Transitional and marginal zone B cells have a high proportion of unmasked CD22: implications for BCR signaling
Claus-Peter Danzer1,3,
Brian E. Collins2,
Ola Blixt2,
James C. Paulson2 and
Lars Nitschke1
1 Institute of Virology and Immunobiology, University of Würzburg, Versbacherstrasse 7, 97078 Würzburg, Germany 2 Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92075, USA 3 Present address: Novartis Pharma, Lichtstrasse 35, 4056 Basel, Switzerland
Correspondence to: L. Nitschke; E-mail: nitschke{at}vim.uni-wuerzburg.de
Transmitting editor: S. Izui
 |
Abstract
|
---|
CD22, a B cell-specific member of the Siglec family, is an important inhibitor of B cell signaling. The first Ig-like domain of CD22 specifically binds to
2,6-linked sialic acids. Through these interactions CD22 can mediate adhesion to other cells in trans, but can also bind endogenous ligands on the B cell surface in cis. Cis binding of CD22 to sialylated ligands enhances the efficiency of inhibition and thereby reduces the BCR signaling strength. In this study we used a newly developed oligomeric streptavidin-based sialylated probe as an artificial CD22 ligand. We found that CD22 is bound to ligands in cis on most B cells. However, there is a proportion of B cells with unbound (unmasked) CD22. The subpopulation with unmasked CD22 is 2-fold increased in transitional and marginal zone B cells in the spleen and on B1 cells in the peritoneum, when compared to mature B cells. Also, B cells with unmasked CD22 have an activated phenotype. Unmasking of CD22 could be functionally involved in lowering the signaling threshold on developmental checkpoints such as transitional B cells and during B cell activation or could be a consequence of such activation processes.
Keywords: B lymphocyte, CD22, sialic acid, Siglec
 |
Introduction
|
---|
CD22, a B cell-specific trans-membrane protein, is a co-receptor and negative regulator of the BCR. Inhibition of BCR signaling by CD22 occurs through recruitment and activation of Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) (13). Accordingly, B cells of CD22-deficient mice are hyper-responsive to BCR stimulation (47). This is most evident by increased Ca2+ mobilization. CD22 belongs to the Siglec family of adhesion receptors. The Siglecs, which are primarily expressed on hematopoetic cells, are characterized by their binding to sialic acids in specific linkages (8). CD22 has high specificity for sialic acids in
2,6 linkage (2,6Sia) (9,10). The CD22 ligand, 2,6Sia, is expressed abundantly as an N-linked sugar on glycoproteins of many cells, among them lymphocytes and cytokine-activated endothelial cells (11,12).
CD22, as most other Siglecs, is bound to ligands in cis, i.e. to ligands on the B cell surface (the masked state). This is deduced from experiments in which human or mouse B cells were stained with a polyacrylamide (PAA)-based 2,6Sia carrying glycoconjugate as synthetic ligand for CD22 (1315). This synthetic ligand could not bind to CD22 on most B cells, unless they were pre-treated with sialidase to remove the cis ligands (unmasked). The terminal sialoside structure recognized by CD22 is synthesized in vivo by the
2,6 sialyl-transferase ST6GalI. Expression of this enzyme is tightly controlled with high levels seen in B cells (1618). With a newly developed mouse CD22-specific probe, Neu5Gc-
2,6GalPAA, we have recently shown that CD22 is constitutively unmasked on B cells of ST6GalI knockout mice (15).
CD22 is also involved in trans interactions to other cells. This can most clearly be demonstrated when CD22 is transfected into heterologous cells, such as COS cells or CHO cells which do not express high levels of ST6Gal1 and hence do not carry 2,6Sia on the same cellular surface. Expressed on the surface of these cells, CD22 can mediate adhesion to other 2,6Sia-carrying cells (9,12). A role for CD22 as an adhesion molecule in bone marrow homing of recirculating B cells has been described. We found that bone marrow sinusoidal endothelium cells specifically express 2,6Sia on their surface which can act as ligand for CD22 when recirculating B cells home to the bone marrow (19). Trans interactions of CD22 to 2,6Sia on target cells can also influence B cell signaling (20).
