HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Santiago-Raber, M.-L. Articles by Kono, D. H. Search for Related Content PubMed PubMed Citation Articles by Santiago-Raber, M.-L. Articles by Kono, D. H. Pubmed/NCBI databases Gene GEO Profiles HomoloGene Nucleotide Protein UniGene Substance via MeSH Medline Plus Health Information Lupus

Characterization of Reciprocal Lmb1–4 Interval MRL-Fas^lpr and C57BL/6-Fas^lpr Congenic Mice Reveals Significant Effects from Lmb3¹

Marie-Laure Santiago-Raber^*, M. Katarina Haraldsson, Argyrios N. Theofilopoulos and Dwight H. Kono^2,

^* Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland; and Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Susceptibility to severe lupus in MRL-Fas^lpr mice requires not only the lpr mutation but also other predisposing genes. Using (MRL-Fas^lpr x B6-Fas^lpr)F2 (where B6 represents C57BL/6) intercrosses that utilize the highly susceptible MRL and poorly susceptible B6 backgrounds, we previously mapped CFA-enhanced systemic lupus-like autoimmunity to four loci, named Lmb1–4, on chromosomes 4, 5, 7, and 10. In the current study, we generated and analyzed reciprocal interval congenic mice for susceptibility to CFA-enhanced autoimmunity at all four Lmb loci. Although all loci had at least a slight effect on lymphoproliferation, only Lmb3 demonstrated a major effect on lymphoproliferation and anti-chromatin Ab levels. Further characterization of Lmb3, primarily by comparing MRL-Fas^lpr with MRL.B6-Lmb3 Fas^lpr congenic mice, revealed that it also played a significant role in spontaneous lupus, modifying lymphoproliferation, IgG and autoantibody levels, kidney disease, and survival. The less susceptible B6 Lmb3 locus was associated with a marked reduction in numbers of CD4⁺ and double-negative (CD4^–CD8^–) T cells, particularly in lymph nodes, as well as reduced T cell proliferation and enhanced T cell apoptosis, both in vivo and in vitro. IFN--producing CD4⁺ T cells were also reduced in MRL.B6-Lmb3 Fas^lpr mice. Further mapping using subinterval congenic mice placed Lmb3 in the telomeric portion of chromosome 7. Thus, Lmb3, primarily through its effects on CD4⁺ and double-negative T cells, appears to be a highly penetrant lupus-modifying locus. Identification of the underlying genetic alteration responsible for this quantitative trait locus should provide new insights into lupus-modifying genes.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
MRL-Fas^lpr mice develop an inherited fatal lupus-like disease with lymphoproliferation, marked expansion of double-negative (DN)³ T cells, hypergammaglobulinemia, abundant and diverse autoantibodies, severe immune complex-mediated glomerulonephritis (GN), ulcerating skin lesions, and mild polyarthritis (reviewed in Ref. 1). Genetic susceptibility is largely due to the apoptosis-impairing recessively transmitted Fas lpr mutation but also to other still undefined predisposing genes. Such background genes have been shown to play a critical role in the severity of both lymphoproliferation and autoimmunity and also, importantly, in the spectrum of lupus manifestations. Thus, elucidation of their identities and specific contributions to systemic autoimmunity is likely to greatly advance our understanding of the etiopathogenesis of this disease.

To accomplish this, we and others mapped lupus-predisposing loci in F2 or N2 crosses of MRL-Fas^lpr with several other strains, including CAST/Ei (2), C3H-Fas^lpr (3, 4, 5, 6), and B6-Fas^lpr (where B6 represents C57BL/6) (7). At least 15 loci located on eight of the 19 autosomal chromosomes were identified linked to various lupus-related manifestations. Several of these, including Lmb2 and Lprm4 on chromosome 5, Lmb3 and Ldrm1 on chromosome 7, and Lmb4 and Asm2 on chromosome 10, map to similar chromosomal intervals, suggesting that they might be identical. Similar to mapping studies involving other lupus-prone strains, findings in MRL crosses were consistent with polygenic inheritance, genetic heterogeneity of lupus-affecting genes, subsets of loci promoting specific traits, and limited contributions from most loci to the overall autoimmune phenotypes.

In our (MRL-Fas^lpr x B6-Fas^lpr)F2 study, four significant quantitative trait loci (QTL) named Lmb1–4 were identified on chromosomes 4, 5, 7, and 10, respectively (7). All contributed to lymphoproliferation in the spleen or lymph node (LN) and three (Lmb1–3) were also linked to elevated Abs to dsDNA. Lmb4 was also linked to GN, but at only at a suggestive significance level. Each locus appeared to contribute to lymphoproliferation in an additive fashion.

