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The Journal of Immunology, 2007, 178: 3272-3280.
Copyright © 2007 by The American Association of Immunologists, Inc.

Expression Profile of Fc{gamma}RIIb on Leukocytes and Its Dysregulation in Systemic Lupus Erythematosus1

Kaihong Su, Hengxuan Yang, Xinrui Li, Xiaoli Li, Andrew W. Gibson, John M. Cafardi, Tong Zhou, Jeffrey C. Edberg and Robert P. Kimberly2

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Fc{gamma}RIIb (CD32B, Online Mendelian Inheritance in Man 604590), an IgG FcR with a tyrosine-based inhibitory motif, plays a critical role in the balance of tolerance and autoimmunity in murine models. However, the high degree of homology between Fc{gamma}RIIb and Fc{gamma}RIIa in humans and the lack of specific Abs to differentiate them have hampered study of the normal expression profile of Fc{gamma}RIIb and its potential dysregulation in autoimmune diseases such as systemic lupus erythematosus (SLE). Using our newly developed anti-Fc{gamma}RIIb mAb 4F5 which does not react with Fc{gamma}RIIa, we found that Fc{gamma}RIIb is expressed on the cell surface of circulating B lymphocytes, monocytes, neutrophils, myeloid dendritic cells (DCs), and at very low levels on plasmacytoid DCs from some donors. Normal donors with the less frequent 2B.4 promoter haplotype have higher Fc{gamma}RIIb expression on monocytes, neutrophils, and myeloid DCs similar to that reported for B lymphocytes, indicating that Fc{gamma}RIIb expression on both myeloid and lymphoid cells is regulated by the naturally occurring regulatory single nucleotide polymorphisms in the FCGR2B promoter. Fc{gamma}RIIb expression in normal controls is up-regulated on memory B lymphocytes compared with naive B lymphocytes. In contrast, in active SLE, Fc{gamma}RIIb is significantly down-regulated on both memory and plasma B lymphocytes compared with naive and memory/plasma B lymphocytes from normals. Similar down-regulation of Fc{gamma}RIIb on myeloid-lineage cells in SLE was not seen. Our studies demonstrate the constitutive regulation of Fc{gamma}RIIb by natural gene polymorphisms and the acquired dysregulation in SLE autoimmunity, which may identify opportunities for using this receptor as a therapeutic target.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The only inhibitory IgG FcR in the classical FcR family (1, 2), Fc{gamma}RIIb (CD32B) has an ITIM in its cytoplasmic domain and counterbalances tyrosine-based activation signals in a variety of cells (3). For example, upon coligation of Fc{gamma}RIIb with the BCR by IgG immune complexes, Fc{gamma}RIIb recruits and activates the SHIP and negatively regulates B cell activation and proliferation, thereby providing a critical mechanism for the feedback inhibition of IgG production (4). On monocytes and macrophages, Fc{gamma}RIIb down-regulates Fc{gamma}RIIa (CD32A)- and Fc{gamma}RIIIa (CD16A)-mediated phagocytosis when coligated with those activating receptors (2, 5, 6). Fc{gamma}RIIb also down-modulates mast cell activation by the high-affinity IgE FcR (7, 8).

On dendritic cells (DCs),3 however, a dual role of Fc{gamma}RIIb has been suggested. Although interfering with the signals mediated by Fc{gamma}RIIb may lead to enhanced myeloid DC (mDC) maturation and immunogenicity (9, 10, 11), several studies suggest that Fc{gamma}RIIb on DCs promotes T-independent humoral responses by presenting native Ag to B cells, and that Fc{gamma}RIIb on follicular DCs facilitates B cell recall responses (12, 13). Thus, Fc{gamma}RIIb may have multiple effects on immune function, each of which points to an important role in immune regulation.

Indeed, the strength of Fc{gamma}RIIb signaling is critical for the induction of immune tolerance or autoimmunity. Mice deficient in FCGR2B with no Fc{gamma}RIIb expression have elevated serum Ig levels and enhanced anaphylactic responses (14). On a susceptible genetic background, mice deficient in FCGR2B develop anti-nuclear autoantibodies, glomerulonephritis, and other lupus-like symptoms (15). Consistent with these studies, natural polymorphisms in the mouse FCGR2B gene promoter, which lead to reduced receptor expression, have been identified in many autoimmune-prone mouse strains (16, 17, 18). Interestingly, 40% more Fc{gamma}RIIb expression on B cells by retroviral delivery is sufficient to restore tolerance and prevent autoimmunity in those mouse strains (19). Thus, in mice, FCGR2B has been proposed as a distal peripheral checkpoint gene for lupus-like autoimmunity.

In humans, the FCGR2B gene is located on Chr.1q23, a systemic lupus erythematosus (SLE) susceptibility locus confirmed by several independent genome-wide scans (20). Polymorphisms in the human FCGR2B gene are associated with the SLE phenotype (21, 22, 23, 24, 25, 26). We have recently demonstrated that polymorphisms in the regulatory region of FCGR2B form two major haplotypes and that the less frequent 2B.4 haplotype is significantly overrepresented in SLE patients (22). A nonsynonymous polymorphism in the transmembrane domain of Fc{gamma}RIIb is also enriched in SLE patients from Japanese and other Asian populations (23, 26, 27). However, due to the lack of specific Abs to distinguish human Fc{gamma}RIIb and its highly homologous family member Fc{gamma}RIIa (which is absent in mice), there is no study to date to examine the expression and regulation of Fc{gamma}RIIb in normal donors and in SLE autoimmunity.

To address this gap in knowledge, we have recently developed a novel mAb that differentiates the cell surface Fc{gamma}RIIb from Fc{gamma}RIIa. Using this unique reagent, we have discovered that, in addition to B cells, Fc{gamma}RIIb is also expressed on monocytes, neutrophils, mDCs, and at very low levels on plasmacytoid DCs (pDCs) from a small percentage of donors. Normal donors with the 2B.4 promoter haplotype have higher Fc{gamma}RIIb expression on myeloid-lineage monocytes, neutrophils, and mDCs compared with donors with the homozygous 2B.1 promoter haplotype, indicating that these promoter variants affect receptor expression in both the lymphoid and myeloid series. Interestingly, while Fc{gamma}RIIb is decreased on memory and plasma B cells from SLE patients, expression on mDCs is not changed, suggesting that there may be differential regulation of expression by humoral factors. SLE is a prototype autoimmune disease characterized by overproduction of autoantibodies and tissue deposition of autoimmune complexes (28, 29). Altered regulation of Fc{gamma}RIIb expression, by both genetic and disease activity related factors, could contribute significantly to the B cell hyperactivity in SLE.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Donors

SLE patients and normal controls were recruited from the University of Alabama at Birmingham-based DISCOVERY and CASSLE cohorts. All SLE patients fulfilled the revised American College of Rheumatology criteria for SLE (30) and were assessed for disease activity using the SLE Disease Activity Index (SLEDAI) (31). The human studies were reviewed and approved by the Institution Review Board, and all donors provided written informed consent.

