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International Immunology, Vol. 14, No. 10, pp. 1179-1191, October 2002
© 2002 Japanese Society for Immunology

Cooperative interaction of Ig{alpha} and Igß of the BCR regulates the kinetics and specificity of antigen targeting

Chang Li1, Karyn Siemasko2, Marcus R. Clark2 and Wenxia Song1

1 Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA 2 Section of Rheumatology, Department of Medicine, University of Chicago, IL 60637, USA

Correspondence to: W. Song; E-mail: ws98{at}umail.umd.edu
Transmitting editor: L. H. Glimcher


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Following the binding of antigens, the BCR transduces signals and internalizes antigens for processing and presentation, both of which are required for initiating an effective antibody response. The BCR, consisting of membrane Ig and Ig{alpha}/Igß heterodimer, facilitates antigen processing by accelerating antigen targeting to the processing compartment. Previous reports showed that Ig{alpha} or Igß alone is competent for internalizing antigens. However, both Ig{alpha} and Igß are required for BCR-enhanced antigen presentation. Using chimeric proteins containing the extracellular and transmembrane domains of human platelet-derived growth factor receptor fused with the cytoplasmic domain of Ig{alpha} or Igß, we studied the roles of the cytoplasmic tails of Ig{alpha} and Igß in BCR-mediated antigen transport. The Igß chimera rapidly moves through the endocytic pathway to lysosomes, while the Ig{alpha} chimera slows down this movement. The Ig{alpha}, but not the Igß chimera, is required for an increase in the turnover rate of the chimeras in response to stimulation. Only when Ig{alpha} and Igß chimeras are co-expressed do the chimeras rapidly and specifically target antigens to the processing compartment. These findings suggest that Ig{alpha} and Igß play distinct roles in BCR trafficking, and the cooperative interaction of Ig{alpha} and Igß controls and regulates the kinetics and specificity of antigen targeting.

Keywords: antibody/antigen receptor, antigen processing, B lymphocytes, Ig{alpha}/Igß heterodimer


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Processing and presentation of antigens by B lymphocytes to T lymphocytes is the central event for the initiation of humoral responses to T cell-dependent antigens. B cells express clonally specific antigen receptors that sense and capture antigens. The binding of an antigen to the BCR initiates signaling cascades (1–3) that provide the first stage of signals for B cell activation. Subsequently, the BCR internalizes and targets the antigen to the processing compartment where complexes of antigenic peptides and MHC class II molecules are assembled (4–6). The interaction of T cells and B cells in the context of antigenic peptide–MHC class II complexes provides the second stage of signals for B cell activation (7,8).

When the immune system initially encounters an antigen, the concentration of the antigen often is low, and the number of antigen-specific B cells and T cells is limited. The antigen-processing efficiency of B cells becomes extremely critical for the induction of a rapid humoral response specific to the antigen. Although antigens internalized through fluid-phase pinocytosis or binding to surface receptors other than the BCR can be processed and presented, the presentation of these antigens is far less efficient than BCR-mediated antigen presentation (9–11). The BCR increases the kinetics and specificity of antigen targeting to facilitate antigen processing (5,6). The BCR captures antigens specifically and internalizes them from the cell surface. Upon entering the endocytic pathway, antigen–BCR complexes transiently pass through early endosomes and reach the MHC class II-containing compartment (MIIC) (4–6). The MIIC is located in the later part of the endocytic system, and contains newly synthesized MHC class II (4,12–15), the catalyst of class II–peptide exchange, DM (15,16), the DM regulator, DO (17,18) and residential proteins of late endosomes, like lysosomal-associated membrane glycoprotein-1 (LAMP-1). Antigen–BCR complexes are degraded in the endosomes and the resulting peptides are loaded onto class II molecules in the MIIC. Although the intracellular trafficking pathway of the BCR has been well characterized, the molecular mechanisms for the specific targeting of the BCR are not well understood.

BCR-initiated signaling plays a major role in regulating antigen processing. Activating the BCR by cross-linking induces a rapid internalization of the BCR and accelerates targeting of the BCR to the MIIC (5). Tyrosine kinase inhibitors that block BCR signaling lower the antigen-presenting efficiency of B cells and inhibit accelerated antigen transport (19,20). In addition, the tyrosine phosphorylation sites in the cytoplasmic tail of the Ig{alpha} chain of the BCR (21,22) and tyrosine kinase, Syk (23), have been shown to be important for BCR-mediated antigen processing. BCR-initiated signaling has also been reported to induce a reorganization and acidification of late endosomes (24). These demonstrate that there are multiple links between BCR signaling and antigen-processing pathways.

The BCR is composed of membrane Ig (mIg) and Ig{alpha}/Igß heterodimer (Ig{alpha}/Igß). The Ig{alpha}/Igß and mIg form a complex through non-covalent interaction. Both Ig{alpha} and Igß are essential for the development (25,26), activation (27, 28) and programmed cell death of B cells (29,30). These two chains have been shown to play distinct and complementary roles in the signal-transduction (31,32) and antigen-processing (22,33) functions of the BCR. Although either the Ig{alpha} or Igß chain appears to be competent for signal transduction and antigen internalization, it has been demonstrated that both chains are required for an optimal level of signaling and a high efficiency of antigen presentation (22,32). Using human platelet-derived growth factor receptor (hPDGFR) chimeras containing the cytoplasmic tail of either Ig{alpha} or Igß, Siemasko et al. (22) recently showed that only when Ig{alpha} and Igß chimeras were co-expressed did the chimeric proteins facilitate antigen presentation as the BCR does, indicating that the interaction between Ig{alpha} and Igß is crucial for the efficiency of antigen presentation. Here we analyzed the intracellular trafficking of the Ig{alpha} and Igß chimeras using three different approaches, including subcellular fractionation, immunofluorescence microscopy and electron microscopy. The results reported here show that Ig{alpha} and Igß play distinct roles in controlling and regulating the kinetics and specificity of antigen targeting.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell culture
The mouse B cell lymphoma A20 IIA1.6 is a H-2d, IgM–, IgG2a+ and FcR– cell line. The A20 cells were cultured in DMEM that was supplemented as described (34) and contained 10% FCS.

