Efficient Peripheral Clonal Elimination of B Lymphocytes in MRL/lpr Mice Bearing Autoantibody Transgenes

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 3-83μδ (anti–H-2K^k,b; reference 37) and MT-K^b (38) transgenes were backcrossed for five generations to the MRL/lpr (H-2^k) background. To prevent central tolerance on this background, mice retaining the B10.D2-derived H-2^d locus were used and interbred, yielding partially inbred MRL/lpr.H-2^d mice with or without the 3-83μδ Ig or MT-K^b antigen transgenes. All the Ig-Tg mice analyzed were hemizygous for the 3-83 μδ transgene. Segregation of transgenes and Fas alleles (lpr or wild-type) was followed using PCR and Southern blot analysis of tail DNA as described (20, 37). The segregation of endogenous H-2^k,d alleles was followed by flow cytometric analysis of peripheral blood lymphocytes using specific antibodies.

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).

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% bovine γ globulin [BGG]) were applied to the plates and incubated overnight. After extensive washing in washing buffer, bound mouse Ig was detected by incubation for 2 h at room temperature with horseradish peroxidase (HRP)-conjugated goat anti–mouse IgG or IgM reagents (Southern Biotechnology Associates), followed by washing in washing buffer and incubation with the colorimetric substrate 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Sigma Chemical Co., St. Louis, MO), in McIlvain's Buffer (84 mM Na₂PO₄ and 48 mM citrate, pH 4.6) containing 0.005% H₂O₂. Absorbance at 410 nm was detected using a spectrophotometer (model 2225; Bio-Rad Laboratories, Hercules, CA). Quantitation of 3-83 clonotype⁺ IgM antibodies was performed as described (37). Standard curves for the anti-idiotype and anti-IgM assays were generated using IgM 3-83 transfectoma (cos linD) supernatant (40). IgG isolated from culture supernatant of the 3-83 hybridoma (41) provided the standard for the anti-IgG assay. Anti-chromatin autoantibody titers were quantitated using microtiter plates coated with 10 μg/ml mouse thymus–derived chromatin (a gift of Dr. L. Wysocki, NJMRC) diluted in PBS. Diluted sera were reacted with the plates for 2 h at room temperature followed by overnight incubation at 4°C. The plates were washed with washing buffer, and bound mouse antibody was detected as described above. Affinity-purified mouse monoclonal IgG anti-dsDNA 3H9 (42) served as a positive control, and as a reference standard curve as described previously (20). Nonspecific binding was assessed with plates coated with 0.2% gelatin in PBS.

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.

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.

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.

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.).

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-2^d 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-2^d and B10.D2 backgrounds (Fig. 1, B and F, respectively). In contrast, antigen-expressing Dbl-Tg MRL/lpr.H-2^d as well as Dbl-Tg MRL/lpr/+.H-2^d 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 3-83μδ/MT-K^b 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-2^d 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-2^d 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-2^d mice.

Efficient clonal elimination of Fas-deficient B cells that were autoreactive to the tissue-specific MT-K^b 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-2^d or B10.D2 genetic background were used to reconstitute lethally irradiated B10.D2 MT-K^b Tg recipients (Fig. 2). Regardless of genetic background, autospecific B cells were deleted from the lymph nodes of Ig-Tg→ MT-K^b 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-K^b recipient lymph nodes was not the result of inefficient reconstitution. These data confirm that the antigen dose on the B10.D2 MT-K^b transgenic background was sufficient to delete Fas-deficient MRL/lpr.H-2^d-derived Ig-Tg B cells.

Since the lupus-like disease of MRL/lpr mice is age dependent, we tested the possibility that Dbl-Tg MRL/ lpr.H-2^d 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 3-83μδ⁺ 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-2^d 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-2^d mice (data not shown). The aged Dbl-Tg MRL/lpr.H-2^d 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-2^d 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-2^d mice.

Using serum ELISA, we verified the prediction that Dbl-Tg MRL/lpr.H-2^d sera were devoid of 3-83 antibodies. As expected, the sera of 3-83μδ Ig-Tg mice lacking the MT-K^b 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-2^d 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-2^d Tg or non-Tg mice contained high titers of anti-chromatin autoantibodies (see below).

Young or aged Ig-Tg and Dbl-Tg MRL/lpr.H-2^d 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-2^d mice that properly controlled transgene-encoded autoreactive B cells, rare B cells reactive to chromatin were functional and specifically activated.

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-2^d 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-2^k) mice die prematurely from autoimmune glomerulonephritis (1), we compared longevity of non-Tg and Tg MRL/lpr.H-2^d mice to the parental (H-2^k) MRL/lpr strain and to the central tolerance model strain, Ig-Tg MRL/lpr (H-2^k) (Fig. 6 B). All lpr mice died prematurely compared with Fas-sufficient controls (not shown), but non-Tg and Ig-Tg MRL/lpr.H-2^d mice lived significantly longer than the MHC-disparate Ig-Tg MRL/ lpr (H-2^k) strain (P = 0.0001) or the parental (H-2^k) MRL/ lpr strain (1). In the non-Tg and Ig-Tg MRL/lpr.H-2^d groups >50% of the mice remained alive at 40 wk. Surprisingly, Dbl-Tg MRL/lpr.H-2^d mice had a median lifespan of only 27 wk which was significantly shorter (P = 0.02) than the lifespans of MRL/lpr.H-2^d 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-2^d 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-2^d 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.

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-2^d mice exhibited greatly elevated numbers of lymph node cells relative to B10.D2 and MRL/lpr/+.H-2^d controls.

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-2^d mice liver damage and inflammation was extensive, yet B cell tolerance to the liver expressed K^b 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-2^d 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-2^d 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-2^d 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-2^d 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-2^d mice are nonautoreactive, they may tend to suppress the rare autoreactive cells. By contrast, in the Dbl-Tg MRL/lpr.H-2^d 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-2^d 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 αβ-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 αβ-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 αβ-T cell helper mechanisms, or may be able to activate appropriate CD4⁺ αβ-T cell help and thereby expand autoreactive clones.

Ig-Tg MRL/lpr (H-2^k) mice had a lifespan comparable to the wild-type MRL/lpr (H-2^k) strain, whereas survival rates of MRL/lpr.H-2^d mice that were non-Tg, Ig-Tg, or MT-K^b-Tg were improved (Fig. 6 B). An obvious difference between Ig-Tg and parental MRL/lpr (H-2^k) strains compared with the Tg mice examined in this study is the substitution of H-2^d for H-2^k at the MHC loci and a prior study found that B6/lpr.H-2^d congenic mice have less severe autoimmune disease than B6/lpr (H-2^b) 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-2^d 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.

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.

ALT

alanine aminotransferase

AST

asparate aminotransferase

BCR

B cell receptor

BGG

bovine γ globulin

Dbl-Tg

double transgenic

FasL

Fas ligand

HRP

horseradish peroxidase

Ig-Tg

immunoglobulin transgenic

MT-K^b

metallothionein-K^b

Tg

transgenic