overnight incubation. Plates were then washed with washing buffer (PBS and 0.05% Tween-20) and blocked using PBS, 0.5% BSA, and 0.4% Tween-20. Mouse sera in diluent (PBS, 0.2% gelatin, and 0.5% bovinePlasma Transaminase Activity.
Blood samples were taken by tail vein sampling or retro-orbital bleed, under Avertin anesthesia (43). Purified sera samples were separated from the clot within 1 h and were retained no more than 24 h at 4°C before analysis by a PARAMAX 720 (Paramax Chemistries AKA Dade-Behring International, Inc., Newark, DE), using Dade Alanine and Asparate Aminotransferase (ALT and AST) Reagent(s), (Dade International, Inc., Aguada, PR; reference 44). Data from serum samples in which the instrument detected hemolysis were excluded.Analysis of Mouse Survival.
Viability of individual mice of the strains indicated in Fig. 6 was followed over time. Mice were maintained under uniform environmental conditions throughout. The study end point event was defined as death. All data on weeks-followed were calculated from the date of birth, and survival probabilities were nonparametrically estimated at event times by the Product-Limit (Kaplan-Meier) method (45) using JMP data analysis software (SAS Institute, Inc., Cary, NC). Mice killed because of sickness (for which death could be anticipated within 24-48 h) were treated as deaths and the lifespans recorded. Data on animals that were killed for any other reason was censored.
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Bone Marrow Chimeric Mice.
Irradiated recipients were prepared as previously described (46), but without antibiotic prophylaxis. Food was withheld from the mice 24 h before irradiation, and returned immediately after irradiation. Bone marrow suspensions were isolated from Ig-Tg donors and prepared sterilely as previously described (46) with some modifications. Cells were isolated, washed in HBSS, depleted of erythrocytes by Gey's lysis buffer, washed, and resuspended at 108 cells/ml in RPMI and 10% FCS. Cell suspensions were incubated for 1 h at 4°C with the following antibodies at a concentration of 1 µg/106 cells: anti-Thy1.1/1.2, T24 (a gift of Uwe Staerz, NJMRC); anti-Thy.1.2, H0-13-4 (47); anti-B220, RA3-3A1 (48); and anti-CDw32, 24G.2 (a gift of J. Cambier, NJMRC). After washing, treated cells were applied to petri dishes (Becton Dickinson) previously coated with goat anti-rat Ig. Dishes were incubated on ice for 40 min, swirled and incubated again for another 30 min. The depleted, nonadherent cell suspensions were collected, washed and resuspended at 5 × 107 cells/ml in sterile HBSS and 0.1 ml was injected intravenously per irradiated recipient.Statistical Analysis.
Significant differences in serum transaminase activities were quantified by a three-way analysis of variance (ANOVA), using a two-tailed test. Significance with respect to survival of MRL/lpr mice was determined using the Log-Rank test. All data were analyzed using JMP analysis software (SAS Institute, Inc.). ![]() |
Results |
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To assess if peripheral B cell tolerance is impaired on the MRL/lpr autoimmune-prone genetic background, we measured depletion of autoreactive B cells in
lymph nodes of 6-8-wk-old Dbl-Tg MRL/lpr.H-2d mice.
Cells bearing the transgene-encoded BCR were quantified by flow cytometry using a clonotype-specific mAb (Fig. 1).
As expected from the specificity of this assay, control non-Tg mice had few cells scoring clonotype+ (Fig. 1, A and
E), and Ig-Tg mice that lacked cognate antigen had high
levels of clonotype+ B cells on both MRL/lpr.H-2d and
B10.D2 backgrounds (Fig. 1, B and F, respectively). In
contrast, antigen-expressing Dbl-Tg MRL/lpr.H-2d as well
as Dbl-Tg MRL/lpr/+.H-2d and Dbl-Tg B10.D2 Fas-sufficient control mice efficiently eliminated clonotype+
lymph node cells (Fig. 1, D, C, and G, respectively). A
characteristic of 3-83µ/MT-Kb Dbl-Tg mice is that they
retain a small population of short-lived 3-83 clonotype+
cells in the spleen (37, 39). Since fewer 3-83 clonotype+
cells remained in the spleens of Dbl-Tg MRL/lpr.H-2d
mice than in Dbl-Tg B10.D2 mice (data not shown), deletion appeared to be at least as efficient on the autoimmune-prone genetic background as on the normal genetic background. In addition, both Dbl-Tg MRL/lpr.H-2d and Dbl-Tg B10.D2 strains lacked clonotype+ B cells in their peritoneal cavities (data not shown). To detect rare Ig-transgenic B cells that may have escaped peripheral tolerance mechanisms at frequencies below the detection level of our
FACS® analyses, we performed an ELISPOT assay designed to reveal clonotype-secreting cells persisting in
lymph node at very low frequencies, but failed to detect
clonotype-secreting cells above background levels (data not
shown). These data indicated that clonal elimination of
transgene-encoded autoreactive B cells proceeded normally
in MRL/lpr.H-2d mice.