Construction of the Ig{alpha} and Igß chimeras
Construction and expression of the chimeras containing the extracellular and transmembrane domains of hPDGFR and the cytoplasmic domain of Ig{alpha} or Igß have been previously described (22,32). The stably transfected A20 cells were thawed from the stocks every month and the expression levels of the chimeras were periodically examined using flow cytometry as previously described (32).

Stimulation of the chimeras
To activate the chimeras, cells expressing different chimeras were incubated with PDGF-BB (100 ng/ml; Zymed, South San Francisco, CA) in DME containing 6 mg/ml BSA and 20 mM MOPS, pH 7.4 (DME/BSA) for 10 min, followed by mouse anti-human PDGFRß mAb (anti-hPDGFRß) (5 µg/ml; Zymed) for 10 min and then anti-mouse IgG1 (5 µg/ml; Zymed) for 20 min at 4°C.

Turnover of the surface-biotinylated chimeras
Cells were washed at 4°C with HBSS lacking phosphate and containing 20 mM Na HEPES, pH 7.4, and incubated in the same buffer containing 0.2 mg/ml sulfosuccinimidyl-6-(biotinamido)hexanoate (Pierce, Rockford, IL) for 15 min at 4°C to label the surface proteins. After 15 min of incubation, a freshly made biotin solution was added and the incubation was extended for another 15 min at 4°C. The cells then were washed with DME/BSA to remove unreacted biotin, treated with DME/BSA alone or PDGF-BB, anti-hPDGFRß and anti-mouse IgG1 antibodies sequentially at 4°C to cross-link the chimeras, and chased at 37°C for varying lengths of time. The cells were lysed with 1% NP-40 lysis buffer (1% NP-40, 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA and protease inhibitors) and the cell lysates were subjected to immunoprecipitation using anti-hPDGFRß antibody. The immunoprecipitates were analyzed by SDS–PAGE and Western blotting. The biotinylated chimeras were detected using horseradish peroxidase–streptavidin and analyzed by densitometry.

Subcellular fractionation
Anti-hPDGFRß antibody was iodinated to a sp. sct. of 1.0–1.5 x 107 c.p.m./µg as previously described (35). More than 95% of [125I]anti-hPDGFRß antibody was precipitated by 10% trichloroacetic acid, indicating little or no free 125I. Cells were washed and incubated with 100 ng/ml PDGF-BB, 2 µg/ml [125I]anti-hPDGFRß and anti-mouse IgG1 (5 µg/ml) antibodies in DME/BSA at 4°C for 60 min. The cells were washed, homogenized and subjected to the Percoll gradient centrifugation as previously described (13). Briefly, cells (~2 x 108) were homogenized in homogenization buffer (10 mM Tris, 1 mM EDTA and 0.25 M sucrose, pH 7.4). The homogenate was centrifuged at 900 g for 15 min to remove nuclei and at 10,000 g for 15 min to remove mitochondria. Then 2 ml of the supernatant was layered onto 9 ml Percoll (1.05 g/ml) and centrifuged at 34,800 gmax for 20 min. Fractions of 0.5 ml were collected. Radioactivity of each fraction was counted and calculated as a percentage of the total cell-associated radioactivity. The total cell-associated radioactivity was 10,000–20,000 c.p.m.

To determine the distribution of molecular markers for different subcellular organelles on the Percoll gradient, the fractions were boiled in equal volumes of the reducing sample-loading buffer of SDS–PAGE, and centrifuged to remove Percoll before subjected to SDS–PAGE and Western blotting. The blots were probed for transferrin (Tf) receptor (TIB219), LAMP-1 (1D4B), mIgG2a or invariant chain (Ii; IN-1) (generous gifts from Dr S. K. Pierce, NIAID). MHC class II molecules were isolated from each fraction by immunoprecipitation using mAb MDK6 or M5/114.15.2 (PharMingen, Franklin Lakes, NJ). The immunoprecipitates were subjected to SDS–PAGE and Western blotting. The blots were probed with mAb MDK6 or M5/114.15.2. The enzymatic activities of {alpha}-mannosidase II and ß-hexosaminidase were measured as previously described (39,40). Tf receptor was used to mark the plasma membrane (PM) and early/recycling endosomes. The activity of {alpha}-mannosidase II marked the Golgi. The dense fractions containing LAMP-1 and ß-hexosaminidase activity were identified as dense late endosomes and lysosomes. The dense fractions where MHC class II molecules and LAMP-1, but not Ii, were detected were characterized as MIIC-like compartments.

