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Genetically determined aberrant down-regulation of FcγRIIB1 in germinal center B cells associated with hyper-IgG and IgG autoantibodies in murine systemic lupus erythematosus

Genetically determined aberrant down-regulation of FcγRIIB1 in germinal center B cells associated... Abstract Systemic lupus erythematosus (SLE) is a multigenic disease associated with IgG hypergammaglobulinemia, IgG anti-nuclear antibodies and immune complex (IC)-type glomerulonephritis. In both human and murine SLE, one susceptibility allele has been mapped to the interval linked to the IgG Fc receptor II (FcγRII) gene on chromosome 1. In spontaneous SLE models of NZB and (NZB × NZW) F1 mice, expression of FcγRIIB1, which acts as a negative regulator for B cells, was abnormally down-regulated in follicular germinal center B cells from aged mice, compared to findings in non-SLE NZW, while levels in non-germinal center B cells were practically identical. Such strain differences were also evident in young mice upon in vivo stimulation with foreign antigens. In the FcγRIIB promoter region, the NZB allele has two deletion sites, including transcription factor-binding sites. Analyses using (NZB × NZW) F1 × NZW backcross mice showed that this NZB allele was significantly linked to hyper-IgG, irrespective of the MHC haplotype, while high levels of IgG antibodies specific for DNA were regulated by a combinatorial effect of the F1-unique MHC haplotype and the NZB FcγRIIB allele. Therefore, the FcγRIIB promoter polymorphism may possibly predispose to SLE through germinal center B cells abnormally down-regulating FcγRIIB1 expression upon autoantigen stimulations and thus escaping negative signals for IgG production. autoimmunity, chromosome 1, disease susceptibility allele, IgG Fc receptors, microsatellite, New Zealand mouse strains, promoter polymorphism ANOVA analysis of variance, FcγR IgG Fc receptor, IC immune complex, PNA peanut agglutinin, QTL quantitative trait loci, SLE systemic lupus erythematosus, SRBC sheep red blood cells Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the development of IgG hypergammaglobulinemia, IgG anti-nuclear antibodies and immune complex (IC)-type glomerulonephritis (lupus nephritis). Studies of families with SLE have documented the importance of genetic predisposition and a complex multigenic mode of inheritance may be involved (1). However, the genetic basis for disease susceptibility is undetermined. One susceptibility allele for SLE has been mapped to a telomeric region on chromosome 1 in humans (2,3) and in mice (4,5). This region in both humans and mice encodes a variety of immunologocally relevant genes, including a family of low-affinity IgG Fc receptors, i.e. FcγRII and FcγRIII. There are allelic variants of FcγR which confer distinct functional capacities to phagocytes (6,7). In this context, a skewing of FcγRIIA and FcγRIIIA allotypes has been reported in patients with SLE, particularly in those with lupus nephritis (8–11), implying that the skewing is linked to abnormal down-regulation of endocytotic ability of phagocytes, which leads to insufficient IC clearance, thus resulting in deposition of IgG IC in renal glomeruli. However, this abnormality does not simply explain the increase in serum levels of monomeric IgG in SLE patients. In this respect, FcγRIIB1 molecules deserve attention, because, unlike other FcγR, they are mainly expressed on B cells and act as a negative regulator for B cells (12–14). Murine FcγRII is encoded by the b gene (FcγRIIB), whereas human FcγRII shows genetic complexity, and is encoded by three genes, a, b and c (FcγRIIA, FcγRIIB and FcγRIIC) (15). There are two known membrane-bound isoforms of FcγRIIB molecules generated by alternative splicing of mRNA transcripts, i.e. FcγRIIB1 and FcγRIIB2. In contrast to FcγRIIB1 molecules which are preferentially expressed on B cells, FcγRIIB2 are expressed on macrophages, monocytes and neutrophils (12–14). The FcγRIIB proteins are basically composed of two Ig-like extracellular domains, one transmembrane domain, and a cytoplasmic tail encoded by three exons. The FcγRIIB1 isoform is composed of products from all exons, whereas the FcγRIIB2 lacks sequences encoded by the first intracytoplasmic exon. This difference determines the endocytotic ability of cells and FcγRIIB2 molecules act to internalize bound IgG IC for antigen presentation, while FcγRIIB1 do not mediate endocytosis, but instead abort B cell activation by inhibiting B cell antigen receptor-elicited activation signals through tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory motifs, encoded by the third intracytoplasmic exon (12–14). Because the negative regulatory effect of FcγRIIB1 influences the extent of IgG antibody responses of B cells (16), we speculated that B cell hyperactivity as noted by IgG hypergammaglobulinemia and IgG autoantibody production observed in SLE might be related to the dysfunction of FcγRIIB1. This notion is consistent with the result of previous genome-wide linkage analyses, which localized one susceptibility allele for hyper-IgG in NZB-related murine model of SLE to the telomeric region on NZB chromosome 1, tightly linked to the FcγRIIB locus (5). However, dysfunction of FcγRIIB1 in NZB cells has not been determined. In the present studies, we focused on this issue and we propose that the NZB-type polymorphic FcγRIIB promoter allele which acts to abnormally down-regulate FcγRIIB1 levels in germinal center B cells is one susceptibility allele for SLE in NZB and (NZB × NZW) F1 mice, and possibly in humans. Methods Mice BALB/c, C57BL/6, NZB, NZW and (NZB × NZW) F1 mice, originally obtained from the Shizuoka Laboratory Animal Center (Shizuoka, Japan), were maintained in our animal facility. Backcross mice were obtained by crossing female (NZB × NZW) F1 mice with male NZW mice. All mice used were housed in the same room and fed an identical diet. Only female mice were analyzed in the present study. If necessary, mice were immunized twice at 6 and 9 weeks of age, by giving an i.p. injection of 1 × 106 sheep red blood cells (SRBC) per mouse. Flow cytometry analysis Aliquots of 1 × 106 spleen cells were stained with phycoerythrin-conjugated 2.4G2, FITC-labeled B cell-specific anti-CD45R/B220 and biotinylated peanut agglutinin (PNA) in PBS containing 1% BSA and 0.1% sodium azide for 30 min at 4°C. After incubation, the cells were washed in PBS with 0.2% BSA and further stained with streptavidin-conjugated allophycocyanin. After washing, the cells were fixed in PBS containing 1% formaldehyde. Fluorescence intensity was measured using a FACStar flow cytometer and CellQuest software (Becton Dickinson, San Jose, CA). RT-PCR analysis of FcγRII and FcγRIII transcripts Total RNA was isolated using ISOGEN (Nippon Gene, Tokyo, Japan). First-strand cDNA was synthesized using an oligo(dT) primer and serially diluted cDNA products were amplified using 5′ and 3′ primers (for FcγRIIB1 and FcγRIIB2: 5′-AAGTCTAGGAAGGACACTGC-3′ and 5′-ATCCTGGCCTTTCTGGCTTGC-3′; for FcγRIII: 5′-AGAC ATGGTGACACTGATG-3′ and 5′-TTGGACAGTGATGGTGACAGGCTTGG-3′, and for FcRγ-chain: 5′-ATCTCAGCCGTGATCTTG-3′ and 5′-TCAGGTCTCTGGCAGCTT-3′). PCR was done at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min for 28 cycles. The products were electrophoresed in 2% agarose gel and visualized after ethidium bromide staining. Amplification of β-actin served as a reference to normalize data for the total amount of RNA input. Primers for β-actin were purchased from Clontech (Palo Alto, CA). Nucleotide sequence analysis The FcγRIIB1 cDNA coding sequence was amplified by RT-PCR and subcloned into the Bluescript plasmid vector (Stratagene, La Jolla, CA). Both strands were sequenced with modified T7 DNA polymerase. The FcγRIIB gene upstream region was amplified from DNA extracted from the mouse tail using the following primers: 5′-AAGACCACAGAGTGTAAGATGG-3′ (position –809 to –788 from the transcription starting point) and 5′-TAAAGGTACCCACAACCACTTACC-3′ (146–123). The PCR products were digested with BamHI, PstI, and KpnI, and restriction fragments (BamHI–PstI and PstI–KpnI) were subcloned and sequenced on both strands. Measurement of serum levels of total IgG and IgG anti-DNA