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The Journal of Immunology, 2008, 180, 793 -799
Copyright © 2008 by The American Association of Immunologists, Inc.
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Live Cell Imaging Reveals that the Inhibitory Fc{gamma}RIIB Destabilizes B Cell Receptor Membrane-Lipid Interactions and Blocks Immune Synapse Formation1

Hae Won Sohn, Susan K. Pierce and Shiang-Jong Tzeng2

Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Fc{gamma}RIIB is a potent regulator of BCR signaling and as such plays a decisive role in controlling autoimmunity. The use of advanced imaging technologies has provided evidence that the earliest events in Ag-induced BCR signaling include the clustering of the BCR, the selective and transient association of the clustered BCR with raft lipids, and the concentration of BCR clusters in an immune synapse. That lipid rafts play a role in Fc{gamma}RIIB’s regulation of BCR signaling was suggested by recent studies showing that a lupus-associated loss of function mutation resulted in the receptor’s exclusion from lipid rafts and the failure to regulate BCR signaling. In this study, we provide evidence from both biochemical analyses and fluorescence resonance energy transfer in conjunction with both confocal and total internal reflection microscopy in living cells that the Fc{gamma}RIIB, when coligated with the BCR, associates with lipid rafts and functions both to destabilize the association of the BCR with raft lipids and to block the subsequent formation of the B cell’s immune synapse. These results define new early targets of Fc{gamma}RIIB inhibitory activity in the Ag-induced B cell activation pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The B cell receptor (BCR)3signaling is initiated following multivalent Ag binding by the phosphorylation of the Ag-clustered BCR by the Src-family kinase Lyn (1, 2). Previous biochemical studies provided evidence that one of the earliest events that accompanies the phosphorylation of the BCR by Lyn is the association of the BCR with detergent insoluble, sphingolipid- and cholesterol-rich membrane microdomains that define lipid rafts (3, 4, 5, 6). Recent studies using fluorescence resonance energy transfer (FRET) to probe the interactions between the BCR and membrane lipids at the nanometer scale in living B cells in the absence of detergent showed that within seconds of Ag binding, the BCR selectively and transiently associated with raft lipids and that this association preceded by several seconds the triggering of Ca2+ (7). A second, recently described early event in Ag-induced B cell activation that follows the BCR’s interaction with Ag on an APC (8) or artificial membrane surface (9) is the formation of an immune synapse. Recent evidence from intravital imaging suggests that B cells engage Ag on the surfaces of APCs (10, 11, 12, 13) and Batista et al. (8) showed that the B cell’s encounter of Ag on an APC in vitro resulted in the formation of an immune synapse at the interface of the engaged B cell and APC. Recent studies using confocal and scanning electron microscopy to investigate the events that follow the B cell’s first encounter with Ag incorporated into a planar lipid bilayer showed that the formation of an immune synapse is preceded by the spreading of the B cell on the Ag-containing bilayer followed by the contraction of the cell, resulting in the collection of the BCR-bound Ag into a central aggregate or immune synapse (9). This Ag collection process that accompanies immune synapse formation was suggested to be critical to the B cell’s ability to discriminate Ag affinities.

Over the last several years, Fc{gamma}RIIB has emerged as a key negative regulator of B cell activation (14). Indeed, deficiencies in Fc{gamma}RIIB result in susceptibility to induced autoimmune disease and, in certain genetic backgrounds, severe autoimmune disease and death (15). When coligated to the BCR by immune complexes, the Fc{gamma}RIIB inhibits BCR signaling by a mechanism that involves the phosphorylation of the tyrosine residues in its ITIM motifs in its cytoplasmic domains by Lyn (16) and the recruitment of the inositol phosphatase SHIP (17).

At present, it is not known whether the Fc{gamma}RIIB regulates either the interaction of the BCR with raft lipids or the subsequent formation of an immune synapse. Earlier studies that explored the role of lipid rafts in the Fc{gamma}RIIB inhibition of BCR signaling showed that following coligation of the Fc{gamma}RIIB to the BCR, the Fc{gamma}RIIB became detergent insoluble as did SHIP (18). Cross-linking Fc{gamma}RIIB was also shown to induce the coalescence and stabilization of lipid raft microdomains independently of Src-family kinase activity by a mechanism that only required the extracellular and transmembrane regions of the Fc{gamma}RIIB (19). The physiological significance of the association of the Fc{gamma}RIIB with lipid rafts in regulating B cell activation was highlighted by recent studies showing that a lupus-associated loss of functional polymorphism in the transmembrane region of the human Fc{gamma}RIIB resulted in the receptor’s exclusion from lipid rafts and failure to regulate BCR signaling (20, 21). This finding strongly suggested that the Fc{gamma}RIIB functions to regulate BCR signaling, at least in part, through a lipid raft-dependent process. The effect of Fc{gamma}RII engagement on immune synapse formation has not been explored. In this study, we provide evidence from both biochemical studies and live cell imaging that both the Ag-induced association of the BCR with raft lipids and the formation of an immune synapse are early targets of Fc{gamma}RIIB regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, cell lines, and Abs

