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The Journal of Immunology, 2001, 167: 2370-2378.
Copyright © 2001 by The American Association of Immunologists

Fas/Fas Ligand Deficiency Results in Altered Localization of Anti-Double-Stranded DNA B Cells and Dendritic Cells1

Michele L. Fields*, Caroline L. Sokol*, Ashlyn Eaton-Bassiri*, Su-jean Seo*, Michael P. Madaio{dagger} and Jan Erikson2,*

* Wistar Institute, Philadelphia, PA 19104; and {dagger} Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Autoantibodies directed against dsDNA are found in patients with systemic lupus erythematosus as well as in mice functionally deficient in either Fas or Fas ligand (FasL) (lpr/lpr or gld/gld mice). Previously, an IgH chain transgene has been used to track anti-dsDNA B cells in both nonautoimmune BALB/c mice, in which autoreactive B cells are held in check, and MRL-lpr/lpr mice, in which autoantibodies are produced. In this study, we have isolated the Fas/FasL mutations away from the autoimmune-prone MRL background, and we show that anti-dsDNA B cells in Fas/FasL-deficient BALB/c mice are no longer follicularly excluded, and they produce autoantibodies. Strikingly, this is accompanied by alterations in the frequency and localization of dendritic cells as well as a global increase in CD4 T cell activation. Notably, as opposed to MRL-lpr/lpr mice, BALB-lpr/lpr mice show no appreciable kidney pathology. Thus, while some aspects of autoimmune pathology (e.g., nephritis) rely on the interaction of the MRL background with the lpr mutation, mutations in Fas/FasL alone are sufficient to alter the fate of anti-dsDNA B cells, dendritic cells, and T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The presence of serum autoantibodies directed against nuclear Ags such as dsDNA is one of the hallmarks of systemic lupus erythematosus (1). Mouse strains with mutations in the genes for Fas and Fas ligand (FasL)3 (i.e., lpr and gld) (2, 3) produce autoantibodies similar to those found in systemic lupus erythematosus patients. lpr and gld mice also develop lymphadenopathy, increased total serum Ig, accelerated mortality, and severe nephritis, although the latter is strain dependent (1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). While these findings implicate Fas/FasL deficiency in autoantibody production, it is uncertain how these mutations lead to the differentiation of autoreactive B cells.

We have used the VH3H9 IgH transgene (Tg) model to track anti-dsDNA B cells in both nonautoimmune-prone (BALB/c) and autoimmune-prone (MRL-lpr/lpr) mice in vivo (16, 17, 18, 19). In VH3H9 BALB/c mice, anti-dsDNA B cells are present with a developmentally arrested phenotype and localize to the interface between the T and B cell areas in the splenic white pulp. Anti-dsDNA Abs are not produced in these mice. In contrast, anti-dsDNA B cells in VH3H9 MRL-lpr/lpr mice have a mature phenotype, populate the splenic B cell follicles, and produce anti-dsDNA Abs.

The purpose of the present study was to identify the specific effects of Fas/FasL mutations on anti-dsDNA B cells, independent of the autoimmune MRL background (4, 9, 11, 20, 21, 22, 23). To this end, the lpr and gld defects were bred onto the VH3H9 BALB/c strain. Unlike their wild-type counterparts, anti-dsDNA B cells in VH3H9 BALB-lpr/lpr and gld/gld mice entered B cell follicles, and by 10 wk of age their Abs were detectable in the serum. Strikingly, as early as 5–6 wk of age, lpr/lpr mice had an increased frequency and altered localization of dendritic cells (DCs), and this was associated with a global increase in CD4 T cell activation. Nevertheless, other aspects of autoimmunity, e.g., nephritis, appear restricted to the MRL-lpr/lpr mouse strain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental mice

Tg- and VH3H9 BALB-lpr/lpr mice were generated by breeding VH3H9 MRL-lpr/lpr mice with BALB/c mice, followed by backcrossing onto the BALB/c background (at least seven times), and then intercrossing to generate homozygous mice. Because it was possible that MRL genes linked to the lpr gene on chromosome 19 (2) might be carried onto the BALB/c background, we took a second approach to studying Fas/FasL deficiency by also examining BALB-gld/gld mice. The gld defect, located on chromosome 1, was originally found as a spontaneous mutation in C3H mice (3, 24, 25). VH3H9 BALB/c mice were mated with BALB-gld/gld mice, followed by intercrosses to generate homozygous gld/gld mice. BALB/c mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). MRL-lpr/lpr, MRL+/+, and BALB-gld/gld (Cpt substrain, backcrossed onto the BALB background for at least 15 generations) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). VH3H9 mice have been previously described (16). The VH3H9 Tg has been backcrossed onto the BALB/c and MRL backgrounds for at least 17 generations.