To further define the role of the Lmb QTL, reciprocal interval congenic mice for each of the Lmb loci were generated and characterized for the development of CFA-enhanced lupus. Strikingly, one of the Lmb QTL, Lmb3 on chromosome 7, was found to have a major effect. Further studies confirmed that it played a significant disease-modifying role in spontaneous lupus, altered T cell proliferation and apoptosis in MRL-Fas^lpr mice, and was located in the distal portion of chromosome 7.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice

MRL-Fas^lpr/MpScr, B6Scr-Fas^lpr, and related reciprocal congenics were bred and maintained at The Scripps Research Institute animal facility (La Jolla, CA) under specific pathogen-free conditions. Reciprocal congenic mice for Lmb1–4 were generated by marker-assisted selection (8) as previously described (9). Markers used to define Lmb intervals are listed on Table I (list of outside markers available on request). Mice were backcrossed for 7–12 generations and then intercrossed to produce homozygous interval congenic lines. In some experiments, 13-wk-old mice were injected with 0.05 ml of CFA supplemented with 10 mg/ml heat-inactivated Mycobacterium tuberculosis H37RA (Difco Laboratories) s.c. at two thoracic sites and sacrificed 5 wk later. All procedures performed were approved by The Scripps Research Institute’s institutional animal care and use committee.

Mice were followed for spontaneous or CFA-accelerated lupus for the indicated durations (7). Serum was obtained and autopsies performed at specified times as previously described (10). Tissues were fixed in Bouin’s or zinc formalin solution and sections were stained with periodic acid-Schiff reagent and hematoxylin. Glomerular IgG deposits were detected by staining frozen kidney sections with anti-IgG-FITC (Vector Laboratories) (11). The severity of GN and deposits was graded blindly on a 0–4 scale (12).

Serology

Ig and autoantibody levels were determined by ELISA as previously reported (9, 13). Microtiter plates were coated with Fc-specific F(ab')₂ goat anti-mouse IgG (5 µg/ml; Jackson ImmunoResearch Laboratories), calf thymus dsDNA (25 µg/ml), ssDNA (25 µg/ml; boiled dsDNA), calf thymus chromatin (5 µg/ml), Sm (5 µg/ml, ImmunoVision), or mouse IgG (BD Biosciences). Bound total IgG or IgG subclasses were detected with alkaline phosphatase-labeled goat Abs (Caltag Laboratories). The presence of IgM rheumatoid factor (RF) was assessed as previously described (14). A mouse serum with known concentrations of Ig (Nordic Immunological Laboratories) was used as the standard.

Flow cytometry

Cells were stained with fluorescent dye-labeled annexin V and Abs to CD3, CD4, CD8, CD19, CD43, CD45R (B220), CD69, IFN-, BrdU, and IgM (BD Biosciences). For surface staining, 10⁶ cells were incubated with Abs for 20 min at 4°C, and then resuspended in 2% FCS PBS after washing. Intracellular staining for IFN- entailed stimulating 10⁶ splenocytes/ml with 50 ng/ml PMA and 500 ng/ml ionomycin for 4 h at 37°C plus 1 µg/ml brefeldin A for the last 2 h (all from Sigma-Aldrich), surface staining with CD3, CD4 and CD8 Abs, fixation with 1% paraformaldehyde PBS for 20 min, and then incubation with anti-IFN- Ab in 0.5% BSA 0.1% saponin PBS for 1 h at room temperature. Staining for BrdU was performed per the manufacturer’s instructions (BrdU flow kit; BD Biosciences). Events were acquired on a FACSCalibur flow cytometer (BD Biosciences) and data were analyzed by CellQuest (BD Biosciences) or Flow Jo (Tree Star).

Cellular studies

In vitro thymidine incorporation following TCR engagement was performed as previously reported (15). Briefly, 1.5 x 10⁵/well sorted CD3⁺ propidium iodide-negative cells in 10% FCS DMEM were incubated in 96-well plates coated with various concentrations of purified anti-CD3 Ab (BD Biosciences) for 3 days. One microcurie of [³H]TdR was added to each well 16 h before harvesting. For BrdU labeling, mice received freshly prepared autoclaved drinking water containing 0.8 mg/ml BrdU (Sigma-Aldrich) every other day for 9 days.