Mouse CH27 cells stably expressing either the recombinant chimeric protein pair Ig{alpha}-cyan fluorescent protein (CFP), containing mouse Ig{alpha} and the monomeric form of CFP and Lyn-yellow fluorescent protein (YFP), containing the first 16 amino acids of mouse Lyn, including the myristoylation and palmitoylation sequence and the monomeric form of YFP were previously described (7) as were Ch27 cells expressing Lyn16-CFP and Ig{alpha}-YFP. B1-8µ transgenic mice, provided by Dr. Mark Shlomchik (Yale University, New Haven, CT), were used to obtain nitrophenyl (NP)-specific splenic B cells. Rabbit Abs specific for mouse G{alpha}i (clone: C-10), Lyn (clone: 44), Igβ (clone: FL-229), and mouse mAbs specific for mouse SHIP (clone: P1C1) were purchased from Santa Cruz Biotechnology. F(ab')2 goat Abs specific for mouse IgG or IgM, F(ab')2 and whole rabbit Abs specific for goat IgG, rabbit Abs specific for mouse IgM and IgG, and HRP-conjugated F(ab')2 goat Abs specific for mouse Ig (H+L) and specific for rabbit IgG were purchased from Jackson ImmunoResearch Laboratory. mAbs specific for mouse IgM (clone: R6–60.2) and CD45 (clone: 6) were purchased from BD Pharmingen. Cy2-conjugated Fabs of rat mAbs specific for mouse IgM (clone II/41) were custom made from rat hybridoma cells (a gift from Dr. Richard Hodes, National Institutes of Health, Bethesda, MD). The phosphotyrosine-specific mouse mAb, 4G10, was purchased from Upstate Biotechnology. Affinity-purified rabbit anti-mouse Ig{alpha} polyclonal Ab (WS2) specific for the cytoplasmic domain (residues 199–219) of Ig{alpha} was generated as described (22). Ab specific for the cytoplasmic domain of mouse Fc{gamma}RIIB (23) was provided by Dr. John Cambier (University of Colorado, Denver, CO).

Confocal imaging, image processing, and FRET analysis

Imaging, image processing, and FRET analysis were performed as described previously (7). In brief, cells were suspended in Medium 199 (Invitrogen Life Technologies) and allowed to attach to poly-L-lysine-coated coverslip chambers (Labtek). The imaging of live cells was performed using a Zeiss 510 Meta confocal microscope equipped with a heated stage, heated air circulation system, and an objective heater adjusted to 37°C (Carl Zeiss). Zeiss Plan-apochromat 63 x oil immersion objective was used exclusively to acquire images. For the time-lapse imaging of BCR alone or BCR-Fc{gamma}RIIB coligation, 200 µl of 200% concentrated stimulating Abs were added to the chamber containing 200 µl of buffer between the fourth and fifth scan and images were obtained in 10 s intervals. Normalized FRET (NFRET) images were obtained and FRET efficiencies were calculated from a region of interest covering the whole plasma membrane in equatorial sections of cells as described (7). BCR cross-linking was accomplished by incubating cells with goat IgG Abs specific for mouse IgM (20 µg/ml) followed by incubation with F(ab')2 rabbit Abs specific for the Fc of goat IgG (40 µg/ml). A lower concentration (20 µg/ml) of F(ab')2 anti-Ig secondary Abs gave similar results. The BCR and Fc{gamma}RIIB were coligated by incubating B cells with goat Abs specific for mouse IgM (20 µg/ml) followed by whole rabbit Abs specific for goat IgG (40 µg/ml). Alternatively, the BCR was cross-linked by incubating cells with a rat mAb specific for mouse IgM (10 µg/ml) followed by goat Abs specific for Fc of rat IgG (20 µg/ml) and then by F(ab')2 rabbit Abs specific for the Fc of goat IgG (40 µg/ml). To coligate the BCR and Fc{gamma}RIIB, B cells were incubated with a rat mAb specific for mouse IgM (10 µg/ml) followed by goat Abs specific for rat IgG (20 µg/ml) followed by whole intact rabbit Abs specific for Fc of goat IgG (40 µg/ml). In each case the Abs were affinity purified to minimize cross reactivity between species. The alternative cross-linking and coligation reagents gave similar results.

Preparation of planar lipid bilayers (PLB)

PLB were prepared that contained biotin lipids to which biotinylated ICAM-1 and BCR and Fc{gamma}RIIB ligands were attached through streptavidin. In brief, phosphorylcholine conjugated BSA PC(10)-BSA (Biosearch Technologies), NP(16)-BSA (24), and the mouse ICAM-1/huFc chimera protein (R&D Systems) were biotinylated with EZ-link Sulfo-NHS-LC-biotin (Pierce). An aliquot of each was labeled with sulfo-NHS-functionalized fluorophores (Molecular Probes) to allow monitoring of mobility of the lipid-anchored proteins in the PLB. Biotin liposomes were prepared by sonication of 1,2-Dioleoyl-sn-Glycero-3-phosphocholine and 1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine-cap-biotin (Avanti Polar Lipids) in a 100:1 molar ratio in PBS at a lipid concentration of 5 mM. The PLBs were formed in Lab-Tek chambers (Nalge Nunc) in which the coverglasses were replaced with nanostrip-washed coverslips. The coverslips were incubated with 0.1 mM biotin liposomes in PBS for 10 min. After washing with 20 ml PBS, the PLB was incubated with 2.5 µg/ml streptavidin for 10 min and excess streptavidin was removed by washing with 20 ml PBS. The PLBs were incubated for 20 min with 0.5 µg/ml biotinylated mouse ICAM-1 and excess ICAM-1 was removed by washing. The streptavidin- and ICAM-1-containing PLBs were incubated with either 4 nM biotinylated PC(10)-BSA or 4 nM biotinylated PC(10)-BSA and 10 nM F(ab')2 fragments of rabbit Abs specific for BSA to cross-link the BCR alone. To coligate the BCR and Fc{gamma}RIIB the streptavidin- and ICAM-1-containing PLBs were incubated with 10 nM rabbit Ab specific for BSA and biotinylated PC(10)-BSA to form Ag-specific immune complexes on the PLB. For the experiments with the splenic B cells, the same amount of NP(16)-BSA was used as Ag instead of PC(10)-BSA. The unbound excess ligands were removed by washing with 20 ml PBS. The mobility of ICAM-1, PC(10)-BSA, and the ICs in the PLBs was checked by analysis of the proteins labeled with fluorescent dyes.