antibodies Serum levels of total IgG and IgG anti-DNA antibodies were determined by ELISA. IgG anti-DNA antibodies were quantified using ELISA plates coated with calf thymus DNA (Sigma, St Louis, MO) and the DNA-binding activities were expressed in units, referring to a standard curve obtained by serial dilutions of a standard serum pool from 7- to 9-month-old (NZB × NZW) F1 mice, containing 1000 U/ml. Genotyping DNA was extracted from the mouse tail. Genotyping for microsatellite markers and the FcγRIIB promoter region was done using PCR. Microsatellite primers were purchased from Research Genetics (Huntsville, AL). Genotyping for the FcγRIIB promoter region was done using 5′ and 3′ primers: 5′-GTTGATCTTCATTTTACAGAC-3′ and 5′-TCTGTGCCCTAGTCCTGAATC-3′ (see Fig. 3A). PCR reactions were run in 96-well plates with 7.5 μl total volume containing 40 ng of genomic DNA. A three-temperature PCR protocol (94, 55 and 72°C) was conducted for 45 cycles in a Geneamp 9600 Thermal Cycler (Perkin-Elmer-Cetus, Norwalk, CT). PCR products were diluted 2-fold with loading buffer consisting of xylene cyanol and bromophenol blue dyes in 50% glycerin, and were run on 15% polyacrylamide gels. After electrophoresis, gels were visualized after ethidium bromide staining. Statistics The linkage of a particular locus with increased levels of serum IgG was estimated using a computer package program of MAPMAKER/EXP and MAPMAKER/QTL (17) to identify chromosomal locations of quantitative trait loci (QTL). Analysis of variance (ANOVA) was used to determine the differences in serum levels of total IgG and IgG anti-DNA antibodies among each group of (NZB × NZW) F1 × NZW backcross mice with different combinations of susceptibility alleles. Results Down-regulation of FcγRIIB1 in germinal center B cells Germinal centers in lymph follicles are the site for IgM to IgG Ig class switch of B cells and for generation of high-affinity IgG antibody responses upon T cell-mediated signals. Thus, it is reasonable to assume that, in autoimmune disease, there is a mechanism that allows pathogenic IgG autoantibodies to be over-produced, through B cells down-regulating their FcγRIIB1 expression in germinal centers and thus escaping the suppressive signals. To address this issue, we first examined levels of FcγR expression on splenic germinal center B cells, as distinguished by expression of high levels of surface binding sites for PNA, and non-germinal center B cells from SLE-prone NZB and (NZB × NZW) F1 and non-SLE-prone NZW mice, using flow cytometric analyses with a monoclonal anti-FcγR antibody 2.4G2 which reacts with FcγRIIB1, FcγRIIB2 and FcγRIII (18). The extent of germinal center formation in lymph follicles increased as animals aged, and mean percentages ± SE of PNAhigh germinal center B cells in total splenic B cells from five each NZB, NZW and (NZB × NZW) F1 mice 8 months of age were 9.1 ± 1.2, 2.3 ± 0.4 and 13.9 ± 4.9 respectively. We found that while 2.4G2 levels in non-germinal center B cells were practically identical, they were down-regulated in germinal center B cells in all the mouse strains examined. It was of particular note, however, that the extent of this down-regulation was much greater in NZB and (NZB × NZW) F1 (~10-fold decrease) than found in NZW (a 4-fold decrease) (Fig. 1A). RT-PCR analyses of mRNAs from FACS-sorted splenic B cell populations from 8-month-old NZB mice showed that, compared to findings in PNAlow non-germinal center B cells which express high amounts of FcγRIIB1 messages, there was ~10-fold decrease in its expression in PNAhigh germinal center B cells (Fig. 2). While the FcγRIIB2 isoform product was detected in peritoneal cavity cells containing a mixture of macrophages and lymphocytes, it was not expressed in FACS-sorted B cells. Similar findings were obtained with the FcγRIII PCR product, but a small amount of messages was detected in PNAhigh, but not PNAlow, B cells (Fig. 2). In contrast to FcγRIIB1 and FcγRIIB2, FcγRIII molecules are expressed as a multimeric complex with homodimeric or heterodimeric complexes with the γ chain, and are not normally expressed on B cells (12–14). Therefore, we examined the expression of FcRγ chain messages in germinal enter B cells and found the presence of a small, but significant amount in PNAhigh, but not PNAlow, B cells (data not shown). However, as amounts of FcγRIII PCR products did not differ significantly between the NZB and NZW strains, the observed decrease in the 2.4G2 expression on germinal center B cells was considered to be mainly due to down-regulation of FcγRIIB1 molecules. Although germinal centers of young mice are not well established, immunizations with particulate or aggregated antigens or deposits of preformed antigen–antibody complexes elicit germinal center formation. Thus, we immunized NZB and NZW mice twice at 6 and 9 weeks of age with SRBC, and FcγRIIB1 expression levels were determined by flow cytometric analysis 1 week after the second immunization. We again found that levels of FcγRIIB1 expression were lower in germinal center B cells than in non-germinal center B cells and that the extent of down-regulation was greater in NZB than in NZW (Fig. 1B). Taken collectively, it was suggested that the strain difference in the extent of FcγRIIB1 down-regulation in germinal center B cells is due to a genetically determined event. Polymorphism of the regulatory region of the FcγRIIB gene To determine the polymorphism between the NZB and NZW FcγRIIB gene, we first PCR amplified and sequenced structural regions. NZB and NZW strains had the same structural polymorphism corresponding to Ly-17.1 allotype (data not shown), as reported by other investigators (19). We then analyzed PCR products of FcγRII promoter regions. As compared with findings in BALB/c, C57BL/6 and NZW, the product of the region (position –194 to –30 from the transcription starting point) from NZB showed a different nucleotide length polymorphism: (NZB × NZW) F1 mice shared both NZB and NZW-type alleles (Fig. 3A). Sequence analyses revealed that NZB had two nucleotide substitutions and two deletion sites in this promoter region, including transcription factor-binding consensus sequences, i.e. Ap-4-binding site (20) and S box (21) (Fig. 3B). Linkage between NZB FcγRIIB allele and hyper-IgG To determine the role of the NZB FcγRIIB1 allele in IgG hypergammaglobulinemia, we examined the association between the NZB FcγRIIB allele and serum IgG levels using 207 (NZB × NZW) F1 × NZW backcross female mice 8 months of age. When the backcross progeny was divided into two groups, i.e. one with heterozygous NZB/NZW (B/W) type for FcγRIIB allele (Fcgr2b) and the other with homozygous NZW/NZW (W/W) type, total serum levels of IgG were significantly higher in the former (Fig. 4A). MAPMAKER/QTL scans of data from backcross progeny using microsatellites and FcγRIIB allele polymorphic between the NZB and NZW strain showed that the NZB locus responsible for high serum IgG, provisionally designated Hig-1 (Hyper-IgG-1), is located on chromosome 1, most significantly linked to the FcγRIIB locus (Fcgr2b) (Fig. 5). Combinatorial effect of FcγRIIB allele and MHC in IgG anti-DNA antibody production When we compared serum levels of IgG anti-DNA antibodies in (NZB × NZW) F1 × NZW backcross progeny between the group with heterozygous B/W type for Fcgr2b and that with homozygous W/W type, neither group showed a significant difference, although the former did tend to be higher (Fig. 4B). As synthesis of IgG anti-DNA antibodies in (NZB × NZW) F1 mice is strictly restricted to H-2d/z heterozygosity (H-2d from NZB and H-2z from NZW) (22), we then separated the backcross progeny into four groups, classified according to combinations of H-2 and Fcgr2b genotypes, i.e. group a, H-2d/z and B/W Fcgr2b genotypes; group b, H-2d/z and W/W Fcgr2b; group c, H-2z/z and B/W Fcgr2b; and group d, H-2z/z and W/W Fcgr2b. Among these four groups, while total serum IgG levels were higher in the progeny with the B/W Fcgr2b genotype than in the progeny with the W/W genotype, irrespective of the H-2 genotypes (Fig. 4C), the IgG anti-DNA antibody levels were in the order of group a, group b, and groups c and d (Fig. 4D), indicating that the high IgG anti-DNA antibody titers seen