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* Department of Pathology, Juntendo University School of Medicine, and
Department of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University School of Medicine, Tokyo, Japan;
Central Laboratory of First Clinical College, China Medical University, Shenyang, China;
Department of Pathology, Ehime University School of Medicine, Ehime, Japan; and
¶ Department of Experimental Immunology and Core Research for Engineering, Science, and Technology of Japan Science and Technology Corporation, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB1 molecules serve as negative feedback regulator for B
cell Ag receptor-elicited activation of B cells; thus, any impaired
FcRIIB1 function may possibly be related to aberrant B cell
activation. We earlier found deletion polymorphism in the
Fcgr2b promoter region among mouse strains in which
systemic autoimmune disease-prone NZB, BXSB, MRL, and autoimmune
diabetes-prone nonobese diabetic, but not NZW, BALB/c, and
C57BL/6 mice have two identical deletion sites, consisting of 13 and 3
nucleotides. In this study, we established congenic C57BL/6 mice for
NZB-type Fcgr2b allele and found that NZB-type allele
down-regulates FcRIIB1 expression levels in germinal center B cells
and up-regulates IgG Ab responses. We did luciferase reporter assays to
determine whether NZB-type deletion polymorphism affects
transcriptional regulation of Fcgr2b gene. Although NZW-
and BALB/c-derived segments from position -302 to +585 of
Fcgr2b upstream region produced significant levels of
luciferase activities, only a limited activity was detected in the
NZB-derived sequence. EMSA and Southwestern analysis revealed that
defect in transcription activity in the NZB-derived segment is likely
due to absence of transactivation by AP-4, which binds to the
polymorphic 13 nucleotide deletion site. Our data imply that because of
the deficient AP-4 binding, the NZB-type Fcgr2b allele
polymorphism results in up-regulation of IgG Ab responses through
down-regulation of FcRIIB1 expression levels in germinal center B
cells, and that such polymorphism may possibly form the basis of
autoimmune susceptibility in combination with other background
contributing genes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB, and FcRIII transduce
opposing activation/inhibition signals upon ligation by IgG immune
complexes (ICs)3
(1, 2, 3, 4). Although FcRIII exists as an oligomeric complex
together with - and 
-chain homo- or heterodimers involved in cell
activation through immunoreceptor tyrosine-based activation motif,
FcRIIB is a monomeric receptor with immunoreceptor tyrosine-based
inhibition motif in the cytoplasmic region. There are known two
membrane-bound isoforms of FcRIIB, FcRIIB1, and FcRIIB2, and
these are encoded by the same gene, but show distinct cell-type
distribution and function. FcRIIB1 molecule, which is predominantly
expressed on B cells, has complete domains from all exons, and upon
cross-linking with B cell Ag receptor (BCR) by IgG ICs, it acts as a
negative feedback regulator by inhibiting BCR-elicited activation
signal through immunoreceptor tyrosine-based inhibition motif. In
contrast, FcRIIB2 is mainly expressed on macrophages and a domain
encoded by the first intracytoplasmic exon is missing, due to
alternative splicing of mRNA transcripts. Such a defect allows
FcRIIB2 to internalize IgG ICs for Ag presentation mainly in
phagocytosis. Susceptibility to autoimmune disease is determined by combined effects of alleles at multiple loci. Several lines of evidence suggest that some could be related to intrinsic hyperresponsiveness of B cells to signals for proliferation and maturation (5). B cell responses are controlled by signaling thresholds through the BCR complex (6). Thus, allelic variants associated with functional deficits in negative feedback regulation of BCR signaling would be worth examining for their association with susceptibility to autoimmune diseases. Genetic studies of our own and others provided evidence that one of the susceptibility alleles to systemic lupus erythematosus (SLE) was mapped in the vicinity of Fcgr2b gene on the telomeric region on chromosome 1, in both murine models (7, 8, 9, 10, 11, 12) and human SLE (13). Thus, we focused on allelic polymorphisms of Fcgr2b as a pivotal genetic element for aberrant B cell activation in SLE.