Total internal reflection fluorescence microscopy (TIRFM) imaging and image analysis

Through-lens TIRFM was performed on an inverted Olympus IX-81 microscope equipped with an Olympus 60 x 1.45 N.A. or a Zeiss 100 x 1.4 objective. A 442 nm laser (Melles Griot) was used for CFP and FRET excitation and images were acquired simultaneously by using a dual image splitter (MAGS Biosystems) equipped with a 505 dichroic beamsplitter and HQ485/30 (CFP) and HQ560/50 (FRET) emission filters (Chroma Technology). The 514 nm line from an argon gas laser (Laser Physics) was used to excite YFP and images were acquired through the same dual image splitter with the HQ560/50 filter. Images were captured with an electron-multiplier charge-coupled device camera (Cascade II, Photometrics). FRET images obtained by TIRFM were analyzed with the sensitized acceptor emission method as described above for confocal microscopy except that FRET was expressed as a FRET ratio normalized with YFP intensity (FRa). For FRa imaging, the simultaneously acquired CFP-FRET images were split into single CFP and FRET images and aligned using the Image Pro Plus software package (Media Cybernetics). The 1.0-micron polystyrene beads that fluoresce in both the CFP and FRET channel (Molecular Probes) were used before each experiment as an alignment reference tool. CFP, FRET, and YFP images were flattened for background and smoothed by a Gauss filter method using Image Pro Plus. Correction factors for CFP bleed-through (β) and YFP cross-talk ({gamma}) in the FRET channel were obtained from single CFP- or YFP-expressing cells present in the same image fields with the experimental cells. FRa was calculated by the following equation: FRa = (FRET -β*CFP -{gamma}*YFP)/{gamma}*YFP. In this TIRFM system, YFP emission bleed-through to the CFP channel during YFP excitation was negligible. For the quantitative data analysis of the immune synapse formation in splenic B cells, TIRF images were acquired at 10 min after cell contact on the PLBs. The relative fluorescence intensities on the line spanning 100 pixel lengths across the surface contact area and the relative distance normalized for the diameter of contact area were obtained using Matlab R2006a (The MathWorks) and the relative fluorescence intensities to the normalized distance plotted using Sigma Plot (Systat Software). For statistic analyses, Student’s t tests were performed and p values were displayed for the significance of difference.

Lipid raft isolation, immunoprecipitation, and immunoblotting

The lipid rafts were isolated as previously described by detergent solubilization and sucrose density gradient separation (4). For immunoprecipitation, 100 µl of each of the detergent insoluble fractions (4, 5, 6) and soluble fractions (10, 11, 12) were pooled. An equal volume of ice-cold 2x lysis buffer was added to a final concentration of 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 150 mM NaCl, 2.5 mM EDTA, 10 mM Tris-HCl (pH 7.4) for 20–30 min on ice before incubating lysates with the specific Abs (2–4 µg) and then protein A or G beads (40–60 µl 50% slurry) at 4°C overnight. The beads were washed three times with radioimmunoprecipitation assay lysis buffer and the bound proteins were eluted by boiling for 5 min in SDS sample buffer. The immunoprecipitates were subjected to SDS-PAGE and immunoblotting probed with the specific Abs indicated detected using HRP-conjugated secondary Abs visualized with ECL (Amersham Pharmacia Biotech) on x-ray films (Kodak).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The Fc{gamma}RIIB blocks the association of the BCR with detergent-insoluble raft lipids

The effect of coligating the Fc{gamma}RIIB to the BCR on the BCR’s Ag-induced association with raft lipids was characterized biochemically in a CH27 B cell line that was also appropriate for FRET confocal microscopy. In addition to the endogenous BCR, this CH27 B cell line also expressed an Ig{alpha}-chain containing in its cytoplasmic domain the FRET acceptor yellow fluorescent protein (Ig{alpha}-YFP). In addition, the cells expressed the FRET donor, CFP, that contained the first 16 residues of the Src-family kinase Lyn (Lyn16-CFP), resulting in its myristoylation and palmitoylation and targeting to the detergent insoluble fraction of the inner leaflet of the plasma membrane (7). Our previously published studies showed that Ig{alpha}-CFP assembles with Igβ- and µ-heavy chains in a stoichiometry of 1 Ig{alpha}:1 Igβ:2 µ-heavy chains to form a functional cell surface expressed BCR (7, 24).

In unstimulated cells, the endogenous Ig{alpha} and Igβ and Ig{alpha}-YFP were present entirely in the detergent soluble fractions (Fig. 1A). The soluble fractions contained CD45 and under these solubilization conditions excluded to some extent the lipid raft markers Lyn, G{alpha}i and Lyn16-CFP (Fig. 1B). Cross-linking the BCR alone resulted in a shift of the endogenous Ig{alpha} and Igβ and Ig{alpha}-YFP into the detergent-insoluble fractions (Fig. 1A) that contained Lyn, G{alpha}i, and Lyn16-CFP (Fig. 1B). Coligating the BCR and the Fc{gamma}RIIB significantly blocked the association of Ig{alpha}, Igβ, and Ig{alpha}-YFP with the detergent insoluble fractions, resulting in only a small fraction of the BCR protein associated with the raft fractions (Fig. 1A). The effect of coligating the Fc{gamma}RIIB with the BCR on the association of the BCR with raft lipids did not appear to be a consequence of an overall, global perturbation of lipid rafts as the detergent solubility of both the raft markers, Lyn, G{alpha}i, Lyn16-CFP, and nonraft marker CD45 were not altered under either the cross-linking or coligation conditions (Fig. 1B).