Apoptosis assays

Spleen cell suspensions were treated with 1 µM dexamethasone (Acros Organics) or radiation (500 rad) and then incubated in 24-well plates at 2 x 10⁶ cells/well for 16 h. Anti-CD3 induced apoptosis was assessed as described previously with some minor modifications (16). Briefly, total LN cells were first activated for 48 h in 24-well plates (3 x 10⁶ cells/well) with 0.5 µg/ml anti-CD3 mAb (BD Pharmingen) and then flow cytometry-isolated T cells (CD3⁺ propidium iodide-negative; 2 x 10⁵ cells/0.2 ml) were incubated for 24 h at in anti-CD3-coated wells (20 µg/ml). Apoptosis was quantified by trypan blue exclusion or annexin V staining.

Statistical analysis

Group comparisons were analyzed by unpaired t test or Mann-Whitney U test. Survival was analyzed by Kaplan-Meier plot and log rank test. p < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Generation and characterization of reciprocal Lmb interval congenic mice

Interval congenic mice, generated for Lmb1–4 on both the MRL-Fas^lpr and B6-Fas^lpr backgrounds using marker-assisted selection, were assessed for susceptibility to CFA-enhanced lymphoproliferation and anti-nuclear Abs, the original traits used in identifying these QTL. Strikingly, Lmb3 had a marked effect on lymphoproliferation and/or anti-chromatin Abs in both reciprocal congenics (Figs. 1 and 2). When compared with B6-Fas^lpr mice, B6.MRL-Lmb3 Fas^lpr congenics had significantly larger spleens and LNs, which was associated with increased splenic CD4⁺ and DN T cells (Fig. 1, A and C). Reduced percentages of B cells were also found, but this was likely compensatory from the relative increase in T cells (see below). Despite these findings, no increase in anti-chromatin Ab levels was detected (Fig. 1B). These traits were also affected but in the opposite direction in MRL.B6-Lmb3 Fas^lpr congenic mice, with significant reductions in spleen and LN weights and percentages of CD4⁺ and DN T cells, compared with MRL-Fas^lpr mice (Fig. 2). In contrast to the B6.MRL-Lmb3 Fas^lpr congenic, however, anti-chromatin Ab levels were also affected and markedly reduced (Fig. 2B). This suggests that Lmb3 may be in epistasis with other autoantibody-promoting QTL.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Lymphoproliferation and anti-chromatin Ab in CFA-treated B6.MRL-Lmb interval congenic strains. A, Spleen and LN weights (log scale). LNs used were mesenteric, inguinal, and axillary. B, IgG anti-chromatin Ab levels (log scale). C, Splenic lymphoid populations (percentage of live-gated cells). B6-Faslpr and B.M-Lmb congenic mice (all Faslpr) were immunized with CFA at 13 wk of age and the indicated phenotypes were determined 5 wk. later. Unimmunized B6 control sera were from 6- to 8-wk-old mice. M, MRL; B, B6.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. Lymphoproliferation and anti-chromatin Ab in CFA-treated MRL.B6-Lmb interval congenic strains. A, Spleen and LN weights (log scale). B, IgG anti-chromatin Ab levels (log scale). C, Splenic lymphoid populations. MRL-Faslpr and M.B-Lmb congenic mice (all Faslpr) were immunized with CFA at 13 wk of age and the indicated phenotypes were determined 5 wk later. Unimmunized MRL- Faslpr control sera were from 4- to 5 mo-old mice. M, MRL; B, B6.

Lmb3 promotes spontaneous systemic autoimmunity

To further define the role of Lmb3 in autoimmunity, MRL.B6-Lmb3 Fas^lpr and B6.MRL-Lmb3 Fas^lpr, were analyzed for spontaneous lupus. Replacement of the MRL with the B6-Fas^lpr Lmb3 interval markedly increased the survival of MRL-Fas^lpr mice (p < 0.001; Fig. 3A). In this cohort, the 50% survival of the MRL-Fas^lpr mice was 8 mo with only one mouse (10%) alive at 12 mo, whereas >60% of congenic MRL.B6-Lmb3 Fas^lpr mice were alive at 12 mo. No effect of Lmb3 on the survival of B6-Fas^lpr mice was observed up to 12 mo of age, with no deaths in either the wild-type or Lmb3 congenic.