Immunofluorescence microscopy
To label the chimeras, cells were incubated sequentially with PDGF-BB (100 ng/ml), anti-hPDGFRß (5 µg/ml) and TRITC–anti-mouse IgG1 (5 µg/ml) antibodies (Zymed) for 40 min at 4°C on polylysine-coated slides (Sigma, St Louis, MO). To label the endogenous BCR, cells were incubated with 10 µg/ml TRITC–goat anti-mouse IgG2a antibody (Southern Biotechnology Associates, Birmingham, AL) at 4°C for 40 min. The cells were washed at 4°C and chased at 37°C for varying lengths of time. After the chase, the cells were fixed with 4% paraformaldehyde and permeabilized by incubation with a permeabilization buffer (1% gelatin, 0.05% saponin, 10 mM glycine and 10 mM HEPES, pH 7.4). They were then incubated with either LAMP-1 (1D4B)- or Tf receptor (TIB219)-specific mAb in the permeabilization buffer, followed by FITC–goat anti-rat IgG (Jackson ImmunoResearch, West Grove, PA) as the secondary antibody. The cells were washed, post-fixed, mounted with Gel/Mount (Biomeda, Foster City, CA) and analyzed using a scanning laser confocal microscope (Zeiss LSM 510). Images were acquired using a x100 oil immersion objective and cropped using Photoshop (Adobe, Mountain View, CA). Optical sections from the middle of cells were selected. No labeling was detectable when untransfected A20 cells were incubated with anti-hPDGFRß and TRITC–anti-mouse IgG1 antibodies. Background labeling using the secondary antibody FITC–goat anti-rat IgG was negligible.

Conventional electron microscopy
Gold-conjugated goat anti-mouse IgG1 (gold–anti-mouse IgG1) and BSA (gold–BSA) were prepared as previously described (36). Cells were incubated sequentially with PDGF-BB (100 ng/ml), anti-hPDGFRß (5 µg/ml) and gold–anti-mouse IgG1 (15 nm) antibodies at 4°C for 40 min, pulsed at 37°C for 15 min, washed at 4°C and chased at 37°C for 15 or 45 min. Gold–BSA (10 nm) was included in the pulse medium. The cells were fixed with 2% glutaraldehyde, post-fixed with 1% osmium tetroxide, dehydrated, infiltrated and embedded in epoxy resin (EM Science, Ft Washington, PA). Thin sections (60–90 nm) of the cells were contrasted with uranyl acetate and lead citrate, and examined in a Zeiss EM10CA electron microscope. To evaluate the results quantitatively, 20 cell profiles were randomly selected from sections generated from three individual experiments. The numbers of different sizes of gold particles in each cell profile were counted. For gold–anti-mouse IgG1 or gold–BSA in each type of structures, percentages of the total cell-associated immunogold particles were calculated.

Immunoelectron microscopy
Cells were incubated with PDGF-BB (100 ng/ml), anti-hPDGFR-antibody (5 µg/ml) and gold–anti-mouse IgG1 (15 nm) sequentially at 4°C for 40 min, pulsed at 37°C for 15 min, washed at 4°C and chased at 37°C for 15 min. The cells were fixed by 2% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4) for 2 h at room temperature and embedded in 7.5% gelatin. The gelatin blocks containing cells were immersed in 2.3 M sucrose in phosphate buffer for 2 h at 4°C and snap-frozen in liquid nitrogen. Ultra-thin cryosections (60–90 nm) were collected on a mixture of sucrose and methylcellulose, and labeled with mouse I-A/I-E (M5/114.15.2)-, Tf receptor (TIB219)- and LAMP-1 (1D4B)-specific mAb, and gold-conjugated rat IgG-specific antibody (6 nm) (Sigma), and examined in a Zeiss EM10CA electron microscope. Background labeling using irrelevant antibodies was negligible.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The chimeras containing different cytoplasmic tails have different turnover rates
Internalized antigens are proteolytically degraded in the endocytic system before being loaded onto MHC class II molecules. To understand how Ig{alpha} and Igß function together to facilitate antigen processing, we determined the turnover rates of chimeric proteins that contain the extracellular and transmembrane domains of hPDGFR{alpha} or ß fused with the cytoplasmic tail of either Ig{alpha} (Ig{alpha} chimera) or Igß (Igß chimera) (Fig. 1). The surfaces of A20 cells expressing Ig{alpha} or Igß chimera alone or expressing both Ig{alpha} and Igß chimeras (Ig{alpha}/Igß chimeras) were biotinylated. The surface-biotinylated cells were treated with either medium alone or PDGF-BB, anti-hPDGFRß and anti-mouse IgG1 antibodies sequentially at 4°C and then chased at 37°C for up to 4 h. PDGF-BB is a dimer that has an equal affinity to PDGFR{alpha} and PDGFRß, and dimerizes the chimeras. Anti-hPDGFRß and anti-mouse IgG1 antibodies further cross-link the dimerized chimeras (Fig. 1). Cells were lysed and the total chimeras in the cell lysates were immunoprecipitated with anti-hPDGFRß antibody. For cells that co-express Ig{alpha} and Igß chimeras, only Ig{alpha} chimeras that contain the extracellular and transmembrane domains of hPDGFRß were isolated by immunoprecipitation. Membrane IgG2a co-purified by Protein A–Sepharose beads served as internal controls. The immunoprecipitates were analyzed using SDS–PAGE and Western blotting. The biotinylated proteins were detected by horseradish peroxidase–streptavidin (Fig. 2A) and quantified by densitometry (Fig. 2B). In the absence of cross-linking, the surface-biotinylated mIgG2a disappeared in a time-dependent manner (Fig. 2). The surface-biotinylated Ig{alpha}/Igß chimeras in untreated cells disappeared at a rate similar to the endogenous mIgG2a. By 2 h, 50% of the biotinylated Ig{alpha}/Igß chimeras remained, comparable to that of mIgG2a (Fig. 2). The reduction of surface-biotinylated Igß chimeras was detected as early as 1 h and faster than that of Ig{alpha}/Igß chimeras. By 2 h, only 35% of the surface-biotinylated Igß remained. In contrast, the biotinylated Ig{alpha} chimeras disappeared much slower than Ig{alpha}/Igß chimeras. No reduction of the biotinylated Ig{alpha} chimeras was detectable until 4 h (Fig. 2). The endogenous mIgG2a in cells expressing different chimeras disappeared at a similar rate (Fig. 2), indicating that the differential turnover rates of the chimeras were not the result of clonal variation. Stimulating the chimeras by dimerization with PDGF-BB, and cross-linking with anti-hPDGFRß and anti-mouse IgG1 antibodies (Fig. 1), which induces the phosphorylation of the chimeras (32), speeded up the disappearance of surface-biotinylated Ig{alpha}/Igß chimeras and Ig{alpha} chimeras. With the stimulation, the Ig{alpha} chimera still disappeared slower than the Ig{alpha}/Igß chimera. In contrast, the stimulation did not affect the disappearance rate of surface-biotinylated Igß chimeras (Fig. 2).