Cell culture

PBMCs were purified from whole blood by density gradient centrifugation using Ficoll-Hypaque. PBMCs were cultured in 12-well plates at 2 x 106 cells/ml in RPMI 1640 medium supplemented with 10% human AB serum (Krackeler Scientific), glutamine, HEPES, and penicillin/streptomycin for 20–24 h in the absence or presence of indicated cytokines before flow cytometry analysis. IL-3, GM-CSF, IL-4, and IL-10 were purchased from PeproTech and used at 100 ng/ml.

Antibodies

The IV.3 and 32.2 hybridomas were purchased from American Type Culture Collection. Their fragments were prepared by Rockland Immunochemicals. The AT10 hybridoma was a gift from Dr. P. Guyre (Dartmouth Medical School, Hanover, NH) (32). mAb 4F5, a murine IgG1, was generated in BALB/c mice using the Escherichia coli-produced recombinant extracellular domain (EC) of human Fc{gamma}RIIb as an immunogen. Approximately 600 mAb clones were screened by ELISA for positive reactivity with the EC of Fc{gamma}RIIb and negative reactivity with the EC of Fc{gamma}RIIa-R (Fc{gamma}RIIa is polymorphic with arg or his at the extracellular amino acid 131 and Fc{gamma}RIIb is monomorphic with arg). The clones were further screened by flow cytometry on Fc{gamma}RIIb, Fc{gamma}RIIa-R, or Fc{gamma}RIIa-H stable transfectants for anti-Fc{gamma}RIIb Abs. mAb 4F5 were purified from ascites fluid using Immunopure Protein A mouse IgG1 Purification kit (Pierce Biotechnology). All mAbs were conjugated with Alexa 488 fluorescence dye using an Alexa488 Protein Labeling kit (Invitrogen Life Technologies-Molecular Probes). The labeling efficiency of 4F5, IV.3, 3G8, AT10, and 32.2, determined following the manufacturer’s instruction, was ~3:1 (three molecules of dyes per protein molecule). The F(ab')2 of mAb 4F5 were made using the ImmunoPure Fab Preparation kit (Pierce Biotechnology).

CD19-allophycocyanin and CD14-Tri-Color mAbs were purchased from Caltag Laboratories. CD27-allophycocyanin mAb was purchased from eBioscience. BDCA1-allophycocyanin and BDCA2-allophycocyanin mAbs were purchased from Miltenyi Biotec. The isotype control mIgG and mIgG F(ab')2 and F(ab')2 goat anti-mouse IgG F(ab')2 were purchased from Jackson ImmunoResearch Laboratories. Goat polyclonal Abs specific for the cytoplasmic domain of Fc{gamma}RIIa/c were purchased from Santa Cruz Biotechnology.

Flow cytometry analysis

PBMCs or whole blood were incubated with the indicated mAbs for 45 min on ice. The cells were washed with 3 ml of ice-cold PBS plus 0.5% BSA and 0.02% NaN3. The RBC were lysed by incubation with 1.5 ml of 1x FACS Lysing Solution (BD Biosciences-BD Pharmingen) at room temperature for 15 min. The cells were washed with PBS and resuspended in PBS plus 1% paraformaldehyde for flow cytometry analysis. Flow cytometry analysis of stable transfectants was performed similarly without the RBC-lysing step.

Stable cell lines

The parental FcR-deficient A20-IIA1.6 murine B cell line was provided by Dr. T. Wade (Dartmouth Medical Center). The Fc{gamma}RIIb-IIA1.6 stable transfectants were established previously (24). The Fc{gamma}RIIa cDNAs (with the "R" or "H" allele at amino acid 131) were subcloned into the pcDNA3 (Invitrogen Life Technologies) expression vector through EcoRI and NotI sites. The plasmids were transfected into A20-IIA1.6 cells using Fugene 6 transfection reagents (Roche Applied Science), and the Fc{gamma}RIIa-R and Fc{gamma}RIIa-H stable transfectants were selected in the presence of 1 mg/ml G418. Fc{gamma}RIIc stable transfectants were established in a similar way. Fc{gamma}RIIc cDNA was subcloned into pcDNA3 vector and the resulting plasmids were transfected into A20IIA1.6 cells.

RT-PCR

The desired leukocyte subsets were purified by FACS sorting after staining with their cell surface markers. pDCs were first enriched by BDCA4 microbeads using the Plasmacytoid Dendritic Cell Isolation kit (Miltenyi Biotec) and then subjected to cell sorting for BDCA2-allophycocyanin-positive cells. The purity of sorted cells was over 96% by subsequent flow cytometry analysis. The total RNA was prepared from the same number of sorted cells using TRIzol Reagents (Invitrogen Life Technologies). The RT-PCR for FCGR2B, FCGR2A, and GAPDH genes was performed using the SuperScript III One-Step RT-PCR kit (Invitrogen Life Technologies) following the manufacturer’s protocol. The Fc{gamma}RIIb-specific sense primer is: 5'-TGTCCAAGCTCCCAACTCTTCACC-3'; the antisense primer is: 5'-GTGTTCTCAGCCCCAACTTTG-3'. The Fc{gamma}RIIa-specific sense primer is: 5'-CACTGTCCAAGTGCCCAGCAT-3'; the antisense primer is: 5'-TTTATCATCGTCAGTAGGTGCCC-3'. The RT-PCR conditions were as follows: 56°C for 30 min, 95°C for 2 min, and 30 cycles of denaturing at 95°C for 15 s, annealing at 56°C for 30 s, and extension at 68°C for 40 s with a final extension at 68°C for 7 min.

Preparation of whole cell lysate and immunoprecipitation assay

Cells were lysed with whole cell lysis buffer at 60 µl/1 x 106 cells as previously described (24). 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 4F5, 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 then washed four times with whole cell lysis buffer and the immunoprecipitates were subjected to Western blot analysis.