All mice were bred and maintained in a specific pathogen-free room at the Wistar Institute animal facility. In all experiments, mice were age matched, and BALB/c mice and Tg- littermates were used as controls. Male and female mice were used with no apparent differences. The presence of the VH3H9 Tg and the lpr and gld mutations was determined by PCR amplification of tail DNA with primers specific for VH3H9 (16, 17, 18, 19), Fas (26), and FasL (a gift of M. Maldonado, Department of Rheumatology, University of Pennsylvania). The primers used for FasL were as follows: FasL wild-type locus, 5'-CTC TGA TCA ATT TTG AGG AAT CTA AGA CGT-3'; FasL mutant locus, 5'-CTC TTG GCC ATT TAA CAT CAG ACA GTT CTT-3'. The PCR conditions used for FasL were: 92°C for 5 min; 92°C for 30 s, 60°C for 1 min, and 72°C for 45 s; for 36 cycles; ending with an extension period at 72°C for 10 min.

Identification of anti-dsDNA B cells

The VH3H9 IgH chain paired with the V{lambda}1 L chain produces an anti-dsDNA Ab (27, 28). The majority (average >95%) of Ig{lambda}+ B cells in VH3H9 BALB-lpr/lpr and BALB-gld/gld mice were Ig{lambda}1+ (data not shown), as was demonstrated previously for VH3H9 BALB/c and MRL-lpr/lpr mice by flow cytometry (17, 18). Therefore, we used pan-anti-Ig{lambda} as well as anti-Ig{lambda}1 reagents to identify anti-dsDNA B cells in VH3H9 mice. In VH3H9 BALB-lpr/lpr and BALB-gld/gld mice at the time points studied (6–12 wk), >95% of the B cells in the mouse expressed surface IgM only, not IgD (data not shown), consistent with the exclusive use of the IgM-only VH3H9 Tg.

Flow cytometric analysis

Spleens were removed from VH3H9 Tg and Tg- mice. Single cell suspensions were prepared, and erythrocytes were lysed (RBC lysing buffer; Sigma, St. Louis, MO). Cells (~1 x 106) were surface stained according to published protocols (29). The following Abs and secondary reagents were used: 1D3 FITC or biotin (anti-CD19), 7G6 FITC (CR2/CR1, anti-CD21/35), Cy34.1 FITC or biotin (anti-CD22), B3B4 FITC (anti-CD23), IM7 FITC (anti-CD44), RA3-6B2 FITC or PE (anti-B220), R11-153 biotin (anti-V{lambda}1), 145-2C11 FITC or PE (anti-CD3), GK1.5 FITC or PE (anti-CD4), 53-6.72 PE (anti-CD8), H1.2F3 FITC (anti-CD69, VEA), 7D4 FITC or PE (anti-IL-2R{alpha}-chain p55, CD25), HC3 FITC or PE (anti-CD11c), 16-10A1 FITC (anti-B7.1/CD80), GL1 PE (anti-B7.2/CD86), 2G9 FITC (anti-I-Ad/I-Ed), and anti-CD40 FITC (Becton Dickinson/PharMingen, San Diego, CA); 33D1 FITC (Leinco Technologies, St. Louis, MO); JC5.1 PE (anti-V{lambda} total, gift of J. Kearney, University of Alabama, Birmingham, AL); GK1.5 (anti-CD4) and RA3-6B2 (anti-B220), which were grown as supernatants and then biotinylated; and streptavidin Red 670 (Life Technologies, Gaithersburg, MD). For CD80 or CD86 staining, FcR were first blocked by incubation with 2.4G2. Cell size was gauged by the forward scatter values of the cells. Samples were collected on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and analyzed using CellQuest software. For B and T cell analyses, 40,000–80,000 events, gated for live lymphocytes based on forward and side scatter, were collected for each sample. For DC analyses, 100,000 events, gated for larger size and granularity, were collected for each sample. To determine absolute numbers within a cell type, their frequency within live gated splenocytes was multiplied by the total number of live splenocytes (determined by trypan blue exclusion).