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  		Fig. 1. The structures of the chimera proteins. The chimeric proteins contain the extracellular and transmembrane domains of human PDGFR{alpha} or PDGFRß fused to the cytoplasmic domain of Ig{alpha} or Igß. Since both PDGFR{alpha} and PDGFRß have the same affinity for PDGF-BB, homodimers can be formed on singly transfected cells (Ig{alpha} or Igß chimeras) and heterodimers can be formed on doubly transfected cells (Ig{alpha}/Igß chimeras). The chimeras can be further aggregated by anti-hPDGFRß antibodies.

 



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  		Fig. 2. Turnover of the surface biotinylated chimeras. The cell surface was biotinylated at 4°C. Then the cells were treated with medium alone (–XL) or PDGF-BB (100 ng), anti-hPDGFRß (5 µg/ml) and anti-mouse IgG1 (5 µg/ml) antibodies sequentially (+XL) to dimerize and cross-link the chimeras at 4°C, and chased at 37°C for times indicated. An equal number of cells from each chase time point were lysed and the total chimeras in the cell lysates were immunoprecipitated with anti-hPDGFRß antibody. In cells expressing both Ig{alpha} and Igß chimeras, only Ig{alpha} chimeras were immunoprecipitated by anti-hPDGFRß antibody. The heavy chain of endogenously expressed mIgG2a [mIgG2a (H)] was co-purified by Protein A-conjugated Sepharose beads. The immunoprecipitates were subjected to SDS–PAGE and Western blotting. The biotinylated proteins were detected by horseradish peroxidase–streptavidin. (A) Representative blots for the surface-biotinylated chimeras and mIgG2a (H) are shown. (B) Data from densitometry analyses were plotted as percentages of total biotinylated chimeras before warming. Averages (±SE) of the results of three independent experiments are shown.

 
These data suggest that the different surface chimeras are degraded at different rates and respond differently to stimulation. Igß chimeras appear to promote the turnover of co-expressed Ig{alpha} chimeras and Ig{alpha} chimeras are required for the increase of the turnover rate in response to stimulation.

The chimeras containing different cytoplasmic tails travel through the endocytic pathway at different kinetics
The different turnover rates of the chimeras probably are the result of their different intracellular trafficking routes or trafficking kinetics. To follow the intracellular trafficking of the chimeras, the chimeras on the cell surface were dimerized by PDGF-BB, labeled, and cross-linked by [125I]anti-hPDGFRß and anti-mouse IgG1 antibodies at 4°C. Then the cells were washed and chased at 37°C for up to 60 min to allow the chimeras to enter cells. To minimize the effect of degradation of the chimeras on the analyses, the cellular distribution of the chimeras was followed in the first hour of chase. The subcellular locations of the chimeras were determined by subcellular fractionation.

The distribution of various subcellular organelles of A20 cells on the Percoll gradient was determined by a set of enzymatic markers and serological reagents. The activity of {alpha}-mannosidase II, as a marker for the Golgi, was detected in light fractions 1–10 (Fig. 3B), and Tf receptor, as an early and recycling endosomal marker, was located in fractions 3–8 (Fig. 3A). The activity of ß-hexosaminidase, marking lysosomes, was detected in dense fractions 17–21 (Fig. 3B). LAMP-1, a marker for late endosomes/lysosomes, was primarily found in dense fractions 17–21. Ii that is mainly located in the endoplasmic reticulum (ER) and Golgi was detected in the fractions 3–10 (Fig. 3A). Thus, the PM, early endosomes, ER and Golgi are distributed in the light fractions 1–10. LAMP-1+ vesicles in the light fractions probably are immature late endosomes and transport vesicles traveling between the Golgi and endosomes. The dense fractions 17–21 contain the dense late endosomes and lysosomes. MHC class II molecules and the endogenous mIgG2a BCR were detected in fractions 3–10 and 15–21. The dense fractions where MHC class II molecules and mIgG2a, but not Ii, were detected are likely to contain the MIIC (13,37). As shown previously in B cell lymphoma CH27 cells (5), cross-linking the chimeras did not significantly alter the distribution of these markers on the Percoll gradient (data not shown).



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  		Fig. 3. Distribution of the subcellular organelles of A20 cells on the Percoll gradient. The distribution of subcellular organelles of A20 cells on the Percoll density gradient was determined using a series of enzymatic markers and serological reagents. The activities of {alpha}-mannosidase II and ß-hexosaminidase were measured as previously described (39,40), and plotted as percentages of total cell-associated activity. Tf receptor, LAMP-1, mIgG2a, MHC class II {alpha} and ß chains, and Ii were detected by SDS–PAGE and Western blotting. {alpha}-Mannosidase II marks the Golgi and ß-hexosaminidase marks the lysosomes. Late endosomes/lysosomes are marked by and LAMP-1. Dense fractions where MHC class II, LAMP-1 but not Ii were detected were identified as the MIIC-like compartment.