GST pull-down experiments

The EC Fc{gamma}RIIb or Fc{gamma}RIIa (R or H allele) was subcloned into the mammalian expression vector pEBG in frame for GST through EcoRI and NotI sites. The plasmids were transiently transfected into 293 T cells using Fugene 6 transfection reagents (Roche). The GST fusion proteins were purified from 293 T cells at 30–48 h after transfection using glutathione Sepharose beads. The purified fusion proteins were incubated with mAbs 4F5 or AT10 on ice for 1 h. After extensive washing, the bound mAbs were subjected to reduced SDS-PAGE electrophoresis and Western blot analysis using HRP-linked F(ab')2 goat anti-mouse IgG (H+L).

Measurement of change in [Ca2+]i

Changes in intracellular [Ca2+]i induced by cross-linking of surface Igs on IIA1.6 transfectants and U937 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 previously described (33). Cells (10 x 106/ml) were loaded with 5 µM indo-1-AM at 37°C for 40 min and preincubated with 5 µg/ml indicated F(ab')2. The cells were washed and cross-linked with 20 µg/ml F(ab')2 goat anti-mouse IgG at the 60 s time point and the data were collected for 300 s.

Statistical analysis

Mean fluorescence intensity (MFI) of mAb 4F5 staining (for the expression levels of Fc{gamma}RIIb) on naive B lymphocytes was compared with that on memory or plasma B lymphocytes from the same participants by the paired Student’s t test, two-tailed. The unpaired Student’s t test, two tailed, was used to compare the MFI of mAb 4F5 staining on naive, memory, or plasma B lymphocytes between normal controls and SLE patients. The range of MFI values for 4F5 staining on myeloid-lineage cells was skewed toward low values (Lilliefors test for normality), and therefore, the Mann-Whitney U test was used to compare the expression levels of Fc{gamma}RIIb on monocytes, neutrophils, or mDCs between 2B.1 and 2B.4 donors and between normal controls and SLE patients. The Spearman rank correlation test was used to assess the degree of correlation of the expression levels of Fc{gamma}RIIb on monocytes, neutrophils, and mDCs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
mAb 4F5 reacts with Fc{gamma}RIIb but not Fc{gamma}RIIa

The EC of human Fc{gamma}RIIb and Fc{gamma}RIIa are ~95% identical due to gene duplication and recombination events in the evolution of FcR cluster (34, 35, 36). Available mAbs that react with the cell surface Fc{gamma}RII (CD32) include pan-reactive Abs, anti-Fc{gamma}RIIa Abs, and allele-specific Abs reacting with the arg allele of Fc{gamma}RIIa and with Fc{gamma}RIIb (9, 32, 37, 38, 39). Studies of Fc{gamma}RIIb expression on primary cells, however, have been greatly hampered by the lack of specific mAbs. To generate mAb that differentiates Fc{gamma}RIIb and Fc{gamma}RIIa, the E. coli-produced recombinant EC of human Fc{gamma}RIIb was used as an immunogen. Approximately 600 mAb clones were first screened by ELISA and further screened by flow cytometry of stably transfected IIA1.6 cells for positive reactivity with Fc{gamma}RIIb and absence of reactivity with the Fc{gamma}RIIa-R or Fc{gamma}RIIa-H (Fc{gamma}RIIa is polymorphic with arg or his at amino acid 131 in its EC while Fc{gamma}RIIb is monomorphic with arg at that site). Clone 4F5 reacted with the cell surface Fc{gamma}RIIb, but not Fc{gamma}RIIa-R or Fc{gamma}RIIa-H, by flow cytometry analysis (Fig. 1A). Similar expression levels of Fc{gamma}RIIb and Fc{gamma}RIIa on the stable transfectants were confirmed with the well-characterized pan-Fc{gamma}RII mAb AT10 (Fig. 1A). Clone 4F5 does react with stably transfected Fc{gamma}RIIc (data not shown), a highly homologous gene which has been reported on NK cells from a small percentage of donors (40).


Figure 1
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  		FIGURE 1. mAb 4F5 reacts with Fc{gamma}RIIb not Fc{gamma}RIIa. A, mAb 4F5 reacts with cell surface Fc{gamma}RIIb but not Fc{gamma}RIIa in flow cytometry analysis. A20IIA1.6 cells stably transfected with Fc{gamma}RIIb or Fc{gamma}RIIa were incubated with Alexa 488-conjugated mAbs mIgG1 (dashed lines), 4F5 (gray solid line), or AT10 (black solid line). Fc{gamma}RIIb is monomorphic at amino acid 131 with an arg residue and Fc{gamma}RIIa is polymorphic at that site with an arg or his residue. Thus, both IIa-R and IIa-H stable transfectants were established and analyzed. B, mAb 4F5 immunoprecipitates Fc{gamma}RIIb but not Fc{gamma}RIIa. Whole cell lysate from Fc{gamma}RIIb, Fc{gamma}RIIa-R, Fc{gamma}RIIa-H stable transfectants were immunoprecipitated by mAb mIgG1, 4F5, or IV.3 and subjected to Western blot analysis using rabbit polyclonal Abs specific for the cytoplasmic domain of Fc{gamma}RIIb (upper) or goat Abs specific for the cytoplasmic domain of Fc{gamma}RIIa/c (lower) as blotting Abs. C, mAb 4F5 interacts with the recombinant EC of Fc{gamma}RIIb but not of Fc{gamma}RIIa. Purified fusion proteins GST-IIb EC or GST-IIa EC (R or H) were incubated with mAb 4F5 or AT10 and the bound proteins were subjected to Western blot analysis by HRP-linked goat anti-mouse IgG Abs. D, mAb 4F5 specifically reacts to denatured Fc{gamma}RIIb in Western blot analysis. The immunoprecipitates with mAb AT10 or mIgG1 from the whole cell lysate of Fc{gamma}RIIb, Fc{gamma}RIIa-R, Fc{gamma}RIIa-H stable transfectants were subjected to Western blot analysis using mAb 4F5 (upper) or goat polyclonal Abs specific for the cytoplasmic domain of Fc{gamma}RIIa/c (lower) as blotting Abs.