Spleen preparations for DC experiments

Spleens were injected with 0.5 ml collagenase solution (100 U/ml Liberase Cl (Boehringer Mannheim, Indianapolis, IN) containing 0.2 mg/ml DNase I (Sigma) in HBSS (Cellgro, Herndon, VA)). Spleens were teased into small fragments and then incubated at 37°C for 30 min in 400 U/ml Liberase Cl in HBSS. Splenic fragments were then pushed through a cell strainer (100 µM). The single cell suspension was mixed with RPMI + 5 mM EDTA (Life Technologies) to inhibit the collagenase. Cells were then treated to lyse erythrocytes and prepared for flow cytometry, as described above.

Anti-nuclear Ab (ANA) and Crithidia luciliae assays

The presence of ANAs and anti-dsDNA Abs in the serum was detected using the ANA and C. luciliae assays, respectively, as previously described (18, 30). Binding was detected using either anti-IgM + IgG FITC (together, to detect total Ig) or anti-Ig{lambda} FITC (all from Southern Biotechnology Associates, Birmingham, AL).

Tissue immunohistochemistry

Spleens from experimental mice were prepared and stained as previously described (18, 31). The following Abs were used: Cy34.1 FITC or biotin (anti-CD22), RA3-6B2 biotin (anti-B220), GK1.5 FITC or biotin (anti-CD4), HC3 FITC (anti-CD11c), M1/70 FITC (anti-CD11b, Mac-1) (PharMingen), and/or anti-Ig{lambda} alkaline phosphatase (AP; Southern Biotechnology Associates). FITC- and biotin-conjugated reagents were detected with the secondary reagents anti-FITC AP (Sigma) or anti-FITC HRP (Chemicon, Temecula, CA), and streptavidin AP or HRP, respectively (Southern Biotechnology Associates). AP and HRP were developed with the substrates Fast-Blue BB base (blue) (Sigma), and 3-amino-9-ethyl-carbazole (red), respectively.

Kidney pathology

Sixteen- to twenty-four-week-old Tg- BALB/c, BALB-lpr/lpr, and MRL-lpr/lpr mice were sacrificed, and kidneys were fixed in 10% buffered Formalin (Fisher Scientific, Pittsburgh, PA), then embedded in paraffin. Kidneys were sectioned to 4-µm thickness and stained with H&E. The presence of renal pathology was determined as described (30, 32) by a single investigator (M. P. Madaio) without knowledge of age or genotype of the mice. Kidneys were graded for severity of disease in three areas (vascular, interstitial, and glomerular) in a range of 0 (for no pathology evident) to 4+ (most severe pathology, end stage disease) (33), and cumulative scores were determined.

Statistics

Statistical significance was determined using an unpaired nonparametric (Wilcoxon or Mann-Whitney) test, Student’s t test, or alternate Welch t test, when appropriate. Statistical significance was ascribed when p values were less than 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lymphoproliferation in BALB-lpr/lpr mice

BALB-lpr/lpr mice were bred to dissect the effects of Fas independent of the MRL background. As was the case for other strains of mice onto which the lpr mutation has been bred (MRL, C57BL/6, C3H, and AKR) (4, 6, 7, 9, 10, 11, 12, 13, 14, 15), by 9–12 wk of age BALB-lpr/lpr mice had a gross increase in spleen weight (Table IGo). This was largely due to significant increases in numbers of B220+CD3+CD4-CD8- double-negative T cells and B220+CD4+ T cells as compared with BALB/c control mice.


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  		Table I. Spleen characteristics from Tg- BALB/c, BALB-lpr/lpr, and MRL-lpr/lpr micea

 
Altered localization and cell surface phenotype of anti-dsDNA B cells in BALB-lpr/lpr and BALB-gld/gld mice

We have previously used VH3H9 IgH Tg mice to study the regulation of anti-dsDNA B cells (16, 27, 34). In vivo, B cells utilize the VH3H9 IgH in combination with a variety of L chains, thus generating a heterogeneous B cell population that includes both anti-DNA and non-DNA B cells (27). The pairing of the VH3H9 IgH with the V{lambda}1 L chain generates an anti-dsDNA Ab (27, 28). This facilitates tracking of anti-dsDNA B cells in vivo in a diverse repertoire by the use of anti-Ig{lambda} reagents (17, 18, 19).

Anti-dsDNA B cells in VH3H9 BALB-lpr/lpr and BALB-gld/gld mice localized in splenic B cell follicles, in contrast to their Fas-sufficient counterparts, which were follicularly excluded (Fig. 1Go). Strikingly, this altered localization of anti-dsDNA B cells in lpr/lpr and gld/gld mice was apparent at an early age (5–6 wk) and persisted in the oldest mice examined (12 wk).