View larger version (45K): [in this window] [in a new window]   FIGURE 3. Spontaneous lupus is reduced in MRL.B6-Lmb3 Faslpr mice. A, Cumulative survival for MRL-Faslpr (n = 10) and MRL.B6-Lmb3 Faslpr (n = 14) mice; p < 0.001. B, Lymphoid organ weights (mean ± SEM); p < 0.05 for wild-type vs Lmb3 congenic MRL-Faslpr or B6-Faslpr mice. C, Glomerular pathology. Representative kidney sections (original magnification, x400) stained with periodic acid-Schiff/hematoxylin (upper panels) or FITC-labeled anti-mouse IgG (lower panels) show reduced glomerular damage and less IgG deposits in congenic mice. D, Kidney weight (grams) and GN scores (mean ± SEM) for parental and Lmb3 congenic mice. Both kidney weight and GN scores are reduced in MRL.B6-Lmb3 Faslpr compared with MRL-Faslpr mice (p < 0.05). All mice were females. In B–D, MRL-Faslpr and MRL.B6-Lmb3 Faslpr mice were 6- to 7-mo old, whereas B6-Faslpr mice and B6.MRL-Lmb3 Faslpr were 12-mo old; n = 3–6 per group. M, MRL; B, B6.

Immune-complex GN was also significantly reduced in MRL.B6-Lmb3 Fas^lpr mice (Fig. 3, C and D). Immune complex deposits of IgG were less and GN scores of 6- to7-mo-old congenic mice were significantly lower than those of wild-type MRL-Fas^lpr mice (1.5 ± 0.2 vs 3.4 ± 0.2, p < 0.05), although not as low as those of B6-Fas^lpr mice (0.50 ± 0.01).

Ig and autoantibodies in MRL.B6-Lmb3 Fas^lpr mice

Congenic MRL.B6-Lmb3 Fas^lpr mice had significantly lower levels of serum IgG primarily affecting the IgG2a and 2b subclasses (p < 0.05; Table II). All IgG subclasses except for IgG2b, however, were still higher than those in B6-Fas^lpr mice, consistent with a multigenic inheritance. IgG autoantibodies to ssDNA, dsDNA, and Sm were likewise all significantly reduced in the congenic MRL.B6-Lmb3 Fas^lpr mice, as were the levels of IgM RF against IgG1 and IgG3 (p < 0.05; Table II). Thus, it appears that the MRL Lmb3 promotes a broad spectrum of autoantibodies.

Analysis of T and B cell subsets revealed several significant changes associated with the Lmb3 locus (Tables III and IV). Most significant was a 2- to 3-fold reduction in the percentage of splenic CD4⁺ and DN T cells of MRL.B6-Lmb3 Fas^lpr mice compared with MRL- Fas^lpr mice at 4 mo along with a concomitant decrease in cells with the activated/memory phenotype (CD44^high), suggesting reduced T cell activation and expansion of the CD4⁺ and DN subsets. CD8⁺ T cells were less affected. In contrast, B6.MRL-Lmb3 Fas^lpr mice (that have the susceptible MRL Lmb3 locus) had increased percentages of CD4⁺ and DN T cells, but not CD8⁺ T cells, compared with B6- Fas^lpr mice at 12 mo. The greater number of CD4⁺ T cells in both the MRL-Fas^lpr and B6.MRL-Lmb3 Fas^lpr mice was partly due to the accumulation of the Fas^lpr–associated B220⁺ subset of CD4⁺ T cells (Table III).

Reduced proliferation of MRL.B6-Lmb3 Fas^lpr T cells

To determine whether the reduced numbers of lymphocytes in MRL.B6-Lmb3 Fas^lpr mice was from decreased proliferation, the in vivo incorporation of BrdU was assessed in wild-type and Lmb3 congenic MRL-Fas^lpr. Indeed, a significant reduction in the percentages of BrdU⁺ T cells and B cells from Lmb3 congenics was observed that involved the CD4⁺, DN, and, to a lesser extent, CD8⁺ T cell subsets (Table V). Reduced proliferation of purified T cells from MRL.B6-Lmb3 Fas^lpr mice was also found after in vitro activation of T cells with the plate-bound anti-CD3 Ab (Fig. 4).

View larger version (11K): [in this window] [in a new window]   FIGURE 4. Ex vivo proliferation of MRL.B6-Lmb3 Faslpr T cells to anti-CD3 Ab. Purified LN CD3+ T cells were stimulated with plate-bound anti-CD3 Ab and harvested 72 h later. Results are mean ± SEM cpm of [3H]TdR of triplicate wells from a pool of three mice per group. Data are representative of two experiments.