 
As shown by Western blot in Fig. 1B, mAb 4F5 immunoprecipitated Fc{gamma}RIIb but not Fc{gamma}RIIa-R or Fc{gamma}RIIa-H from the whole cell lysate of stable transfectants (Fig. 1B, lanes 2, 5, and 8). As a comparison, mAb IV.3 preferentially immunoprecipitated Fc{gamma}RIIa-R and Fc{gamma}RIIa-H (Fig. 1B, lanes 6 and 9), with a small amount of Fc{gamma}RIIb also evident (Fig. 1B, lane 3). To further establish the specific interaction between mAb 4F5 and the EC of Fc{gamma}RIIb, we used a GST-pull down paradigm. GST proteins fused with the EC of Fc{gamma}RIIa or Fc{gamma}RIIb were incubated with mAb 4F5 or AT-10. The Abs immunoprecipitated by the GST fusion proteins were visualized by goat anti-mouse IgG Western blot analysis. The GST-Fc{gamma}RIIb EC but not Fc{gamma}RIIa-R/H EC fusion proteins pulled down mAb 4F5 (Fig. 1C, lanes 1–3). As a positive control, GST-IIb and GST-IIa R/H fusion proteins pulled down the pan-mAb AT10 (Fig. 1C, lanes 4–6). In summary, our data demonstrated that mAb 4F5 reacts with native Fc{gamma}RIIb but not with Fc{gamma}RIIa (R or H) on the cell surface, in the cell lysate, and as recombinant proteins.

We also tested the ability of mAb 4F5 to recognize denatured Fc{gamma}RIIb in Western blot analysis. Pan-mAb AT10 or its isotype control mIgG1 were used to immunoprecipitate Fc{gamma}RII from the whole cell lysate of A20IIA1.6 stable transfectants. The immunoprecipitates were subjected to Western blot analysis using either mAb 4F5 or polyclonal anti-Fc{gamma}RIIa/c as blotting Abs. mAb 4F5 only blotted Fc{gamma}RIIb immunoprecipated by mAb AT10 or directly from the whole cell lysate of Fc{gamma}RIIb-transfectants (Fig. 1D, upper panel, lanes 2 and 7). Western blots using polyclonal anti-Fc{gamma}RIIa/c demonstrated that there were comparable amounts of Fc{gamma}RIIa-R or Fc{gamma}RIIa-H proteins immunoprecipitated by mAb AT10 or directly from the whole cell lysate of Fc{gamma}RIIa transfectants (Fig. 1D, upper panel, lanes 4, 6, 8, and 9). Together, our data suggest that mAb 4F5 also reacts with denatured Fc{gamma}RIIb but not Fc{gamma}RIIa in Western blot analysis.

Cross-linking of Fc{gamma}RIIb by mAb 4F5 induces an inhibitory signal for BCR and Fc{gamma}RIa-mediated intracellular Ca2+ flux ([Ca2+]i)

Fc{gamma}RIIb negatively regulates the activation signals mediated by BCR and by activating FcRs. We next examined the ability of mAb 4F5 to modulate the Ca2+ responses induced by BCR and the activating Fc{gamma}RIa. Cross-linking of mouse IgG (mIgG)-BCR alone by F(ab')2 goat anti-mIgG induced a brisk rise in [Ca2+]i in the IIA1.6-Fc{gamma}RIIb stable transfectants (Fig. 2A). Coengagement of human Fc{gamma}RIIb and mIgG-BCR by preincubation with mAb 4F5 F(ab')2 significantly decreased the change in [Ca2+]i (Fig. 2A). Similarly, in human monocytic U937 cells, cross-linking of Fc{gamma}RIa by preincubation with mAb 32.2 F(ab')2 induced a rise in [Ca2+]i (Fig. 2B). However, coengagement of Fc{gamma}RIIb and Fc{gamma}RIa by preincubation with mAbs 4F5 F(ab')2 and 32.2 F(ab')2 induced a significant decrease in the Ca2+ responses (Fig. 2B). As a control, coengagement of the activating Fc{gamma}RIIa and Fc{gamma}RIa by preincubation with mAbs IV.3 Fab and 32.2 F(ab')2 induced more rapid and stronger Ca2+ responses (Fig. 2B). Collectively, our data suggest that co-cross-linking of Fc{gamma}RIIb by mAb 4F5 induces an inhibitory signal for the Ca2+ responses mediated by BCR and the activating Fc{gamma}RIa.


Figure 2
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  		FIGURE 2. Cross-linking of Fc{gamma}RIIb by mAb 4F5 induces an inhibitory signal for Ca2+ influxes mediated by BCR and Fc{gamma}RIa (CD64). A, Cross-linking of Fc{gamma}RIIb by mAb 4F5 inhibited the BCR-mediated Ca2+ influxes. A20IIA1.6-Fc{gamma}RIIb stable transfectants were preincubated with 4F5 F(ab')2 (gray line) or its isotype control mIgG1 F(ab')2 (black line). The endogenous mIgG-BCR and transfected human Fc{gamma}RIIb were co-cross-linked by F(ab')2 goat anti-mouse IgG. The Ca2+ responses were recorded using an SLM 8000 spectrofluorometer and presented as the changes in intracellular Ca2+ concentration. B, Cross-linking of Fc{gamma}RIIb by mAb 4F5 inhibited the Fc{gamma}RI-mediated Ca2+ influxes. U937 cells were preincubated with primary 32.2 F(ab')2, IV.3 Fab, and/or 4F5 F(ab')2 and cross-linked with secondary F(ab')2 goat anti-mouse IgG for the engagement of Fc{gamma}RIa alone (black solid line), coengagement of Fc{gamma}RIa and Fc{gamma}RIIa (gray dashed line), or coengagement of Fc{gamma}RIa and Fc{gamma}RIIb (gray solid line).

 
mAb 4F5 partially competes with heat-aggregated human IgG for Fc{gamma}RIIb binding

To further investigate the binding property of mAb 4F5 on Fc{gamma}RIIb, we performed cross-competition binding experiments with 4F5 and heat-aggregated human IgG (ahIgG) that mimics the ligand binding of FcRs. Fc{gamma}RIIb stable transfectants were preincubated with 4F5 F(ab')2, IV.3 Fab, or isotype control mIgG1 F(ab')2 then stained with Alexa 488-conjugated ahIgG. As expected, flow cytometry analysis demonstrated that IV.3 Fab did not affect the binding of ahIgG to Fc{gamma}RIIb transfectants, but 4F5 F(ab')2 did partially block the binding of ahIgG to Fc{gamma}RIIb (Fig. 3A, top panel). As a control, the same experiment was performed on Fc{gamma}RIIa-R transfectants. IV.3 Fab almost completely blocked the binding of ahIgG to Fc{gamma}RIIa, while 4F5 F(ab')2 did not affect the binding of ahIgG to Fc{gamma}RIIa (Fig. 3A, bottom panel). These results are in agreement with previous findings that mAb IV.3 competes with the ligand binding of CD32. In a reciprocal paradigm, preincubation of Fc{gamma}RIIb-transfectants with ahIgG, but not similarly prepared heat-aggregated human IgA, partially blocked the binding of 4F5 to Fc{gamma}RIIb (Fig. 3B). Similar competition experiments between mAbs 4F5 and AT10 demonstrated that 4F5 did not affect the binding of Alexa 488-conjugated mAb AT10 to Fc{gamma}RIIb (Fig. 3C). Overall, our data suggest that mAb 4F5 partially competes with ahIgG but not with the pan-mAb AT10 for receptor binding.


