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A Promoter Haplotype of the Immunoreceptor Tyrosine-Based Inhibitory Motif-Bearing FcRIIb Alters Receptor Expression and Associates with Autoimmunity. II. Differential Binding of GATA4 and Yin-Yang1 Transcription Factors and Correlated Receptor Expression and Function^1,2

Division of Clinical Immunology and Rheumatology, Departments of Medicine and Microbiology, University of Alabama, Birmingham, AL 35294

	Abstract

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The immunoreceptor tyrosine-based inhibitory motif-containing FcRIIb modulates immune function on multiple cell types including B cells, monocytes/macrophages, and dendritic cells. The promoter for the human FCGR2B is polymorphic, and the less frequent 2B.4 promoter haplotype is associated with the autoimmune phenotype of systemic lupus erythematosus. In the present study, we demonstrate that the 2B.4 promoter haplotype of FCGR2B has increased binding capacity for GATA4 and Yin-Yang1 (YY1) transcription factors in both B lymphocytes and monocytes, and that overexpression of GATA4 or YY1 enhances the FCGR2B promoter activity. The 2B.4 haplotype leads to elevated expression of the endogenous receptor in heterozygous donors by 1.5-fold as assessed on EBV-transformed cells, primary B lymphocytes, and CD14⁺ monocytes. This increased expression accentuates the inhibitory effect of FcRIIb on B cell Ag receptor signaling, measured by Ca²⁺ influx and cell viability in B cells. Our results indicate that transcription factors GATA4 and YY1 are involved in the regulation of FcRIIb expression, and that the expression variants of FcRIIb lead to altered cell signaling, which may contribute to autoimmune pathogenesis in humans.

	Introduction

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The IgG FcRs play an important role in regulating immune system by bridging the humoral and cellular immune responses (1, 2, 3, 4). On mouse follicular dendritic cells (FDCs),⁵ FcRIIb is the highly expressed IgG FcR and can mediate the retention and conversion of immune complexes on FDCs to a highly immunogenic form (5, 6), which may play a role in affinity maturation and memory B cell development (7, 8). Similarly, on Langerhans cells, FcRIIb mediates Ag internalization and presentation (9, 10, 11). On B cells, at least in part by recruitment of phosphatases to its immunoreceptor tyrosine-based inhibitory motif, FcRIIb engagement can shape the Ab repertoire through modulation of B cell Ag receptor (BCR)-mediated cell activation and proliferation (12, 13), through signals for apoptosis independent of BCR (14), and through down-regulation of pre-BCR-mediated apoptosis (15). On myeloid lineage cells, FcRIIb down-regulates Ab-mediated phagocytosis and inflammatory responses when clustered with the activating FcRs, such as FcRIa, FcRIIa, and FcRIIIa (16, 17). Thus, through its roles in facilitating Ag presentation and in regulating B cell survival and proliferation, FcRIIb has a significant role in maintaining immune homeostasis, which makes FcRIIb an attractive functional candidate for autoimmune diseases.

We have demonstrated that a functional promoter haplotype in the human FCGR2B gene is associated with systemic lupus erythematosus (SLE) (18), suggesting that FcRIIb contributes to susceptibility for autoimmune disease. To address the underlining molecular mechanism in relation to the in vivo function of these FCGR2B haplotypes, we have explored the transcription factor-binding capability of the polymorphic sites within the FCGR2B promoter haplotypes. Computer-based searches suggested that the single-nucleotide polymorphisms (SNPs) were located in putative GATA family and Yin-Yang1 (YY1) transcription factor-binding elements. Direct assessment of binding indicated that the allelic variants from the less frequent 2B.4 haplotype have increased binding capacity for both GATA4 and YY1 transcription factors in B lymphocytes and monocytes. Overexpression of either GATA4 or YY1 up-regulates FcRIIb promoter activity, suggesting that GATA4 and YY1 are involved in the regulation of FcRIIb expression. Among genotyped donors, the 2B.4 haplotype leads to higher expression of endogenous FcRIIb on both primary B lymphocytes and monocytes. This increased receptor expression accentuates the FcRIIb function as measured by BCR-induced Ca²⁺ influx and cell viability in B cells. Thus, our data indicate that the FCGR2B promoter SNPs occur in transcription factor-binding elements and alter transcription factor binding, that GATA4 and YY1 transcription factors regulate FcRIIb expression, and that the resultant change in expression can alter cell function. Given the several roles that FcRIIb may play in the pathogenesis of autoimmunity, the specific function for FcRIIb may vary according to the nature and stage of the disease.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Donors

Caucasian SLE patients and disease-free controls were recruited as part of the University of Alabama at Birmingham-based DISCOVERY cohort, a population-based case-control study. The studies were reviewed and approved by the Institutional Review Board, and all donors were provided written informed consent.

Reagents

AT-10-FITC was purchased from Serotec (Raleigh, NC). The IV.3 hybridoma cell line was purchased from American Type Culture Collection (Manassas, VA), and purified IV.3 Ab was conjugated with FITC with FITC-labeling kit (Sigma-Aldrich, St. Louis, MO). Anti-CD19-allophycocyanin, anti-CD14-TRI-COLOR, anti-CD56-PE, and anti-CD3-PE mAb were purchased from Caltag Laboratories (Burlingame, CA). The FcRIIb-specific polyclonal Ab was generated by immunization of rabbits with GST fusion protein containing the unique cytoplasmic domain of FcRIIb. Goat anti-FcRIIa/c polyclonal Ab, anti-YY1, anti-GATA1, -2, -3, -4, and -6 Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-HisG and anti-Xpress tag Abs were purchased from Invitrogen (Carlsbad, CA). The A20-IIA1.6 cell line was kindly provided by Dr. T. Wade at Dartmouth Medical Center (Lebanon, NH) (19).

We have performed flow cytometry using mAb IV.3 and AT-10 to compare their staining patterns on FcRIIa or FcRIIb transfectants. For FcRIIb, mAb AT-10 stains 10 times stronger than mAb IV.3 (data not shown). In contrast, for both FcRIIa alleles (H131 and R131), mAbs AT-10 and IV.3 have comparable reactivity (<2-fold difference between the two mAbs). Thus, mAb IV.3 weakly recognizes FcRIIb when highly expressed in transfected cell lines.