Strikingly, Lmb3-congenic MRL-Fas^lpr mice also had significantly increased percentages of early apoptotic (annexin V⁺) CD4⁺ and DN splenic T cells, indicating that Lmb3 may also affect the survival of Fas^lpr T cells (Table VI). Additional ex vivo studies showed reduced resistance of Lmb3-congenic Fas^lpr T cells to activation-induced cell death induced by religation of the Ag-receptor that affected the CD4⁺, CD8⁺, and DN subsets (Fig. 5A). When susceptibility to apoptosis either spontaneously or mediated by irradiation (500 rads) or dexamethasone was examined by trypan blue exclusion, a greater overall cell death of total splenocytes from Lmb3-congenic MRL-Fas^lpr mice was observed spontaneously or with either agent after 16 h of culture (Fig. 5B). Further analysis by annexin V staining showed greater spontaneous apoptosis of CD4⁺ and DN T cell subsets from the Lmb3 congenics, as well as slightly greater apoptosis of B and T cells exposed to radiation or a corticosteroid (Fig. 5B). The apparent effects of radiation and dexamethasone on T cells, however, is likely related to the greater spontaneous apoptosis in Lmb3 congenics.

View larger version (26K): [in this window] [in a new window]   FIGURE 5. MRL.B6-Lmb3 Faslpr T cells are less resistant to apoptosis in vitro. A, Anti-CD3 religation-induced apoptosis. LN cells were incubated with anti-CD3 Ab for 48 h and then isolated T cells were restimulated with plate-bound anti-CD3 for 24 h. The frequency of Annexin V+ CD4, CD8, and DN (CD4–CD8–) T cells is shown. B, Spontaneous and induced apoptosis. Spleen cells were incubated in medium alone (Med), after 500 rads (500R), or in 1 mM dexamethasone (DEX) for 16 h then analyzed by trypan blue or flow cytometry for dead (Trypan Blue+) or apoptotic cells (Annexin V+). Results are from a pool of three mice per group and are representative of one of two experiments.

IFN--producing CD4⁺ T cells in MRL.B6-Lmb3 Fas^lpr mice

The frequency of IFN-⁺ T cells in 4 mo-old Lmb3-congenic MRL-Fas^lpr mice was also examined, because IFN- is required for the development of lupus in MRL-Fas^lpr mice (17, 18, 19). The presence of the B6 Lmb3 interval resulted in a substantially lower frequency of IFN--producing CD4⁺ T cells in the MRL.B6-Lmb3 Fas^lpr mice to, remarkably, levels similar to those of the less lupus-susceptible B6-Fas^lpr mice (Fig. 6). In contrast, there was no difference in the frequency of IFN-⁺ cells in the CD8⁺ subset, and IFN- was not detectable in DN T cells (data not shown).

View larger version (15K): [in this window] [in a new window]   FIGURE 6. IFN--producing CD4+ T cells are reduced in MRL.B6-Lmb3 Faslpr (M.B-Lmb3 Faslpr) mice. Total splenocytes were stimulated in vitro with PMA/ionomycin for 4 h then analyzed by flow cytometry for CD4 and intracellular IFN-. The mean ± SD from two 4-mo-old mice is indicated.

Three subinterval congenic MRL-Fas^lpr mice were used to more precisely define the location of Lmb3. M7B(57/211) covered the centromeric and M7B(147/101) the telomeric parts of chromosome 7, whereas M7B(323/101) included fragments from both (Fig. 7A). Using reduced lymphoid organs weights and enhanced survival as the criteria for the B6 Lmb3 phenotype, it could be clearly seen that the B6 Lmb3 allele was present in the two subinterval lines with telomeric B6 intervals, M7B(147/101) and M7B(323/101), but not in M7B(57/211) with only the centromeric B6 interval (Fig. 7, B and C). This places Lmb3 between D7Mit350 (84 megabases (Mb)) and D7Mit109 (136 Mb). Interestingly, there was also a statistically significant difference in the cumulative survival of MRL-Fas^lpr mice and the centromeric interval-containing M7B(57/211) subline, but this was not reflected in the severity of lymphoproliferation.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. Subinterval MRL.B6-Lmb3 Faslpr (M.B-Lmb3 Faslpr) congenic mice place Lmb3 on distal chromosome 7. A, Genotypes of subinterval mice. B6 (black), MRL (no color), and undetermined (gray) genomic regions are indicated. Mb distances are based on the Ensembl (www.ensembl.org) and MGI (www.informatics.jax.org) databases. Lmb3 trait was determined by lymphoid organ weight and survival (see below). B, LN and spleen weights. Ages were 4–5 mo for MRL-Faslpr and M7B(57/211) mice and 6–9 mo for others; n = 14–20 (subinterval congenics), 13 (MRL-Faslpr), and 3 (M.B-Lmb3 Faslpr); *, p < 0.0001 vs MRL-Faslpr for both LN and spleen. C, Cumulative survival; n = 19–25 (subinterval congenics), 14 (MRL-Faslpr), and 3 (M.B-Lmb3 Faslpr); p < 0.0001 for M7B(57/211), M7B(147/101), and M7B(323/101) vs MRL-Faslpr.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