Figure 3
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  		FIGURE 3. mAb 4F5 partially competes with ahIgG but not with mAb AT10 for receptor binding. A, mAb 4F5 partially competes with ahIgG for receptor binding. Upper panel, IIA1.6-Fc{gamma}RIIb stable transfectants were preincubated with 30 µg/ml 4F5 F(ab')2 (thick gray line), IV.3 Fab (thin gray line), or control mIgG1 F(ab')2 (thick black line). The cells were then washed, stained with 10 µg/ml Alexa 488-conjugated ahIgG, and subjected to flow cytometry analysis. Direct staining of the cells with Alexa 488-conjugated mIgG1 was used as a control (dashed black line). Lower panel, The same experiment was performed on Fc{gamma}RIIa-R IIA1.6 stable transfectants. B, ahIgG partially competes with mAb 4F5 for receptor binding. The IIA1.6-Fc{gamma}RIIb stable transfectants were preincubated with 30 µg/ml ahIgG (thick gray line), ahIgA (thin gray line), or no Ab (thick black line). The cells were then washed, stained with 10 µg/ml Alexa 488-conjugated 4F5, and subjected to flow cytometry analysis. Direct staining of the cells with mIgG1-Alexa 488 was used as a control (dashed black line). C, mAb 4F5 does not compete with mAb AT10 for receptor binding. The Fc{gamma}RIIb-IIA1.6 stable transfectants were preincubated with 30 µg/ml unlabeled mAb 4F5 (thick gray line), AT10 (thin gray line), or no Ab (thick black line). The cells were then washed, stained with 10 µg/ml Alexa 488-conjugated AT10, and subjected to flow cytometry analysis. Direct staining of the cells with mIgG1-Alexa 488 was used as a control (dashed black line).

 
The expression profile of Fc{gamma}RIIb on circulating leukocytes

Using mAb 4F5 and other lineage-specific markers, we were able to examine the cell surface expression of Fc{gamma}RIIb on circulating leukocyte subpopulations. We performed multicolor flow cytometry analysis on whole blood or Ficoll-purified PBMCs. B lymphocytes (CD19+) express high levels of Fc{gamma}RIIb as evidenced by mAb AT10 and 4F5 reactivity (Fig. 4A). Circulating T cells (CD3+) do not express any detectable levels of Fc{gamma}RIIb as determined by negative 4F5 staining (data not shown). The CD14+ monocytes and neutrophils (polymorphonuclear neutrophils (PMNs)) express Fc{gamma}RIIb on the cell surface although at a much lower level than Fc{gamma}RIIa (Fig. 4, B and C). A major population of mDCs in human blood is defined as CD19–BDCA1+ (41), and those mDCs express both Fc{gamma}RIIa and Fc{gamma}RIIb (Fig. 4D). Of note, the level of Fc{gamma}RIIb expression on mDCs is heterogeneous both between donors and within a single donor, which may reflect the heterogeneity of mDCs at different maturation and/or differentiation stages.


Figure 4
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  		FIGURE 4. The cell surface expression profile of Fc{gamma}RIIb on circulating leukocytes. PBMCs (A, B, D, and E) or whole blood (C) was incubated with lineage-specific markers and Alexa 488-conjugated mAbs 4F5 (black line), AT10 (dark gray line), IV.3 (light gray line), or mIgG1 isotype control (dashed line) and subjected to multicolor flow cytometry analysis. Left panels, The gating of the cell subpopulations analyzed in the right panel histograms. B lymphocytes are defined as CD19+, mDCs as CD19–BDCA1+, monocytes as CD14+, and pDCs as BDCA2+; neutrophils are defined by side- and forward-scatter.

 
pDCs in human blood can be identified by a single marker, BDCA2 (41). Previous studies have suggested that Fc{gamma}RIIa is the only FcR expressed on pDCs (10, 42). Because pDCs are a major producer of type I IFN (IFN-{alpha}/IFN-beta) and may be involved in the "IFN signature" in SLE (43), we examined the expression of Fc{gamma}RIIb on circulating pDCs from a number of donors. We found that Fc{gamma}RIIb is expressed at very low levels on circulating pDCs in ~10% donors, but in most donors, Fc{gamma}RIIb is not expressed at detectable levels (Fig. 4E, top and bottom panels, respectively). In summary, our data demonstrate the cell surface expression of Fc{gamma}RIIb on myeloid-lineage cells including monocytes, neutrophils, and mDCs. Our data also suggest that Fc{gamma}RIIb is expressed on circulating pDCs from a small percentage of normal donors.

To confirm the expression profile of Fc{gamma}RIIb in leukocyte subpopulations, we performed semiquantitative RT-PCR for Fc{gamma}RIIb and Fc{gamma}RIIa messages from FACS-purified leukocyte subsets. Fc{gamma}RIIb has two major splice variants, IIb1 which is more abundantly expressed in B lymphocytes and IIb2 which is more abundantly expressed in myeloid lineage cells (1). Fc{gamma}RIIb2 lacks the 19 aa of the first exon encoding the cytoplasmic domain and is more capable of endocytosis (44). PCR primers for Fc{gamma}RIIb were designed to amplify both IIb1 and IIb2 messages with amplicons distinguishable on the basis of size. The specificity of the primers and PCR were confirmed by PCRs using Fc{gamma}RIIb1, Fc{gamma}RIIb2, or Fc{gamma}RIIa cDNA-containing plasmids as the template (Fig. 5, lanes 1–3). In agreement with previous literature (45), CD19+ B cells express more IIb1 message than IIb2 and do not express detectable IIa message while CD14+ monocytes express more IIb2 than IIb1 and abundant IIa messages (Fig. 5, lanes 4 and 5). Purified mDCs (CD19–BDCA1+) and PMNs express more Fc{gamma}RIIb2 than IIb1 messages and abundant Fc{gamma}RIIa message (Fig. 5, lanes 7 and 9). Because pDCs are very rare in PBMCs (~0.1–0.5%), we first used BDCA4 microbeads (Miltenyi Biotec) to enrich pDCs and then sorted BDCA2+ cells. pDCs from donor 1 express IIb2, IIb1, and IIa messages while pDCs from donor 2 do express IIa message but no detectable IIb1 or IIb2 messages (Fig. 5, lanes 6 and 8), findings in agreement with the cell surface staining by mAbs 4F5 and IV.3 (Fig. 4E). Taken together, we have demonstrated the expression profile of Fc{gamma}RIIb on PBL subpopulations at both message and cell surface protein levels.