In previous studies, we found that mouse strains can be divided in two
groups, based on the presence or absence of allelic polymorphisms in
the Fcgr2b promoter region, consisting of 13 and 3
nucleotide deletion sites (14). Intriguingly, spontaneous
animal models of autoimmune diseases, i.e., SLE-prone NZB, BXSB, MRL,
and diabetes-prone nonobese diabetic (NOD), all shared identical
deletion sites in the Fcgr2b promoter region. The
polymorphic promoter region is located in the region containing the
reported regulatory element for transcription of Fcgr2b gene
in B cells (15), and a 13 nucleotide deletion site in the
NZB-type Fcgr2b promoter region includes putative
transcription factor-binding sequences, i.e., the AP-4-binding site
(16) and S box (17). Of note was the finding
that the FcRIIB1 expression in mice having this polymorphism was
abnormally down-regulated in B cells, particularly in follicular
germinal centers (GCs), areas of class switching and affinity
maturation of B cells, while expression in non-GC B cells was
practically identical (10, 14). The importance of this
finding was supported by genetic studies using (NZB x
NZW)F1 x NZW backcross mice, indicating that the NZB-type
Fcgr2b allele was significantly linked to hyper-IgG and IgG
anti-DNA Abs in quantitative trait loci analysis (10).
Therefore, the FcRIIB promoter polymorphism may possibly predispose
to SLE through GC B cells down-regulating FcRIIB1 expression upon
autoantigen stimulations, thus escaping negative signals for IgG
production. Nevertheless, there is no direct evidence that the
polymorphic regions are indeed contributing to transcriptional
regulation of Fcgr2b gene. In the present study, we
highlighted this issue and did luciferase reporter assays, together
with EMSAs and Southwestern analysis to detect nuclear binding protein,
and to confirm the pivotal role of promoter region polymorphisms in
transcriptional regulation of the Fcgr2b gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As the 129Sv strain of mice was reported to have NZB-type
fcgr2b allele (18), C57BL/6 (B6) congenic mice
for this allele were generated by introducing Fcgr2b allele
from 129Sv with selective back-crossing of (129Sv x
B6)F1 to B6 for 10 generations. FcRII-deficient B6 mice
were obtained by back-crossing the originally constructed deficient
mice on a hybrid (129Sv x B6) background to B6 for 12 generations
(19). All mice used were housed under identical
conditions. If necessary, mice were immunized twice at 10 and 13 wk of
age by giving an i.p. injection of 100 µg per mouse of keyhole limpet
hemocyanin (KLH) in CFA and IFA, respectively.
Genotyping
DNA was extracted from the mouse tail tissue. Genotyping for
microsatellite markers and the FcRIIB promoter region was done using
PCR. Microsatellite primers were purchased from Research Genetics
(Huntsville, AL). Genotyping for FcRIIB promoter region was done
using 5' and 3' primers: 5'-GTAAGTGGTTGTGGGTACCTTTATT-3' and
5'-CGCAGCTCAGAAGTCATTTGCCTCA-3'. PCRs were run in 20 µl total
volume containing 40 ng of genomic DNA. A three temperature PCR
protocol (94°C, 65°C, and 72°C) was used for 35 cycles in a
GeneAmp 9600 Thermal Cycler (PerkinElmer/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 18%
polyacrylamide gels. After electrophoresis, gels were visualized with
ethidium bromide staining.
Flow cytometry analysis
For analysis of FcRIIB1 expression levels on B cells,
aliquots of 1 x 106 spleen cells were first
stained with PE-conjugated mAb 2.4G2 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 then stained with FITC-labeled B
cell-specific anti-B220 (6B2) mAb and biotinylated peanut
agglutinin (PNA). After washing, the cells were further stained with
streptavidin-conjugated allophycocyanin. For FcRIIB2 expression
levels on peritoneal macrophages, peritoneal exudate cells were
double-stained with Alexa 488-conjugated mAb F4/80 and PE-conjugated
2.4G2 in PBS containing 1% BSA and 0.1% sodium azide for 30 min at
4°C. After the cells were washed and fixed in PBS containing 1%
formaldehyde, fluorescence intensity was measured using a FACStar flow
cytometer and CellQuest software (BD Biosciences, Mountain View,
CA).
Measurement of serum Ab levels
Serum levels of IgG anti-KLH Abs were quantified using ELISA plates coated with 5 µg/ml of KLH (Sigma-Aldrich, St. Louis, MO). KLH-binding activities were expressed in units, referring to a standard curve obtained by serial dilutions of a standard serum pool from hyperimmunized NZB mice, containing 1000 U activities/ml.