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Efficient clonal elimination of Fas-deficient B cells that
were autoreactive to the tissue-specific MT-Kb antigen
raised the consideration that expression of this antigen was
altered on the MRL/lpr genetic background compared
with the B10.D2 background. Therefore, we confirmed
the results of the double transgenic approach in short-term
bone marrow chimeras in which Ig-Tg bone marrow cells
of the MRL/lpr.H-2d or B10.D2 genetic background were
used to reconstitute lethally irradiated B10.D2 MT-Kb Tg
recipients (Fig. 2). Regardless of genetic background, autospecific B cells were deleted from the lymph nodes of Ig-Tg MT-Kb chimeras tested at 3-4 wk after reconstitution, whereas control, Ig-Tg
non-Tg lymph nodes had
abundant 3-83 clonotype+ cells. The bone marrows of all
mice were shown to contain an abundance of 3-83 clonotype+ cells, indicating that the lack of B cells in the MT-Kb
recipient lymph nodes was not the result of inefficient reconstitution. These data confirm that the antigen dose on
the B10.D2 MT-Kb transgenic background was sufficient to
delete Fas-deficient MRL/lpr.H-2d-derived Ig-Tg B cells.
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Since the lupus-like disease of MRL/lpr mice is age
dependent, we tested the possibility that Dbl-Tg MRL/
lpr.H-2d mice had an age-dependent defect in peripheral B
cell tolerance by analyzing lymph nodes of 5-7-mo-old
mice for the appearance of 3-83 clonotype+ B cells. The
percentages of transgenic B cells observed in lymph nodes of
aged mice are indicated in Fig. 3 and the absolute 3-83µ+
B cell numbers in all mouse age groups analyzed are shown
in Fig. 4. Similar to the results obtained in young Dbl-Tg
MRL/lpr.H-2d mice, lymph nodes of the older mice were
virtually devoid of 3-83 clonotype+ cells (Fig. 3). Additionally, ELISPOT assay failed to reveal any rare 3-83 antibody-forming cells and 3-83-positive B cells were absent
from the peritoneums of these aged Dbl-Tg MRL/lpr.H-2d
mice (data not shown). The aged Dbl-Tg MRL/lpr.H-2d
also had no increase in the percentage of clonotype+ B cells
in their spleens compared with Dbl-Tg B10.D2 mice. A small, but variable population of B cells expressing very
low levels of transgenic Ig was observed in livers of Dbl-Tg
mice of both autoimmune and nonautoimmune genetic
backgrounds (data not shown). Collectively, these data suggest that throughout the life of the Dbl-Tg MRL/lpr.H-2d
mouse B cells bearing the 3-83 BCR are generated in the
bone marrow and are subsequently deleted upon contact
with peripheral autoantigen. As we show below, this occurs in spite of the ongoing inflammatory and anti-nuclear
antibody responses of Dbl-Tg MRL/lpr.H-2d mice.
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Using serum ELISA, we verified the prediction that Dbl-Tg MRL/lpr.H-2d sera were devoid of 3-83 antibodies. As
expected, the sera of 3-83µ Ig-Tg mice lacking the MT-Kb
antigen transgene contained easily detectable levels of 3-83 clonotype antibodies, with equivalent serum concentrations in young mice of both genetic backgrounds. Interestingly, idiotype concentrations in aged MRl/lpr.H-2d Ig-Tg
were increased several-fold over B10.D2 Ig-Tg controls (Table 1). More importantly, most of these same sera from
older MRL/lpr.H-2d Tg or non-Tg mice contained high
titers of anti-chromatin autoantibodies (see below).
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Young or aged Ig-Tg and Dbl-Tg MRL/lpr.H-2d mice had high levels of IgG anti-chromatin, whereas Fas-sufficient MRL Dbl-Tg or B10.D2 background mice lacked anti-chromatin autoantibodies (Fig. 5). These increases in autoantibody titers were associated with a modest degree of IgG hypergammaglobulinemia (Table 1). Thus, in Dbl-Tg MRL/lpr.H-2d mice that properly controlled transgene-encoded autoreactive B cells, rare B cells reactive to chromatin were functional and specifically activated.
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Since anti-nuclear autoantibodies are diagnostic of both murine and human lupus nephritis (49, 50), we assessed kidney function by measuring levels of proteinuria. Older MRL/lpr.H-2d mice had elevated levels of protein in the urine (data not shown), indicating that this increase was correlated with Fas-insufficiency rather than transgene genotype.