Figure 5
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  		FIGURE 5. The expression of Fc{gamma}RIIb1 and IIb2 messages in leukocyte subpopulations. Semiquantitative RT-PCR for Fc{gamma}RIIb and Fc{gamma}RIIa messages was performed using RNAs isolated from 20,000 purified CD19+ B cells, CD14+ monocytes, CD19–BDCA1+ mDCs, or BDCA2+ pDCs of normal donors (lanes 4–9). The PCR using Fc{gamma}RIIb1, Fc{gamma}RIIb2, or Fc{gamma}RIIa cDNA-containing pcDNA3 plasmids as the templates were served as specificity control for primers and PCR conditions (lanes 1–3). The RT-PCR for housekeeping GAPDH gene was used as a loading control.

 
The expression of Fc{gamma}RIIb on pDCs is induced by cytokines

Because of the potential importance of Fc{gamma}RIIb expression by pDCs, we explored the possibility that Fc{gamma}RIIb on pDCs could be up-regulated by cytokines. PBMCs were cultured in RPMI 1640 with 10% human AB serum in the absence or presence of cytokines for 22 h and the expression of Fc{gamma}RIIb was examined by mAb 4F5 staining. Because IL-4 plus IL-10 synergistically up-regulate Fc{gamma}RIIb on monocyte-derived DCs (39), we tested the effect of IL-4 plus IL-10 and IL-3 plus GM-CSF on Fc{gamma}RIIb expression by pDCs. Starting with a normal donor who did not show detectable levels of Fc{gamma}RIIb on pDCs before culture (Fig. 6A), ~8% of the pDCs were Fc{gamma}RIIb-positive after in vitro culture in pooled AB serum. Approximately 20% of the pDCs were Fc{gamma}RIIb-positive with IL-3 plus GM-CSF treatment, and ~30% of the pDCs were Fc{gamma}RIIb-positive with IL-4 plus IL-10 treatment (Fig. 6B). Based on five independent experiments, our data suggest that the expression of Fc{gamma}RIIb can be up-regulated by cytokines, involving 10–30% of pDCs. The expression of the inhibitory Fc{gamma}RIIb on pDCs could provide an important control for immune complex-induced IFN-{alpha} production.


Figure 6
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  		FIGURE 6. The expression of Fc{gamma}RIIb on pDCs is induced by cytokines. A, PBMCs purified from a representative normal donor were stained with BDCA2-allophycocyanin and 4F5-Alexa 488. Dot plot shows the percentage of pDCs positive for mAb 4F5. B, PBMCs from the same person were cultured in RPMI 1640 plus 10% human AB serum medium with or without indicated cytokines for 22 h and then stained with BDCA2-allophycocyanin and 4F5-Alexa 488.

 
The expression of Fc{gamma}RIIb is regulated by its promoter haplotypes

Of the two major promoter haplotypes in the human FCGR2B gene, the less frequent 2B.4 haplotype leads to increased Fc{gamma}RIIb expression on B lymphocytes (21, 22). Because myeloid lineage cells express both Fc{gamma}RIIa and Fc{gamma}RIIb, the unique cell surface expression of Fc{gamma}RIIb cannot be determined using pan-Fc{gamma}RII mAbs. Using anti-Fc{gamma}RIIb mAb 4F5, we next examined the cell surface expression of Fc{gamma}RIIb on circulating monocytes, neutrophils, and mDCs from normal donors with homozygous 2B.1 or heterozygous 2B.1/2B.4 haplotype. Donors with the 2B.4 haplotype have increased mAb 4F5 staining on CD14+ monocytes compared with 2B.1 homozygous donors (Fig. 7A, p < 0.002, two-tailed, Mann-Whitney U test), which is in agreement with the previously reported expression of Fc{gamma}RIIb in monocytes by Western blot analysis (21). Similarly, donors with the 2B.4 haplotype have increased 4F5 staining levels on neutrophils and CD19–BDCA1+ mDCs (Fig. 7, B and C, p < 0.013 and p < 0.001, respectively, two-tailed, Mann-Whitney U test). Analysis of mAb IV.3-staining levels on the same cells did not reveal any difference between the 2B.4 and 2B.1 donors (data not shown).


Figure 7
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  		FIGURE 7. The 2B.4 haplotype leads to increased Fc{gamma}RIIb expression on monocytes, neutrophils, and mDCs. PBMCs from normal donors with the 2B.4 haplotype and with homozygous 2B.1 haplotype were stained with lineage-specific markers and mAb 4F5 for the expression levels of Fc{gamma}RIIb. A, Normal donors with the 2B.4 haplotype have increased 4F5 staining on CD14+ monocytes than 2B.1 donors. Left panel, A histogram overlay showing 4F5 staining on CD14+ monocytes from a representative 2B.1 donor (gray line) and a representative 2B.4 donor (black line). The dashed lines are mIgG1 isotype controls. Right panel, A summary of the MFI of 4F5 staining on monocytes from 22 2B.1 donors and 5 2B.4 donors (p < 0.002, two-tailed, Mann-Whitney U test). B, Normal donors with the 2B.4 haplotype have increased 4F5 staining on neutrophils compared with 2B.1 donors (p < 0.013, two-tailed, Mann-Whitney U test). Similar analyses were performed as shown in A. C, Donors with the 2B.4 haplotype have increased 4F5 staining on CD19–BDCA1+ mDCs compared with 2B.1 donors (p < 0.001, two-tailed, Mann-Whitney U test). Similar analyses were performed as in A.