Serum levels of IgG class autoantibodies to dsDNA were also determined using ELISA plates coated with 5 µg/ml of dsDNA derived from the calf thymus (Sigma-Aldrich). Binding activities were expressed in units, referring to a standard curve obtained by serial dilution of a standard serum pool from (NZB x NZW)F1 mice >8 mo of age, containing 1000 U activities/ml.
Plasmid construction and nucleotide sequence analysis
The 5'-flanking sequences of Fcgr2b gene (nucleotides from position -302 to +76 or position -302 to +585 relative to the transcription start site) were generated by PCR from genomic DNA, using appropriate primers derived from the sequences published by Hogarth et al. (16). The PCR products were cloned into the upstream of the luciferase coding region of the pGL3-Basic vector (Promega, Madison, WI) to yield luciferase constructs carrying each mouse Fcgr2b gene promoter.
For sequencing, we used these PCR products and the dideoxy chain termination method with Taq dye primer cycle sequencing kits (Applied Biosystems, Foster City, CA), according to the manufacturer’s instruction.
Luciferase assays
Bal17 (murine B cell line) and P388D1 (murine macrophage-like cell line) cells were kindly provided by Dr. T. Tsubata (Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan), and Dr. K. Okumura (Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan), respectively. For stable transfections, cells grown to subconfluence in RPMI 1640 with 10% FBS were washed twice with PBS and resuspended at the density of 1.5 x 107 cells/ml. A total of 700 µl of cells were transfected with 20 µg of the 2.9-kb SalI-SacI fragment derived from each luciferase construct and 1 µg of the 5.7-kb EcoRI fragment derived from pSV2-neo (20), including the neomycin-resistant gene by electroporation using Gene pulser (Bio-Rad, Hercules, CA) (350 V/950 µF for Bal17 cells, 400 V/950 µF for P388D1 cells). Two days after electroporation, medium was replaced with that containing 1 mg/ml (Bal17 cells) or 400 µg/ml (P388D1 cells) of G418 (Calbiochem, La Jolla, CA). After a 14-day selection period, luciferase assays were done. Luciferase activity was measured for a 20-s time course with the use of a luminometer (Lumat LB 9507; Berthold, Wildbad, Germany), and then normalized by gene dosage estimated by Southern blot hybridization using the 1.7-kb NcoI-XbaI fragment from the pGL3-Basic vector as a probe.
Preparation of nuclear protein extracts and EMSA
Nuclear protein extracts were prepared as described by Schreiber
et al. (21). The oligonucleotides were chemically
synthesized, annealed after being heated to 100°C, and gel purified.
The resultant probes (Table I
) were
labeled with [-32P]ATP using T4
polynucleotide kinase. Binding reactions and electrophoresis were done
essentially according to Fujihara et al. (22). Nuclear
protein extracts (5 µg) with 2 µg of poly(dI-dC) (Amersham
Pharmacia Biotech, Piscataway, NJ) and labeled DNA (100,000 cpm) were
mixed in 25 µl of binding buffer (10 mM Tris-HCl (pH 7.5), 40 mM
NaCl, 1 mM EDTA, 1 mM 2-ME, 4% glycerol, 0.1% Nonidet P-40, 1 mM DTT,
1 mg/ml BSA) then incubated for 30 min on ice after mixing. The
DNA-protein complexes were separated on a native 4.5% polyacrylamide
gel in 0.25 x Tris-borate EDTA buffer, then the gels were
fixed, vacuum dried, and exposed for autoradiography.
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A total of 50 µg of nuclear extracts obtained from P388D1 cells were separated on 10% SDS-polyacrylamide gels at 4°C for 120 min (20 mA). The separated proteins were then electrically transferred overnight to a polyvinylidene difluoride (PVDF) membrane at 4°C (30 V) in buffer containing 25 mM Tris, 190 mM glycine, and 20% methanol. After transfer, the membrane was soaked in Z' buffer (25 mM HEPES-KOH (pH 7.6), 12.5 mM MgCl2, 20% glycerol, 0.1% Nonidet P-40, 0.1 M KCl, 10 µM ZnSO4, 1 mM DTT) with 6 M guanidine-HCl, and was washed five times with Z' buffer containing graded concentrations of guanidine-HCl to obtain a final concentration of 0.1 M guanidine-HCl. The PVDF membrane was finally washed twice with Z' buffer without guanidine-HCl, blocked with 3% skim milk in Z' buffer for 30 min, and preequilibrated in binding buffer (0.25% skim milk in Z' buffer) for 5 min before addition of a probe. dsDNA oligonucleotide probe, end-labeled with 1 x 107 cpm 32P, was diluted in 2 ml binding buffer, and the PVDF membrane was incubated with probes for 30 min at 4°C with continuous agitation. The membrane was washed three times with Z' buffer and then three times with 0.3 M KCl in Z' buffer, each time for 5 min. After air drying, the membrane was autoradiographed.