in (NZB × NZW) F1 mice are regulated by a combinatorial effect of H-2d/z and the NZB FcγRIIB allele. Discussion In T cell-dependent humoral immune responses, antigen-activated B cells accumulate in the germinal centers where Ig class switching and generation of high-affinity IgG antibodies somatically mutated in Ig variable regions occur (23). As pathogenic IgG anti-DNA antibodies in SLE are also affinity selected (24,25), the process of the generation of these autoantibodies also appears operative in germinal centers. In the present studis, we found that the NZB strain has a unique FcγRIIB promoter allele which is associated with abnormal down-regulation of FcγRIIB1 levels in germinal center B cells. Thus, it is reasonable to assume that this FcγRIIB1 down-regulation is a mechanism which allows high-affinity IgG autoantibodies to be over-produced through germinal center B cells escaping negative signals for IgG production. Structural regions of the NZB FcγRIIB gene did not differ from the NZW gene. The difference in the extent of FcγRIIB1 expression levels between NZB and non-SLE NZW strains was linked to sequence differences in the FcγRIIB promoter region. The two deletion sites observed in the NZB allele, one including the Ap-4-binding site (20) and S box (21) (Fig. 3B), are located in the region containing the element which regulates cell-type-specific FcγRIIB expression and drives the transcription of FcγRIIB gene in a B cell, but not a mast cell, line (26). Lack of differential FcγRIIB1 expression in the non-germinal center resting B cells between NZB and NZW strains suggests that the deletion sites regulate FcγRIIB1 expression in the process of T cell-mediated B cell activation. Thus, the constitutive FcγRIIB1 expression in resting B cells must be differentially controlled by other regulatory elements, such as those located in the third intron containing a series of purine/pyrimidine-rich regions, NF-κB-like binding motif and elements with silencer activity (20,26). On the other hand, the decrease in the FcγRIIB1 level in germinal center B cells from aged (NZB × NZW) F1 mice was much closer to NZB than to NZW. At present, we have no explanation for the reason of the dominant negative effect of the down-regulatory NZB allele in the F1 mice. Combinatorial effects of other elements also appear to be involved in this regulation. A suggested role for FcγRIIB1 in the hyperproduction of IgG was supported by the significant association between high serum level of IgG and the NZB-type promoter polymorphism in (NZB × NZW) F1 × NZW backcross progeny. A tight linkage in QTL analyses between the NZB FcγRIIB locus and the extent of hyper-IgG further supported the notion (Fig. 5). We do not exclude the possibility, however, that the hyper-IgG can be in part attributed to a decrease in the expression of FcγRIIB2 on phagocytes due to the NZB FcγRIIB promoter polymorphism, the result being a reduction in IgG IC clearance and thus leading to accumulation of IC in the circulation. Indeed, our preliminary studies showed that levels of the FcγRIIB2 isoform on macrophages from NZB mice are also down-regulated, as compared with the finding in NZW mice. It is possible that this abnormality is related to the pathogenesis of IC-type glomerulonephritis in NZB and (NZB × NZW) F1 mice. However, the defect in FcγRIIB2 expression on macrophages does not simply explain the increase in monomeric IgG in the blood. This notion was supported by our preliminary results that mice with the NZB FcγRIIB allele had much higher secondary IgG anti-SRBC antibody responses than found in mice lacking this allele. In the present studies, we found that germinal center B cells express small, but significant amounts of FcγRIII, which are not normally expressed on B cells. In contrast to FcγRIIB1, FcγRIII molecules are expressed on NK cells, mast cells, neutrophils and macrophages, and mediate a variety of functions, such as antibody-dependent cell-mediated cytotoxicity, degranulation, endocytosis, phagocytosis and antigen presentation (12–14). Further studies are required to elucidate the role of FcγRIII on germinal center B cells. Nonetheless, because the amounts of FcγRIII PCR products in germinal center B cells did not differ between the NZB and NZW strains, the observed decrease in 2.4G2 expression in germinal center B cells is mainly due to down-regulation of FcγRIIB1. Although the role of NZB-type FcγRIIB allele in hyper-IgG and IgG anti-DNA antibody production appears significant, the mechanism is more complex, because our earlier studies revealed that serum levels of both total IgG and IgG anti-DNA antibodies in (NZB × NZW) F1 mice are much higher than these levels in NZB mice and that a complimentary effect of both NZB and NZW genes determines these two traits in the F1 hybrid mice (22, 27). Our present progeny studies were carried out using (NZB × NZW) F1 × NZW backcross mice, in which NZB genes segregate in the progeny bearing all NZW alleles in the genetic background. Therefore, although the observed hyper-IgG seemed to be regulated by the polymorphic NZB FcγRIIB promoter allele alone, other NZW genetic contributions are likely to be involved. In this respect, in contrast to the serum levels of total IgG, IgG anti-DNA antibodies were combinatorially regulated by the FcγRIIB and H-2 complex alleles (Fig. 4). This observation is consistent with our earlier findings that production of high-affinity IgG anti-DNA antibodies in (NZB × NZW) F1 mice is highly restricted to H-2d/z heterozygosity (22), suggesting that the mixed haplotype class II molecules, most likely Aαdβz (28,29), serve as a restriction element for autoreactive T cells. As serum hyper-IgG occurred in the backcross progeny, irrespective of the H-2 haplotypes, this trait is largely due to the increase in polyclonal IgG that are mainly produced under the restriction of homozygous d/d and/or z/z, but not heterozygous d/z, A and/or E class II molecules. A report by Luan et al. (30) showed that the NOD mouse strain, a spontaneous model of autoimmune diabetes, also has hyper-IgG in association with a defect in the promoter region of FcγRIIB gene, very close to the sites detected in NZB. These same authors noted the down-regulation of FcγRIIB2 in macrophages, but not FcγRIIB1 in B cells. Our preliminary studies on NOD mice, however, showed that FcγRIIB1 expression in NOD germinal center B cells is also markedly down-regulated. Taken collectively, it is highly possible that the observed regulatory defect in FcγRIIB1 expression also predisposes to other autoantibody-mediated autoimmune diseases. Allelic polymorphisms of FcγRIIA and FcγRIIIA have been associated with human SLE (8–11), although the role of FcγRIIA alleles in Caucasian SLE patients is controversial (9,31). Considering the regulatory role of FcγRIIB1 on IgG synthesis (16), linkage of the chromosome 1 region to human SLE may be partly due to linkage to the polymorphic FcγRIIB allele, as observed in the murine model. We propose that this NZB-type FcγRIIB promoter polymorphism is one susceptibility allele for murine and possibly human SLE. Fig. 1. View largeDownload slide Comparisons of FcγRIIB1 expression levels between splenic PNAhigh germinal center B cells and PNAlow non-germinal center B cells from NZB, NZW and (NZB × NZW) F1 mice. (A) Spleen cells from 8-month-old NZB, NZW and (NZB × NZW) F1 mice were stained with phycoerythrin-labeled anti-FcγRII/III (2.4G2) mAb, FITC-labeled B cell-specific anti-CD45R/B220 mAb and biotinylated PNA, followed by streptavidin–allophycocyanin. Levels of 2.4G2 expression on PNAhigh and PNAlow splenic CD45R/B220+ B cell populations were compared on the histograms. The closed area indicates the background staining. Because RT-PCR analysis showed that B cells expressed mainly FcγRIIB1 (Fig. 2), 2.4G2 expression on B cells reflects the level of FcγRIIB1 isoform. (B) NZB and NZW mice were immunized twice at 6 and 9 weeks of age with SRBC and spleen cells were obtained 1 week after the second immunization. Spleen cells were analyzed in the same manner as shown in (A). PNAhigh B cell population was not detected in non-treated young mice. A representative result of five experiments is shown in (A) and (B). Fig. 1. View largeDownload