Plasmid construction

For luciferase-based constructs, various human FcRIIb promoter fragments were amplified by PCR from genomic DNA and subcloned into the luciferase reporter vector pGL3-Basic (Promega, Madison, WI). The alternative alleles were introduced at the polymorphic sites of the FcRIIb promoter using QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). For the mammalian expression of GATA4 and YY1 transcription factors, the cDNA of GATA4 or YY1 was amplified by RT-PCR from BJAB and Hela cells, respectively, and subcloned into pcDNA3His expression vector (Invitrogen). The expressed protein was N-terminally tagged with His₆Gly and Xpress epitopes. For transient expression of human FcRIIa (both H131 and R131 alleles), the cDNA was amplified by RT-PCR from peripheral mononuclear cells isolated from whole blood of an FcRIIa H131/R131 heterozygous donor. The FcRIIa cDNA was subcloned into pcDNA3His vector for expression in COS-7 cells. All of the constructs were confirmed by direct DNA sequencing.

The PCR primers for the cloning of human GATA4 cDNA were as follows: sense, 5'-GCAGGTACCCATGTATCAGAGCTTGGCCATG-3'; and antisense, 5'-GAAGAATTCAGATTACGCAGTGATTATGTCCC-3'. The PCR primers for the cloning of human YY1 cDNA were as follows: sense, 5'-CGCGGATCCACCATGGCCTCGGGCGACACC; and antisense, 5'-CGGAATTCTCACTGGTTGTTTTTGGCCTTAG-3'. The PCR primers for the cloning of human FcRIIa cDNA were as follows: sense, 5'-TCGGAATTCATGGCTATGGAGACCCAAATGTC-3'; and antisense, 5'-CTGTCTAGATTAGTTATTACTGTTGACATG GTCG-3'.

RT-PCR

The total RNA was prepared from different types of cells using TRIzol reagents (Invitrogen). The cDNAs were synthesized using SuperScript Preamplification System (Invitrogen). The gene-specific PCR was performed in a 9600 PCR System with 2 µl of cDNA, 200 nM each primer, and 2.5 U of DNA polymerase from Failsafe PCR system (Epicenter Technologies, Madison, WI) starting with 94°C for 2 min, 28 cycles of denaturing at 98°C for 20 s, annealing at 58°C for 30 s, and extension at 68°C for 90 s, with a final extension at 68°C for 7 min. The PCR product was purified using QIAquick Gel Extraction kit (Qiagen, Chatsworth, CA).

EMSAs

The oligonucleotide probes for EMSA were labeled by Klenow fill-in with [-³²P]dCTP. Nuclear extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL). EMSA was performed with 6 µg of nuclear extract and 20,000 cpm ³²P-labeled probe in 20 µl of binding buffer (10 mM HEPES (pH 7.5), 50 mM KCl, 5% glycerol, 2 mM MgCl₂, 0.2 mM EDTA, 0.2 mg/ml BSA, 1 µg of polydeoxyinosinic-deoxycytidylic acid, 1 mM DTT, and 1 mM Pefabloc). The labeled probe was incubated with nuclear extract at room temperature for 20 min. Bound and free DNA probe were then resolved by electrophoresis through a 6% polyacrylamide gel in 0.5x Tris-borate-EDTA buffer at 200 V for 2 h. The gel was dried and exposed to film for autoradiography. For competition and supershift assays, before the addition of the labeled probe, a 200-fold molar excess of the indicated unlabeled oligonucleotides or 4 µg of Abs was added to the nuclear extracts and incubated at 4°C for 1 h. The labeled probe was then added and incubated at room temperature for additional 20 min followed by electrophoresis.

Transient transfections

For luciferase assays, reporter plasmid pGL-2B (10 µg) was cotransfected with the reference plasmid pRL-SV40 (150 ng) and the GATA4 or YY1 expression vector pcDNA (1 µg) into 10 x 10⁶ BJAB cells by electroporation at 200 V and 960 µF. For U937 cells, reporter plasmid pGL-2B (0.5 µg) was cotransfected with the reference plasmid pRL-SV40 (30 ng) and the GATA4 or YY1 expression vector pcDNA (50 ng) into 5 x 10⁵ U937 cells in 12-well plates using 1.5 µl of Fugene 6 reagent according to the manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, IN). The luciferase activities were measured at 40 h after transfection using the Dual Luciferase Reporter Assay System (Promega). The firefly luciferase activity was normalized by Renilla luciferase activity to yield the relative luciferase activity.

For COS-7 transfections, cells (60–80% confluent) in 10-cm plates were transfected with 6 µg of plasmids and 18 µl of Fugene 6 reagent according to the manufacturer’s instructions. Cells were harvested for preparation of nuclear extracts or whole-cell lysate at 30 h posttransfection.

Preparation of whole-cell lysate and immunoprecipitation assay

Cells were lysed with whole-cell lysis buffer (19) at 20 µl per 1 x 10⁶ cells for EBV cells and monocytes, or 60 µl per 1 x 10⁶ cells for COS-7 and A20-IIA1.6-FcRIIb transfectants (19). The samples were vortexed for 10 s and incubated on ice for 30 min with a brief vortexing every 10 min. The samples were then centrifuged at 15,000 rpm at 4°C for 15 min, and the supernatant was collected.

For immunoprecipitation, mAbs 32.2, IV.3, or AT-10 were added to the whole-cell lysate and incubated at 4°C for 2 h with mixing. Protein G-Sepharose beads were added to each sample, and the samples were further incubated at 4°C for 1 h with mixing. The beads were washed four times with whole-cell lysis buffer, and the immunoprecipitates were subjected to Western blot analysis.