RT-PCR analysis for AP-4 transcripts
Total RNA was isolated from cell lines Bal17 and P388D1, and T cell-depleted splenic B cells from 2- and 10-mo-old NZB mice using ISOGEN (Nippon Gene, Tokyo, Japan). To obtain a T cell-depleted B cell population, spleen cells were treated with a mixture of the rat mAbs to CD4, CD8, and Thy-1.2 plus rabbit complement at 37°C for 45 min. First-strand cDNA was synthesized using a random hexamer and Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA). The specific PCR products for AP-4 were amplified using primers (5'-GATGATGCTGGAAGAGCAGGTGCGC-3' and 5'-CCCTCAATGTGCTGGATGGCCTGCAC-3') in three-temperature system (94°C, 57°C, and 72°C) for 35 cycles. Amplification of GAPDH served as a reference to normalize data for the total amount of RNA input. Primers for GAPDH were 5'-GGACTGCCCAGAACATCATCCCTGC-3' and 5'-TTAGTGGGCCCCTGGATGCCAGCTT-3'. The products were electrophoresed in 2% agarose gel and visualized after ethidium bromide staining.
Statistics
Statistical analysis was done using Student’s t test. A value of p < 0.05 was considered to have a statistical significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB
expression levels in immune cells and IgG Ab responses
To provide evidence that the marked down-regulation of FcRIIB1
expression levels on GC B cells is linked to the NZB-type
Fcgr2b promoter polymorphism, but not to other background
genetic elements, we established B6 mice congenic for NZB-type
Fcgr2b allele. As the 129Sv strain shares the NZB-type
Fcgr2b allele (18), congenic mice were
generated by selective back-crossing of (129Sv x
B6)F1 to B6 for 10 generations. The 129Sv interval on
chromosome 1 introduced into B6 mice extended from 
88 (D1
Mit15)-92 (Fcgr2b) cM, which were verified to be
negative for the 129Sv allele of Fcer1g, Cd48, Ly9, Fcer1a, Crp,
Sap, Ifi202, Ifi203, Ifi204, Cr2, and Adprp
(Fig. 1). These congenic B6 mice,
together with control B6 and B6.FcRIIB-/-
mice at 10 and 13 wk of age, were immunized twice with KLH. One week
after the second immunization, expression levels of FcRIIB1 on
B220-positive splenic B cells were examined, using 2.4G2 mAb
(FcRIIB/III; Ref. 23). As shown in Fig. 2A, both
PNAlow non-GC B cells and
PNAhigh GC B cells from
B6.FcRIIB-/- mice were negative for 2.4G2
staining; thus, fluorescence intensity of 2.4G2 on B cells represents
the expression of FcRIIB1, but not FcRIII.
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RIIB1 expression levels of GC B cells in B6
mice were up-regulated, those in congenic B6 mice were markedly
down-regulated, as compared with levels on non-GC B cells, which were
virtually identical between the two strains (Fig. 2
A). Based
on the mean channel of 2.4G2 fluorescence intensity, the FcRIIB1
expression level on GC B cells was 10 times lower in congenic B6
than in control B6 mice. Fig. 3 compares
dual-color immunofluorescent staining patterns of splenic tissue
sections with 2.4G2 mAb and PNA, representing marked differences in the
staining intensity of 2.4G2 on GC B cells between control B6 and
congenic B6 mice, high in the former and low in the latter. As
for follicular dendritic cells (FDCs), intensities of 2.4G2 were almost
identical between B6 and congenic B6 mice and negative in
B6.FcRIIB-/- mice. This indicates that FDCs
in these mice are positive for either FcRIIB1 or FcRIIB2, or
both, and negative for FcRIII, at least at the level of tissue
immunofluorescence.
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B compares 2.4G2 staining levels on
F4/80-positive peritoneal macrophages among three strains of mice. As
there was no remarkable difference in 2.4G2 staining intensities on
macrophages from nonimmunized B6, congenic B6, and
B6.FcRIIB-/-, the staining appeared to
mainly reflect FcRIII expression, although small amounts of
FcRIIB2 molecules would also be expressed on macrophages from B6 and
congenic B6 mice. Levels of FcRIIB2 in congenic B6 macrophages were
slightly lower than seen in B6 mice. This difference was clearer in
macrophages from immunized mice.