slide Comparisons of FcγRIIB1 expression levels between splenic PNAhigh germinal center B cells and PNAlow non-germinal center B cells from NZB, NZW and (NZB × NZW) F1 mice. (A) Spleen cells from 8-month-old NZB, NZW and (NZB × NZW) F1 mice were stained with phycoerythrin-labeled anti-FcγRII/III (2.4G2) mAb, FITC-labeled B cell-specific anti-CD45R/B220 mAb and biotinylated PNA, followed by streptavidin–allophycocyanin. Levels of 2.4G2 expression on PNAhigh and PNAlow splenic CD45R/B220+ B cell populations were compared on the histograms. The closed area indicates the background staining. Because RT-PCR analysis showed that B cells expressed mainly FcγRIIB1 (Fig. 2), 2.4G2 expression on B cells reflects the level of FcγRIIB1 isoform. (B) NZB and NZW mice were immunized twice at 6 and 9 weeks of age with SRBC and spleen cells were obtained 1 week after the second immunization. Spleen cells were analyzed in the same manner as shown in (A). PNAhigh B cell population was not detected in non-treated young mice. A representative result of five experiments is shown in (A) and (B). Fig. 2. View largeDownload slide A representative result of RT-PCR amplifications of FcγRIIB1, FcγRIIB2 and FcγRIII messages from splenic PNAhigh germinal center (GC) B cells and PNAlow non-germinal center (nonGC) B cells from NZB mice. Total RNA was isolated from FACS-sorted PNAhigh and PNAlow splenic B cells obtained from 8-month-old NZB mice, and used for RT-PCR. As a control, RNA was isolated from peritoneal cavity cells (PerC) containing macrophages. Amplification of β-actin served to check RNA amounts. PCR products were electrophoresed on 2% agarose gels and visualized after ethidium bromide staining. The number of the top of each lane indicates the reciprocal of the dilution of cDNA for FcγRIIB1, FcγRIIB2, FcγRIII and β-actin. Numbers of amplified bases are as follows; 481 bp for FcγRIIB1, 343 bp for FcγRIIB2 and 453 bp for FcγRIII. Note that ~10-fold decrease in FcγRIIB1 PCR product is observed in PNAhigh B cells, compared to findings in PNAlow B cells. Fig. 2. View largeDownload slide A representative result of RT-PCR amplifications of FcγRIIB1, FcγRIIB2 and FcγRIII messages from splenic PNAhigh germinal center (GC) B cells and PNAlow non-germinal center (nonGC) B cells from NZB mice. Total RNA was isolated from FACS-sorted PNAhigh and PNAlow splenic B cells obtained from 8-month-old NZB mice, and used for RT-PCR. As a control, RNA was isolated from peritoneal cavity cells (PerC) containing macrophages. Amplification of β-actin served to check RNA amounts. PCR products were electrophoresed on 2% agarose gels and visualized after ethidium bromide staining. The number of the top of each lane indicates the reciprocal of the dilution of cDNA for FcγRIIB1, FcγRIIB2, FcγRIII and β-actin. Numbers of amplified bases are as follows; 481 bp for FcγRIIB1, 343 bp for FcγRIIB2 and 453 bp for FcγRIII. Note that ~10-fold decrease in FcγRIIB1 PCR product is observed in PNAhigh B cells, compared to findings in PNAlow B cells. Fig. 3. View largeDownload slide Nucleotide deletions in the FcγRIIB promoter region in the NZB strain. (A) Genomic organization of FcγRIIB gene (Fcgr2b) and length polymorphism of PCR products of FcγRIIB promoter region. Exons are numbered and indicated by boxes, untranslated regions by open boxes, and coding regions by filled boxes which are also labeled by the region they encode, i.e. EC for extracellular domains, TM for transmembrane domain and IC for intracytoplasmic domains. Primer positions for PCR are indicated by arrows. The position 0 is defined as the transcription starting point. Tail DNA from a single mouse of each strain was used as a template for PCR, and the amplified fragments were run on 15% polyacrylamide gels and visualized by staining with ethidium bromide. Size difference in PCR product indicates the deletion in FcγRIIB promoter region in NZB strain. (B) Nucleotide sequence of PCR-amplified FcγRIIB promoter region in BALB/c, NZW and NZB strains. Horizontal bar indicates sequence identity. Asterisk depicts nucleotide deletions in NZB strain. Transcription factor-binding consensus sequences in the deleted region are indicated. Fig. 3. View largeDownload slide Nucleotide deletions in the FcγRIIB promoter region in the NZB strain. (A) Genomic organization of FcγRIIB gene (Fcgr2b) and length polymorphism of PCR products of FcγRIIB promoter region. Exons are numbered and indicated by boxes, untranslated regions by open boxes, and coding regions by filled boxes which are also labeled by the region they encode, i.e. EC for extracellular domains, TM for transmembrane domain and IC for intracytoplasmic domains. Primer positions for PCR are indicated by arrows. The position 0 is defined as the transcription starting point. Tail DNA from a single mouse of each strain was used as a template for PCR, and the amplified fragments were run on 15% polyacrylamide gels and visualized by staining with ethidium bromide. Size difference in PCR product indicates the deletion in FcγRIIB promoter region in NZB strain. (B) Nucleotide sequence of PCR-amplified FcγRIIB promoter region in BALB/c, NZW and NZB strains. Horizontal bar indicates sequence identity. Asterisk depicts nucleotide deletions in NZB strain. Transcription factor-binding consensus sequences in the deleted region are indicated. Fig. 4. View largeDownload slide Associations of FcγRIIB gene (Fcgr2b) and major histocompatibility complex (H-2) genotypes with serum levels of total IgG and IgG anti-DNA antibodies in 207 female (NZB × NZW) F1 × NZW backcross mice aged 8 months. Mice were separated into two (A and B) and four groups (C and D), classified according to genotypes of Fcgr2b and a combination of genotypes for H-2 and Fcgr2b respectively. Genotypes of B/W and W/W for Fcgr2b depict NZB/NZW heterozygotes and NZW/NZW homozygotes respectively. Genotypes of d/z and z/z for H-2 depict heterozygotes with H-2d from NZB and H-2z from NZW, and homozygotes with H-2z/z respectively. The number of mice examined is shown in parentheses and serum levels of antibodies are expressed by mean ± SE in mice at 8 months of age. Significant difference by ANOVA is shown. Fig. 4. View largeDownload slide Associations of FcγRIIB gene (Fcgr2b) and major histocompatibility complex (H-2) genotypes with serum levels of total IgG and IgG anti-DNA antibodies in 207 female (NZB × NZW) F1 × NZW backcross mice aged 8 months. Mice were separated into two (A and B) and four groups (C and D), classified according to genotypes of Fcgr2b and a combination of genotypes for H-2 and Fcgr2b respectively. Genotypes of B/W and W/W for Fcgr2b depict NZB/NZW heterozygotes and NZW/NZW homozygotes respectively. Genotypes of d/z and z/z for H-2 depict heterozygotes with H-2d from NZB and H-2z from NZW, and homozygotes with H-2z/z respectively. The number of mice examined is shown in parentheses and serum levels of antibodies are expressed by mean ± SE in mice at 8 months of age. Significant difference by ANOVA is shown. Fig. 5. View largeDownload slide MAPMAKER/QTL scans on chromosome 1 for hyper-IgG in 207 female (NZB × NZW) F1 × NZW backcross mice aged 8 months. The lod score curve is shown on the right with scale on the bottom. Map positions of microsatellite markers are arranged from centromere to telomere on the left of the chromosome line. Genotyping for FcγRIIB (Fcgr2b) was done using the promoter region primer pair shown in Fig. 3(B). Maximum lod score (3.4) at Fcgr2 indicates a significant linkage (32). Fig. 5. View largeDownload slide MAPMAKER/QTL scans on chromosome 1 for hyper-IgG in 207 female (NZB × NZW) F1 × NZW backcross mice aged 8 months. The lod score curve is shown on the right with scale on the bottom. Map positions of microsatellite markers are arranged from centromere to telomere on the left of the chromosome line. Genotyping for FcγRIIB (Fcgr2b) was done using the promoter region primer pair shown in Fig. 3(B). Maximum lod score (3.4) at Fcgr2 indicates a significant linkage (32). Transmitting editor: K. Okumura We thank M. Ohara for language assistance. 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Genetically determined aberrant down-regulation of FcγRIIB1 in germinal center B cells associated with hyper-IgG and IgG autoantibodies in murine systemic lupus erythematosus