 
Using subcellular fractionation, the trafficking of the chimeras from the PM to the dense late endosomes/lysosomes was determined by movement of the surface-labeled chimeras from the light (1–10) to dense fractions (17–21) (Fig. 4). After being chased at 37°C for 15 min, the majority of the chimeras were in the light fractions containing the PM and early endosomes, and a small portion of the chimeras was detected in the dense fractions. The relative amount of Igß chimeras (16%) in the dense fractions was higher than those of Ig{alpha} (6%) or Ig{alpha}/Igß (10%) chimeras. By 30 min, the amount of all chimeras in the dense fractions was increased (Fig. 4). By 60 min, ~20% of the chimeras was detected in the dense fractions and there was no significant difference among different chimeras (Fig. 4). When cells were chased at 37°C for a time longer than 60 min, the total cell-associated radioactivity was dramatically decreased, especially in Igß chimera-expressing cells, but the percentage of radioactivity in the dense fractions did not increase (data not shown), suggesting the degradation of the chimeras in the dense fractions. The result from the subcellular fractionation study shows that Igß chimeras move from the light fractions to the dense fractions slightly faster than Ig{alpha} and Ig{alpha}/Igß chimeras.



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  		Fig. 4. Subcellular distribution of the chimeras. Cells were incubated sequentially with PDGF-BB (100 ng/ml), [125I]anti-hPDGFRß (2 µg/ml) and anti-mouse IgG1 (5 µg/ml) antibodies at 4°C, washed, and chased at 37°C for the times indicated to allow the radiolabeled chimeras to internalize and enter different subcellular organelles. The cells were then cooled to 4°C and subjected to subcellular fractionation (13). Fractions (0.5 ml) were collected. The amount of radioactivity in each fraction was measured. The radioactivity in the fractions 1–10 was summed as the light fractions and the radioactivity in the fractions 17–21 was summed as the dense fractions. The radioactivity of the light and dense fractions was plotted as a percentage of total cell-associated radioactivity. The total cell-associated radioactivity for each sample was 10,000–20,000 c.p.m. Shown are the average results (±SD) of three independent experiments.

 
To further analyze the trafficking kinetics and characterize the trafficking pathways of different chimeras, we followed the intracellular trafficking of the chimeras using immunofluorescence microscopy. The chimeras on the cell surface were labeled with anti-hPDGFRß and TRITC-conjugated anti-mouse IgG1 antibodies in the presence of PDGF-BB at 4°C. The endogenous BCR was labeled with TRITC–anti-mouse IgG2a as a control. The cells were washed and chased at 37°C for varying lengths of time. Early and recycling endosomes were labeled with a Tf receptor-specific mAb (Fig. 5) and late endosomes/lysosomes were labeled with a LAMP-1-specific mAb (Fig. 6). Before chase, the surface-labeled BCR and chimeras were all found at the outer rim of cells, where they partially co-localized with Tf receptor (Fig. 5A–D) but not with LAMP-1 (Fig. 6A–D). Upon warming to 37°C, the endogenous BCR started to move into the cells. By 30 min, the BCR appeared as punctate staining scattered through cells and partially co-localized with Tf receptor (Fig. 5D') and LAMP-1 (Fig. 6D'), suggesting that the BCR moves from the PM to early endosomes and is on its way to late endosomes. After 60 min of chase, the majority of the BCR staining clustered in the perinuclear area and co-localized with LAMP-1 extensively, an indication of its late endosomal and lysosomal location (Fig. 6D''). The staining patterns of Ig{alpha} chimeras that were co-expressed with Igß chimeras at all chase times analyzed were similar to those of the endogenous BCR (Figs 5C–C'' and 6C–C''). Interestingly, while their staining pattern was similar to the endogenous BCR, Igß chimeras only briefly co-localized with LAMP-1 around 30 min (Fig. 6B'), and by 60 min no significant co-localization between Igß chimeras and LAMP-1 was detected (Fig. 6B''). No significant amount of Ig{alpha} chimera staining was found inside cells until 60 min (Figs 5A–A'' and 6A–A''). After 60 min of chase, Ig{alpha} chimeras partially co-localized with LAMP-1 (Fig. 6A''), but a large portion of Ig{alpha} chimeras remained co-localized with Tf receptor (Fig. 5A'').



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  		Fig. 5. Co-localization of the chimeras with Tf receptor. Cells were incubated sequentially on ice with 100 ng/ml PDGF–BB, 5 µg/ml anti-hPDGFRß and 5 µg/ml TRITC–anti-mouse IgG1 antibodies without pre-starvation. For the endogenous BCR, cells were incubated with TRITC–goat anti-mouse IgG2a antibody at 4°C for 30 min. After washes, cells were chased at 37°C for varying lengths of time, washed, fixed and permeabilized. Then the cells were stained with a mAb specific for Tf receptor (TIB219) and FITC–goat anti-rat IgG as the secondary antibody. Single optical sections from the middle of cells were acquired using a scanning laser confocal microscope. Shown are the representative images from three or four independent experiments. Bar, 10 µm.

 


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  		Fig. 6. Co-localization of the chimeras with LAMP-1. The chimeras and the endogenous BCR were labeled as described in Fig. 5. After fixation and permeabilization cells were stained with LAMP-1-specific mAb (1D4B) and FITC–goat anti-rat IgG as the secondary antibody. Single optical sections from the middle of the cells were acquired using a scanning laser confocal microscope. Shown are the representative images from three or four independent experiments. Bar, 10 µm.