In contrast, the Lmb3 QTL had a marked effect on both the MRL-Fas^lpr and B6-Fas^lpr backgrounds, resulting in significant disease reduction in the MRL-Fas^lpr and enhancement of lymphoproliferation in the B6-Fas^lpr mice In the MRL-Fas^lpr mice, replacement of the MRL with the B6 Lmb3 interval resulted in significantly reduced lymphoproliferation, lower levels of total IgG, particularly IgG2a and IgG2b, reductions in all autoantibody specificities tested, including antinuclear and RF Abs, less GN, and substantial improvement in 1-year survival. The reduced lymphoproliferation was most dramatically observed in the LNs and involved primarily T cells, particularly the CD4⁺ and DN subsets. B cells were also affected, with minor changes in the bone marrow of MRL.B6-Lmb3 Fas^lpr mice and significant reductions in the total number of B cells and the percentage of recently activated (CD69⁺) cells. These findings suggest that Lmb3 acts primarily on T lymphocytes and to a lesser extent on B cells, although the reduced activation and number of B cells may be secondary to alterations in T cell function.

Additional in vitro and in vivo studies demonstrating that mice with the MRL Lmb3 allele have greater T cell proliferation and less apoptosis than mice with the B6 Lmb3 allele further support the possibility that Lmb3 has an intrinsic effect on T cells. Interestingly, a New Zealand White (NZW)-derived, lupus-predisposing locus, Sle3, is also located on chromosome 7 and is similarly associated with enhanced T cell activation and reduced apoptosis (20). In this instance, however, the cause of the T cell hyperactivity is not T cell intrinsic but is due to enhanced Ag presentation and proinflammatory activity by dendritic cells and macrophages (21). This suggests that the Lmb3 and Sle3 susceptibility genes are probably different. Two other loci were also mapped to the vicinity of Lmb3. Aem2, localized near D7Mit30 (81.4 Mb; 37.0 centimorgans (cM)) using (B6 x NZB)F1 x NZB backcrosses, is a NZB susceptibility locus for anti-RBC Ab, whereas Myo1, a BXSB susceptibility locus for myocardial infarction, was mapped in NZW x (NZW x BXSB)F1 backcrosses to around D7Mit14 (141.2 Mb; 69.0 cM). The relationship of Lmb3 to these loci remains to be determined.

Several genetic alterations in mice have resulted in enhanced T cell activation and systemic autoimmunity, including the Roquin (Rc3h1) M199R mutation, the linker for the activation of T cells (Lat) Y236F mutation, transgenic expression of LIGHT (Tnfsf14) in T cells, and deficiencies of CTLA-4 (Cd152), PD-1 (Pdcd1), TGF-1 (Tgfb1), Cbl-b, p21^Cip1/Waf1 (Cdkn1a), Gadd45 and , Gpr132, mannoside acetyl glucosaminyltransferase 5 (Mgat5), E2F2, and protein-L-isoaspartate (D-aspartate) O-methyltransferase 1 (Pcmt1) (reviewed in Ref. 1). Of these, only Lat is located within the Lmb3 interval (126.2 Mb); however, the MRL and B6 Lat cDNA sequences are identical and, by semiquantitative PCR, are expressed at similar levels in young mice (data not shown). Similarly, mutations or transgenic expression leading to defective apoptosis of lymphocytes and lupus-like disease have been described for Bim (Bcl2l11), Bcl-2, TSAd (Sh2d2a), IL-2, IL-2R and , Flip (Cflar), Pten, IEX-1 (Ier3), Ras GRP1, and Stra13 (reviewed in Ref. 1). None of these genes, however, are situated within the Lmb3 interval.

Among the various cytokines implicated in the pathogenesis of systemic lupus erythematosus (SLE), IFN- has been uniformly shown to play a critical pathogenic role in all major lupus-prone strains, including the MRL-Fas^lpr (17, 18, 19). Moreover, complete inhibition of IFN- is not required for a significant beneficial effect (11, 19). In MRL.B6-Lmb3 Fas^lpr mice, replacement of the MRL with the B6 Lmb3 interval resulted in a substantial decrease in the number of IFN--producing CD4⁺ T cells. This reduction, which indicates lower production of IFN-, is likely one of the main reasons for the lower levels of Th1-induced polyclonal and autoantibody IgG subclasses and almost certainly a factor in the attenuation of disease severity.