Figure 1
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  		FIGURE 1. The Fc{gamma}RIIB blocks the association of the BCR with detergent-insoluble lipid raft membranes. A, Mouse CH27 cells (2 x 108) expressing Ig{alpha}-YFP and Lyn16-CFP were either untreated or treated to either cross-link the BCR (BCR X-linked) or to coligate the BCR and Fc{gamma}RIIB (BCR-Fc{gamma}RIIB coligated) as described in Materials and Methods. The cells were washed, lysed in ice-cold buffer containing 0.5% Triton X-100, and the detergent lysates were separated on a sucrose density gradient. The gradient fractions containing the detergent-insoluble (lanes 1 and 2) and detergent-soluble membranes (lanes 3 and 4) were subjected to immunoprecipitation (IP) and immunoblotting (IB) as follows: Ig{alpha}: IP anti-Ig{alpha}, IB anti-Ig{alpha}; Igβ: IP anti- Ig{alpha}, IB anti-Igβ; Ig{alpha}-YFP: IP anti-Ig{alpha}, IB anti-GFP; Fc{gamma}RIIB: IP anti-Fc{gamma}R (2.4G2), IB anti-Fc{gamma}RIIB (cytoplasmic domain). B, A 25-µl sample of each of the gradient fractions in A was subjected to SDS-PAGE and immunoblotting probing with Abs to CD45, Lyn, G{alpha}i, or GFP.

 
It was also of interest to determine the membrane microenvironment of the clustered Fc{gamma}RIIB. In untreated cells or cells in which the BCR was cross-linked, the Fc{gamma}RIIB was entirely in the soluble membrane fractions (Fig. 1A). A portion of the Fc{gamma}RIIB became detergent insoluble when it was coligated to the BCR (Fig. 1A). Thus, the conditions under which the Fc{gamma}RIIB blocked the BCR’s association with detergent insoluble membranes resulted in the association of a portion of the Fc{gamma}RIIB with insoluble membranes.

Coligating the Fc{gamma}RIIB and the BCR blocks the association of the BCR with raft lipids as measured by FRET confocal microscopy in living cells

The results of the biochemical analysis of the detergent solubility of the BCR and Fc{gamma}RIIB above indicated a change in the local lipid environment of the BCR during signaling that was blocked by coligation with Fc{gamma}RIIB. To further explore the effect of the Fc{gamma}RIIB on the association of the BCR with lipid rafts, we took advantage of high-resolution FRET imaging to quantify BCR-raft lipid interactions in live B cells over the time and length scales necessary to capture the earliest events in B cell activation (24, 25, 26). We compared the interaction of the BCR and raft lipids in cells that expressed Ig{alpha}-CFP and the raft lipid marker Lyn16-YFP by FRET confocal microscopy following cross-linking of the BCR alone or coligation of the BCR and Fc{gamma}RIIB. FRET between CFP and YFP was quantified by sensitized acceptor emission using a Zeiss Meta 510 spectral laser scanning confocal microscope to collect fluorescence from three channels: namely, CFP (CFP excitation and CFP emission), FRET (CFP excitation and YFP emission), and YFP (YFP excitation and YFP emission) from a region of interest selected as the plasma membrane.

As shown previously (7), FRET between Ig{alpha}-CFP and Lyn16-YFP increased immediately, within 10 s, following BCR cross-linking alone and then gradually decreased, resulting in background levels of FRET by 150 s. The FRET images of the cells taken every 30 s are shown and the quantitation of NFRET for all images taken every 10 s is also provided (Fig. 2A). The maximal change in FRET for several individual cells following BCR cross-linking and BCR-Fc{gamma}RIIB coligation is given, showing that the difference in FRET between the BCR and the lipid raft probe is highly significant (Fig. 2B). Similar studies were conducted using a CH27 cell line expressing Ig{alpha}-YFP and Lyn16-CFP. The quantitation and averaging of the images from several cells showed that the coligation of the BCR and the Fc{gamma}RIIB completely blocked BCR FRET with raft lipids (Fig. 2C). Taken together with the biochemical evidence presented above, these results provide evidence that coligating the Fc{gamma}RIIB to the BCR blocks or destabilizes the interaction of the BCR with raft lipids.


Figure 2
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  		FIGURE 2. Quantitative FRET imaging reveals Fc{gamma}RIIB-mediated inhibition of BCR association with lipid rafts in live B cells. A, Cells expressing Ig{alpha}-CFP and Lyn16-YFP were stimulated at 37°C with Abs (40 µg/ml) to cross-link the BCR alone or to coligate the BCR and Fc{gamma}RIIB. Shown are merged images of CFP and YFP fluorescence (upper) and NFRET (lower) as a percentage scale in a color-coded mode displayed at 30-s intervals of a representative cell after BCR cross-linking (BCR X-linked) or BCR and Fc{gamma}RIIB coligation (BCR-Fc{gamma}RIIB). NFRET were calculated from the region of interest taken as the plasma membrane at 10-s intervals after the addition of Abs at time 0 to cross-link the BCR (left axis) or to coligate the BCR and Fc{gamma}RIIB (right axis). Scale bar, 10 µm. B, Fc{gamma}RIIB cross-linking blocks FRET between BCR and raft lipids. Shown is the maximum increase in percent FRET occurring between 0 and 120 s in individual cells expressing Ig{alpha}-CFP and Lyn16-YFP following BCR cross-linking alone (BCR X-linked) (Figure 2) and BCR-Fc{gamma}RIIB coligation (BCR-Fc{gamma}RIIB coligated) ({triangledown}). The mean of the distribution is given by bars. The p values from t tests comparing the FRET values obtained from cells for each experimental condition to FRET values obtained from cells treated to cross-link the BCR alone are also given. Shown are the results from 10 to 15 cells from one of three independent experiments. Similar results were seen using CH27 cells from expressing Ig{alpha}-YFP and Lyn16-CFP. C, NFRET in cells expressing Lyn16-CFP and Ig{alpha}-YFP with time after the addition of Abs to cross-link the BCR alone (•) compared with NFRET in cells expressing Lyn16-CFP and Ig{alpha}-YFP with time after the addition of Abs to coligate the BCR and the Fc{gamma}RIIB ({circ}). Each point represents the means + SD for 16 cells for BCR cross-linking alone and 13 cells for BCR-Fc{gamma}RIIB coligation.