Fig. 4A compares serum IgG
anti-KLH Ab levels among KLH-immunized B6, congenic B6, and
B6.FcRIIB-/- mice. As compared with the
levels in control B6 mice, those in congenic B6 mice showed
significantly higher anti-KLH Ab responses. The levels in
B6.FcRIIB-/- mice were highest. Similar
results were obtained with serum levels of spontaneously produced IgG
anti-DNA Abs in these three strains of mice at 4 mo of age (Fig. 4B).
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To determine whether NZB-type deletion polymorphisms of the
Fcgr2b promoter region contribute to transcriptional
regulation of the Fcgr2b gene, luciferase reporter assay was
done using an NZW-derived Fcgr2b upstream gene fragment
connected in front of the luciferase reporter gene of pGL3-Basic vector
(Fig. 5A). For stable
transfections, B cell line (Bal17) and macrophage-like cell
line (P388D1) were transfected with the 2.9-kb
SalI-SacI fragment containing the
Fcgr2b upstream gene fragment and luciferase reporter gene.
As it was reported that the Fcgr2b gene fragment from
position -229 to +70 was fully active in the A20/2J B cell line in a
short-term transfection assay (15), luciferase activity
was first examined with a construct containing the Fcgr2b
upstream fragment from position -302 to +76. However, this fragment
was not long enough, and a longer one from position -302 to +585 drove
transcription activities in stable transfectants of both
Bal17 B cell and P388D1 macrophage-like cell lines.
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). In addition to the
previously detected 13 and 3 nucleotide deletion sites in the
Fcgr2b promoter region in NZB and 129Sv strains, these
strains have an insertion of 16 nucleotides in the first intron in
close vicinity to exon 2, as compared with BALB/c, B6, and NZW strains.
Only a limited number of single nucleotide polymorphism was detected
among mouse strains.
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B). These findings indicate that
polymorphic regions in the Fcgr2b upstream fragment appear
to contribute to transcriptional regulation of Fcgr2b gene
in both B cells and macrophages. Absence of transcription factor binding to the NZB-type Fcgr2b promoter region
To demonstrate if a decreased transcription activity in the
NZB-type Fcgr2b upstream gene is due to a defect in binding
interactions with nuclear proteins, EMSA was done. Oligonucleotide
probes with sequences of the polymorphic promoter region and first
intron were synthesized to identify which regions are involved in
nuclear factor binding (Table I). First, the promoter probe derived
from NZW sequence (NZW(P)) or the promoter probe from NZB sequence with
13 and 3 nucleotide deletion sites (NZB(P)) was radiolabeled, mixed
with extracted nuclear proteins from P388D1 cell lines, and
electrophoresed on acrylamide gels. In addition to the free probe, the
specific band reflecting probe-nuclear factor complex was detected
using a radiolabeled NZW(P) probe, in which a cold NZW(P), but not a
NZB(P), probe inhibited formation of this specific band. However, when
radiolabeled NZB(P) probe was used, this specific band was not detected
(Fig. 7A). Thus, the nuclear
factor-binding site appeared to be deleted in NZB(P) probe. The same
result was obtained by using nuclear proteins extracted from B cell
line Bal17 (data not shown).
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B, the
same band pattern was detected in NZW(I) and NZB(I) probes, and was
inhibited by both cold NZB(I) and NZW(I) competitors. Thus, this band
formation seems to be derived from common sequence (probably
corresponding to putative GAL4 binding sequence as shown in Table I)
involved in NZB(I) and NZW(I), and is not specific for NZB-type
insertion site.
As the 13-nucleotide deletion site in the NZB promoter region includes
the putative AP-4 binding site (16), we then asked if the
nuclear factor detected in the above EMSA is AP-4. As shown in Fig. 8A, specific bands with the
same molecular mass were observed using a synthesized radiolabeled
probe corresponding to AP-4 binding consensus sequence (AP-4) (Table I;
Ref. 24) and the NZW(P) probe in EMSA, and these bands
were inhibited by both AP-4 and NZW(P) cold competitors. To further ask
if the involved nuclear factor is indeed AP-4, Southwestern assay was
done (Fig. 8B). When the nuclear extract was electrophoresed
and hybridized with a radiolabeled AP-4 probe (AP-4), a specific band
of 48 kDa was detected. A band of the same size was detected using a
radiolabeled NZW(P), but not a NZB(P), probe. Taken collectively, it
seems likely that AP-4 binds to the polymorphic Fcgr2
promoter region corresponding to the 13-nucleotide deletion site in
NZB-type Fcgr2b allele and promotes transcription activity.