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  		FIGURE 1. Altered localization of anti-dsDNA B cells in lpr/lpr and gld/gld mice. Spleen sections from VH3H9 BALB/c, VH3H9 BALB-lpr/lpr, and VH3H9 BALB-gld/gld mice were stained with Abs to Ig{lambda} (blue), and either CD22 or CD4 (red). Anti-dsDNA B cells localize at the T/B interface in VH3H9 BALB/c mice, but populate B cell follicles in VH3H9 BALB-lpr/lpr and BALB-gld/gld mice. n >= 8 mice for each genotype.

 
In all VH3H9 mouse genotypes examined, the anti-dsDNA B cells had decreased surface levels of Ig and CD21/35 (Fig. 2GoA), a phenotype that we and others have attributed to continual Ag encounter (17, 18, 30, 35, 36, 37, 38, 39, 40, 41, 42). At all ages examined, anti-dsDNA B cells from VH3H9 BALB-lpr/lpr mice had increased levels of B220, as compared with Fas-sufficient VH3H9 BALB/c mice (Fig. 2GoB); these levels were similar to those in VH3H9 MRL-lpr/lpr mice (18). In terms of the other maturation/activation markers studied (CD22, CD44, and cell size), the anti-dsDNA B cells from VH3H9 BALB-lpr/lpr mice fell into two groups that segregated with age (Fig. 2GoC). Anti-dsDNA B cells from young (<=8-wk) VH3H9 BALB-lpr/lpr mice were similar to those in Fas-sufficient VH3H9 BALB/c mice, expressing low levels of CD22 and increased levels of CD44 and cell size. However, in older VH3H9 BALB-lpr/lpr mice, (>=8 wk of age), the anti-dsDNA B cells up-regulated CD22, down-regulated CD44, and were smaller. This latter phenotype is like that seen in VH3H9 MRL-lpr/lpr mice of all ages (18). The phenotypic patterns observed in young and old VH3H9 BALB-gld/gld mice were comparable with those in age-matched VH3H9 BALB-lpr/lpr mice (data not shown, n = 6 at <=8 wk; n = 7 at >=8 wk).



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  		FIGURE 2. The phenotype of anti-dsDNA B cells in VH3H9 BALB-lpr/lpr or BALB-gld/gld mice. Splenocytes were stained with Abs against CD19, Ig{lambda}, CD21/35, B220, CD22, and CD44. Histograms are gated on the Ig{lambda}- B cell population from a Tg- BALB/c mouse (thin black line) and Ig{lambda}+ cells in each mouse (gray line). Shown are representative phenotypes of Ig{lambda}+ B cells in Tg- BALB/c (5–12 wk (17 18 ) and n = 7, this study), VH3H9 BALB/c (5–12 wk (17 18 ) and n = 7, this study), VH3H9 BALB-lpr/lpr mice (5–8 wk, n = 8), and VH3H9 BALB-lpr/lpr mice (8–12 wk, n = 13). The underlaid histograms (thin black line) were scaled down to allow for comparison with the Ig{lambda}+ cells, which only comprise ~10% of the B cell population in VH3H9 mice. A, Anti-dsDNA B cells in VH3H9 mice have decreased levels of Ig and CD21/35. Notably, older (8- to 12-wk) VH3H9 BALB-lpr/lpr mice have a reduced frequency and number of CD19+Ig{lambda}+ cells compared with younger (5–8 wk) VH3H9 BALB-lpr/lpr mice: 2.79% ± 1.22 vs 6.90% ± 0.96, p < 0.0001; absolute number 2.76 x 106 vs 4.74 x 106, p = 0.0049. B, Anti-dsDNA B cells from VH3H9 BALB-lpr/lpr mice have increased levels of B220, in contrast to VH3H9 BALB/c mice. C, Anti-dsDNA B cells from young (<8-wk) VH3H9 BALB-lpr/lpr mice are similar to VH3H9 BALB/c mice in terms of CD22 and CD44 expression, and cell size. However, in older VH3H9 BALB-lpr/lpr mice, >8 wk of age, anti-dsDNA B cells have up-regulated CD22 and down-regulated CD44, and decreased in cell size. Fifty percent of the VH3H9 BALB-lpr/lpr mice studied at 8 wk fall into each group. Ig{lambda}+ B cell phenotypes in Tg- and VH3H9 BALB/c mice are age independent (up to 12 wk); thus, they were not divided into age groups.