 
Coligating the Fc{gamma}RIIB and BCR blocks the interaction of the BCR with raft lipids and the formation of an immune synapse following B cell engagement of Ag on a planar lipid bilayer

To determine the effect of the ligation of the Fc{gamma}RIIB on the interaction with raft lipids and BCR’s concentration of Ag and immune synapse formation, we turned to TIRFM in conjunction with FRET, an imaging technique that allowed the interaction of individual Ag-induced BCR clusters with a raft lipid probe to be resolved. CH27 B cells expressing Ig{alpha}-YFP and Lyn16-CFP were placed on a planar lipid bilayer that contained lipid anchored ICAM-1 and either the BCR Ag, PC(10)-BSA, alone, to engage the BCR or PC(10)-BSA and intact Abs specific for BSA, forming Ag-containing immune complexes, to coligate the BCR and Fc{gamma}RIIB (Fig. 3A). Alternatively, the BCR was cross-linked using PC(10)-BSA and F(ab')2 BSA-specific Abs with identical results to PC(10)-BSA cross-linking alone. The B cells first contacted the bilayer at several points (0.5 min frame) that likely represent B cell membrane protrusion reaching for the bilayer. The B cell membranes in these contact points contain both Lyn16-CFP and Ig{alpha}-YFP and notably are in close molecular proximity and show FRET. A merged image of the FRa and Ig{alpha}-YFP images at 0.5 min showed that the FRa and Ig{alpha}-YFP overlap indicating that the majority of BCRs in the initial contact points of the B cell with the bilayer are in close molecular proximity with the raft lipid probe, Lyn16-CFP. By 2 min, the B cell spread on the bilayer and the B cell membrane in contact with the bilayer contained both Lyn16-CFP and Ig{alpha}-YFP. FRET was detected between CFP and YFP. The merged image of FRa and YFP shows that the majority of Ig{alpha}-YFP overlaps with FRa indicating close molecular proximity of the majority of BCR and the raft lipid probe, Lyn16-CFP. By 10 min, the B cell is fully spread on the bilayer and the Ig{alpha}-YFP is more concentrated in the center of the contact area. FRET is detected, but a merged image of Ig{alpha}-YFP and FRa shows considerable FRET in the periphery outside the area where the BCR is most concentrated (Fig. 3A and supplementary video 1)4, presumably where BCRs continue to make their first contact with Ag. Both the movement of the BCR to the center to form an immune synapse and the interaction with the raft lipid probe as measured by FRET required Ag. In control experiments in which B cells were placed on lipid bilayer containing ICAM-1 alone, no FRET was detected and the BCR did not cluster in an immune synapse (data not shown).


Figure 3
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  		FIGURE 3. Fc{gamma}RIIB blocks BCR-raft lipid interactions as imaged by TIRFM in combination with FRET. B cells expressing Lyn16-CFP and Ig{alpha}-YFP were incubated on lipid bilayers containing either ICAM-1 and Ag, PC(10)-BSA alone or ICAM-1 and PC(10)-BSA and BSA-specific Abs to form Ag-containing ICs to coligate the BCR and the Fc{gamma}RIIB. ICAM-1, PC(10)-BSA, and the ICs were shown to be mobile in the bilayers by analysis of fluorescently labeled ICAM-1 and PC(10)-BSA. CFP, YFP, and FRET images were acquired every 2 s for 10 min by objective-type TIRFM on an inverted IX-81 Olympus microscope equipped with a 60 x 1.45 N.A. objective and laser TIRF illumination optics as detailed in Materials and Methods. A, Shown are individual frames of Lyn16-CFP, Ig{alpha}-YFP, and FRET efficiency expressed as FRa at 0.5, 2, and 10 min after addition of the B cells to the bilayers. Also shown are the merged FRa and YFP images at the same time points. B, The quantitative analyses of FRET efficiencies at 5–10 min after B cells contacted the bilayers. B cells were incubated with the bilayers containing ICAM-1, PC(10)-BSA, and F(ab')2 rabbit Abs specific for the BSA to cross link the BCR. To coligate BCR and Fc{gamma}RIIB, B cells were placed on bilayers that contained ICAM-1, PC(10)-BSA, and intact BSA-specific Abs to form Ag-containing ICs. Each data point represents the average FRa value of a single cell obtained from FRa images as described above (135 cells for PC(10)-BSA and F(ab')2 rabbit Abs specific for the BSA; 120 cells for PC(10)-BSA and intact BSA-specific Abs). The significance of difference was analyzed by paired t test.