 
In addition, the cellular distribution of LAMP-1 varied among cells expressing different chimeras. Cross-linking the chimeras in cells expressing both Ig{alpha} and Igß chimeras induced the movement of the LAMP-1+ vesicles from the cell periphery to the perinuclear area (Fig. 6C'') as seen in cells treated with anti-mouse IgG2a antibody (Fig. 6D''). However, cross-linking the chimeras in cells expressing either Ig{alpha} or Igß chimeras alone failed to induce the redistribution of the LAMP-1+ compartment (Fig. 6A'' and B''), which is similar to what Siemasko et al. showed previously (24).

Taken together, all the chimeras appear to travel through a similar pathway, but at different kinetics. Ig{alpha} chimeras move from the PM to late endosomes and lysosomes slower than Igß and Ig{alpha}/Igß chimeras.

Ultrastructural analysis of the subcellular compartments containing the chimeras
The Igß chimera transiently passed though the LAMP-1+ compartment and was accumulated in LAMP-1– compartments, suggesting that different cytoplasmic tails target the chimeras to different compartments. To characterize subcellular structures that the different chimeras were targeted to, we analyzed the ultrastructures of the endocytic compartments containing pulse-labeled chimeras using conventional electron microscopy. The chimeras on the cell surface were dimerized by PDGF-BB, cross-linked by anti-hPDGFRß antibody and labeled by gold–anti-mouse IgG1 (15 nm) at 4°C. Cells were pulsed for 15 min at 37°C, washed at 4°C and chased at 37°C for 15 or 45 min. To mark the endocytic pathway, gold–BSA (10 nm) was simultaneously taken up during the pulse. Previously, using immunoelectron microscopy, Kleijmeer et al. (15) characterized the endocytic compartments of A20 cells in great detail. Based on their analysis, five types of morphologically distinct structures in transfected A20 cells that contained gold–BSA were identified. The numbers of gold–anti-mouse IgG1 for the chimeras or gold–BSA in each type of structures were counted and calculated as percentages of the total cell-associated immunogold particles. Type I structures were small or tubular vesicles that were located in the cell periphery and contained 22% of cell-associated gold–BSA after 15 min of chase (Fig. 7A and B, and Table 1). The morphology and the presence of the pulsed BSA after a 15-min chase suggest that the type I structures are early endosomes. Type II structures were relatively bigger, electron-lucent vesicles containing a few internal vesicles (Fig. 7B). A portion of the pulsed gold–BSA (18%) reached the type II structures after 15 min of chase (Fig. 7B and Table 1). The type II structures appeared to be the intermediates between early endosomes and multi-vesicular bodies (MVB) or early MVB. Type III structures had the typical morphology of the MVB (Fig. 7C). Type IV structures were morphologically similar to the type III structures, but they were in close proximity to the electron-dense structures, type V structures (Fig. 7D and E). The type V structures were the densest organelles detected in A20 cells and contained tightly stacked membrane lamellas (Fig. 7D–F), which are the characteristic of lysosomes. After 45 min of chase, gold–BSA primarily located in type III (28%), type IV (25%) and type V (29%) structures (Table 1), suggesting that they are the late endosomes and lysosomes.



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  		Fig. 7. Ultrastructural analysis of the endocytic compartments accessed by the chimeras. Cells were incubated sequentially on ice with PDGF-BB (100 ng/ml), anti-hPDGFRß (5 µg/ml) and gold–anti-mouse IgG1 (15 nm) antibodies with gold–BSA (10 nm). The cells were pulsed at 37°C for 15 min, washed at 4°C, and chased at 37°C for 15 and 45 min. The cells were then processed for electron microscopy. (A and B) Ig{alpha} chimeras. (C and D) Ig{alpha}/Igß chimeras. (E and F) Igß chimeras. Roman letters indicate the structural types. G, Golgi; N, nuclei; arrows, chimeras. Bar, 100 nm.

 

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  		Table 1. Quantitative analysis of the subcellular distribution of chimeras and BSAa
 
Next we determined which type of structures different chimeras were targeted to. By 15 min, Ig{alpha}/Igß chimeras concentrated in the type II (43%) structures, the early MVB, and the type III (23%) structures, the MVB (Fig. 7C and Table 1), while Ig{alpha} chimeras (51%) were primarily located in the type I structures (Fig. 7A and Table 1). Meanwhile Igß chimeras (57%) had already accumulated in the type V structures (Fig. 7F and Table 1). After 45 min of chase, Igß chimeras continued accumulating in the type V structures, reaching 65%. The majority of Ig{alpha}/Igß chimeras were found in the type III (54%) and type IV (26%) structures, the MVB. By now, Ig{alpha} chimeras had entered the type III structures (50%). However, a major portion of Ig{alpha} chimeras (29%) still remained in the type II structures, the early MVB, and no significant amount of Ig{alpha} chimera was detected in the type IV and V structures (Table 1). Thus, Igß chimeras are rapidly targeted to the type V structure and Ig{alpha}/Igß chimeras are accumulated in MVB. The rate of Ig{alpha} chimeras moving to the MVB is significantly reduced.

The type IV structures have drawn our special attention. The type IV structures were MVB closely tethered to the type V structures. The close proximity of these two structures implies that they undergo tethering, fusion and/or content exchanges. Moreover, at both chase times examined, the amount of either Ig{alpha} (1 and 4%) or Igß (2 and 16%) chimera in the type IV compartments was much less than the amount of Ig{alpha}/Igß chimera (10 and 26%). Significantly, in pairs of the attached type IV and V structures, Ig{alpha}/Igß chimeras were preferentially located in the MVB side (Fig. 7D) and Igß chimeras were in the side of the type V structure, lysosomes (Fig. 7E). This suggests that Ig{alpha}/Igß chimeras, but not Igß chimeras, have the ability to remain in the MVB to delay the movement to lysosomes.