 
Autoantibody production in lpr/lpr mice

By 10 wk of age, 100% of BALB-lpr/lpr mice produced ANAs with patterns indistinguishable from those of MRL-lpr/lpr mice (Fig. 3Go). BALB-gld/gld mice also developed ANAs with similar patterns and frequencies (n = 8, data not shown). ANA titers in BALB-lpr/lpr and MRL-lpr/lpr mice were not significantly different, and at 10 wk of age, the titer and incidence of ANAs were not affected by the presence of the VH3H9 Tg (Table IIGo). Sera from older (9- to 12-wk-old) ANA+ Fas/FasL-deficient BALB/c mice contained Ig{lambda}+ anti-dsDNA Abs (for VH3H9 BALB-lpr/lpr mice, n = 10/10; for VH3H9 BALB-gld/gld mice, n = 8/8, data not shown). Consistent with the serum data, staining of spleens from 9- to 12-wk-old Fas/FasL-deficient VH3H9 mice revealed darkly staining Ig{lambda}+ cells in the T cell area as well as in the bridging channels to the red pulp, which coincided with staining for syndecan-1, a marker of Ab-forming cells (data not shown and (18, 43)).



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  		FIGURE 3. Mice deficient in Fas/FasL develop lupus autoantibodies. Sera were tested for the presence of total ANAs by immunofluorescence. Shown are representative nuclear staining patterns seen in the ANA assay of Tg- MRL-lpr/lpr and BALB-lpr/lpr mice. At 10 wk of age, >95% of Tg- MRL-lpr/lpr mice (Ref. 18 and n = 5, this study) and 100% of BALB-lpr/lpr mice (n = 12) are serum ANA positive.

 

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  		Table II. Mean ANA titers in BALB-lpr/lpr and MRL-lpr/lpr mice are not statistically differenta

 
Altered DC localization and increased frequency/numbers in lpr/lpr mice

DCs express Fas, although its role on these cells is controversial (44, 45, 46, 47, 48, 49, 50). Whereas several studies have illustrated Fas-mediated apoptosis of DCs (45, 46, 47), recent data suggest not only that DCs resist Fas-induced death, but also that signals through Fas may induce DC maturation (50). To evaluate DCs in Fas/FasL-deficient mice, spleen sections were stained with Abs to the integrin CD11c (51). Immunohistochemical staining demonstrated striking differences in the splenic localization of DCs in Tg- MRL-lpr/lpr and BALB-lpr/lpr mice compared with MRL+/+ and BALB/c mice (Fig. 4Go). As previously reported (49, 51, 52, 53, 54), splenic DCs were clustered primarily in the bridging channels to the red pulp in Fas-sufficient mice (Fig. 4Go). In contrast, DCs were spread throughout the T cell zone (periarteriolar lymphoid sheath, PALS) in spleens of Fas/FasL-deficient mice. This altered localization was evident in lpr/lpr mice as early as 5–6 wk of age and persisted up to 12 wk of age, the latest time point examined. BALB-gld/gld mice also exhibited this altered DC localization (n = 3, data not shown). Furthermore, the lpr mutation resulted in an increased frequency and number of DCs in both BALB/c and MRL strains (Fig. 5Go, A and B). Additionally, older lpr/lpr mice had an increased absolute number of DCs compared with younger lpr/lpr mice.



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  		FIGURE 4. DCs in lpr/lpr mice have an altered localization compared with those in Fas-sufficient mice. Spleen sections from Tg- BALB/c, BALB-lpr/lpr, MRL+/+, and MRL-lpr/lpr mice were stained with Abs to CD11c (blue) and CD22 (red). While CD11c+ DCs are concentrated in the bridging channels in Fas-sufficient mice (arrows), they are spread throughout the PALS in lpr/lpr mice. This altered localization is observed at ages 5–12 wk. n >= 6 mice for each genotype.

