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The Journal of Immunology, 2001, 166: 736-740.
Copyright © 2001 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Differential Sequestration of Plasma Membrane-Associated B Cell Antigen Receptor in Mature and Immature B Cells into Glycosphingolipid-Enriched Domains1

James B. Chung*, Mark A. Baumeister{dagger} and John G. Monroe2,{dagger}

* Division of Rheumatology, Department of Medicine and {dagger} Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glycosphingolipid-enriched domains (GEDs) are believed to act as platforms for transduction of B cell Ag receptor (BCR)-induced signals from the cell surface. We sought to study whether differential sequestration of BCR into GEDs may contribute to the described intrinsic signaling differences between mature and immature B cells. In this study we found that mature B cells copolarize the BCR with GEDs following BCR aggregation, whereas transitional immature B cells do not. Although anti-BCR treatment leads to receptor aggregation by immature stage B cells, the aggregated complexes do not colocalize with GEDs. We found this difference to be independent of the isotype of the receptor, thereby associating this difference in BCR-GED colocalization to the developmental stage of the B cell. These findings suggest a structural basis for the developmentally regulated differences observed in Ag receptor-mediated signal transduction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
B cell Ag receptor (BCR)3 cross-linking and aggregation in immature B cells has been shown to lead to cell cycle arrest (1) and death by apoptosis (2) in vitro. In contrast, the same stimulus in mature B cells leads to activation and proliferation (3). This differential response to BCR-induced signaling is believed to purge the B cell repertoire of the potentially self-reactive BCR-bearing cells as they are developing (4, 5), while allowing fully mature B cells to take part in effector functions. Studies suggest that intrinsic signaling differences between immature and mature B cells may account for these varied functional responses. For example, although mature B cells respond to BCR cross-linking by increasing phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and elevating intracellular calcium levels, immature B cells increase intracellular calcium levels in the relative absence of PIP2 hydrolysis (6, 7). Mature and immature B cells also differ in their BCR-induced expression of c-myc and B7.2 (8) (J. B. Chung, R. A. Sater, M. L. Fields, J. Erikson, and J. G. Monroe, manuscript in preparation). The mechanism of these developmental differences in signal transduction is not well understood.

It is increasingly appreciated that cell signaling occurs within the context of discrete, specialized domains on the cell membrane. Recently identified glycosphingolipid-enriched domains (GEDs), commonly referred to as lipid rafts, are proposed to function as platforms for signal transduction and membrane trafficking (9). Isolated based on their insolubility in nonionic detergents such as Triton X-100, and buoyant density on sucrose gradients (10), a variety of proteins involved in lymphocyte signal transduction have been shown to be associated with them. Src family protein tyrosine kinases Lck, Lyn, and Fyn (11), and the Zap-70 family protein tyrosine kinase, Syk (12), as well as lipids involved in signal transduction such as PIP2 (13) have been described as residents in GEDs. Receptor ligation results in the recruitment and clustering of various signal transduction proteins, a process that is presumed to lead to activation and subsequent downstream signaling (14, 15). Upon cross-linking, BCR in the mouse B cell lymphoma CH27 rapidly translocated into the buoyant density fractions containing the phosphorylated form of Ig{alpha} and the Src family kinase, Lyn. The phosphatase CD45R was found to be excluded, thus optimizing signaling activity in these defined platforms (16). Weintraub et al. recently made the intriguing observation that tolerant B cells from the anti-hen egg lysozyme (HEL)-soluble HEL double-transgenic mice fail to efficiently partition the BCR into detergent-insoluble cell fractions, leading to reduced tyrosine kinase activation and calcium flux in response to Ag (17). These findings led us to hypothesize that the differences in signal transduction between immature and mature B cells may, in part, be explained by developmentally regulated translocation of the BCR into GEDs upon surface ligation. We sought to directly observe, through immunofluorescence microscopy of primary B cells, the ability of BCR in mature and immature B cells to colocalize with the specialized lipid domains.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell preparation

Female BALB/c mice were used between the ages of 8 and 12 wk for all experiments. Mature and transitional B cells were purified from the spleens of unirradiated mice or mice 13 or 14 days after sublethal irradiation with 500 rad, respectively, as previously described (5, 18). We used 493 Abs (19) to deplete immature B cells from unirradiated adult spleens. Typical preparations were 90–95% pure for B cells.