 
The FRET pattern between the BCR and raft lipids for B cells placed on the ICAM-1 and immune complex containing-bilayers to coligate the BCR and Fc{gamma}RIIB differed dramatically from that on the bilayers that contained ICAM-1 and Ag. Although the cells contacted and spread on these lipid bilayers, no FRET was detected between the BCR Ig{alpha}-YFP and Lyn16-CFP at any time (Fig. 3A and supplementary video 2). The quantification of the FRa for a large number of cells in which the BCR was cross-linked vs coligated to the Fc{gamma}RIIB showed that the difference in FRa was highly significant (Fig. 3B).

In addition to the failure to show FRET, the B cells in which the BCR and Fc{gamma}RIIB were coligated appeared to fail to concentrate the BCR clusters in the center of the contact area in an immune synapse. Rather, the BCR clusters remained in the periphery of the contact area. The distribution of the BCR and the formation of an immune synapse in cells in which the BCR was cross-linked vs coligated to the Fc{gamma}RIIB were explored further. TIRFM of the Ig{alpha}-YFP-expressing CH27 cells showed the initial contact (1 min), spreading (2–3 min), and immune synapse formation (4–5 min) of the B cell on an Ag coated planar lipid bilayer (Fig. 4A). The behavior of the BCR on the immune complex (IC)-containing bilayer was dramatically different. The BCR appears to cluster and to trigger the spreading response (beginning at 2 min) but the clusters never accumulated in the center to form an immune synapse. Rather, the BCR clusters remain in the periphery of the cell. The percent of cells that formed immune synapses was determined under conditions where the BCR was cross- linked with PC(10)-BSA alone or as a control with PC(10)-BSA plus F(ab')2 anti-BSA and the BCR and the Fc{gamma}RIIB were coligated using PC(10)-BSA and intact anti-BSA Abs. Several hundred cells in randomly selected fields were examined. The results show that a significantly smaller percent of cells formed immune synapses when the BCR was coligated to the Fc{gamma}RIIB vs cross-linked (Fig. 4B). The effect of coligation of the BCR to the Fc{gamma}RIIB was also analyzed in mouse splenic B cells (Fig. 5). The distribution of the fluorescence across the cell was determined and the average for several cells is given. The fluorescence patterns for cells in which the BCR was cross-linked vs coligated to the Fc{gamma}RIIB were significantly different indicating an inability of B cells to form a synapse when the BCR was coligated to the Fc{gamma}RIIB.


Figure 4
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  		FIGURE 4. BCRs fail to form immune synapses when coligated to the Fc{gamma}RIIB. CH27 B cells expressing Ig{alpha}-YFP were incubated on bilayers to cross-link BCR alone or to coligate the BCR and Fc{gamma}RIIB. A, Shown are TIRFM images obtained every 2 s and the individual images at 1, 3, and 5 min after the B cells contacted bilayers containing ICAM-1 and PC(10)-BSA to cross link the BCR or PC(10)-BSA and intact BSA-specific Abs to colligate the BCR and the Fc{gamma}RIIB. Real-time videos (33 frames/s) of the cells are also available in supplementary video 3 for BCR and supplementary video 4 for BCR-Fc{gamma}RIIB. The relative fluorescence intensities of Ig{alpha}-YFP across the synapse (yellow line, horizontal) are also given (bottom). B, The quantitative analyses of the percent of cells that formed immune synapses between 5 and 10 min after the cells contacted the bilayer. For BCR cross-linking, cells were incubated with the bilayers containing ICAM-1 and PC(10)-BSA alone (Ag) or as a control PC(10)-BSA and F(ab')2 rabbit Abs specific for BSA (Ag + F(ab')2 anti-BSA). For BCR and Fc{gamma}RIIB coligation, bilayers contained ICAM-1, PC(10)-BSA, and intact BSA-specific Abs (Ag + IgG anti-BSA). The percent of cells showing failure of immune synapse formation in randomly selected fields (n = 200–300) are given as mean + SEM from five different experiments.

 

Figure 5
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  		FIGURE 5. The effect of Fc{gamma}RIIB coligation on the immune synapse formation in primary B cells. Mouse splenic B cells from B1-8µ transgenic mice which express a NP-specific IgM BCR were used to quantitate the effect of Fc{gamma}RIIB-BCR coligation on the synapse formation. Cells were labeled with Cy2-conjugated Fab of rat Ab specific for mouse IgM and then incubated for 5–10 min on bilayers containing either ICAM1 and NP(16)-BSA to cross-link the BCR or ICAM-1 and NP(16)-BSA and BSA-specific rabbit Abs to coligate the BCR and Fc{gamma}RIIB. Shown are TIRF images of a representative cell in each condition (left) and the plot of the relative fluorescence intensities of IgM vs the relative distance normalized for the diameter of contact area (right). The graph given by mean + SEM was obtained as described in Materials and Methods from 60 BCR-cross-linked cells and BCR-Fc{gamma}RIIB-coligated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The Fc{gamma}RIIB has emerged as an important determinant of autoimmune disease due to its potent regulation of BCR signaling and B cell activation (14). The Fc{gamma}RIIB has been shown to block Ag-induced BCR signaling when coligated to the BCR through Ag-containing immune complexes through a SHIP- and ITIM-dependent mechanism (17). In this study, we provide evidence that coligation of the BCR and Fc{gamma}RIIB interferes with one of the earliest events in BCR signaling, namely the association of the BCR with raft lipids and the subsequent formation of an immune synapse.