Purification of CD14⁺ monocytes from whole blood

PBMCs were isolated by density gradient centrifugation using Ficoll-Hypaque followed by CD14 Magnetic MicroBeads (Miltenyi Biotec, Auburn, CA). The CD14⁺ monocytes were purified on positive-selection columns (MS⁺). Multicolor flow cytometry (anti-CD19-allophycocyanin for B lymphocytes, anti-CD3-TRI-COLOR for T lymphocytes, anti-CD56-PE for NK cells, and mAb IV.3-FITC for monocytes) was performed on the separated cell populations to determine the purity (>90%) and recovery (50–70%).

Measurement of change in intracellular Ca²⁺ concentration ([Ca²⁺]_i)

Changes in [Ca²⁺]_i induced by cross-linking of surface Ig on EBV-transformed B cells were determined using an SLM 8000 spectrofluorometer monitoring the simultaneous 405/490-nm fluorescence emission ratio of the calcium-binding indo-1 fluorophore, as previous described (20). Cells (10 x 10⁶/ml) were loaded with 5 µM indo-1-AM at 37°C for 40 min and stimulated with 10 µg/ml goat IgG anti-human or an equal-molar concentration of goat F(ab')₂ anti-human (Southern Biotechnology Associates, Birmingham, AL) at the 60-s time point.

Cell viability assay

EBV-transformed B cell lines from genotyped donors were treated with 6.7 µg/ml goat F(ab')₂ anti-human IgM or 10 µg/ml goat IgG anti-human IgM for 60 h. The ATPlite assay was performed in 96-well assay plates with 200 cells/well, and each condition was performed in triplicate. The cells were lysed and assayed for the amount of ATP on a Packard TopCount Microplate Scintillation and Luminescence Counter following the manufacturer’s directions (Packard, Meriden, CT).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
SNPs on the 2B.4 haplotype have increased binding capacity for GATA4 and YY1 transcription factors

We have identified and characterized two functional haplotypes (–386G-120T and –386C-120A) in the proximal promoter region of human FCGR2B gene (18). Our case control studies have suggested that the gain-of-function 2B.4 haplotype (–386C-120A) is associated with SLE phenotype (18). To explore the molecular basis for the differential function of the two promoter haplotypes, we performed EMSAs to determine the capability of –120 T/A SNP and –386 G/C SNP regions to bind transcription factors.

Computer-based searches revealed that a GATA-binding motif is located 12–15 nt 5' to the –120T/A SNP, and that the –120A allele creates a second GATA-binding motif, thus forming palindromic binding sites for GATA (Fig. 1A). EMSAs using U937 nuclear extracts showed that a –120A probe had a much higher binding capacity for transcription factors than a –120T probe (Fig. 1B, lanes 1–5). A 200-fold excessive of unlabeled –120A oligonucleotides, but not nonspecific oligonucleotides, effectively blocked binding of labeled probe (Fig. 1B, lanes 6 and 7). The protein binding to the –120A probe was partially competed away by unlabeled GATA1-binding oligonucleotides derived from human -globin promoter (21), but –120 oligonucleotides with "GATA" motif mutated were ineffective in competition experiments (Fig. 1B, lanes 8 and 9). These data suggested that the transcription factor was a GATA family member. Unlabeled –120T oligonucleotides (containing only one "GATA" motif) less efficiently competed the binding consistent with its lower binding capacity for the transcription factor relative to the –120A oligonucleotides (Fig. 1B, lane 10).

View larger version (68K): [in this window] [in a new window]   FIGURE 1. The –120A allele has increased binding capacity for transcription factor GATA4. A, The sequence of the probes used in the EMSAs. Polymorphic alleles are presented in bold lowercase, and mutant sites are indicated in bold uppercase. Arrows indicate GATA-binding motifs. The GATA-binding probe "gGATA" is derived from the human A -globin gene promoter (21 ). B, EMSAs were performed with nuclear extracts (NE) from U937 cells, BJAB cells, or COS-7 transfectants, and 32P-radiolabeled –120T and –120A probes. Two hundred-fold unlabelled probe (NS, nonspecific probes) or 4 µg of Abs was added to the reaction as indicated.

To further demonstrate that –120T/A alleles have differential binding capacity for the GATA4 transcription factor, we transiently overexpressed human GATA4 in COS-7 cells using the pcDNA3His vector. EMSAs using nuclear extracts from these transfectants showed that –120A probe has increased binding capacity for GATA4 compared with the –120T probe (data not shown) and Abs against GATA4, His₆G, and Xpress tags all supershifted the binding complex (Fig. 1B, lanes 23–26). Taken together, our data suggest that the variant –120A allele has increased binding capacity for GATA4 transcription factor in both B and monocyte cell lines and provide additional evidence that palindromic GATA-binding motifs have much higher binding capacity for the transcription factor than a single GATA-binding site (21).

For –386 G/C SNP, computer-based searches revealed that the less frequent allele –386C created a binding motif for a universal transcription factor YY1 (Fig. 2A). EMSA experiments showed that two DNA-protein complexes formed only on –386C probe but not at detectable level on –386G probe (Fig. 2B, lanes 1–5, indicated by "YY1" double arrows). This binding was specific because unlabeled –386C oligonucleotides, but not nonspecific oligonucleotides, effectively competed for binding (Fig. 2B, lanes 6 and 7). Binding was also blocked by known YY1-binding oligonucleotides "YY1" (derived from human gp91^phox gene promoter (22)) but not by –386 mutant oligonucleotides with 3 nt critical for YY1 binding mutated or by –386G oligonucleotides containing no YY1 binding motif (Fig. 2B, lanes 8–10). These data suggested that the complexes contain YY1.

View larger version (63K): [in this window] [in a new window]   FIGURE 2. The –386C allele has increased binding capacity for transcription factor YY1. A, The sequence of the probes used in the EMSAs. Polymorphic alleles are presented in bold lowercase, and mutant sites are indicated in bold uppercase. Arrows indicate the YY1-binding motif. The YY1-binding probe "YY1" is derived from the human gp91phox gene promoter (22 ). B, EMSAs were performed with nuclear extracts (NE) from U937 cells or COS-7 transfectants and 32P-radiolabeled –386G and –386C probes. Two hundred-fold unlabelled probe or 4 µg of Abs was added to the reaction as indicated.