Abs used in anti-BCR stimulation and immunofluorescence microscopy

Rabbit anti-IgM F(ab')2 prepared from IgG collected from rabbits immunized with an IgMa Ab as previously described (20), rat anti-mouse IgM (B76, rat IgG1, µ specific), and mouse anti-mouse IgD (IgG2b, {delta} specific). The following secondary Abs were purchased from Jackson ImmunoResearch (West Grove, PA): AMCA-conjugated F(ab')2 donkey anti-rabbit IgG, AMCA-conjugated F(ab')2 donkey anti-rat IgG, and AMCA-conjugated F(ab')2 goat anti-mouse IgG (Fc{gamma} fragment specific).

Fluorescence microscopy and data analysis

Purified B cells (107/ml) incubated with cholera toxin B-FITC (Sigma, St. Louis, MO) at 8 µg/ml on ice for 20 min. After washing in PBS, the cells were resuspended in ice-cold RPMI 1640. The cells (106) were added to FACS tubes and warmed to 37°C for 20 min. Anti-Ig was added for the indicated times, and the reaction was stopped with the addition of cold PBS/0.1% BSA/0.02% azide (staining buffer). The cells were incubated with AMCA-conjugated secondary Abs and fixed in 1% formaldehyde. The cells were loaded onto CytoSpin (Shandon, Pittsburgh, PA) for adherence onto glass slides then mounted with ProLong (Molecular Probes, Eugene, OR) antifade agent. An epifluorescence microscope (Leica, Deerfield, IL) was used to view the samples. Images were captured using OpenLab image analysis software (Improvision). For Fig. 1Go, the confocal imaging module of the Open Lab software was used to remove out-of-focus portions of the image using a mathematical "point spread function" algorithm. The experiments were performed in triplicate and independently confirmed by another investigator blinded to the identity of the samples. The mean values among the various groups were compared using JMPIN 3 statistical software (SAS Institute, Cary, NC) and a Student’s t test was performed for each pair to calculate the p value.



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  		FIGURE 1. Immature and mature B cells differentially sequester ligated BCR into GEDs after anti-BCR treatment. Representative immunofluorescent images of sIg and GEDs on immature and mature B cells before and 5 min after treatment with anti-BCR.

 
Flow cytometry

Cytometry was performed with Abs according to standard techniques using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA). The following Abs were used: PE-conjugated antiheat-stable Ag (anti-HSA; PharMingen, San Diego, CA), APC-conjugated anti-B220 (PharMingen), FITC-conjugated anti-IgDa (PharMingen), biotinylated anti-IgM (µ specific, B76) followed by streptavidin:Red-670 (PharMingen).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mature and immature B cells differ in their association of BCR with GED after anti-BCR-induced polarization

Peripheral immature (transitional) B cells are HSAhigh and surface IgM (sIgM)high and comprise 5–20% of the adult splenic B cell population (4). We isolated an enriched population of newly generated immature B cells by sublethally irradiating BALB/c mice. The spleens of these mice autoreconstitute in a uniform wave such that by day 13 or 14, ~98% of B220+ cells that result are identical with the HSAhigh cells of the normal adult spleen (4).

We used immunofluorescence microscopy to examine the relative locations of the BCR with respect to GEDs before and after anti-BCR stimulation. We labeled the primary anti-BCR used to bind the sBCR with AMCA-conjugated secondary Abs, and visualized GEDs with the GM1-binding cholera toxin B subunit conjugated to FITC (CTxB-FITC). Without anti-BCR stimulation, mature and immature B cells exhibited a relatively homogeneous surface expression pattern of BCR (Fig. 1Go). When mature B cells were incubated with anti-BCR for 5 min at 37°C, the sBCR polarized to the same location as the corresponding polarized GEDs. In contrast, immature B cells also polarized the BCR after anti-BCR treatment but did not copolarize GEDs to the same area in an analogous manner (Fig. 1Go).

Although immunofluorescence allows an examination of colocalization at the individual cell level, there is heterogeneity in the individual B cells’ responses to experimental conditions. These may be due to the existence of subsets and contaminating cells within our purified mature or immature B cell populations. For example, although the autoreconstituted spleen is comprised of >98% HSAhigh immature B cells, the mature B cell preparation contains a small but nevertheless significant population of HSAhigh expressing immature B cells (Fig. 2GoA). Even among the HSAhigh immature B cell population, the existence of distinct subpopulations is increasingly appreciated (21). Therefore, we performed the experiment in triplicate and examined >100 B cells at high power magnification for each experimental sample to more rigorously quantify our immunofluorescence image data.