The potential physiological significance of the observation presented in this study that Fc{gamma}RIIB perturbs the association of the BCR with raft lipids was provided by two reports of the biochemical analysis of a lupus-associated loss of functional polymorphism of the Fc{gamma}RIIB. This polymorphism was due to a single amino acid interchange (Ile 232 Thr) in the transmembrane region of the Fc{gamma}RIIB (Fc{gamma}RIIBT232). In one study, Fc{gamma}RIIBT232 was shown to be deficient in blocking Fc{gamma}RI triggered functions in monocyte cell lines and in primary human macrophages and BCR-triggered proliferation (21). In myeloid cells, Fc{gamma}RIIBT232 showed membrane proximal signaling defects involving its own phosphorylation and recruitment of the phosphatase SHIP. In a second study, the Fc{gamma}RIIBT232 was shown to be defective in its own phosphorylation and recruitment of SHIP, leading to a failure to block BCR-induced signaling as measured by Akt and PLC{gamma}2 activation and Ca2+ mobilization (20). In both studies, biochemical analyses of the detergent solubility of the FcRs showed that the lupus-associated allele was excluded from lipid rafts. The authors (20, 21) concluded that the failure of Fc{gamma}RIIBT232 to associate with lipid rafts resulted in a failure in its inhibitory function. Moreover, it was speculated that the expression of Fc{gamma}RIIBT232 would reduce the threshold for Ag presentation by dendritic cells, compromising peripheral tolerance and facilitating the activation of autoreactive B cells (21). Together, these alterations in immune function would predispose individuals to the development of SLE.

At present, we do not fully understand the mechanisms by which either the BCR or the Fc{gamma}RIIB, when cross-linked, associate with raft lipids or how the association of the Fc{gamma}RIIB with raft lipids perturbs the association of the BCR with raft lipids. Earlier biochemical studies provided evidence that the association of both the Fc{gamma}RIIB and the BCR with lipid rafts was independent of their signaling functions and was simply a repercussion of the physical cross-linking of the receptors. The cross-linked Fc{gamma}RIIB was shown to coalesce lipid rafts independently of Src-family kinase activity in a process that was dependent only on the extracellular and transmembrane domains of the Fc{gamma}RIIB (19). These authors (19) concluded that the lateral coalescence of lipid rafts after Fc{gamma}RIIB cross-linking represented the earliest signal-generating process that triggered the subsequent Fc{gamma}RIIB signaling cascade. Similarly, association of the BCR with lipid rafts was shown to occur in the absence of the initiation of BCR signaling as raft association was not blocked by the Src-family kinase inhibitor PP2 and did not require the Ig{alpha}/Igβ signaling complex of the BCR (22). Subsequent FRET confocal microscopy provided evidence that the association of the BCR with raft lipids occurred within seconds of BCR cross-linking and preceded by 30–40 s the induced Ca2+ flux (7). In this study, we provided evidence that coligating the Fc{gamma}RIIB to the BCR resulted in the association of the Fc{gamma}RIIB with insoluble membranes and in a block in BCR raft association. We speculate that the cross-linked Fc{gamma}RIIB by coalescing raft lipids around itself may alter the dynamics of the plasma membrane so as to preclude BCR coalescence of raft lipids. The raft perturbation defines a new step in the Fc{gamma}RIIB inhibitory effect on B cell activation. Given the evidence cited above, that the Fc{gamma}RIIB’s association with lipid rafts in human cells is critical to its function, it will be of interest to investigate the Fc{gamma}RIIB lipid raft destabilizing function described in this study for mouse B cells for the human Fc{gamma}RIIB.

We also provided evidence that the coligation of the Fc{gamma}RIIB and a BCR blocked the ability of the BCR to form an immune synapse. Recent studies by Fleire et al. (9) suggested that the concentration of Ag-bound BCR clusters in the center of the B cell contact area during immune synapse formation was a critical process in the mechanism by which B cells discern the affinity of an Ag. As the ability to discern the affinity of an Ag is critical to self-nonself discrimination, it may be that the Fc{gamma}RIIB’s block of immune synapse formation plays a role in autoimmune B cell responses. It will also be of interest to determine the relationship between synapse formation and BCR internalization and how the Fc{gamma}RIIB might influence this process.


   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 research was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases. Back

2 Address correspondence and reprint requests to Dr. Shiang-Jong Tzeng, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook II, 12441 Parklawn Drive, Room 213, Rockville, MD 20852. E-mail address: sjtzeng{at}niaid.nih.gov Back

3 Abbreviations used in this paper: BCR, B-cell receptor; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescence protein; NP, nitrophenyl; NFRET, normalized FRET; FRa, FRET ratio normalized with acceptor; TIRFM, total internal fluorescence reflection microscopy; IC, immune complex. Back