Because MRL/lpr (H-2k) mice die prematurely from autoimmune glomerulonephritis (1), we compared longevity of non-Tg and Tg MRL/lpr.H-2d mice to the parental (H-2k) MRL/lpr strain and to the central tolerance model strain, Ig-Tg MRL/lpr (H-2k) (Fig. 6 B). All lpr mice died prematurely compared with Fas-sufficient controls (not shown), but non-Tg and Ig-Tg MRL/lpr.H-2d mice lived significantly longer than the MHC-disparate Ig-Tg MRL/ lpr (H-2k) strain (P = 0.0001) or the parental (H-2k) MRL/ lpr strain (1). In the non-Tg and Ig-Tg MRL/lpr.H-2d groups >50% of the mice remained alive at 40 wk. Surprisingly, Dbl-Tg MRL/lpr.H-2d mice had a median lifespan of only 27 wk which was significantly shorter (P = 0.02) than the lifespans of MRL/lpr.H-2d non-Tg mice or Tg mice bearing only one of either of the transgenes. It appears that potential autoreactivity of the B cells or the B cell lymphopenia in these Dbl-Tg mice contributed to their shortened lifespan.
We examined the livers of Dbl-Tg MRL/lpr.H-2d and control mice because this tissue was the predicted site at which transgene-encoded autoreactive B cells could potentially cause pathology and because MRL/lpr mice develop inflammation in many organ systems as part of their autoimmune disease (1). All histologically examined lpr livers exhibited moderate to severe disease, including the presence of infiltrating inflammatory cells with small foci of large lymphocytes and neutrophils throughout the parenchyma, along with a highly diffuse perivascular response, all indicating disseminated hepatitis and massive damage (data not shown; Henson, P., personal communication). To quantify liver damage in the mouse strains of interest, we chose to measure plasma concentrations of the liver enzymes ALT and AST. Whereas young MRL/lpr.H-2d mice exhibited enzyme activities no different than B10.D2 mice, the circulating levels of either or both AST (P = 0.002) and ALT (P = 0.04) were elevated in aged MRL mice, regardless of Tg genotype (Table 2), confirming the presence of liver damage in these mice, and suggesting that this damage was not specifically exacerbated in the Dbl-Tg individuals.
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Mice of the various experimental groups were tested for evidence of lymphoid hyperplasia (Fig. 6 A), which is a reproducible characteristic of older lpr mice (1). Aged non-Tg and Tg MRL/lpr.H-2d mice exhibited greatly elevated numbers of lymph node cells relative to B10.D2 and MRL/lpr/+.H-2d controls.
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Discussion |
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Despite the indications from other studies that Fas-mediated cell death can play an important role in B and T cell peripheral immune tolerance and the clear ability of the lpr mutation to cause accelerated lupus-like autoimmunity, in this study, we find that B cell clonal elimination induced by liver-expressed membrane protein was not impaired in the absence of Fas. Similar studies have indicated that both B cell anergy and central tolerance are barely affected in lpr mice (19, 20). In our prior analysis of central tolerance (20) and in this study lpr mice tolerant with respect to the Ig transgene-encoded antigen specificity nevertheless manifested high titer autoantibodies to nuclear antigens as well as tissue destruction and premature death. Since in Dbl-Tg MRL/lpr.H-2d mice liver damage and inflammation was extensive, yet B cell tolerance to the liver expressed Kb molecule was efficient, it would appear that tissue damage and inflammation alone cannot rescue autoreactive B cells from peripheral tolerance. This observation places severe constraints on models of autoimmunity that rely on the concept of tissue damage- or danger-induced adjuvanticity (51), at least as these concepts relate to B lymphocytes.
Complete ablation of the B cell compartment in JH- deficient MRL/lpr mice has been shown to rescue the animals from their autoimmunity (12), suggesting that B cells or their antibody products are required for lupus disease. At variance with prior studies involving lpr mice that were Ig transgenic or B cell deficient (12, 19), our experiments using single and Dbl-Tg MRL/lpr.H-2d mice demonstrate that restriction of the peripheral B cell repertoire by Ig transgenesis does not significantly rescue these animals from lupus-like disease and, as demonstrated by the reduced survival of the Dbl-Tg MRL/lpr.H-2d mice, may in fact accelerate death. Because in these latter mice B cells of the 3-83 Tg specificity appeared to be appropriately tolerized in all tissues examined, we suspect that the lifespan of the MRL/ lpr.H-2d Dbl-Tg is somehow further shortened as an indirect consequence of its B cell lymphopenia, rather than as a result of secretion of 3-83 autoantibody.