A related congenic B6 strain (B6.MRLc7) containing an introgressed centromeric region of the MRL genome extending from at least D7Mit178 (Mb not mapped in current Ensembl m36 mouse assembly, 0.5 cM) up to D7Mit147 (74.2 Mb, 37 cM) was also previously studied in mice with and without the Fas^lpr defect (22). In this instance, the MRLc7 fragment enhanced and accelerated autoantibody production, kidney disease, T cell activation, and expansion of DN T cells only in Fas^lpr mice. Although this study suggested that the MRLc7 interval was Lmb3 and there are, indeed, many effects of this interval on lupus and the immune system similar to Lmb3, more precise mapping from our study indicates that there is no overlap of MRLc7 with the Lmb3 interval. Thus, MRLc7 may be another susceptibility locus with weaker but significant effects. Our finding that the survival of M7B(57/211) subinterval congenic mice, which have the centromeric MRL chromosome 7 fragment, was slightly but significantly better than that of MRL-Fas^lpr mice certainly supports this possibility.

Finally, the results herein confirm the presence of the Lmb loci and provide the foundation to further map and then clone the underlying gene or genes. This is especially true for Lmb3, which has robust surrogate phenotypes that can be used to distinguish the presence of MRL or B6 alleles in relatively young mice. Identification of the specific genes should yield new insights into the pathogenesis of SLE as well as novel molecules or pathways to target intervention.

	Acknowledgments

 
We thank Kat Occhipinti for editorial assistance and Nate Dela Paz, Jason Kuan, Jim Lee, and Cynthia Thompson for outstanding technical support.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This is publication 18691-IMM from the Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. The work reported herein was supported by National Institutes of Health Grants AR39555, AR42242, AR31203, and AI051977.

² Address correspondence and reprint requests to Dr. Dwight H. Kono, Department of Immunology/IMM3, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: dkono{at}scripps.edu

³ Abbreviations used in this paper: DN, double negative (CD4^–CD8^–); GN, glomerulonephritis; B6, C57BL/6; cM, centimorgan; LN, lymph node; Mb, megabase; NZW, New Zealand White; QTL, quantitative trait locus; RF, rheumatoid factor; SLE, systemic lupus erythematosus.