 
Because monocytes, neutrophils, and mDCs have the same myeloid origin, we next examined the potential correlation of Fc{gamma}RIIb expression levels on these three cell types. Although the expression levels of Fc{gamma}RIIb varied over a several-fold range between normal donors, there was a significant correlation among the expression levels of Fc{gamma}RIIb on monocytes, mDCs, and neutrophils (Spearman rank correlation coefficients of 0.692, 0.535, and 0.674 for monocytes vs neutrophils, monocytes vs mDCs, and neutrophils vs mDCs, respectively; p values for all the correlations are <0.002). Our data suggest that Fc{gamma}RIIb is coordinately expressed across primary monocytes, neutrophils, and mDCs from normals in the constitutive state.

Fc{gamma}RIIb expression is dysregulated on B lymphocytes in active SLE patients

Recent studies in mice have highlighted the important role of Fc{gamma}RIIb in the development of lupus autoimmunity (19, 46). Therefore, we compared Fc{gamma}RIIb expression on circulating leukocytes from SLE patients and normal controls. We first examined Fc{gamma}RIIb expression on circulating B cell subpopulations: naive (CD19+CD27–), memory (CD19+CD27+), and a subgroup of plasma (CD19lowCD27high) B cells. We observed that in patients with active SLE, there is a significant increase in the number and percentage of CD19lowCD27high plasmablast B cells: 12.9% in a representative SLE patient vs 2.1% in a normal donor (Fig. 8A). The number of naive B cells was substantially decreased and the percentage of memory B cells was relatively increased in SLE patients (Fig. 8A), as previously reported (47, 48, 49). In individual normal donors, Fc{gamma}RIIb expression on memory B lymphocytes appeared to be up-regulated relative to naive B lymphocytes (Fig. 8B, left panel), a finding confirmed in a group of 30 normal controls with ~20% more expression on memory B cells than on their naive B cells (Fig. 8C; p < 0.0001, two-tailed, paired, Student’s t test). The expression levels of Fc{gamma}RIIb on CD27high plasma B cells of normal controls were not different from those on the naive B compartments (Fig. 8C). However, in 19 active SLE patients (SLEDAI ≥2), Fc{gamma}RIIb expression was decreased on memory and plasma B lymphocytes compared with their own naive B compartments (Fig. 8, B and C; p < 0.05 and p < 0.04, respectively, two-tailed, paired, Student’s t test). The Fc{gamma}RIIb expression levels on active SLE memory B lymphocytes was more significantly decreased when compared with those on normal memory B lymphocytes (~30% less; Fig. 8C; p < 0.0001, two-tailed, unpaired, Student’s t test). Similarly, the Fc{gamma}RIIb expression levels on active SLE plasma B cells was also decreased compared with normal plasma B cells (~15% less; Fig. 8C; p < 0.03, two-tailed, unpaired, Student’s t test). However, the expression levels of Fc{gamma}RIIb on memory or plasma B lymphocytes from five SLE patients in remission (SLEDAI = 0) were not significantly different from those on their naive B compartments, nor from those on normal memory or plasma B lymphocytes (Fig. 8C). The expression levels of Fc{gamma}RIIb on naive B compartments were not different between normals, active SLE, or SLE in remission (Fig. 8C). In summary, our data strongly suggest that the expression of Fc{gamma}RIIb is regulated during normal B cell differentiation and activation and that this regulation is abnormal in patients with active SLE.


Figure 8
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  		FIGURE 8. The expression of Fc{gamma}RIIb is dysregulated on B lymphocytes from active SLE patients. A, Active SLE patients have increased percentage of plasma B lymphocytes compared with normal controls. The percentages of naive B (CD19+CD27–), memory B (CD19+CD27+), and plasma B cells (CD19lowCD27high) from a representative normal donor (left) and an active SLE patient (right) are shown in the dot plots. B and C, The expression of Fc{gamma}RIIb on memory and plasma B cells from active SLE patients is significantly decreased compared both to their own naive B lymphocytes and to memory/plasma B lymphocyte counterparts from normal controls. Histograms in B display the staining of mAb 4F5 on naive and memory B lymphocytes from a representative normal donor (left) and an active SLE patient (right). C, A summary of the MFI of mAb 4F5 staining on naive (N), memory (M), and plasma (P) B lymphocytes from 30 normal controls, 19 active SLE patients (SLEDAI ≥2), and 5 SLE patients in remission (SLEDAI = 0). Two-tailed Student’s t tests were performed to compare the expression levels of Fc{gamma}RIIb on B cell subpopulations (normal memory B compared with their naive B lymphocytes: p < 0.0001; active SLE memory or plasma B compared with their own naive B lymphocytes: p < 0.05 and p < 0.04, respectively; active SLE memory or plasma B lymphocytes compared with memory or plasma B lymphocyte counterparts from normals: p < 0.0001 and p < 0.03, respectively). There was an increase of 2B.4 haplotype in SLE patients compared with normals (15.8 and 9%, respectively) and the analysis was comparable when the 2B.4 donors were removed from both groups.

 
Comparison of Fc{gamma}RIIb expression levels on myeloid-lineage cells in normal controls and SLE patients

In contrast, the expression of Fc{gamma}RIIb on myeloid-lineage cells including monocytes, neutrophils, and mDCs was not altered in SLE. Analyses of 26 normal controls and 14 active SLE patients revealed no difference in the expression levels of Fc{gamma}RIIb on CD19–BDCA1+ mDCs (Fig. 9). Similarly, there was no significant difference in the expression levels of Fc{gamma}RIIb on CD14+ monocytes and neutrophils between normal controls and active SLE patients despite an increased expression of Fc{gamma}RIa (CD64) on monocytes (data not shown) as previously described (50).


Figure 9
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  		FIGURE 9. The expression of Fc{gamma}RIIb is not decreased on mDCs from active SLE patients. The same normal controls and active SLE patients as analyzed in Fig. 8 were also compared for Fc{gamma}RIIb expression on mDCs by mAb 4F5 staining (p > 0.05, two-tailed, Mann-Whitney U test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The ITIM-bearing Fc{gamma}RIIb is expressed on lymphoid- and myeloid-lineage cells and plays an important role in immune regulation (2). Studies in mice have suggested that the expression levels of Fc{gamma}RIIb greatly influence humoral immune responses and the development of autoimmunity. Mice with reduced Fc{gamma}RIIb expression exhibit enhanced IgG responses and an autoimmune-prone phenotype (16, 17, 18). Partial restoration of Fc{gamma}RIIb expression on B cells in lupus-prone mouse strains is sufficient to restore tolerance and prevent autoimmunity (19). On a susceptible genetic background, FCGR2B-deficient mice develop a lupus-like autoimmune disease (15). Reciprocally, recent studies suggest that Fc{gamma}RIIb on DCs promote T-independent humoral responses and B cell recall responses (12, 13). In each model, the data strongly suggest that Fc{gamma}RIIb is critical for the modulation of immune responses.