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Oxford University Press
Copyright
© 1999 Japanese Society for Immunology
ISSN
0953-8178
eISSN
1460-2377
DOI
10.1093/intimm/11.10.1685
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Abstract

Abstract Systemic lupus erythematosus (SLE) is a multigenic disease associated with IgG hypergammaglobulinemia, IgG anti-nuclear antibodies and immune complex (IC)-type glomerulonephritis. In both human and murine SLE, one susceptibility allele has been mapped to the interval linked to the IgG Fc receptor II (FcγRII) gene on chromosome 1. In spontaneous SLE models of NZB and (NZB × NZW) F1 mice, expression of FcγRIIB1, which acts as a negative regulator for B cells, was abnormally down-regulated in follicular germinal center B cells from aged mice, compared to findings in non-SLE NZW, while levels in non-germinal center B cells were practically identical. Such strain differences were also evident in young mice upon in vivo stimulation with foreign antigens. In the FcγRIIB promoter region, the NZB allele has two deletion sites, including transcription factor-binding sites. Analyses using (NZB × NZW) F1 × NZW backcross mice showed that this NZB allele was significantly linked to hyper-IgG, irrespective of the MHC haplotype, while high levels of IgG antibodies specific for DNA were regulated by a combinatorial effect of the F1-unique MHC haplotype and the NZB FcγRIIB allele. Therefore, the FcγRIIB promoter polymorphism may possibly predispose to SLE through germinal center B cells abnormally down-regulating FcγRIIB1 expression upon autoantigen stimulations and thus escaping negative signals for IgG production. autoimmunity, chromosome 1, disease susceptibility allele, IgG Fc receptors, microsatellite, New Zealand mouse strains, promoter polymorphism ANOVA analysis of variance, FcγR IgG Fc receptor, IC immune complex, PNA peanut agglutinin, QTL quantitative trait loci, SLE systemic lupus erythematosus, SRBC sheep red blood cells Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the development of IgG hypergammaglobulinemia, IgG anti-nuclear antibodies and immune complex (IC)-type glomerulonephritis (lupus nephritis). Studies of families with SLE have documented the importance of genetic predisposition and a complex multigenic mode of inheritance may be involved (1). However, the genetic basis for disease susceptibility is undetermined. One susceptibility allele for SLE has been mapped to a telomeric region on chromosome 1 in humans (2,3) and in mice (4,5). This region in both humans and mice encodes a variety of immunologocally relevant genes, including a family of low-affinity IgG Fc receptors, i.e. FcγRII and FcγRIII. There are allelic variants of FcγR which confer distinct functional capacities to phagocytes (6,7). In this context, a skewing of FcγRIIA and FcγRIIIA allotypes has been reported in patients with SLE, particularly in those with lupus nephritis (8–11), implying that the skewing is linked to abnormal down-regulation of endocytotic ability of phagocytes, which leads to insufficient IC clearance, thus resulting in deposition of IgG IC in renal glomeruli. However, this abnormality does not simply explain the increase in serum levels of monomeric IgG in SLE patients. In this respect, FcγRIIB1 molecules deserve attention, because, unlike other FcγR, they are mainly expressed on B cells and act as a negative regulator for B cells (12–14). Murine FcγRII is encoded by the b gene (FcγRIIB), whereas human FcγRII shows genetic complexity, and is encoded by three genes, a, b and c (FcγRIIA, FcγRIIB and FcγRIIC) (15). There are two known membrane-bound isoforms of FcγRIIB molecules generated by alternative splicing of mRNA transcripts, i.e. FcγRIIB1 and FcγRIIB2. In contrast to FcγRIIB1 molecules which are preferentially expressed on B cells, FcγRIIB2 are expressed on macrophages, monocytes and neutrophils (12–14). The FcγRIIB proteins are basically composed of two Ig-like extracellular domains, one transmembrane domain, and a cytoplasmic tail encoded by three exons. The FcγRIIB1 isoform is composed of products from all exons, whereas the FcγRIIB2 lacks sequences encoded by the first intracytoplasmic exon. This difference determines the endocytotic ability of cells and FcγRIIB2 molecules act to internalize bound IgG IC for antigen presentation, while FcγRIIB1 do not mediate endocytosis, but instead abort B cell activation by inhibiting B cell antigen receptor-elicited activation signals through tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory motifs, encoded by the third intracytoplasmic exon (12–14). Because the negative regulatory effect of FcγRIIB1 influences the extent of IgG antibody responses of B cells (16), we speculated that B cell hyperactivity as noted by IgG hypergammaglobulinemia and IgG autoantibody production observed in SLE might be related to the dysfunction of FcγRIIB1. This notion is consistent with the result of previous genome-wide linkage analyses, which localized one susceptibility allele for hyper-IgG in NZB-related murine model of SLE to the telomeric region on NZB chromosome 1, tightly linked to the FcγRIIB locus (5). However, dysfunction of FcγRIIB1 in NZB cells has not been determined. In the present studies, we focused on this issue and we propose that the NZB-type polymorphic FcγRIIB promoter allele which acts to abnormally down-regulate FcγRIIB1 levels in germinal center B cells is one susceptibility allele for SLE in NZB and (NZB × NZW) F1 mice, and possibly in humans. Methods Mice BALB/c, C57BL/6, NZB, NZW and (NZB × NZW) F1 mice, originally obtained from the Shizuoka Laboratory Animal Center (Shizuoka, Japan), were maintained in our animal facility. Backcross mice were obtained by crossing female (NZB × NZW) F1 mice with male NZW mice. All mice used were housed in the same room and fed an identical diet. Only female mice were analyzed in the present study. If necessary, mice were immunized twice at 6 and 9 weeks of age, by giving an i.p. injection of 1 × 106 sheep red blood cells (SRBC) per mouse. Flow cytometry analysis Aliquots of 1 × 106 spleen cells were stained with phycoerythrin-conjugated 2.4G2, FITC-labeled B cell-specific anti-CD45R/B220 and biotinylated peanut agglutinin (PNA) in PBS containing 1% BSA and 0.1% sodium azide for 30 min at 4°C. After incubation, the cells were washed in PBS with 0.2% BSA and further stained with streptavidin-conjugated allophycocyanin. After washing, the cells were fixed in PBS containing 1% formaldehyde. Fluorescence intensity was measured using a FACStar flow cytometer and CellQuest software (Becton Dickinson, San Jose, CA). RT-PCR analysis of FcγRII and FcγRIII transcripts Total RNA was isolated using ISOGEN (Nippon Gene, Tokyo, Japan). First-strand cDNA was synthesized using an oligo(dT) primer and serially diluted cDNA products were amplified using 5′ and 3′ primers (for FcγRIIB1 and FcγRIIB2: 5′-AAGTCTAGGAAGGACACTGC-3′ and 5′-ATCCTGGCCTTTCTGGCTTGC-3′; for FcγRIII: 5′-AGAC ATGGTGACACTGATG-3′ and 5′-TTGGACAGTGATGGTGACAGGCTTGG-3′, and for FcRγ-chain: 5′-ATCTCAGCCGTGATCTTG-3′ and 5′-TCAGGTCTCTGGCAGCTT-3′). PCR was done at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min for 28 cycles. The products were electrophoresed in 2% agarose gel and visualized after ethidium bromide staining. Amplification of β-actin served as a reference to normalize data for the total amount of RNA input. Primers for β-actin were purchased from Clontech (Palo Alto, CA). Nucleotide sequence analysis The FcγRIIB1 cDNA coding sequence was amplified by RT-PCR and subcloned into the Bluescript plasmid vector (Stratagene, La Jolla, CA). Both strands were sequenced with modified T7 DNA polymerase. The FcγRIIB gene upstream region was amplified from DNA extracted from the mouse tail using the following primers: 5′-AAGACCACAGAGTGTAAGATGG-3′ (position –809 to –788 from the transcription starting point) and 5′-TAAAGGTACCCACAACCACTTACC-3′ (146–123). The PCR products were digested with BamHI, PstI, and KpnI, and restriction fragments (BamHI–PstI and PstI–KpnI) were subcloned and sequenced on both strands. Measurement of serum levels of total IgG and IgG anti-DNA antibodies