In addition, the cellular distribution of the type III, IV and V structures in cells expressing both Ig{alpha} and Igß chimeras was pronouncedly different from that seen in cells expressing either Ig{alpha} or Igß chimeras alone. In cells expressing Ig{alpha}/Igß chimeras, these structures clustered in a greater number (Fig. 8A) than those in cells expressing Ig{alpha} or Igß chimeras alone (Fig. 8B). This is consistent with our immunofluorescence microscopy studies that showed that cross-linking the endogenous BCR or Ig{alpha}/Igß chimeras, but not Ig{alpha} chimeras or Igß chimeras, induced the clustering of the LAMP-1+ vesicles.



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  		Fig. 8. Aggregation of Ig{alpha}/Igß chimeras induces the clustering of late endosomes and lysosomes. Cells were treated and processed as described in Fig. 7. (A) Cell expressing both Ig{alpha} and Igß. (B) Cell expressing Igß chimeras only. Bar, 100 nm.

 
To confirm that the type III and IV structures where Ig{alpha}/Igß chimeras are targeted to are the MIIC or MIIC-like compartment, the co-localization of the chimeras with MHC class II molecules, LAMP-1 or Tf receptor was analyzed using immunoelectron microscopy. In addition to the multi-vesicular or multi-lamellar morphology, the MIIC contains MHC class II molecules and LAMP-1, but not Tf receptor. Cells were pulsed with PDGF-BB, anti-hPDGFRß and gold–anti-mouse IgG1 (10 nm) antibodies, and chased at 37°C for 15 min as described above. Then cells were fixed, embedded, infiltrated and snap-frozen in liquid nitrogen. The cryo-thin sections of cells were labeled with either MHC class II-, LAMP-1- or Tf receptor-specific mAb and gold-conjugated secondary antibodies (6 nm). After 15 min of chase, Ig{alpha}/Igß chimeras co-localized with class II molecules (Fig. 9A) and LAMP-1 (Fig. 9B) in the MVB, but not with Tf receptor (data not shown). In contrast, no significant co-localization of Igß chimeras with class II (Fig. 9C) or LAMP-1 (data no shown) was detected. Thus, Ig{alpha}/Igß chimeras, but not Igß chimeras, are targeted to the MIIC-like compartment.



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  		Fig. 9. Co-localization of the chimeras with MHC class II molecules. Cells were incubated sequentially on ice with PDGF-BB (100 ng/ml), anti- hPDGFRß (5 µg/ml) and gold–anti-mouse IgG1 (15 nm) antibodies. The cells were pulsed at 37°C for 15 min, washed at 4°C and chased at 37°C for 15 min. The cells were fixed by 2% paraformaldehyde, embedded in 7.5% gelatin and snap-frozen in liquid nitrogen. Ultra-thin cryosections were collected and labeled with anti-mouse I-A/I-E (M5/114.15.2) (A and C) or LAMP-1 (1D4B) (B) antibodies, and gold–anti-rat IgG antibody (6 nm). (A and B) Cell expressing both the Ig{alpha} and Igß chimeras. (C) Cell expressing Igß chimeras. Arrows, MHC class II (A) or LAMP-1 (B). Bar, 100 nm.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The binding of antigens to the BCR induces signaling cascades and rapid internalization, processing and presentation of the antigens. The BCR facilitates antigen processing and presentation by specifically targeting antigens to the processing compartment (5,6). Here, using a chimeric protein system, we found that the chimeras containing the cytoplasmic tail of Ig{alpha} or Igß moved through the endocytic pathway and were degraded at different rates. Only when Ig{alpha} and Igß chimeras were co-expressed were the chimeras targeted to the MIIC-like compartment. Our findings suggest that the cytoplasmic domains of Ig{alpha} and Igß play distinct roles in the intracellular trafficking of the BCR, and the interaction of Ig{alpha} and Igß determines the kinetics of BCR traveling through the endocytic pathway and the endocytic compartment that the BCR is targeted to.

The antigen-processing compartment, MIIC, has been characterized as a conventional endocytic compartment that is located in the late part of the endocytic pathway (15). One of the important roles of the BCR in antigen processing is to accelerate the intracellular movement of antigens to the MIIC. Our results generated from three different approaches, including subcellular fractionation, immunofluorescence and electron microscopy, consistently showed that the chimeras containing the different cytoplasmic tails of the BCR move through the endocytic pathway at different speeds. The cytoplasmic tail of Ig{alpha} slows down, and the cytoplasmic tail of Igß speeds up, the movement of the chimeras through the endocytic pathway, indicating that the cytoplasmic tails of Ig{alpha} and Igß play distinct roles in controlling the trafficking kinetics of the BCR. Ig{alpha} chimeras co-expressed with Igß chimeras move through the endocytic pathway at a speed faster than that of Ig{alpha} chimeras alone and slower than that of Igß chimeras alone, suggesting that Ig{alpha} and Igß cooperatively control the trafficking kinetics of the BCR. The cytoplasmic tails of Ig{alpha}/Igß potentially carry the targeting signals for the BCR. The interaction between Ig{alpha} and Igß chains may control the exposure of right targeting signals in response to BCR activation. At present, the targeting signals carried by Ig{alpha}/Igß have not been completely identified.

The cytoplasmic tails of Ig{alpha} and Igß appear to be one of the important factors that control the turnover rate of the BCR. Surface-labeled chimeras with different cytoplasmic tails are degraded at different rates. Compared to that of Ig{alpha}/Igß chimeras, the reduction of the surface-biotinylated Igß chimeras was detected at least 30 min earlier and the reduction of the surface-biotinylated Ig{alpha} chimeras was detected until 2 h later. The trafficking kinetics of the chimeras is apparently correlated to their turnover rate. The faster the chimeras reach late endosomes and lysosomes, the more rapidly they are degraded. Among the three chimeras tested here, the Igß chimera has the highest and the Ig{alpha} the lowest trafficking kinetics and turnover rate. However, the Igß chimera is not able to facilitate antigen processing and presentation to a level similar to Ig{alpha}/Igß chimeras (22), suggesting that accelerated intracellular movement and degradation of antigens do not necessarily lead to a higher efficiency of antigen processing and presentation.