However, studies of Fc{gamma}RIIb in the human system have been hampered by the lack of mAbs able to differentiate between the highly homologous Fc{gamma}RIIa and Fc{gamma}RIIb. In this study, we have developed mAb 4F5 that reacts with human Fc{gamma}RIIb but not Fc{gamma}RIIa under both native and denatured conditions. mAb 4F5 partially blocks the ligand-binding of Fc{gamma}RIIb and mediates an inhibitory signal by co-cross-linking Fc{gamma}RIIb with activating FcRs. Thus, mAb 4F5 not only enables study of the expression, regulation, and function of this important receptor, but also may point to the therapeutic applications where the modulation of Fc{gamma}RIIb is required.

Using mAb 4F5, we have determined the cell surface expression profile of Fc{gamma}RIIb on different leukocyte subpopulations. In addition to expression on B lymphocytes, monocytes, neutrophils, and myeloid DCs, Fc{gamma}RIIb is also constitutively expressed at low levels on pDCs from some donors, and this expression on pDCs can be up-regulated by incubation with certain cytokines. Because pDCs are a major producer of type I IFNs (IFN-{alpha}/IFN-beta) and enhanced expression of a range of IFN-{alpha}-regulated genes ("IFN-{alpha}-signature") has been observed in active SLE patients (43, 51, 52), the expression of the inhibitory Fc{gamma}RIIb on pDCs could provide an important control for immune complex-mediated production of IFN-{alpha}. Modulation of Fc{gamma}RIIb expression on pDCs might provide a novel therapeutic pathway to induce SLE remission.

We have previously identified two major promoter haplotypes in the human FCGR2B gene: 2B.1 and 2B.4 (21, 22). The less frequent 2B.4 haplotype has higher binding capacity for GATA4 and YYI transcription factors and leads to higher expression in a luciferase reporter system. Donors with the 2B.4 haplotype have increased Fc{gamma}RIIb expression on B lymphocytes, and in this study, we have used the newly developed anti-Fc{gamma}RIIb mAb 4F5 to demonstrate that donors with the 2B.4 haplotype also have increased Fc{gamma}RIIb expression on myeloid-lineage cells, including monocytes, neutrophils, and mDCs. Our data suggest that the expression of Fc{gamma}RIIb is regulated by its natural promoter polymorphisms in both lymphoid and myeloid-lineage cells. However, our data showing differential regulation of Fc{gamma}RIIb between normals and SLE patients in some, but not all, cell subsets also suggest that Fc{gamma}RIIb expression may have tissue-specific properties.

Our comparison of Fc{gamma}RIIb expression in normal donors and SLE patients suggests a dysregulation of Fc{gamma}RIIb on SLE B lymphocytes. In normal donors, the expression of Fc{gamma}RIIb is up-regulated on memory B cells compared with naive B cells, perhaps as a means to negatively control humoral responses. In contrast, the expression of Fc{gamma}RIIb in active SLE patients is significantly down-regulated on memory B cells compared with their own naive B cells and memory B cells from normal donors. Similarly, the expression of Fc{gamma}RIIb is decreased on SLE plasma cells compared with their naive B cells and normal plasma B cells. Our observation of decreased expression of Fc{gamma}RIIb on SLE memory and plasma B lymphocytes together with previous observation of defective Fc{gamma}RIIb signaling in SLE B cells may provide an important mechanism for the overproduction of autoantibodies and other B cell abnormalities in SLE (53). In a transgenic mouse model with anti-DNA BCR, Fc{gamma}RIIb is shown to mediate peripheral tolerance by limiting the cell number of anti-DNA plasma cells (46). Further studies to examine the function of Fc{gamma}RIIb in B cell differentiation and development will be particularly interesting because they will not only help to understand the normal control of humoral immune responses but also shed light on the pathogenesis of autoimmune diseases. In addition, the mechanism for Fc{gamma}RIIb dysregulation in SLE B cells may represent a more general defect in the B cell development and differentiation in patients with SLE.

The expression level of Fc{gamma}RIIb on myeloid-lineage cells showed a broad distribution among normal donors and SLE patients, a pattern which was pronounced on mDCs and may reflect stages of differentiation. Although monocytes from SLE patients showed higher levels of Fc{gamma}RIa (CD64), consistent with an IFN effect (50, 51, 52), comparison of Fc{gamma}RIIb expression on monocytes, neutrophils, and mDCs between SLE patients and normal donors did not reveal a significant difference. This result may be due, in part, to the relative small sample size and, in part, to the multiple factors of activation and differentiation states of cells within the myeloid compartment. Indeed, this suggests that Fc{gamma}RIIb may be regulated in a lymphoid and myeloid lineage-specific manner under certain circumstances and the role of Fc{gamma}RIIb in autoimmunity may depend upon the specific cell types involved.

In summary, the expression profile of human Fc{gamma}RIIb is regulated in both the lymphoid and myeloid compartments not only by its natural gene polymorphisms but also by active autoimmunity exemplified by SLE. Our data further suggest several pathways through which FCGR2B may be an important disease susceptibility gene for SLE and implicate Fc{gamma}RIIb as a potential therapeutic target for human diseases.


   Acknowledgments
 
We thank Drs. G. S. Alarcón, H. Bastian, W. Chatham, and B. Fessler for identification assessment of SLE patients and Susan Kimberly for assistance in patient recruitment.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported in part by Grants P01 AR49084, R01 AR42476, R01 AR33062, and N01-AI40068 from the National Institutes of Health and in part by the University of Alabama at Birmingham Rheumatic Diseases Core Center (P30 AR48311) and the General Clinical Research Center (M01 RR-00032). Back

2 Address correspondence and reprint requests to Dr. Robert P. Kimberly, University of Alabama at Birmingham, 1530 Third Avenue South, Shelby Interdisciplinary Biomedical Research Building 172D, Birmingham, AL 35294-2182. E-mail address: rpk{at}uab.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; mDC, myeloid DC; pDC, plasmacytoid DC; SLE, systemic lupus erythematosus; SLEDAI, SLE Disease Activity Index; EC, extracellular domain; MFI, mean fluorescence intensity; [Ca2+]i, intracellular Ca2+ concentration; mIgG, mouse IgG; ahIgG, heat-aggregated human IgG; PMN, polymorphonuclear neutrophil. Back

Received for publication February 14, 2006. Accepted for publication December 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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