How can reducing the transgenic B cell population shorten the lifespan of the MRL/lpr.H-2d Dbl-Tg mice? One possibility is that this exacerbates lupus autoimmunity. In some circumstances the presence of an excess of nonautoreactive B cells can inhibit the survival of autoreactive B cells (52, 53). It is also known that resting, but not activated, B cells can tolerize T cells to MHC class II-restricted antigens (54). If most B cells generated in the Ig-Tg or non-Tg MRL/lpr.H-2d mice are nonautoreactive, they may tend to suppress the rare autoreactive cells. By contrast, in the Dbl-Tg MRL/lpr.H-2d mice, such a pool of "inert" nonautoreactive cells is lacking. We speculate that the reduction in nonautoreactive B cell numbers in Dbl-Tg MRL/lpr.H-2d mice may improve the fitness or enhance the generation of the autoreactive B cells that eventually develop.
Autoantibody production in the MRL/lpr mouse relies
upon a cognate, MHC-restricted T cell-B cell interaction (55)
suggesting that the peripheral T lymphocyte repertoire in
MRL/lpr mice includes T cells that are able to provide help to
B cells with antinuclear specificities. If peripheral T cell tolerance in these mice is otherwise intact, B cells that are autoreactive for other self-antigens such as H-2K molecules may
be tolerized by the absence of T cell help. However, a number of studies have shown that -T cell-independent mechanisms may drive some aspects of lupus-like autoimmune
disease, not only in the MRL mouse (56), but on nonautoimmune murine genetic backgrounds as well (57, 58). Therefore, peripheral B cell reactivity to certain self-antigens in the
MRL strains could be independent of
-T cell help. Furthermore, a recent study by Chan and Shlomchik (59) suggests that B cells may act as APC and activate naive CD4+ T
cells in MRL/lpr mice. Therefore, it is possible that the limited B cell repertoire in our peripheral tolerance model contains subpopulations with antinuclear specificities that may be
independent of classical
-T cell helper mechanisms, or may
be able to activate appropriate CD4+
-T cell help and
thereby expand autoreactive clones.
Ig-Tg MRL/lpr (H-2k) mice had a lifespan comparable to the wild-type MRL/lpr (H-2k) strain, whereas survival rates of MRL/lpr.H-2d mice that were non-Tg, Ig-Tg, or MT-Kb-Tg were improved (Fig. 6 B). An obvious difference between Ig-Tg and parental MRL/lpr (H-2k) strains compared with the Tg mice examined in this study is the substitution of H-2d for H-2k at the MHC loci and a prior study found that B6/lpr.H-2d congenic mice have less severe autoimmune disease than B6/lpr (H-2b) mice (60). However, it should be noted that we failed to observe a direct correlation between survival and anti-chromatin antibody titers.
In summary, we have demonstrated that there is no global defect in the ability of MRL/lpr mice to eliminate autoreactive B cells. However, in spite of ongoing deletion of self-reactive B cells newly exported from the bone marrow, aging MRL/lpr.H-2d Dbl-Tg mice develop various characteristics of lupus-like autoimmune disease, including the production of anti-chromatin autoantibodies, indicating the presence of autoreactive B cells. Others have shown that anti-DNA-Tg B cells were not only present and persistent as a functionally inactivated population in the periphery of nonautoimmune mice (61, 62), but also that anti-DNA-Tg B cells experienced selective clonal expansion in the autoimmune MRL/lpr genetic background (32). Perhaps MRL/lpr anti-DNA B cells not only circumvent central and peripheral tolerance mechanisms, but also exploit some activation process that is not useful to other autoreactive B cells that are sensitive to tolerance mechanisms.
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Footnotes |
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Address correspondence to David Nemazee, Rm. K1023, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Phone: 303-398-1623; Fax: 303-398-1225; E-mail address: nemazeed{at}njc.org
Received for publication 20 April 1998 and in revised form 10 June 1998.
We are indebted to Drs. B. Kotzin and L. Wysocki for their critical reading of this manuscript and suggestions regarding the research, Dr. P. Henson for advice regarding histological analysis, W. Townend and S. Sobus for technical help with FACS® instrumentation, D. Smith for help with serological analysis, M. Holka and colleagues for advice and technical support regarding transaminase assays, and D. McCormick for helpful dialogue with respect to statistical analysis.
This work was supported by the Arthritis Foundation and National Institutes of Health grants R01 GM/AI 44809 and K04 AI 0116.
Abbreviations used in this paper
ALT, alanine aminotransferase;
AST, asparate aminotransferase;
BCR, B cell receptor;
BGG, bovine globulin;
Dbl-Tg, double transgenic;
FasL, Fas ligand;
HRP, horseradish peroxidase;
Ig-Tg, immunoglobulin transgenic;
MT-Kb, metallothionein-Kb;
Tg, transgenic.
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