Received for publication January 22, 2007. Accepted for publication March 29, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Kono, D. H., A. N. Theofilopoulos. 2006. Genetics of SLE in mice. Springer Semin. Immunopathol. 28: 83-96. [Medline]
2. Watson, M. L., J. K. Rao, G. S. Gilkeson, P. Ruiz, E. M. Eicher, D. S. Pisetsky, A. Matsuzawa, J. M. Rochelle, M. F. Seldin. 1992. Genetic analysis of MRL-lpr mice: relationship of the Fas apoptosis gene to disease manifestations and renal disease-modifying loci. J. Exp. Med. 176: 1645-1656. [Abstract/Free Full Text]
3. Wang, Y., M. Nose, T. Kamoto, M. Nishimura, H. Hiai. 1997. Host modifier genes affect mouse autoimmunity induced by the lpr gene. Am. J. Pathol. 151: 1791-1798. [Abstract]
4. Nishihara, M., M. Terada, J. Kamogawa, Y. Ohashi, S. Mori, S. Nakatsuru, Y. Nakamura, M. Nose. 1999. Genetic basis of autoimmune sialadenitis in MRL/lpr lupus-prone mice: additive and hierarchical properties of polygenic inheritance. Arthritis Rheum. 42: 2616-2623. [Medline]
5. Qu, W., T. Miyazaki, M. Terada, L. Lu, M. Nishihara, A. Yamada, S. Mori, Y. Nakamura, H. Ogasawara, C. Yazawa, et al 2000. Genetic dissection of vasculitis in MRL/lpr lupus mice: a novel susceptibility locus involving the CD72^c allele. Eur. J. Immunol. 30: 2027-2037. [Medline]
6. Kamogawa, J., M. Terada, S. Mizuki, M. Nishihara, H. Yamamoto, S. Mori, Y. Abe, K. Morimoto, S. Nakatsuru, Y. Nakamura, M. Nose. 2002. Arthritis in MRL/lpr mice is under the control of multiple gene loci with an allelic combination derived from the original inbred strains. Arthritis Rheum. 46: 1067-1074. [Medline]
7. Vidal, S., D. H. Kono, A. N. Theofilopoulos. 1998. Loci predisposing to autoimmunity in MRL-Fas^lpr and C57BL/6-Fas^lpr mice. J. Clin. Invest. 101: 696-702. [Medline]
8. Markel, P., P. Shu, C. Ebeling, G. A. Carlson, D. L. Nagle, J. S. Smutko, K. J. Moore. 1997. Theoretical and empirical issues for marker-assisted breeding of congenic mouse strains. Nat. Genet. 17: 280-284. [Medline]
9. Haraldsson, M. K., N. G. dela Paz, J. G. Kuan, G. S. Gilkeson, A. N. Theofilopoulos, D. H. Kono. 2005. Autoimmune alterations induced by the New Zealand Black Lbw2 locus in BWF1 mice. J. Immunol. 174: 5065-5073. [Abstract/Free Full Text]
10. Kono, D. H., R. W. Burlingame, D. G. Owens, A. Kuramochi, R. S. Balderas, D. Balomenos, A. N. Theofilopoulos. 1994. Lupus susceptibility loci in New Zealand mice. Proc. Natl. Acad. Sci. USA 91: 10168-10172. [Abstract/Free Full Text]
11. Lawson, B. R., G. J. Prud’homme, Y. Chang, H. A. Gardner, J. Kuan, D. H. Kono, A. N. Theofilopoulos. 2000. Treatment of murine lupus with cDNA encoding IFN-R/Fc. J. Clin. Invest. 106: 207-215. [Medline]
12. Andrews, B. S., R. A. Eisenberg, A. N. Theofilopoulos, S. Izui, C. B. Wilson, P. J. McConahey, E. D. Murphy, J. B. Roths, F. J. Dixon. 1978. Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains. J. Exp. Med. 148: 1198-1215. [Abstract/Free Full Text]
13. Burlingame, R. W., R. L. Rubin, R. S. Balderas, A. N. Theofilopoulos. 1993. The genesis and evolution of anti-chromatin autoantibodies in murine lupus implicates T-dependent immunization with self antigen. J. Clin. Invest. 91: 1687-1696. [Medline]
14. Feeney, A. J., B. R. Lawson, D. H. Kono, A. N. Theofilopoulos. 2001. Terminal deoxynucleotidyl transferase deficiency decreases autoimmune disease in MRL-Fas^lpr mice. J. Immunol. 167: 3486-3493. [Abstract/Free Full Text]
15. Santiago-Raber, M. L., B. R. Lawson, W. Dummer, M. Barnhouse, S. Koundouris, C. B. Wilson, D. H. Kono, A. N. Theofilopoulos. 2001. The role of cyclin kinase inhibitor p21 in systemic autoimmunity. J. Immunol. 167: 4067-4074. [Abstract/Free Full Text]
16. Lawson, B. R., R. Baccala, J. Song, M. Croft, D. H. Kono, A. N. Theofilopoulos. 2004. Deficiency of the cyclin kinase inhibitor p21(WAF-1/CIP-1) promotes apoptosis of activated/memory T cells and inhibits spontaneous systemic autoimmunity. J. Exp. Med. 199: 547-557. [Abstract/Free Full Text]
17. Peng, S. L., J. Moslehi, J. Craft. 1997. Roles of interferon- and interleukin-4 in murine lupus. J. Clin. Invest. 99: 1936-1946. [Medline]
18. Haas, C., B. Ryffel, H. M. Le. 1997. IFN- is essential for the development of autoimmune glomerulonephritis in MRL/Ipr mice. J. Immunol. 158: 5484-5491. [Abstract]
19. Balomenos, D., R. Rumold, A. N. Theofilopoulos. 1998. Interferon- is required for lupus-like disease and lymphoaccumulation in MRL-lpr mice. J. Clin. Invest. 101: 364-371. [Medline]
20. Mohan, C., Y. Yu, L. Morel, P. Yang, E. K. Wakeland. 1999. Genetic dissection of Sle pathogenesis: Sle3 on murine chromosome 7 impacts T cell activation, differentiation, and cell death. J. Immunol. 162: 6492-6502. [Abstract/Free Full Text]
21. Zhu, J., X. Liu, C. Xie, M. Yan, Y. Yu, E. S. Sobel, E. K. Wakeland, C. Mohan. 2005. T cell hyperactivity in lupus as a consequence of hyperstimulatory antigen-presenting cells. J. Clin. Invest. 115: 1869-1878. [Medline]
22. Kong, P. L., L. Morel, B. P. Croker, J. Craft. 2004. The centromeric region of chromosome 7 from MRL mice (Lmb3) is an epistatic modifier of Fas for autoimmune disease expression. J. Immunol. 172: 2785-2794. [Abstract/Free Full Text]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Santiago-Raber, M.-L. Articles by Kono, D. H. Search for Related Content PubMed PubMed Citation Articles by Santiago-Raber, M.-L. Articles by Kono, D. H. Pubmed/NCBI databases Gene GEO Profiles HomoloGene Nucleotide Protein UniGene Substance via MeSH Medline Plus Health Information Lupus