4 The online version of this article contains supplemental material. Back

Received for publication July 7, 2007. Accepted for publication October 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Reth, M., J. Wienands. 1997. Initiation and processing of signals from the B cell antigen receptor. Annu. Rev. Immunol. 15: 453-479. [Medline]
  2. Gauld, S. B., J. M. Dal Porto, J. C. Cambier. 2002. B cell antigen receptor signaling: roles in cell development and disease. Science 296: 1641-1642. [Abstract/Free Full Text]
  3. Dykstra, M., A. Cherukuri, H. W. Sohn, S. J. Tzeng, S. K. Pierce. 2003. Location is everything: lipid rafts and immune cell signaling. Annu. Rev. Immunol. 21: 457-481. [Medline]
  4. Cheng, P. C., M. L. Dykstra, R. N. Mitchell, S. K. Pierce. 1999. A role for lipid rafts in BCR signaling and antigen targeting. J. Exp. Med. 190: 1549-1560. [Abstract/Free Full Text]
  5. Petrie, R. J., P. P. M. Schnetkamp, K. D. Patel, M. Awasthi-Kalia, J. P. Deans. 2000. Transient translocation of the B cell receptor and src homology 2 domain-containing inositol phosphatase to lipid rafts: evidence toward a role in calcium regulation. J. Immunol. 165: 1220-1227. [Abstract/Free Full Text]
  6. Aman, M. J., K. S. Ravichandran. 2000. A requirement for lipid rafts in B cell receptor induced Ca2+ flux. Curr. Biol. 10: 393-396. [Medline]
  7. Sohn, H. W., P. Tolar, T. Jin, S. K. Pierce. 2006. Fluorescence resonance energy transfer in living cells reveals dynamic membrane changes in the initiation of B cell signaling. Proc. Natl. Acad. Sci. USA 103: 8143-8148. [Abstract/Free Full Text]
  8. Batista, F. D., D. Iber, M. S. Neuberger. 2001. B cells acquire antigen from target cells after synapse formation. Nature 411: 489-494. [Medline]
  9. Fleire, S. J., J. P. Goldman, Y. R. Carrasco, M. Weber, F. D. Bray, F. D. Batista. 2006. B cell ligand discrimination through a spreading and contraction response. Science 312: 738-741. [Abstract/Free Full Text]
  10. Qi, H., J. G. Egen, A. Y. C. Huang, R. Germain. 2006. Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science 312: 1672-1676. [Abstract/Free Full Text]
  11. Allen, C. D. C., T. Okada, H. L. Tang, J. G. Cyster. 2007. Imaging of germinal center selection events during affinity maturation. Science 315: 528-531. [Abstract/Free Full Text]
  12. Schwickert, T. A., R. L. Lindquist, G. Shakhar, G. Livshits, D. Skokos, M. H. Kosco-Vilbois, M. L. Dustin, M. C. Nussenzweig. 2007. In vivo imaging of germinal centres reveals a dynamic open structure. Nature 446: 83-87. [Medline]
  13. Bergtold, A., D. D. Desai, A. Gavhane, R. Clynes. 2005. Cell surface recycling of internalized antigen permits dendritic cell priming of B cells. Immunity 23: 503-514. [Medline]
  14. Nimmerjahn, F., J. V. Ravetch. 2006. Fc{gamma} receptors: Old friends and new family members. Immunity 24: 19-28. [Medline]
  15. Bolland, S., J. V. Ravetch. 2000. Spontaneous autoimmune disease in Fc{gamma}RIIB-deficient mice results from strain-specific epistasis. Immunity 13: 277-285. [Medline]
  16. Ravetch, J. V., S. Bolland. 2001. IgG Fc receptors. Annu. Rev. Immunol. 19: 275-290. [Medline]
  17. Bolland, S., R. N. Pearse, T. Kurosaki, J. V. Ravetch. 1998. SHIP modulates immune receptor responses by regulating membrane association of Btk. Immunity 8: 509-516. [Medline]
  18. Aman, M. J., A. C. Tosello-Trampont, K. Ravichandran. 2001. Fc{gamma}RIIB1/SHIP-mediated inhibitory signaling in B cells involves lipid rafts. J. Biol. Chem. 276: 46371-46378. [Abstract/Free Full Text]
  19. Kono, H., T. Suzuki, K. Yamamoto, M. Okada, T. Yamamoto, Z. Honda. 2002. Spatial raft coalescence represents an initial step in Fc{gamma}R signaling. J. Immunol. 169: 193-203. [Abstract/Free Full Text]
  20. Kono, H., C. Kyogoku, T. Suzuki, N. Tsuchiya, H. Honda, K. Yamamoto, K. Tokunaga, Z. Honda. 2005. Fc{gamma}RIIB IIe232Thr transmembrane polymorphism associated with human systemic lupus erythematosus decreases affinity to lipid rafts and attenuates inhibitory effects on B cell receptor signaling. Hum. Mol. Genet. 14: 2881-2892. [Abstract/Free Full Text]
  21. Floto, R. A., M. R. Clatworthy, K. R. Heilbronn, D. R. Rosner, P. A. MacAry, A. Rankin, P. J. Lehner, W. H. Ouwehand, J. M. Allen, N. A. Watkins, K. G. C. Smith. 2005. Loss of function of a lupus-associated Fc{gamma}RIIb polymorphism through exclusion from lipid rafts. Nat. Med. 11: 1056-1058. [Medline]
  22. Cheng, P., B. K. Brown, W. Song, S. K. Pierce. 2001. Translocation of the B cell antigen receptor into lipid rafts reveals a novel step in signaling. J. Immunol. 166: 3693-3701. [Abstract/Free Full Text]
  23. Fong, D. C., A. Brauweiler, S. A. Minskoff, P. Bruhns, I. Tamir, I. Mellman, M. Daeron, J. C. Cambier. 2000. Mutational analysis reveals multiple distinct sites within Fc{gamma} receptor IIB that function in inhibitory signaling. J. Immunol. 165: 4453-4462. [Abstract/Free Full Text]
  24. Tolar, P., H. W. Sohn, S. K. Pierce. 2005. The initiation of antigen-induced BCR signaling viewed in living cells by FRET. Nat. Immunol. 6: 1168-1176. [Medline]
  25. Barda-Saad, M., A. Braiman, R. Titerence, S. C. Bunnell, V. A. Barr, L. E. Samelson. 2005. Dynamic molecular interactions linking the T cell antigen receptor to the actin cytoskeleton. Nat. Immunol. 6: 80-89. [Medline]
  26. Zal, T., M. A. Zal, N. R. J. Gascoigne. 2002. Inhibition of T cell receptor-coreceptor interactions by antagonist ligands visualized by live FRET imaging of the T-hybridoma immunological synapse. Immunity 16: 521-534. [Medline]



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