Fig. 9 shows RT-PCR analysis for
detecting AP-4 transcripts, demonstrating that the transcripts were
detected in both Bal17 and P388D1 cell lines and splenic B
cells from NZB mice. The levels were much higher in P388D1 than in
Bal17. Splenic B cells from aged NZB mice showed higher
levels of AP-4 transcripts than those from young NZB mice, in parallel
with numerous spontaneous GC formation in aged NZB mice
(10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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48 kDa
determined by Southwestern analysis strongly suggests that AP-4 is the
possible transcription factor (24). In addition to
deletion polymorphisms in the promoter region, our sequence analysis
identified an insertion polymorphism of 16 nucleotides in the first
intron in mice with the NZB-type Fcgr2b allele. This
insertion site did not have consensus sequences for known transcription
factors, and indeed did not show any binding activity to nuclear
proteins in the present studies. Bonnerot et al. (15) used A20/2J B cell line and MMC-1 mast cell line in chloramphenicolacetyl transferase assays, and found that different promoter fragments determine transcription of the Fcgr2b gene in these two cell lines. The fragment of positions from -229 to +70 relative to the transcription start site was shown to be able to drive the transcription of Fcgr2b gene in B cell line, but not in mast cell line. In our studies, the fragment of positions from -302 to +76 was not long enough and a longer one from -302 to +585 did gain the transcription activity in both Bal17 B cell and P388D1 macrophage-like cell lines. The reason for this discrepancy is unclear, but may relate to differences in assay systems in which the short-term transcription assay used by Bonnerot et al. (15) and a stable transfection assay by our own, and also in cell lines used. In any instance, the polymorphic promoter region among mouse strains is located in the fragment containing the regulatory element, and appears to function for transcription of the Fcgr2b gene in both B cells and macrophages.
Establishment of the B6 strain congenic for the NZB-type
Fcgr2b allele made it feasible to examine the in vivo effect
of the Fcgr2b allele polymorphism in the same B6 genetic
background. For this purpose, we took advantage of the B6 congenic
strain carrying the 129Sv-derived Fcgr2b allele, since the
upstream Fcgr2b region gene sequence was almost identical
between NZB and 129Sv, except for one single nucleotide polymorphism in
the second intron (Fig. 6). When immunized with KLH, FcRIIB1
expression levels on non-GC B cells were high and identical between
both strains of mice, yet those on activated GC B cells were markedly
down-regulated in congenic B6 mice and up-regulated in control B6 mice,
as compared with findings in non-GC B cells. This down-regulation of
FcRIIB1 expression levels was associated with the up-regulation of
IgG anti-KLH Ab responses. FcRIIB2 expression levels on
macrophages from congenic B6 mice were also low, as compared with
levels in control B6 mice. However, the effect of FcRIIB2 expression
levels on macrophage function is controversial. There is a report of
the possibility that impairment of FcRIIB2 expression on macrophages
from mice with the NZB-type Fcgr2b allele contributes to
elevated serum levels of IgG through the diminished catabolism of ICs
(25). In contrast, another report showed an increase in
the phagocytosis of opsonized SRBC by macrophages from mice with
the NZB-type Fcgr2b allele (18). Thus, the
exact role of FcRIIB2 molecules in macrophage function remains to be
determined. As far as the role in B cells is concerned, we propose that
the NZB-type polymorphic Fcgr2b promoter region is related
to enhanced IgG responses through down-regulation of FcRIIB1
expression levels on activated B cells in GCs, where affinity
maturation and Ig class switch occur, thus escaping negative signals
for BCR-elicited activation. In vivo studies also showed that the
polymorphic region of Fcgr2b allele does not affect
FcRIIB1 expression levels on resting non-GC B cells, which suggests
that an unidentified different transcriptional regulation mechanism
is operative in resting stage B cells.