The finding here raises an interesting question why the fast runner, the Igß chimera, is not able to facilitate antigen processing and presentation. Our ultrastructural analyses provide an explanation for this question. Ig{alpha}/Igß chimeras, but not Igß chimeras, are targeted to the MIIC-like compartment that contains MHC class II and LAMP-1, and has multi-vesicular morphology. The movement of Ig{alpha} chimeras to the MIIC-like compartment is delayed by their slow kinetics. Igß chimeras transiently pass through the LAMP-1+ compartment and MVB, and are targeted to electron-dense vesicles that do not contain MHC class II, LAMP-1 or Tf receptor. Their morphology and degradative properties suggest that these dense vesicles are the mature lysosomes. It is unclear why LAMP-1 is not located in these mature lysosomes. Significantly, while Ig{alpha}/Igß chimeras reach the MVB as fast as Igß chimeras, Ig{alpha}/Igß chimeras are able to remain in the MVB side of the clusters of the MVB and dense vesicles. In contrast, Igß chimeras quickly move through the MVB down to the dense vesicles. This suggests that Ig{alpha}/Igß chimeras not only move to the MIIC-like compartments quickly, but also are capable of remaining there, which probably provides a sufficient amount of time for antigen fragmentation and peptide loading.

Signals transduced through Ig{alpha}/Igß are important regulating factors for BCR trafficking. Ig{alpha} and Igß chains play different roles in signal transduction. Using the same set of chimeric proteins, Luisiri et al. (32) showed that when expressed individually, Ig{alpha} and Igß chimeras all induce a low level of protein tyrosine phosphorylation in response to stimulation; however, the phosphorylation of the Igß chimera is very transient. When co-expressed, Ig{alpha}/Igß chimeras induce a much higher level of protein phosphorylation than Ig{alpha} or Igß chimeras alone, and the Ig{alpha} tails of Ig{alpha}/Igß chimeras are dominantly phosphorylated. Here we found that Ig{alpha} and Ig{alpha}/Igß chimeras, but not Igß chimeras, increase their turnover rate in response to stimulation, suggesting that the signal-transducing ability of the chimeras correlates with their turnover rate, and that signaling induced by Ig{alpha} chimeras or Ig{alpha}/Igß chimeras is sufficient to promote the degradation of the chimeras. Since the Ig{alpha} tail is dominantly phosphorylated upon activation, it appears that Ig{alpha}, but not Igß, plays a major role in coordinating the signaling and antigen-targeting functions of the BCR.

Various chimeras have been used for studies of functions of Ig{alpha} and Igß, and these studies have shown that Ig{alpha} and Igß play important roles in BCR-mediated antigen processing (23,33,38). Our studies using the chimeras consisting of the extracellular and transmembrane domains of PDGFR fused with the cytoplasmic tail of Ig{alpha} or Igß extend the previous finding by demonstrating a cooperative role of Ig{alpha} and Igß chains in antigen targeting. Since PDGFR functions as a dimer, the Ig{alpha} and Igß chimeras have the potential to form homodimers when they are expressed individually or to form heterodimers when they are co-expressed. These chimeras may not completely represent the native BCR since they do not interact with mIg, and the Ig{alpha} and Igß chimeras may not all form heterodimers when treated with PDGF-BB. In this study, the behavior of Ig{alpha} chimeras in cells co-expressing Ig{alpha} and Igß chimeras was followed. Therefore, the results reflect the combination of Ig{alpha}/Igß chimera heterodimers and Ig{alpha} chimera homodimers. Cross-linking with an antibody specific for hPDGFRß brings the heterodimers and homodimers together. The exact percentage of chimeras that form heterodimers is unknown. However, our results showed that the co-expression of Igß chimeras with Ig{alpha} chimeras increases the trafficking kinetics and turnover rate of Ig{alpha} chimeras, and allows Ig{alpha} chimeras to enter and remain in the MIIC. This shows that Ig{alpha} and Igß chimeras do interact with each other when they are co-expressed, and suggests that the interaction of Ig{alpha} and Igß generates synergetic and cooperative actions that control and regulate the trafficking kinetics, targeting specificity and turnover rate of the BCR. The fact that the Ig{alpha}/Igß chimeras are functionally similar to the BCR supports the notion that Ig{alpha}/Igß and their interaction are required and sufficient for accelerated and specific antigen targeting.


   Acknowledgements
 
This work was supported by National Institute of Health Grants AI42093 (to W. S.) and GM52736 (to M. R. C.). K. S. was supported by a Postdoctoral Fellowship from the Cancer Research Institute. We thank Dr Susan K. Pierce for providing antibodies and the Laboratory for Biological Ultrastructure at University of Maryland for technical support (Contribution no. 90).


   Abbreviations
 
ER—endoplasmic reticulum

Ig{alpha}/Igß—Ig{alpha}/Igß heterodimer

Ii—invariant chain

LAMP-1—lysosomal associated membrane glycoprotein-1

MIIC—MHC class II-containing compartment

mIg—membrane Ig

hPDGFR—human platelet-derived growth factor receptor

MVB—multi-vesicular body

PM—plasma membrane

Tf—transferrin

XL—cross-linking



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   References
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 Introduction
 Methods
 Results
 Discussion
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