Serum levels of total IgG and IgG anti-DNA antibodies were determined by ELISA. IgG anti-DNA antibodies were quantified using ELISA plates coated with calf thymus DNA (Sigma, St Louis, MO) and the DNA-binding activities were expressed in units, referring to a standard curve obtained by serial dilutions of a standard serum pool from 7- to 9-month-old (NZB × NZW) F1 mice, containing 1000 U/ml. Genotyping DNA was extracted from the mouse tail. Genotyping for microsatellite markers and the FcγRIIB promoter region was done using PCR. Microsatellite primers were purchased from Research Genetics (Huntsville, AL). Genotyping for the FcγRIIB promoter region was done using 5′ and 3′ primers: 5′-GTTGATCTTCATTTTACAGAC-3′ and 5′-TCTGTGCCCTAGTCCTGAATC-3′ (see Fig. 3A). PCR reactions were run in 96-well plates with 7.5 μl total volume containing 40 ng of genomic DNA. A three-temperature PCR protocol (94, 55 and 72°C) was conducted for 45 cycles in a Geneamp 9600 Thermal Cycler (Perkin-Elmer-Cetus, Norwalk, CT). PCR products were diluted 2-fold with loading buffer consisting of xylene cyanol and bromophenol blue dyes in 50% glycerin, and were run on 15% polyacrylamide gels. After electrophoresis, gels were visualized after ethidium bromide staining. Statistics The linkage of a particular locus with increased levels of serum IgG was estimated using a computer package program of MAPMAKER/EXP and MAPMAKER/QTL (17) to identify chromosomal locations of quantitative trait loci (QTL). Analysis of variance (ANOVA) was used to determine the differences in serum levels of total IgG and IgG anti-DNA antibodies among each group of (NZB × NZW) F1 × NZW backcross mice with different combinations of susceptibility alleles. Results Down-regulation of FcγRIIB1 in germinal center B cells Germinal centers in lymph follicles are the site for IgM to IgG Ig class switch of B cells and for generation of high-affinity IgG antibody responses upon T cell-mediated signals. Thus, it is reasonable to assume that, in autoimmune disease, there is a mechanism that allows pathogenic IgG autoantibodies to be over-produced, through B cells down-regulating their FcγRIIB1 expression in germinal centers and thus escaping the suppressive signals. To address this issue, we first examined levels of FcγR expression on splenic germinal center B cells, as distinguished by expression of high levels of surface binding sites for PNA, and non-germinal center B cells from SLE-prone NZB and (NZB × NZW) F1 and non-SLE-prone NZW mice, using flow cytometric analyses with a monoclonal anti-FcγR antibody 2.4G2 which reacts with FcγRIIB1, FcγRIIB2 and FcγRIII (18). The extent of germinal center formation in lymph follicles increased as animals aged, and mean percentages ± SE of PNAhigh germinal center B cells in total splenic B cells from five each NZB, NZW and (NZB × NZW) F1 mice 8 months of age were 9.1 ± 1.2, 2.3 ± 0.4 and 13.9 ± 4.9 respectively. We found that while 2.4G2 levels in non-germinal center B cells were practically identical, they were down-regulated in germinal center B cells in all the mouse strains examined. It was of particular note, however, that the extent of this down-regulation was much greater in NZB and (NZB × NZW) F1 (~10-fold decrease) than found in NZW (a 4-fold decrease) (Fig. 1A). RT-PCR analyses of mRNAs from FACS-sorted splenic B cell populations from 8-month-old NZB mice showed that, compared to findings in PNAlow non-germinal center B cells which express high amounts of FcγRIIB1 messages, there was ~10-fold decrease in its expression in PNAhigh germinal center B cells (Fig. 2). While the FcγRIIB2 isoform product was detected in peritoneal cavity cells containing a mixture of macrophages and lymphocytes, it was not expressed in FACS-sorted B cells. Similar findings were obtained with the FcγRIII PCR product, but a small amount of messages was detected in PNAhigh, but not PNAlow, B cells (Fig. 2). In contrast to FcγRIIB1 and FcγRIIB2, FcγRIII molecules are expressed as a multimeric complex with homodimeric or heterodimeric complexes with the γ chain, and are not normally expressed on B cells (12–14). Therefore, we examined the expression of FcRγ chain messages in germinal enter B cells and found the presence of a small, but significant amount in PNAhigh, but not PNAlow, B cells (data not shown). However, as amounts of FcγRIII PCR products did not differ significantly between the NZB and NZW strains, the observed decrease in the 2.4G2 expression on germinal center B cells was considered to be mainly due to down-regulation of FcγRIIB1 molecules. Although germinal centers of young mice are not well established, immunizations with particulate or aggregated antigens or deposits of preformed antigen–antibody complexes elicit germinal center formation. Thus, we immunized NZB and NZW mice twice at 6 and 9 weeks of age with SRBC, and FcγRIIB1 expression levels were determined by flow cytometric analysis 1 week after the second immunization. We again found that levels of FcγRIIB1 expression were lower in germinal center B cells than in non-germinal center B cells and that the extent of down-regulation was greater in NZB than in NZW (Fig. 1B). Taken collectively, it was suggested that the strain difference in the extent of FcγRIIB1 down-regulation in germinal center B cells is due to a genetically determined event. Polymorphism of the regulatory region of the FcγRIIB gene To determine the polymorphism between the NZB and NZW FcγRIIB gene, we first PCR amplified and sequenced structural regions. NZB and NZW strains had the same structural polymorphism corresponding to Ly-17.1 allotype (data not shown), as reported by other investigators (19). We then analyzed PCR products of FcγRII promoter regions. As compared with findings in BALB/c, C57BL/6 and NZW, the product of the region (position –194 to –30 from the transcription starting point) from NZB showed a different nucleotide length polymorphism: (NZB × NZW) F1 mice shared both NZB and NZW-type alleles (Fig. 3A). Sequence analyses revealed that NZB had two nucleotide substitutions and two deletion sites in this promoter region, including transcription factor-binding consensus sequences, i.e. Ap-4-binding site (20) and S box (21) (Fig. 3B). Linkage between NZB FcγRIIB allele and hyper-IgG To determine the role of the NZB FcγRIIB1 allele in IgG hypergammaglobulinemia, we examined the association between the NZB FcγRIIB allele and serum IgG levels using 207 (NZB × NZW) F1 × NZW backcross female mice 8 months of age. When the backcross progeny was divided into two groups, i.e. one with heterozygous NZB/NZW (B/W) type for FcγRIIB allele (Fcgr2b) and the other with homozygous NZW/NZW (W/W) type, total serum levels of IgG were significantly higher in the former (Fig. 4A). MAPMAKER/QTL scans of data from backcross progeny using microsatellites and FcγRIIB allele polymorphic between the NZB and NZW strain showed that the NZB locus responsible for high serum IgG, provisionally designated Hig-1 (Hyper-IgG-1), is located on chromosome 1, most significantly linked to the FcγRIIB locus (Fcgr2b) (Fig. 5). Combinatorial effect of FcγRIIB allele and MHC in IgG anti-DNA antibody production When we compared serum levels of IgG anti-DNA antibodies in (NZB × NZW) F1 × NZW backcross progeny between the group with heterozygous B/W type for Fcgr2b and that with homozygous W/W type, neither group showed a significant difference, although the former did tend to be higher (Fig. 4B). As synthesis of IgG anti-DNA antibodies in (NZB × NZW) F1 mice is strictly restricted to H-2d/z heterozygosity (H-2d from NZB and H-2z from NZW) (22), we then separated the backcross progeny into four groups, classified according to combinations of H-2 and Fcgr2b genotypes, i.e. group a, H-2d/z and B/W Fcgr2b genotypes; group b, H-2d/z and W/W Fcgr2b; group c, H-2z/z and B/W Fcgr2b; and group d, H-2z/z and W/W Fcgr2b. Among these four groups, while total serum IgG levels were higher in the progeny with the B/W Fcgr2b genotype than in the progeny with the W/W genotype, irrespective of the H-2 genotypes (Fig. 4C), the IgG anti-DNA antibody levels were in the order of group a, group b, and groups c and d (Fig. 4D), indicating that the high IgG anti-DNA antibody titers seen in (NZB × NZW) F1 