To further support our EMSA and supershift data, we performed RT-PCR to confirm the expression of GATA4 and YY1 transcription factors in BJAB and U937 cells. Gene-specific RT-PCR for YY1 and each GATA family member demonstrated that YY1 is universally expressed, and GATA4 is the predominant GATA family member expressed in BJAB cells (Fig. 3). U937 cells express GATA4 and, to a lesser extent, GATA3. Primary tonsillar cells express both GATA3 and GATA4 (Fig. 3).

View larger version (22K): [in this window] [in a new window]   FIGURE 3. YY1 and GATA4 are expressed in BJAB and U937 cells. Gene-specific RT-PCR for YY1 and six GATA family members were performed from RNA prepared from BJAB and U937 cell lines, and primary tonsil cells. The PCR specificity was confirmed by directly sequencing the PCR products.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Overexpression of GATA4 and/or YY1 transcription factors leads to increased FCGR2B promoter activity. The FCGR2B promoter reporter constructs pGL-2B.1 or pGL-2B.4 were cotransfected with the reference plasmid pRL-SV40 (SV40 promoter drives Renilla luciferase gene) and the GATA4 and/or YY1 expression vector pcDNA3 into BJAB or U937 cells. Dual Luciferase assay was performed 40 h after transfection. The firefly luciferase activity was normalized by Renilla luciferase levels, and the ratio is designated as relative luciferase activity (RLA). The results represent the mean ± SEM from three independent experiments (p < 0.0001 by ANOVA).

2B.4 haplotype leads to elevated FcRIIb expression on B lymphocytes

Recognizing that the 2B.1 and 2B.4 haplotypes occur naturally in human donors, we explored possible differential expression levels of FcRIIb of both transformed cells and primary cells ex vivo. We have used mAb AT-10 in flow cytometry to determine the FcRIIb expression levels on B cells. Binding of mAb IV.3 to both EBV-transformed B cells and peripheral B lymphocytes was indistinguishable from that of isotype control (data not shown), suggesting that these B cells do not express detectable levels of FcRIIa in agreement with others (4, 23). Using EBV-transformed B cells from 18 2B.1/2B.1 homozygous, 17 2B.1/2B.4 heterozygous, and 1 2B.4/2B.4 homozygous donors, we demonstrated that the 2B.1/2B.4 heterozygous donors had 1.5-fold increased FcRIIb expression on EBV-B cells and the single 2B.4 homozygous donor had 2.5-fold increased FcRIIb expression compared with the 2B.1 homozygous donors (Fig. 5, A and B). Furthermore, we developed a rabbit polyclonal Ab that specifically recognizes the unique cytoplasmic domain of FcRIIb. We have shown that both mAb IV.3 and AT-10 immunoprecipitates from COS-7-FcRIIa transient transfectants were not recognized by our rabbit anti-FcRIIb sera (Fig. 5C, panel I, lanes 1–3); however, they were recognized by a goat anti-FcRIIa/c cytoplasmic domain Ab (panel II, lanes 1–3). The mAb AT-10 immunoprecipitates from A20-IIA1.6-FcRIIb stable transfectants (19) were recognized by rabbit anti-FcRIIb sera (Fig. 5C, panel I, lane 6), but not by goat anti-FcRIIa/c Ab (panel II, lane 6). These data demonstrate that our rabbit anti-FcRIIb Ab is FcRIIb specific and can be used to specifically detect the expression of FcRIIb by Western blot. Western blot analysis using the FcRIIb-specific antisera, after normalization for protein loading with an anti-Lyn Ab, detected a 2.3-fold increased expression levels of FcRIIb in the EBV-transformed cells from the 2B.4-containing donors compared with that from the 2B.1 homozygous donors (Fig. 5D).

View larger version (30K): [in this window] [in a new window]   FIGURE 5. Haplotype 2B.4 leads to higher expression of endogenous FcRIIb on EBV-transformed and peripheral blood B lymphocytes. A, The EBV-B cells derived from 2B.1 homozygous (thin gray line), 2B.1/2B.4 heterozygous (thick black line), and 2B.4 homozygous (thick gray line) donors were stained with murine IgG1 isotype control (dotted line) or mAb AT-10, followed by staining with FITC-conjugated goat anti-mouse IgG. The binding of the isotype control to the three cell lines was identical, and only one is shown for clarity. No binding of mAb IV.3 above the isotype control was observed on any line (data not shown). B, A summary of FcRIIb expression levels on EBV-transformed cells derived from 18 2B.1 homozygous (), 17 2B.1/2B.4 heterozygous (), and 1 2B.4 homozygous () donors by flow cytometry using mAb AT10. (*, p < 0.0155, Kruskal-Wallis test; MFI, mean fluorescence intensity). The average MFI of the isotype control among the three groups was not different (2B.1/2B.1: 3.6 ± 0.2, n = 18; 2B.1/2B.4: 3.6 ± 0.3, n = 17; 2B.4/2B.4: 3.9, n = 1). C, Whole-cell lysate was prepared from COS-7 cells transiently transfected with FcRIIa or A20-IIA1.6 cells stably transfected with FcRIIb and immunoprecipitated with mAb 32.2 (as a negative control), IV.3, or AT-10, and subjected to Western blot analysis using rabbit anti-FcRIIb sera (panel I) or goat anti-FcRIIa/c Abs (panel II). D, Whole-cell lysate from EBV-transformed cells derived from four 2B.1 homozygous donors (lanes 1–4) and four 2B.4-containing donors (lanes 5–8; three 2B.1/2B.4 heterozygous and one 2B.4 homozygous donors) was subjected to Western blot analysis using rabbit anti-FcRIIb cytoplasmic domain Ab (panel I). The membrane was stripped and reprobed with anti-Lyn Ab as a protein loading control (panel II). E, Whole blood from a 2B.1 homozygous (thin gray line) and a 2B.1/2B.4 (thick black line) normal donor was stained with mAb AT-10-FITC and anti-CD19-allophycocyanin (for B lymphocytes) Abs and analyzed by flow cytometry. The binding of the isotype control to the two cell lines was identical, and only one is shown for clarity. No binding of mAb IV.3 above the isotype control was observed on any line (data not shown). F, A summary of the expression levels of FcRIIb on peripheral B lymphocytes from 12 2B.1 homozygous () and 8 2B.1/2B.4 heterozygous () normal donors (**, p < 0.0003). The average MFI of the isotype control between the two groups was not different (2B.1/2B.1: 4.1 ± 0.3, n = 12; 2B.1/2B.4: 3.9 ± 0.4, n = 8).