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  		FIGURE 2. Quantifying the differences in BCR-GED copolarization between mature and immature B cells. A, The purity of immature and mature B cells in the B cell preparations used in the experiment. HSA staining of B220+ cells shows that almost all of the immature B cells exhibit high levels of HSA expression, whereas the mature B cell preparation contains mostly HSAlow cells. However, there is a small but significant percentage of immature HSAhigh cells in the mature B cell preparation. B, Percentage of immature and mature B cells that formed BCR caps within 1 and 5 min after treatment with anti-Ig. Error bars represent the SEM from triplicate experiments. C, Of the B cells that formed caps in (B), the percentage in which the GED colocalized with the BCR cap.

 
BCR polarization occurred as early as 1 min after ligation of the BCR in both the mature and immature B cells (Fig. 2GoB). The percentage of BCR polarization in the two populations was similar, whereas the degree of BCR-GED copolarization after anti-BCR treatment varied significantly between mature and immature B cells. Of the mature B cells that polarized the Ag receptor in response to incubation with anti-BCR, a mean of 69 and 76% showed the copolarization of the BCR with GEDs by 1 and 5 min, respectively (Fig. 2GoC). Of the immature B cells in which BCR polarization occurred, a mean of 34% at 1 min and 19% at 5 min was found to exhibit BCR-GED copolarization.

By 30–60 min after anti-BCR stimulation, the morphology of the polarized BCR had changed dramatically on the surface of mature and immature B cells. Most of the sBCR were endocytosed, leaving only a punctate pattern of BCR staining. Nevertheless, the small area of BCR staining corresponded to polarized GEDs in mature B cells but not in immature B cells (Fig. 3Go).



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  		FIGURE 3. BCR-GED coaggregates are maintained after 60 min of anti-BCR treatment in mature B cells. Representative immunofluorescence images of BCR and GEDs in immature and mature B cells 60 min after anti-BCR treatment. Note the small, punctate pattern of BCR expression that colocalizes to GED aggregates in mature, but not in immature, B cells.

 
Sequestration of BCR into GEDs is independent of Ag receptor isotype

Complicating the analysis of these anti-BCR-mediated studies is the disproportionate expression of the IgM and IgD forms of the BCR on mature and immature B cells. Immature B cells are initially IgM+IgD- then progressively acquire IgD as they pass through the transitional immature compartments. Coincident with the 5- to 10-fold increase in IgD levels, the IgM levels begin to decline as the B cells become fully mature (4). The correlation between changes in IgM and IgD expression and decreased susceptibility to tolerance induction suggests that there may be isotype-specific signaling differences that lead to tolerance induction or protection. Indeed, Gold et al. found that in mature splenic B cells that express both IgM and IgD, cross-linking either IgM or IgD stimulated tyrosine phosphorylation only of the MB-1 related proteins associated with that receptor (22). And although CH33 and WEHI-231, both B cell lymphoma cell lines, undergo growth arrest and apoptosis in response to IgM stimulation, stimulation through IgD in {delta}-transfectants protected the cells from the same fates (23, 24, 25). In opposition to this view, anti-HEL-transgenic mice expressing either IgM or IgD retain the ability to generate activation to foreign Ags as well as tolerance to self-Ags (26). Previous work in our laboratory has supported the latter view by showing that both sIgM- and sIgD-mediated signals induce apoptosis of immature and activation of mature murine B cells (5).

To confirm that the difference in BCR-GED copolarization was also dependent on the maturational state of the B cell and not on the Ig isotype associated with the BCR, we separately ligated sIgM or sIgD on the mature or transitional immature B cell preparations. Importantly, as previously shown (4, 5), both B cell populations express both forms of the BCR. But as discussed above, the relative levels of the two forms differs between these two stages of development. Selectively ligating sIgM showed that copolarization with GEDs was still dependent upon the maturation state of the B cell. Treatment of immature B cells with µ-specific mAb did not lead to the same degree of copolarization with GEDs as was observed for mature B cells (Fig. 4GoA). Although we treated both immature and mature B cells with {delta}-specific anti-Ig, we were only able to visualize BCR polarization and copolarization with GEDs in mature B cells (Fig. 4GoB). This is likely due to the low level of IgD expression on immature B cells. Flow cytometry analysis showed that although sIgM levels are higher in immature B cells compared with mature B cells, and sIgD levels are higher in mature compared with immature B cells, the relative differences in the levels of expression of the two isotypes are far greater for sIgD (Fig. 5GoA). Quantifying the degree of sIgM-GED copolarization 5 min after ligation with µ-specific Abs showed that the difference in copolarization was not isotype specific but dependent on the maturational state of the B cell (Fig. 5GoB). {delta}-specific sIgD ligation in mature B cells led to a high degree of BCR-GED copolarization but, as previously noted, we could not assess the expression pattern of IgD on immature B cells due to their low level of expression.