 


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  		FIGURE 5. DC frequency and numbers are elevated in lpr/lpr mice. Each circle represents one mouse. {circ}, Mice age 5–9 wk; •, mice age 10–15 wk. A, Frequency was assessed as percentage of B220-CD11c+ cells of live splenocytes. Over the entire age range (5–15 wk), mean frequencies are: BALB/c, 0.95%; BALB-lpr/lpr, 1.45%; MRL+/+, 0.68%; and MRL-lpr/lpr, 1.11%. The lpr mutant mice had significantly increased frequencies of DCs over Fas-sufficient mice. B, Mean values for the absolute number of DCs over the entire age range (5–15 wk) are: BALB/c, 1.09 x 106; BALB-lpr/lpr, 6.16 x 106; MRL+/+, 9.78 x 105; and MRL-lpr/lpr, 2.27 x 106. The lpr mutant mice had significantly increased numbers of DCs over Fas-sufficient mice. Statistically significant p values are shown on the graph.

 
Unaltered DC subtypes and maturation/activation status in lpr/lpr mice

DCs in murine secondary lymphoid organs have been classified into two main subtypes: myeloid and lymphoid (51, 55, 56, 57, 58, 59). These two DC types have been proposed to take opposing roles in T cell activation and tolerance, although this remains controversial (60, 61, 62, 63, 64). Myeloid DCs are marked by expression of CD11c, CD11b, and 33D1, and within the spleen resting myeloid DCs are concentrated at the bridging channels to the red pulp (51, 56). These DCs enter the PALS upon activation and terminal maturation (49, 65, 66). Lymphoid DCs are identified by expression of CD11c, NLDC145 (DEC-205), and the CD8{alpha}-{alpha} homodimer, and within the spleen they are normally found in the PALS (51, 52, 57). We examined the subtype of the DCs that located in the PALS of lpr/lpr mice. Immunohistological staining with CD11b established that myeloid DCs represent the majority of DCs in the PALS of BALB-lpr/lpr mice, whereas most myeloid DCs were at the bridging channels in BALB/c mice (Fig. 6Go). The same pattern was seen in MRL-lpr/lpr mice compared with MRL+/+ mice (data not shown, n = 4 and 3, respectively). While these differences in localization of myeloid DCs were seen, the percentages of DCs classified as myeloid were statistically equivalent within backgrounds, regardless of the presence of functional Fas (Table IIIGo). The majority of CD11c+ DCs in both Tg- BALB-lpr/lpr and BALB/c mice were of the myeloid phenotype. In MRL-lpr/lpr and MRL+/+ mice, the majority of DCs were also of the myeloid phenotype; however, the frequency of myeloid DCs was significantly less than in the BALB/c strains. The reason for the strain difference is unclear.



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  		FIGURE 6. Myeloid DCs are spread throughout the PALS in BALB-lpr/lpr mice. Spleen sections from Tg- BALB/c and BALB-lpr/lpr mice were stained with Abs to CD22 (red) and either CD11c (top) or CD11b (bottom) (blue). Myeloid DCs are indicated by their CD11c and CD11b coexpression. n > 3 for BALB/c and BALB-lpr/lpr mice.

 

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  		Table III. Frequencies of myeloid-type (33D1+) and lymphoid-type (CD8{alpha}+) DCs (gated on B220-CD11c+) in Fas-sufficient and lpr/lpr micea

 
Since myeloid DCs were accumulating in the PALS of lpr/lpr mice, we tested whether these DCs were activated. This was not the case. Ex vivo splenic DCs from Tg- BALB-lpr/lpr and MRL-lpr/lpr mice expressed levels of CD80 (B7.1), CD86 (B7.2), CD40, and MHC class II that were equivalent to those found on DCs from nonimmunized BALB/c mice (Fig. 7Go and data not shown).



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  		FIGURE 7. Maturation/activation status of DCs ex vivo. Spleen cells were stained with Abs to B220, CD11c, and either CD80, CD86, CD40, or I-Ad. Histograms (thin black lines) were gated on splenic B220-CD11c+ cells taken ex vivo. The maturation/activation phenotype of DCs from Tg- BALB/c and BALB-lpr/lpr spleens looks similar. n > 4 mice for each genotype. For comparison, the top row of histograms (BALB/c) is overlaid (gray line) with staining of activated DCs from BALB/c spleens that have been cultured for 6 h in 1 µg/ml LPS (n = 3), which resulted in up-regulation of the markers shown here.