2B.4 haplotype leads to higher expression of FcRIIb on CD14⁺ monocytes

Because the differential promoter activity of the two haplotypes is evident in both B and monocytic cell lines, we next examined the FcRIIb expression on freshly isolated monocytes from genotyped non-SLE normal donors. High levels of FcRIIa expression on CD14⁺ monocytes precluded our ability to detect any expression difference of FcRIIb by mAb AT-10 using flow cytometry (data not shown). Therefore, to determine the FcRIIb expression levels on peripheral monocytes, we purified CD14⁺ monocytes from Ficoll-separated mixed mononuclear cell by anti-CD14 Magnetic MicroBeads. Multicolor flow cytometry was performed on the separated cell populations to confirm the purity (>90%) of the monocytes using markers for B (CD19) and T (CD3) lymphocytes, NK cells (CD56), and monocytes (IV.3) (data not shown). The purified monocytes were lysed, and equal amount of whole-cell lysate was applied to Western blot analysis using the FcRIIb-specific polyclonal Ab. Our data showed elevated FcRIIb expression on peripheral monocytes from 2B.1/2B.4 heterozygous donors compared with 2B.1/2B.1 homozygous donors (Fig. 6).

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Haplotype 2B.4 leads to higher expression of FcRIIb on peripheral CD14+ monocytes. CD14+ monocytes were purified from whole blood from four 2B.1/2B.1 (lanes 1–4) and four 2B.1/2B.4 (lanes 5–8) normal donors. Whole-cell lysate prepared from those monocytes was subjected to Western blot analysis using specific rabbit anti-FcRIIb sera followed by an anti-Lyn Ab as a protein loading control.

Differential inhibitory function of FcRIIB from genotyped donors

To determine whether the differential FcRIIb expression among the donors could have differential inhibitory effects, we assayed BCR-induced intracellular Ca²⁺ fluxes by F(ab')₂ or whole IgG anti- stimulation. We used EBV-transformed cell lines from three 2B.1/2B.1 and three 2B.4-containing donors (two 2B.1/2B.4 heterozygous and one 2B.4 homozygous donor). Ca²⁺ influx was induced by engagement of BCR and down-regulated by coengagement of BCR and FcRIIb (Fig. 7A). The FcRIIb from the 2B.4-containing donors had 1.5-fold higher inhibitory effects on the BCR-mediated Ca²⁺ influx than that from the 2B.1 homozygous donors (Fig. 7B).

View larger version (25K): [in this window] [in a new window]   FIGURE 7. The FcRIIb from 2B.4-containing donors has higher inhibitory effects on BCR-induced Ca2+ influx. A, EBV-transformed cells from genotyped donors were stimulated with either goat IgG anti-human (thick line) or goat F(ab')2 anti-human (thin line). The relative inhibition of BCR-induced Ca2+ influx is presented as the ratio of the [Ca2+]i change induced by engagement of BCR alone and the [Ca2+]i change induced by coengagement of both BCR and FcRIIb. B, A summary of the relative inhibition of BCR-induced Ca2+ influx by FcRIIb from three 2B.1 homozygous () and three 2B.4-containing donors (; one 2B.4 homozygous and two 2B.1/2B.4 heterozygous donors; * p < 0.0055; results represent the mean ± SEM from three experiments).

View larger version (23K): [in this window] [in a new window]   FIGURE 8. The FcRIIb from 2B.4-containing donors has higher inhibitory effects on anti-BCR-induced decrease in cell viability. EBV cells from five 2B.1 homozygous () and five 2B.4-containing donors (; one 2B.4 homozygous and four 2B.1/2B.4 heterozygous donors) were untreated or stimulated with goat F(ab')2 anti-human IgM or goat IgG anti-human IgM for 60 h. The relative inhibition of anti-BCR-mediated decrease in cell viability is presented as the ratio of the ATP levels by engagement of BCR alone and coengagement of both BCR and FcRIIb (*, p < 0.023).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have identified and characterized the genetic variations affecting FcRIIb expression and function in humans. We have shown the association of the less frequent 2B.4 FCGR2B promoter haplotype with SLE (18). In this study, we further characterized the two promoter haplotypes of human FCGR2B gene that differ in both their in vitro and ex vivo activities. The 2B.4 FCGR2B promoter haplotype has an increased binding capacity for GATA4 and YY1 transcription factors compared with the more frequent 2B.1 haplotype. Donors with one copy of 2B.4 haplotype have 1.5-fold elevated FcRIIb receptor expression compared with 2B.1 homozygous donors when assessed on EBV-transformed cells, fresh peripheral B lymphocytes, and CD14⁺ monocytes. The FcRIIb from 2B.4-containing donors has accentuated inhibitory function compared with that from 2B.1 donors on BCR-induced Ca²⁺ influx and on cell viability. Such differences could have significant biological consequences in vivo. For example, subtle differences in BCR function, modulated by 20% differential CD19 expression, have a strong influence on the development of an autoimmune phenotype (28).

GATA4 and the universally expressed YY1 are responsible for the differential promoter activity of the FCGR2B haplotypes in B cells and monocytes. In vitro EMSA assays demonstrate that the variant –120A allele has increased binding capacity for GATA4, and that the variant –386C has increased binding capacity for YY1. Overexpression of YY1 and GATA4 enhances the FCGR2B promoter activity, and the enhancement is more dramatic with the 2B.4 promoter haplotype than the common haplotype. The increased luciferase reporter expression by YY1 and/or GATA4 in the context of the low-binding 2B.1 haplotype, although lower than the 2B.4 haplotype, was more than expected. Although this may reflect the stoichiometry and mass action of overexpressed transcription factors binding to the polymorphic site, we searched for additional potential binding elements for GATA and YY1 within the 1-kb promoter region of FCGR2B. In addition to the polymorphic sites, three or four putative monomorphic elements are present, which may explain the more modest differential up-regulation of the luciferase expression by GATA4 and YY1 overexpression in the context of the two haplotypes (1.6-fold) compared with their differential binding capacity indicated in the EMSAs (at least 3- to 5-fold).