mice are regulated by a combinatorial effect of H-2d/z and the NZB FcγRIIB allele. Discussion In T cell-dependent humoral immune responses, antigen-activated B cells accumulate in the germinal centers where Ig class switching and generation of high-affinity IgG antibodies somatically mutated in Ig variable regions occur (23). As pathogenic IgG anti-DNA antibodies in SLE are also affinity selected (24,25), the process of the generation of these autoantibodies also appears operative in germinal centers. In the present studis, we found that the NZB strain has a unique FcγRIIB promoter allele which is associated with abnormal down-regulation of FcγRIIB1 levels in germinal center B cells. Thus, it is reasonable to assume that this FcγRIIB1 down-regulation is a mechanism which allows high-affinity IgG autoantibodies to be over-produced through germinal center B cells escaping negative signals for IgG production. Structural regions of the NZB FcγRIIB gene did not differ from the NZW gene. The difference in the extent of FcγRIIB1 expression levels between NZB and non-SLE NZW strains was linked to sequence differences in the FcγRIIB promoter region. The two deletion sites observed in the NZB allele, one including the Ap-4-binding site (20) and S box (21) (Fig. 3B), are located in the region containing the element which regulates cell-type-specific FcγRIIB expression and drives the transcription of FcγRIIB gene in a B cell, but not a mast cell, line (26). Lack of differential FcγRIIB1 expression in the non-germinal center resting B cells between NZB and NZW strains suggests that the deletion sites regulate FcγRIIB1 expression in the process of T cell-mediated B cell activation. Thus, the constitutive FcγRIIB1 expression in resting B cells must be differentially controlled by other regulatory elements, such as those located in the third intron containing a series of purine/pyrimidine-rich regions, NF-κB-like binding motif and elements with silencer activity (20,26). On the other hand, the decrease in the FcγRIIB1 level in germinal center B cells from aged (NZB × NZW) F1 mice was much closer to NZB than to NZW. At present, we have no explanation for the reason of the dominant negative effect of the down-regulatory NZB allele in the F1 mice. Combinatorial effects of other elements also appear to be involved in this regulation. A suggested role for FcγRIIB1 in the hyperproduction of IgG was supported by the significant association between high serum level of IgG and the NZB-type promoter polymorphism in (NZB × NZW) F1 × NZW backcross progeny. A tight linkage in QTL analyses between the NZB FcγRIIB locus and the extent of hyper-IgG further supported the notion (Fig. 5). We do not exclude the possibility, however, that the hyper-IgG can be in part attributed to a decrease in the expression of FcγRIIB2 on phagocytes due to the NZB FcγRIIB promoter polymorphism, the result being a reduction in IgG IC clearance and thus leading to accumulation of IC in the circulation. Indeed, our preliminary studies showed that levels of the FcγRIIB2 isoform on macrophages from NZB mice are also down-regulated, as compared with the finding in NZW mice. It is possible that this abnormality is related to the pathogenesis of IC-type glomerulonephritis in NZB and (NZB × NZW) F1 mice. However, the defect in FcγRIIB2 expression on macrophages does not simply explain the increase in monomeric IgG in the blood. This notion was supported by our preliminary results that mice with the NZB FcγRIIB allele had much higher secondary IgG anti-SRBC antibody responses than found in mice lacking this allele. In the present studies, we found that germinal center B cells express small, but significant amounts of FcγRIII, which are not normally expressed on B cells. In contrast to FcγRIIB1, FcγRIII molecules are expressed on NK cells, mast cells, neutrophils and macrophages, and mediate a variety of functions, such as antibody-dependent cell-mediated cytotoxicity, degranulation, endocytosis, phagocytosis and antigen presentation (12–14). Further studies are required to elucidate the role of FcγRIII on germinal center B cells. Nonetheless, because the amounts of FcγRIII PCR products in germinal center B cells did not differ between the NZB and NZW strains, the observed decrease in 2.4G2 expression in germinal center B cells is mainly due to down-regulation of FcγRIIB1. Although the role of NZB-type FcγRIIB allele in hyper-IgG and IgG anti-DNA antibody production appears significant, the mechanism is more complex, because our earlier studies revealed that serum levels of both total IgG and IgG anti-DNA antibodies in (NZB × NZW) F1 mice are much higher than these levels in NZB mice and that a complimentary effect of both NZB and NZW genes determines these two traits in the F1 hybrid mice (22, 27). Our present progeny studies were carried out using (NZB × NZW) F1 × NZW backcross mice, in which NZB genes segregate in the progeny bearing all NZW alleles in the genetic background. Therefore, although the observed hyper-IgG seemed to be regulated by the polymorphic NZB FcγRIIB promoter allele alone, other NZW genetic contributions are likely to be involved. In this respect, in contrast to the serum levels of total IgG, IgG anti-DNA antibodies were combinatorially regulated by the FcγRIIB and H-2 complex alleles (Fig. 4). This observation is consistent with our earlier findings that production of high-affinity IgG anti-DNA antibodies in (NZB × NZW) F1 mice is highly restricted to H-2d/z heterozygosity (22), suggesting that the mixed haplotype class II molecules, most likely Aαdβz (28,29), serve as a restriction element for autoreactive T cells. As serum hyper-IgG occurred in the backcross progeny, irrespective of the H-2 haplotypes, this trait is largely due to the increase in polyclonal IgG that are mainly produced under the restriction of homozygous d/d and/or z/z, but not heterozygous d/z, A and/or E class II molecules. A report by Luan et al. (30) showed that the NOD mouse strain, a spontaneous model of autoimmune diabetes, also has hyper-IgG in association with a defect in the promoter region of FcγRIIB gene, very close to the sites detected in NZB. These same authors noted the down-regulation of FcγRIIB2 in macrophages, but not FcγRIIB1 in B cells. Our preliminary studies on NOD mice, however, showed that FcγRIIB1 expression in NOD germinal center B cells is also markedly down-regulated. Taken collectively, it is highly possible that the observed regulatory defect in FcγRIIB1 expression also predisposes to other autoantibody-mediated autoimmune diseases. Allelic polymorphisms of FcγRIIA and FcγRIIIA have been associated with human SLE (8–11), although the role of FcγRIIA alleles in Caucasian SLE patients is controversial (9,31). Considering the regulatory role of FcγRIIB1 on IgG synthesis (16), linkage of the chromosome 1 region to human SLE may be partly due to linkage to the polymorphic FcγRIIB allele, as observed in the murine model. We propose that this NZB-type FcγRIIB promoter polymorphism is one susceptibility allele for murine and possibly human SLE. Fig. 1. View largeDownload slide Comparisons of FcγRIIB1 expression levels between splenic PNAhigh germinal center B cells and PNAlow non-germinal center B cells from NZB, NZW and (NZB × NZW) F1 mice. (A) Spleen cells from 8-month-old NZB, NZW and (NZB × NZW) F1 mice were stained with phycoerythrin-labeled anti-FcγRII/III (2.4G2) mAb, FITC-labeled B cell-specific anti-CD45R/B220 mAb and biotinylated PNA, followed by streptavidin–allophycocyanin. Levels of 2.4G2 expression on PNAhigh and PNAlow splenic CD45R/B220+ B cell populations were compared on the histograms. The closed area indicates the background staining. Because RT-PCR analysis showed that B cells expressed mainly FcγRIIB1 (Fig. 2), 2.4G2 expression on B cells reflects the level of FcγRIIB1 isoform. (B) NZB and NZW mice were immunized twice at 6 and 9 weeks of age with SRBC and spleen cells were obtained 1 week after the second immunization. Spleen cells were analyzed in the same manner as shown in (A). PNAhigh B cell population was not detected in non-treated young mice. A representative result of five experiments is shown in (A) and (B). Fig. 1. View largeDownload slide Comparisons