 
T cell activation in lpr/lpr mice

Consistent with previous reports (8, 33, 67, 68), the number of activated T cells in lpr/lpr mice was increased compared with BALB/c mice (Fig. 8Go). The B220-CD4+ T cell population in both MRL-lpr/lpr and BALB-lpr/lpr mice had significantly elevated percentages that were CD25+ and/or CD69+ in the spleen compared with BALB/c mice (Fig. 8GoB). In BALB-lpr/lpr mice, the percentages of CD4 T cells that up-regulated CD69 and CD25 were similar. However, in MRL-lpr/lpr mice, the proportion of CD4 T cells expressing CD69 was higher than the proportion expressing CD25. Interestingly, CD69+CD4 T cells have been isolated from the synovial fluid and membrane of chronic rheumatoid arthritis patients (69), and strikingly, these included an unusual CD69+/CD25- T cell subset (70). CD4 T cell activation was apparent in the youngest animals examined (6 wk) and did not show a significant increase as the mice aged up to 12 wk. In MRL-lpr/lpr and BALB-lpr/lpr mice, a significant fraction of the CD4+ T cells that also expressed B220 had up-regulated CD69, but not CD25 (Fig. 8GoC). The VH3H9 Tg did not alter the state of T cell activation in the mice examined (n = 6, VH3H9 BALB/c; n = 7, VH3H9 MRL-lpr/lpr; n = 12, VH3H9 BALB-lpr/lpr, data not shown).



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  		FIGURE 8. T cell activation in 10-wk-old Fas/FasL-deficient mice. Spleen cells were stained with Abs to B220, CD4, and either CD25 or CD69. Numbers in the histograms are means ± SD. A, Dot plots were gated on B220-CD4+ cells and B220+CD4+ cells. B, Both MRL-lpr/lpr (n = 15) and BALB-lpr/lpr (n = 12) mice have more CD4 T cells that are CD69+ and/or CD25+ compared with BALB/c mice (n = 9) (for CD25, p < 0.0001 for both strains; for CD69, p < 0.0001 for both strains). In BALB-lpr/lpr mice, percentages of CD4+ T cells that were CD69+ or CD25+ were not significantly different (p = 0.45); however, in MRL-lpr/lpr mice, the proportion of CD4 T cells expressing CD69 is significantly higher than the proportion expressing CD25 (p = 0.0011). C, A significant proportion of B220+CD4+ cells in MRL-lpr/lpr (n = 4) and BALB-lpr/lpr (n = 6) mice expresses CD69, but not CD25 (compared with BALB/c mice, p = 0.0381 and 0.0260, respectively; for CD69 compared with CD25, p = 0.0286 and 0.0022, respectively). Twenty percent of the few B220+CD4+ cells present in BALB/c mice (n = 6) express both CD69 and CD25.

 
Nephritis in Fas/FasL-deficient BALB/c mice

Despite the presence of high titer ANAs, BALB-lpr/lpr mice developed minimal histological evidence of nephritis (Fig. 9Go). The degree of nephritis was unaffected by the VH3H9 Tg (data not shown; for VH3H9 BALB/c, n = 4; VH3H9 MRL-lpr/lpr, n = 4; and VH3H9 BALB-lpr/lpr, n = 4). Furthermore, other signs of autoimmune disease, such as failure to groom and skin lesions, evident in MRL-lpr/lpr mice by 12–16 wk, were not apparent in BALB-lpr/lpr or BALB-gld/gld mice up to 24 wk of age (n > 10 for each, data not shown).



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  		FIGURE 9. Nephritis in Tg- lpr/lpr mice. Kidneys from mice at 16–24 wk of age were scored for glomerulonephritis (0–4+), interstitial nephritis (0–4+), and vasculitis (0–4+). Each circle represents the cumulative score for one mouse. The lines represent the mean values for that mouse strain. *, Indicates that MRL-lpr/lpr mice (mean score 10.36) developed significantly greater nephritis than either BALB/c (mean score 2.62; p = 0.0061) or BALB-lpr/lpr (mean score 3.20; p < 0.0001) mice. Disease in the BALB-lpr/lpr mice was not significantly different from that in BALB/c mice (p = 0.81).

 

   Discussion
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 Abstract
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 Materials and Methods
 Results
 Discussion
References
 
lpr or gld mutations bred onto nonautoimmune-prone strains of mice demonstrate that Fas/FasL deficiency results in autoantibody production and lymphoproliferation (1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). We extend these studies by documenting that Fas/FasL deficiency, alone, is sufficient for anti-dsDNA B cells to bypass follicular exclusion and produce autoantibodies. Furthermore, this is accompanied by an alteration in the localization of DCs that is evident as early as 5 wk of age and persists into adulthood.