The observation that GATA4 is the predominant GATA member bound on FCGR2B promoter in B lymphocytes and monocytes is surprising. Of the six known GATA family members, GATA1, GATA2, and GATA3 are expressed predominantly in hemopoietic cells, whereas GATA4, GATA5, and GATA6 are predominantly expressed in the developing heart and several endodermal lineages (29). Mutations in GATA4 have recently been shown to cause human congenital heart defects (30). However, our gene-specific RT-PCR for GATA1, -2, -3, -4, -5, and -6 demonstrates that GATA4 is the major GATA expressed in B lymphoid BJAB cells and myeloid U937 cells. Our results are in agreement with previous findings that GATA4 are expressed in primary monocytes by Western blot analysis and immunohistochemistry (31). Little is known about GATA expression in B lymphocytes, and our Western blot analysis using anti-GATA1 and GATA2 Abs suggest that neither of these family members is expressed in BJAB cells (data not shown). Gene-specific RT-PCR in EBV-transformed B cells and primary tonsil cells detected GATA3 and GATA4 messages, supporting GATA4 expression in B cells. Interestingly, synergistic effects of GATA and YY1 on gene transcription have been reported. Human FcRI -chain gene expression is synergistically up-regulated by GATA1 and YY1 (32). The cardiac B-type natriuretic peptide promoter is cooperatively activated by GATA4 and YY1 (33). This synergistic effect also exists for FCGR2B gene in the context of 2B.4 (–386C-120A) haplotype.

The 2B.4 FCGR2B promoter haplotype leads to increased receptor expression on both B lymphocytes and monocytes. Donors with heterozygous haplotypes have 1.5-fold elevated receptor expression compared with donors with homozygous common haplotype on EBV-transformed and fresh peripheral B lymphocytes. One donor with homozygous variant haplotype has 2.5-fold increased receptor expression on EBV cells. Similar differential FcRIIb expression is seen on CD14⁺ monocytes. Thus, it may be reasonable to speculate that, on dendritic cells, derived from either lymphoid or myeloid lineages, the levels of FcRIIb are regulated by similar mechanisms, and that these promoter haplotypes will lead to similar differences among individuals.

The role of FcRIIb in autoimmunity may be more intricate than that of a negative regulator, because it subserves multiple functions on different cell types. For example, on mononuclear phagocytes, FcRIIb can decrease the uptake and clearance of immune complexes (16), which might prolong circulation of autoantigens, increase availability of such Ags for processing at other sites, and enhance the tissue deposition of immune complexes with subsequent tissue injury. On FDCs, FcRIIb promotes the maturation of FDC reticulum and may enhance the Ab recall responses of memory B cells (5, 6, 7, 8, 34). FcRIIb also mediates Ag internalization and presentation on other dendritic cell types (9, 10, 35). From these perspectives, a relative increase in FcRIIb expression and function might decrease the clearance of antigenic, apoptotic material by macrophages and increase DC-mediated processing and presentation of these autoantigens.

On B cells, FcRIIb plays an important regulatory role in BCR signaling and Ab production. Coengagement of FcRIIb by IgG complexes down-modulates B cell activation and provides a negative feedback mechanism for IgG production. One can speculate that FcRIIb polymorphisms play a role in the regulation of Ab responses, and indeed, low-expression FcRIIb polymorphisms may lead to higher humoral immune responses in mice (36). Complete deficiency of FcRIIb, combined with a permissive genetic background, leads to an autoimmune phenotype. In humans, however, no FcRIIb deficiency has been identified yet, and the expression levels of FcRIIb in SLE patients are complicated by the disease stages and activity. Our genetic association studies suggest that the gain-of-function FcRIIb polymorphisms are enriched in SLE patients compared with ethnically matched controls. Perhaps, altered FcRIIb expression may influence negative selection occurring in immature B lymphocytes in bone marrow and in transitional B cells in the peripheral lymphoid organs. These two stages of negative selection are critical in maintaining immune tolerance to self-Ags. Although largely unexplored, FcRIIb negatively regulates pre-BCR-mediated signaling for apoptosis in the pre-B cell stage (15). In our cell viability assays, FcRIIb negatively regulates IgM BCR-induced decrease in cell viability in EBV-transformed peripheral blood B cells. Although our observation may reflect a combination of effects on cell proliferation and cell death, apoptosis-specific annexin V and activated caspase-3 staining suggested that apoptosis did occur during this process (data not shown). Thus, elevated FcRIIb expression may provide a mechanism for leakiness in the negative selection of autoreactive B cells. Indeed, it has been shown that estrogen and prolactin promote autoimmunity by altering thresholds for B cell apoptosis and rescuing the autoreactive B cells that would normally be deleted (37, 38, 39). Overexpression of the antiapoptotic Bcl-2 in several transgenic mouse models enhances survival of the bone marrow-derived autoreactive B cells and of autoreactive B cells that arise in germinal centers following somatic mutation (40, 41, 42). Thus in considering modulation of FcRIIb expression as a therapeutic target, it may be important to consider cell type-specific targeting according to the disease stage and characteristics. It may be that the predominant, pathophysiologically important cell type to target will vary according to the nature and stage of the autoimmune process.

Taken together, our data characterized the two FCGR2B promoter haplotypes that affect endogenous receptor expression on primary B lymphocytes and monocytes. The association of the high-expression haplotype with human SLE demonstrates a role for FcRIIb in autoimmune susceptibility. Cell type-specific modulation of FcRIIb expression with consideration of mechanisms of pathogenesis may be important in the treatment of autoimmune diseases. Furthermore, FCGR2B promoter genotypes may also play an important role in the variations of human responses to vaccines and to Ab-based therapeutic drugs.

	Acknowledgments

 
We thank Dr. S. McKenzie for discussion regarding the genomic structure of FcR cluster.