We previously mapped an allele susceptible for hyper-IgG, including IgG
anti-DNA Abs, Hig-1, to close vicinity of the
Fcgr2b gene on NZB chromosome 1, and proposed that the
NZB-type Fcgr2b promoter polymorphism forms the basis of one
aspect of SLE susceptibility in NZB and (NZB x NZW)
F1 mice (5, 10). This is consistent
with the present finding that serum levels of IgG anti-DNA Abs were
significantly higher in Fcgr2b-congenic B6 than in control
B6 mice, albeit the levels being significantly lower than in
B6.FcRIIB-/- mice. At present, it is not
clear if congenic B6 mice with the NZB-type Fcgr2b allele
develop SLE, and the study is under investigation in our laboratory.
Significance of the NZB-type Fcgr2b promoter polymorphism in
the pathogenesis of autoantibody-mediated autoimmune diseases may
require further investigations. 129Sv mice neither produce IgG
anti-DNA Abs nor develop the autoimmune disease, despite carrying
the NZB-type Fcgr2b allele. Contribution of the NZB-type
Fcgr2b allele to the pathogenesis of NOD diabetes is
unknown, as the pathogenesis is thought to be mainly mediated by T
cells, although there are reports showing a possible involvement of Abs
in diabetes (26, 27). Bolland and Ravetch
(28) reported that B6.FcRIIB-/-
mice with aging spontaneously develop SLE-like autoimmune disease;
however, the disease does not occur in
BALB.FcRIIB-/- mice. All these findings are
probably related to the premise that autoimmune disease is a complex
multigenic disease in which susceptibility arises from the combined
effects of multiple contributing genes, and different autoimmune
manifestations are controlled separately by different sets of
susceptibility alleles (29, 30, 31). Function of the
Fcgr2b gene appears to be a kind of immune regulatory gene,
and it is feasible to speculate that the NZB-type Fcgr2b
promoter polymorphism could be selected evolutionally for natural
defense against pathogens. Such polymorphism may in turn form the basis
of autoantibody-mediated autoimmune diseases as one of multiple
susceptibility alleles.
In earlier genetic studies, it was shown that the telomeric chromosome 1 interval from not only NZB and BXSB, but also NZW and SWR, strains contributes to IgG autoantibody production (7, 8, 9, 10, 11, 12), and that several potential candidate genes, such as Cr2, Adprp, and Ifi202 may be involved (32, 33, 34). In our congenic B6 mice with the NZB-type Fcgr2b allele, the interval introduced into B6 mice does not include these genes, thus indicating that, at least in our congenic B6 mice, the observed enhanced IgG responses are probably due to the NZB-type Fcgr2b allele and not other suggested candidate genes. Studies by Morel et al. (33) using congenic B6 mice carrying telomeric chromosome 1 segments derived from NZM2410 mice, a recombinant inbred strain generated from crosses of NZB and NZW strains by Rudofsky et al. (35), yielded evidence that three loci, Sle 1a, Sle 1b, and Sle 1c, but not the interval containing the Fcgr2b gene, are independently associated with loss of tolerance to chromatin. However, it is to be noted that the chromosomal region containing Fcgr2b gene in NZM mice is derived from the NZW, but not the NZB strain. It is highly feasible that there are several other important candidate genes related to the susceptibility to SLE in the vicinity of the Fcgr2b gene on chromosome 1, and intensive congenic dissection will be useful to search for other susceptibility allele located in this region of chromosome 1.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sachiko Hirose, Department of Pathology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail address: sacchi{at}med.juntendo.ac.jp
3 Abbreviations used in this paper: IC, immune complex; BCR, B cell Ag receptor; FDC, follicular dendritic cell; GC, germinal center; KLH, keyhole limpet hemocyanin; PNA, peanut agglutinin; SLE, systemic lupus erythematosus; NOD, nonobese diabetic; PVDF, polyvinylidene difluoride.
Received for publication May 9, 2002. Accepted for publication August 12, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB1 in germinal center B cells associated with hyper-IgG and IgG autoantibodies in murine systemic lupus erythematosus. Int. Immunol. 11:1685.
FcR gene control its transcription. Immunobiology 185:222.[Medline]
Fc receptor II gene. J. Immunol. 146:369.[Abstract]
RII. Curr. Biol. 10:227.[Medline]
RII-deficient mice. Nature 379:346.[Medline]
mRNA expression involves lack of phosphorylation of I
B in a murine macrophage-like cell line, P388D1. J. Leukocyte Biol. 68:267.RII gene expression in macrophages of NOD mice. J. Immunol. 157:4707.[Abstract]
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