DCs in the spleens of MRL-lpr/lpr, BALB-lpr/lpr, and BALB-gld/gld mice were spread throughout the PALS. This localization was due to an influx of myeloid DCs, which are typically found in the PALS after activation. In this case, however, the localization was not associated with DC maturation/activation, as indicated by basal expression levels of CD80, CD86, CD40, and MHC class II. Given that DCs express Fas (44), one potential explanation for their unique localization in lpr/lpr and gld/gld mice is that they escape death from Fas-induced apoptosis and thus accumulate in the splenic T cell areas. Supporting this idea, there are an increased frequency and number of DCs in lpr/lpr mice. In this regard, it is intriguing that a defect in another apoptosis-related gene, caspase 10, resulted in an accumulation of DCs in the T cell zone of the lymph node of one patient with autoimmune lymphoproliferative syndrome type II (71). Another possibility arises from the recent suggestion that Fas is not a death receptor for DCs, but rather acts to induce their maturation (50). Therefore, the phenotype we have documented in this work could result from defective maturation/activation due to the absence of Fas. Experiments are underway to distinguish between these two possibilities.

In agreement with previous publications (8, 33, 67, 68), CD4 T cell activation was apparent in the youngest lpr/lpr animals examined (6 wk). The changes in the DCs and T cells in lpr/lpr mice most likely feed back upon one another as DC-T cell cross-talk occurs. For instance, activated T cells may influence DC localization in lpr/lpr mice by secreting DC chemoattractants (reviewed in Ref. 72) in the PALS. Furthermore, since DCs can potentially kill T cells via FasL (73), the absence of Fas/FasL interactions between T cells and DCs most likely contributes to the presence of activated T cells in lpr/lpr mice.

Finally, BALB-lpr/lpr mice, like other lpr/lpr and gld/gld strains (4, 9, 10, 11, 13), did not develop severe nephritis. The BALB-lpr/lpr mice are unique, however, in that they produce autoantibodies that appear indistinguishable from those in MRL-lpr/lpr mice. It is notable that CD4+ T cells from MRL+/+ mice are hyperresponsive (23). Furthermore, older MRL+/+ mice develop ANAs and nephritis (4, 9, 11) consistent with the hypothesis that other factors contribute to the full-blown nephritis phenotype. For example, MRL-lpr/lpr mice may produce autoantibodies that more readily form immune deposits or are more likely to elicit an inflammatory response. Alternatively, the renal inflammatory response to deposited Ab may be more vigorous in MRL mice. BALB/c mice, on the other hand, may be protected from kidney pathology by virtue of their Th2-like nature (74, 75, 76, 77). In this vein, it has been demonstrated that BALB/c mice, but not C57BL/6 mice, are resistant to damage induced by a vigorous immune response to peroral infection with Toxoplasma gondii, even though both strains produce IFN-{gamma} in the response (78).

In summary, we have shown that Fas/FasL deficiency, on the nonautoimmune-prone BALB/c background, results in several alterations in anti-dsDNA B cells. As early as 6 wk of age, they populate the splenic B cell follicle, and by 10–12 wk of age show signs of terminal differentiation into Ab-forming cells. In addition, 6-wk-old lpr/lpr mice, compared with wild-type mice, have an altered localization and increased frequency of CD11c+ DCs as well as a significant amount of activated T cells. We hypothesize that alterations in the DCs, as well as a lack of FasL-mediated T cell killing by DCs, may lead to the presence of activated, autoreactive CD4 T cells, which are critical for autoantibody production (79, 80, 81).


   Acknowledgments
 
We thank Ryan G. Fields, Kathryn M. Potts, and Jodi L. Buckler for critical reading of the manuscript, and Dr. Laura Mandik-Nayak for initiating the BALB-lpr/lpr studies.


   Footnotes
 
1 M.L.F. is supported by a Howard Hughes Medical Institute predoctoral fellowship grant and by a Gina Finzi Memorial Student Summer Fellowship from the Lupus Foundation of America; M.P.M. is supported by National Institutes of Health Grant DK33694; and J.E. is supported by National Institutes of Health Grant 5R01 AI32137-10, the Lupus Foundation of America, and the Arthritis Foundation. Back

2 Address correspondence and reprint requests to Dr. Jan Erikson, Wistar Institute, Room 276, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address: jan{at}wistar.upenn.edu Back

3 Abbreviations used in this paper: FasL, Fas ligand; ANA, anti-nuclear Ab; AP, alkaline phosphatase; DC, dendritic cell; PALS, periarteriolar lymphoid sheath; Tg, transgene, transgenic. Back

Received for publication April 11, 2001. Accepted for publication June 5, 2001.


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