	Footnotes

 
¹ This work was supported by Grants R01 AR42476, P50 AR45231, P01 AR49084, and R01 33062 from the National Institutes of Health. The University of Alabama at Birmingham (UAB) Pittman General Clinical Research Center is supported by Grant MO1 RR00032 from the National Center for Research Resources at the National Institutes of Health. The FACS Core Facility of the UAB Arthritis and Musculoskeletal Center was supported by Rheumatic Diseases Core Center (P30 AR48311).

² The authors declare that they have no competing financial interests.

³ Current address: Department of Pediatrics, University of Iowa College of Medicine, Iowa City, IA 52242.

⁴ Address correspondence and reprint requests to Dr. Robert P. Kimberly, Tinsley Harrison Tower 429, 1900 University Boulevard, University of Alabama, Birmingham, AL 35294-0006. E-mail address: rpk{at}uab.edu

⁵ Abbreviations used in this paper: FDC, follicular dendritic cell; BCR, B cell Ag receptor; SLE, systemic lupus erythematosus; SNP, single-nucleotide polymorphism; YY1, Yin-Yang1; [Ca²⁺]_i, intracellular Ca²⁺ concentration.

Received for publication November 26, 2003. Accepted for publication February 2, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Takai, T.. 2002. Roles of Fc receptors in autoimmunity. Nat. Rev. Immunol. 2:580.[Medline]
2. Ravetch, J. V., S. Bolland. 2001. IgG Fc receptors. Annu. Rev. Immunol. 19:275.[Medline]
3. Dijstelbloem, H. M., J. G. van de Winkel, C. G. Kallenberg. 2001. Inflammation in autoimmunity: receptors for IgG revisited. Trends Immunol. 22:510.[Medline]
4. Hulett, M. D., P. M. Hogarth. 1994. Molecular basis of Fc receptor function. Adv. Immunol. 57:1.[Medline]
5. Qin, D., J. Wu, K. A. Vora, J. V. Ravetch, A. K. Szakal, T. Manser, J. G. Tew. 2000. Fc receptor IIB on follicular dendritic cells regulates the B cell recall response. J. Immunol. 164:6268.[Abstract/Free Full Text]
6. Tew, J. G., J. Wu, M. Fakher, A. K. Szakal, D. Qin. 2001. Follicular dendritic cells: beyond the necessity of T-cell help. Trends Immunol. 22:361.[Medline]
7. Haberman, A. M., M. J. Shlomchik. 2003. Reassessing the function of immune-complex retention by follicular dendritic cells. Nat. Rev. Immunol. 3:757.[Medline]
8. Kosco-Vilbois, M. H.. 2003. Are follicular dendritic cells really good for nothing?. Nat. Rev. Immunol. 3:764.[Medline]
9. Esposito-Farese, M. E., C. Sautes, H. de la Salle, S. Latour, T. Bieber, C. de la Salle, P. Ohlmann, W. H. Fridman, J. P. Cazenave, J. L. Teillaud, et al 1995. Membrane and soluble FcRII/III modulate the antigen-presenting capacity of murine dendritic epidermal Langerhans cells for IgG-complexed antigens. J. Immunol. 155:1725.[Abstract]
10. Amigorena, S.. 2002. Fc receptors and cross-presentation in dendritic cells. J. Exp. Med. 195:F1.
11. Guermonprez, P., J. Valladeau, L. Zitvogel, C. Thery, S. Amigorena. 2002. Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev. Immunol. 20:621.[Medline]
12. Bolland, S., J. V. Ravetch. 1999. Inhibitory pathways triggered by ITIM-containing receptors. Adv. Immunol. 72:149.[Medline]
13. Vivier, E., M. Daeron. 1997. Immunoreceptor tyrosine-based inhibition motifs. Immunol. Today 18:286.[Medline]
14. Pearse, R. N., T. Kawabe, S. Bolland, R. Guinamard, T. Kurosaki, J. V. Ravetch. 1999. SHIP recruitment attenuates FcRIIB-induced B cell apoptosis. Immunity 10:753.[Medline]
15. Kato, I., T. Takai, A. Kudo. 2002. The pre-B cell receptor signaling for apoptosis is negatively regulated by FcRIIB. J. Immunol. 168:629.[Abstract/Free Full Text]
16. Pricop, L., P. Redecha, J. L. Teillaud, J. Frey, W. H. Fridman, C. Sautes-Fridman, J. E. Salmon. 2001. Differential modulation of stimulatory and inhibitory Fc receptors on human monocytes by Th1 and Th2 cytokines. J. Immunol. 166:531.[Abstract/Free Full Text]
17. Hunter, S., Z. K. Indik, M. K. Kim, M. D. Cauley, J. G. Park, A. D. Schreiber. 1998. Inhibition of Fc receptor-mediated phagocytosis by a nonphagocytic Fc receptor. Blood 91:1762.[Abstract/Free Full Text]
18. Su, K., J. Wu, J. C. Edberg, X. Li, P. Ferguson, G. S. Cooper, C. D. Langefeld, R. P. Kimberly. 2004. A promoter haplotype of the immunoreceptor tyrosine-based inhibitory motif-bearing FcRIIb alters receptor expression and associates with autoimmunity. I. Regulatory FCGR2B polymorphisms and their association with systemic lupus erythematosus. J. Immunol. 172:7186.[Abstract/Free Full Text]
19. Li, X., J. Wu, R. H. Carter, J. C. Edberg, K. Su, G. S. Cooper, R. P. Kimberly. 2003. A novel polymorphism in the Fc receptor IIB (CD32B) transmembrane region alters receptor signaling. Arthritis Rheum. 48:3242.[Medline]
20. Wu, J., J. C. Edberg, P. B. Redecha, V. Bansal, P. M. Guyre, K. Coleman, J. E. Salmon, R. P. Kimberly. 1997. A novel polymorphism of FcRIIIa (CD16) alters receptor function and predisposes to autoimmune disease. J. Clin. Invest. 100:1059.[Medline]