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  		FIGURE 4. Sequestration of BCR into GEDs is independent of Ag receptor isotype. Representative immunofluorescent images of sIg and GEDs on immature and mature B cells before and after treatment for 5 min with µ-specific (A) and {delta}-specific (B) anti-Ig.

 


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  		FIGURE 5. Quantifying the Ig isotype-independent copolarization of BCR with GEDs in mature and immature B cells. A, Flow cytometry overlap histogram of sIgM and sIgD on immature and mature B cells. Immature B cells express higher levels of sIgM and lower levels of sIgD compared with mature B cells. The relative differences between immature and mature B cells are greater for IgD than for IgM. B, Of the immature and mature B cells that formed BCR caps 5 min after treatment with µ- or {delta}-specific anti-Ig, the percentage in which BCR-GED colocalization was observed. sIgD capping could not be visualized in immature B cells; therefore, the degree of colocalization could not be quantified (*).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The lateral organization of plasma membranes allows those proteins involved in signal transduction to be confined spatially within different lipid domains, and a model is emerging for signal transduction in which the signaling elements are aided by a structural framework. GEDs probably function to concentrate the surface receptors and ligands with the effectors, thus optimizing associations during signaling. Just as importantly, they may prevent inappropriate cross-talk with other signaling pathways. An important element of such a system must be the ability to modulate the association and exclusion of relevant proteins. It is not currently known how the various proteins acquire affinities for the GEDs.

In this report, we show that mature and immature B cells differentially sequester the ligated BCR within polarized GEDs. This phenomenon is isotype independent and may partly explain the signaling differences between mature and immature B cells that lead to varied functional response to BCR-mediated stimulation. Previous studies have used biochemical methods to follow the relative location of proteins within buoyant fractions of sucrose density gradients after treatment with nonionic detergents such as Triton X-100. The large number of cells required for such studies has limited the study of B cells mostly to transformed cell lines. In addition, the behavior of individual cells was inferred from the composite results of bulk cell lysates. We used primary B cells from BALB/c mice to better preserve the physiologic signaling processes compared with cell lines. Our use of immunofluorescence to examine individual B cells allowed us, in a manner that sucrose density gradient assays cannot, to study the topology of the colocalization at the individual cell level.

We found that, at later time points, the polarized BCR on the surface of both mature and immature B cells became dramatically smaller, exhibiting a punctate pattern. In mature B cells, the GEDs remained prominent and copolarized with the BCR. This suggests that once rafts have been mobilized, they can maintain their reorganized state even after the dispersion or endocytosis of the ligands or receptors that initiated their reorganization. The resulting stabilization and maintenance of signals in such platforms may translate into the different functional outcomes seen in mature and immature B cells in response to anti-BCR treatment.

It is generally assumed that receptors such as BCR reside outside of rafts and are recruited into rafts once a stimulus has been given. However, there is data to support the idea that proteins constitutively associated with individual rafts are brought together with a cross-linking ligand, leading to a passive coalescence of rafts and their associated signaling molecules. Pralle et al. used photonic force microscopy to determine that single rafts, 50 nm in size, can stably associate with proteins for minutes (27). Also, pre-BCR and pre-TCR were found to localize to the rafts constitutively without ligation (28, 29). It is currently unclear how single small rafts form clustered rafts upon receptor ligation. Our results in immature B cells demonstrate that BCR cross-linking by itself does not necessarily lead to associated raft coalescence, and that an additional developmentally regulated step lacking in immature B cells may increase the affinity of the BCR complex for rafts. As the components of the BCR signaling complex and their relationship with specialized lipid domains are better defined, future efforts will be directed toward better defining the spatial and temporal interplay of the BCR with associated signal-transducing tyrosine kinases and adaptor proteins.


   Acknowledgments
 
We thank Dr. Fred Finkelman for the mouse anti-IgD Ab and Dr. Fritz Melchers for the gift of 493 mAb.


   Footnotes
 
1 This work was partially supported by National Institutes of Health Grants AI32592, AI23568, and AI43620 (to J.G.M.), and by the Arthritis Foundation’s Physician Scientist Development Award (to J.B.C.). Back

2 Address correspondence and reprint requests to Dr. John G. Monroe, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 311 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104. Back

3 Abbreviations used in this paper: BCR, B cell Ag receptor; GED, glycosphingolipid-enriched domain; PIP2, phosphatidylinositol 4,5-bisphosphate; HEL, hen egg lysozyme; AMCA, aminomethylcoumarin; HSA, heat-stable Ag; s, surface. Back

Received for publication October 17, 2000. Accepted for publication November 8, 2000.


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R. J. Benschop, E. Brandl, A. C. Chan, and J. C. Cambier
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