of FcγRIIB1 expression levels between splenic PNAhigh germinal center B cells and PNAlow non-germinal center B cells from NZB, NZW and (NZB × NZW) F1 mice. (A) Spleen cells from 8-month-old NZB, NZW and (NZB × NZW) F1 mice were stained with phycoerythrin-labeled anti-FcγRII/III (2.4G2) mAb, FITC-labeled B cell-specific anti-CD45R/B220 mAb and biotinylated PNA, followed by streptavidin–allophycocyanin. Levels of 2.4G2 expression on PNAhigh and PNAlow splenic CD45R/B220+ B cell populations were compared on the histograms. The closed area indicates the background staining. Because RT-PCR analysis showed that B cells expressed mainly FcγRIIB1 (Fig. 2), 2.4G2 expression on B cells reflects the level of FcγRIIB1 isoform. (B) NZB and NZW mice were immunized twice at 6 and 9 weeks of age with SRBC and spleen cells were obtained 1 week after the second immunization. Spleen cells were analyzed in the same manner as shown in (A). PNAhigh B cell population was not detected in non-treated young mice. A representative result of five experiments is shown in (A) and (B). Fig. 2. View largeDownload slide A representative result of RT-PCR amplifications of FcγRIIB1, FcγRIIB2 and FcγRIII messages from splenic PNAhigh germinal center (GC) B cells and PNAlow non-germinal center (nonGC) B cells from NZB mice. Total RNA was isolated from FACS-sorted PNAhigh and PNAlow splenic B cells obtained from 8-month-old NZB mice, and used for RT-PCR. As a control, RNA was isolated from peritoneal cavity cells (PerC) containing macrophages. Amplification of β-actin served to check RNA amounts. PCR products were electrophoresed on 2% agarose gels and visualized after ethidium bromide staining. The number of the top of each lane indicates the reciprocal of the dilution of cDNA for FcγRIIB1, FcγRIIB2, FcγRIII and β-actin. Numbers of amplified bases are as follows; 481 bp for FcγRIIB1, 343 bp for FcγRIIB2 and 453 bp for FcγRIII. Note that ~10-fold decrease in FcγRIIB1 PCR product is observed in PNAhigh B cells, compared to findings in PNAlow B cells. Fig. 2. View largeDownload slide A representative result of RT-PCR amplifications of FcγRIIB1, FcγRIIB2 and FcγRIII messages from splenic PNAhigh germinal center (GC) B cells and PNAlow non-germinal center (nonGC) B cells from NZB mice. Total RNA was isolated from FACS-sorted PNAhigh and PNAlow splenic B cells obtained from 8-month-old NZB mice, and used for RT-PCR. As a control, RNA was isolated from peritoneal cavity cells (PerC) containing macrophages. Amplification of β-actin served to check RNA amounts. PCR products were electrophoresed on 2% agarose gels and visualized after ethidium bromide staining. The number of the top of each lane indicates the reciprocal of the dilution of cDNA for FcγRIIB1, FcγRIIB2, FcγRIII and β-actin. Numbers of amplified bases are as follows; 481 bp for FcγRIIB1, 343 bp for FcγRIIB2 and 453 bp for FcγRIII. Note that ~10-fold decrease in FcγRIIB1 PCR product is observed in PNAhigh B cells, compared to findings in PNAlow B cells. Fig. 3. View largeDownload slide Nucleotide deletions in the FcγRIIB promoter region in the NZB strain. (A) Genomic organization of FcγRIIB gene (Fcgr2b) and length polymorphism of PCR products of FcγRIIB promoter region. Exons are numbered and indicated by boxes, untranslated regions by open boxes, and coding regions by filled boxes which are also labeled by the region they encode, i.e. EC for extracellular domains, TM for transmembrane domain and IC for intracytoplasmic domains. Primer positions for PCR are indicated by arrows. The position 0 is defined as the transcription starting point. Tail DNA from a single mouse of each strain was used as a template for PCR, and the amplified fragments were run on 15% polyacrylamide gels and visualized by staining with ethidium bromide. Size difference in PCR product indicates the deletion in FcγRIIB promoter region in NZB strain. (B) Nucleotide sequence of PCR-amplified FcγRIIB promoter region in BALB/c, NZW and NZB strains. Horizontal bar indicates sequence identity. Asterisk depicts nucleotide deletions in NZB strain. Transcription factor-binding consensus sequences in the deleted region are indicated. Fig. 3. View largeDownload slide Nucleotide deletions in the FcγRIIB promoter region in the NZB strain. (A) Genomic organization of FcγRIIB gene (Fcgr2b) and length polymorphism of PCR products of FcγRIIB promoter region. Exons are numbered and indicated by boxes, untranslated regions by open boxes, and coding regions by filled boxes which are also labeled by the region they encode, i.e. EC for extracellular domains, TM for transmembrane domain and IC for intracytoplasmic domains. Primer positions for PCR are indicated by arrows. The position 0 is defined as the transcription starting point. Tail DNA from a single mouse of each strain was used as a template for PCR, and the amplified fragments were run on 15% polyacrylamide gels and visualized by staining with ethidium bromide. Size difference in PCR product indicates the deletion in FcγRIIB promoter region in NZB strain. (B) Nucleotide sequence of PCR-amplified FcγRIIB promoter region in BALB/c, NZW and NZB strains. Horizontal bar indicates sequence identity. Asterisk depicts nucleotide deletions in NZB strain. Transcription factor-binding consensus sequences in the deleted region are indicated. Fig. 4. View largeDownload slide Associations of FcγRIIB gene (Fcgr2b) and major histocompatibility complex (H-2) genotypes with serum levels of total IgG and IgG anti-DNA antibodies in 207 female (NZB × NZW) F1 × NZW backcross mice aged 8 months. Mice were separated into two (A and B) and four groups (C and D), classified according to genotypes of Fcgr2b and a combination of genotypes for H-2 and Fcgr2b respectively. Genotypes of B/W and W/W for Fcgr2b depict NZB/NZW heterozygotes and NZW/NZW homozygotes respectively. Genotypes of d/z and z/z for H-2 depict heterozygotes with H-2d from NZB and H-2z from NZW, and homozygotes with H-2z/z respectively. The number of mice examined is shown in parentheses and serum levels of antibodies are expressed by mean ± SE in mice at 8 months of age. Significant difference by ANOVA is shown. Fig. 4. View largeDownload slide Associations of FcγRIIB gene (Fcgr2b) and major histocompatibility complex (H-2) genotypes with serum levels of total IgG and IgG anti-DNA antibodies in 207 female (NZB × NZW) F1 × NZW backcross mice aged 8 months. Mice were separated into two (A and B) and four groups (C and D), classified according to genotypes of Fcgr2b and a combination of genotypes for H-2 and Fcgr2b respectively. Genotypes of B/W and W/W for Fcgr2b depict NZB/NZW heterozygotes and NZW/NZW homozygotes respectively. Genotypes of d/z and z/z for H-2 depict heterozygotes with H-2d from NZB and H-2z from NZW, and homozygotes with H-2z/z respectively. The number of mice examined is shown in parentheses and serum levels of antibodies are expressed by mean ± SE in mice at 8 months of age. Significant difference by ANOVA is shown. Fig. 5. View largeDownload slide MAPMAKER/QTL scans on chromosome 1 for hyper-IgG in 207 female (NZB × NZW) F1 × NZW backcross mice aged 8 months. The lod score curve is shown on the right with scale on the bottom. Map positions of microsatellite markers are arranged from centromere to telomere on the left of the chromosome line. Genotyping for FcγRIIB (Fcgr2b) was done using the promoter region primer pair shown in Fig. 3(B). Maximum lod score (3.4) at Fcgr2 indicates a significant linkage (32). Fig. 5. View largeDownload slide MAPMAKER/QTL scans on chromosome 1 for hyper-IgG in 207 female (NZB × NZW) F1 × NZW backcross mice aged 8 months. The lod score curve is shown on the right with scale on the bottom. Map positions of microsatellite markers are arranged from centromere to telomere on the left of the chromosome line. Genotyping for FcγRIIB (Fcgr2b) was done using the promoter region primer pair shown in Fig. 3(B). Maximum lod score (3.4) at Fcgr2 indicates a significant linkage (32). Transmitting editor: K. Okumura We thank M. Ohara for language assistance. 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International ImmunologyOxford University Press

Published: Oct 1, 1999

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