21. Trainor, C. D., J. G. Omichinski, T. L. Vandergon, A. M. Gronenborn, G. M. Clore, G. Felsenfeld. 1996. A palindromic regulatory site within vertebrate GATA-1 promoters requires both zinc fingers of the GATA-1 DNA-binding domain for high-affinity interaction. Mol. Cell. Biol. 16:2238.[Abstract]
22. Jacobsen, B. M., D. G. Skalnik. 1999. YY1 binds five cis-elements and trans-activates the myeloid cell-restricted gp91^phox promoter. J. Biol. Chem. 274:29984.[Abstract/Free Full Text]
23. Cassel, D. L., M. A. Keller, S. Surrey, E. Schwartz, A. D. Schreiber, E. F. Rappaport, S. E. McKenzie. 1993. Differential expression of FcRIIA, FcRIIB and FcRIIC in hematopoietic cells: analysis of transcripts. Mol. Immunol. 30:451.[Medline]
24. Bolland, S., J. V. Ravetch. 2000. Spontaneous autoimmune disease in FcRIIB-deficient mice results from strain-specific epistasis. Immunity 13:277.[Medline]
25. Takai, T., M. Ono, M. Hikida, H. Ohmori, J. V. Ravetch. 1996. Augmented humoral and anaphylactic responses in FcRII-deficient mice. Nature 379:346.[Medline]
26. Jiang, Y., S. Hirose, M. Abe, R. Sanokawa-Akakura, M. Ohtsuji, X. Mi, N. Li, Y. Xiu, D. Zhang, J. Shirai, et al 2000. Polymorphisms in IgG Fc receptor IIB regulatory regions associated with autoimmune susceptibility. Immunogenetics 51:429.[Medline]
27. Pritchard, N. R., A. J. Cutler, S. Uribe, S. J. Chadban, B. J. Morley, K. G. Smith. 2000. Autoimmune-prone mice share a promoter haplotype associated with reduced expression and function of the Fc receptor FcRII. Curr. Biol. 10:227.[Medline]
28. Sato, S., M. Hasegawa, M. Fujimoto, T. F. Tedder, K. Takehara. 2000. Quantitative genetic variation in CD19 expression correlates with autoimmunity. J. Immunol. 165:6635.[Abstract/Free Full Text]
29. Patient, R. K., J. D. McGhee. 2002. The GATA family (vertebrates and invertebrates). Curr. Opin. Genet. Dev. 12:416.[Medline]
30. Garg, V., I. S. Kathiriya, R. Barnes, M. K. Schluterman, I. N. King, C. A. Butler, C. R. Rothrock, R. S. Eapen, K. Hirayama-Yamada, K. Joo, et al 2003. GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5. Nature 424:443.[Medline]
31. Caramori, G., S. Lim, K. Ito, K. Tomita, T. Oates, E. Jazrawi, K. F. Chung, P. J. Barnes, I. M. Adcock. 2001. Expression of GATA family of transcription factors in T-cells, monocytes and bronchial biopsies. Eur. Respir. J. 18:466.[Abstract/Free Full Text]
32. Nishiyama, C., M. Hasegawa, M. Nishiyama, K. Takahashi, Y. Akizawa, T. Yokota, K. Okumura, H. Ogawa, C. Ra. 2002. Regulation of human FcRI -chain gene expression by multiple transcription factors. J. Immunol. 168:4546.[Abstract/Free Full Text]
33. Bhalla, S. S., L. Robitaille, M. Nemer. 2001. Cooperative activation by GATA-4 and YY1 of the cardiac B-type natriuretic peptide promoter. J. Biol. Chem. 276:11439.[Abstract/Free Full Text]
34. Rao, S. P., K. A. Vora, T. Manser. 2002. Differential expression of the inhibitory IgG Fc receptor FcRIIB on germinal center cells: implications for selection of high-affinity B cells. J. Immunol. 169:1859.[Abstract/Free Full Text]
35. Yada, A., S. Ebihara, K. Matsumura, S. Endo, T. Maeda, A. Nakamura, K. Akiyama, S. Aiba, T. Takai. 2003. Accelerated antigen presentation and elicitation of humoral response in vivo by FcRIIB- and FcRI/III-mediated immune complex uptake. Cell. Immunol. 225:21.[Medline]
36. Xiu, Y., K. Nakamura, M. Abe, N. Li, X. S. Wen, Y. Jiang, D. Zhang, H. Tsurui, S. Matsuoka, Y. Hamano, et al 2002. Transcriptional regulation of Fcgr2b gene by polymorphic promoter region and its contribution to humoral immune responses. J. Immunol. 169:4340.[Abstract/Free Full Text]
37. Bynoe, M. S., C. M. Grimaldi, B. Diamond. 2000. Estrogen up-regulates Bcl-2 and blocks tolerance induction of naive B cells. Proc. Natl. Acad. Sci. USA 97:2703.[Abstract/Free Full Text]
38. Grimaldi, C. M., J. Cleary, A. S. Dagtas, D. Moussai, B. Diamond. 2002. Estrogen alters thresholds for B cell apoptosis and activation. J. Clin. Invest. 109:1625.[Medline]
39. Peeva, E., D. Michael, J. Cleary, J. Rice, X. Chen, B. Diamond. 2003. Prolactin modulates the naive B cell repertoire. J. Clin. Invest. 111:275.[Medline]
40. Hande, S., E. Notidis, T. Manser. 1998. Bcl-2 obstructs negative selection of autoreactive, hypermutated antibody V regions during memory B cell development. Immunity 8:189.[Medline]
41. Hartley, S. B., M. P. Cooke, D. A. Fulcher, A. W. Harris, S. Cory, A. Basten, C. C. Goodnow. 1993. Elimination of self-reactive B lymphocytes proceeds in two stages: arrested development and cell death. Cell 72:325.[Medline]
42. Smith, K. G., A. Light, L. A. O’Reilly, S. M. Ang, A. Strasser, D. Tarlinton. 2000. bcl-2 transgene expression inhibits apoptosis in the germinal center and reveals differences in the selection of memory B cells and bone marrow antibody-forming cells. J. Exp. Med. 191:475.[Abstract/Free Full Text]

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