By solving the crystal structure of sialoadhesin (Siglec-1) and site-directed mutagenesis of the ligand binding site of both sialoadhesin and CD22, it was found that the primary molecular contacts of the two Siglecs occur with the sialic acid moiety of the carbohydrate (21,22). The affinity of CD22 for sialic acid is very low (104 M) (23). CD22 can bind to a number of sialylated proteins on the cellular surface, among them prominently CD45, as was demonstrated by CD22Fc binding and protein precipitation (24). However, a recent surface plasmon resonance study showed that the affinity of CD22 for native CD45, another sialylated protein or a synthetic 2,6Sia-carrying glycoconjugate did not differ greatly (23). This means that the protein backbone of the glycan does not contribute to ligand binding of CD22, and it is only the presence and density of 2,6Sia that determines binding.
Recently, two sets of experiments have demonstrated that cis-interactions of CD22 on the B cell surface control signaling. In one approach, a CD22 protein with a mutated 2,6Sia-binding domain was expressed in a B cell line (25); in another approach, we used a CD22-specific sialic acid analog which inhibited ligand binding with high affinity (26). In both cases CD22 was less tyrosine phosphorylated, recruited less SHP-1 protein and B cell Ca2+ mobilization was increased after BCR stimulation. Thus, ligand binding in cis directly affects CD22 tyrosine phosphorylation and signal inhibition. These studies directly imply that B cell subpopulations in which CD22 is not ligand bound should show differences in BCR signaling, compared to B cells where CD22 is bound. To identify and characterize B cell populations with unmasked CD22, a new streptavidin-based NeuGc
2,6Gal probe was utilized. This new synthetic ligand for murine CD22 was used to analyze B cells with unmasked CD22 in detail.
 |
Methods
|
---|
Mice
C57BL/6 and CD22-deficient mice (4) which are on a pure (100%) C57BL/6 background, were bred and housed in the animal facilities of our institute. All mice used were aged 610 weeks.
Antibodies
Antibodies used in flow cytometry were anti-mouse B220phycoerythrin (PE) (clone RA3-6B2; PharMingen), anti-mouse CD21biotin (clone 7E9), anti-mouse IgMPE [goat (Fab)2; Medac] anti-mouse CD22FITC (clone Cy34.1; PharMingen), anti-mouse IgDbiotin (clone 11-26C), anti-mouse CD23PE (clone B3-B4; PharMingen), anti-mouse B7-2PE (clone GL1; PharMingen), anti-mouse CD69biotin (clone H1.2F3; Phar Mingen), anti-mouse CD5PE (clone 53-7.3; PharMingen) and anti-mouse MHC-II (I-Ab, clone 25-9-17; PharMingen). Biotinylated antibodies were detected using streptavidinCyChrome (PharMingen). CD22d13Fc was purified from transfected COS cells (a generous gift from A. van der Merwe and P. Crocker). CD22Fc was detected with donkey anti-human IgGFITC (Jackson).
Preparation of NeuGc
2,6GalSAAPFITC
The synthesis and coupling of the sialoside NeuGc
2,6Galß1,4GlcNAcbiotin was described in detail elsewhere (27). Some of the NeuGc
2,6Galß1,4GlcNAcbiotin sialoside was provided by the Consortium for Functional Glycomics. To generate NeuGc
2,6GalSAAPFITC, streptavidinalkaline phosphatase (SAAP) was coupled to FITC. First, 250 µl SAAP (0.5 mg/ml; Sigma) was dialyzed against borate buffer, pH 9 for 24 h, then 6.25 µl FITC (4 mg/ml in DMSO; Molecular Probes) was added and the coupling reaction was allowed to proceed for 4 h in the dark. The FITC-labeled conjugate was dialyzed against PBS, 0.01% NaN3, pH 9 for 48 h. To generate NeuGc
2,6GalSAAPFITC, 30 µl SAAPFITC (0.5 mg/ml) was incubated with 15 µl of 55 µM of the sialoside NeuGc
2,6Galß1,4GlcNAc, biotinylated using Sulfo-NHS-LC-LCbiotin (Pierce) for 30 min at room temperature. This ratio of biotinylated sialoside and SAAPFITC represents a 16-fold molar excess (double the amount of biotin-binding sites on dimeric streptavidin). For the control compound (linkerSAAPFITC), Sulfo-NHS-LC-LCbiotin was hydrolyzed in diethanolamine and then bound to SAAPFITC in the same molar ratio as the sialoside. Both SAAP conjugates were extensively dialyzed, and stored with 1% BSA and 0.1% NaN3 at 80°C.
Cell preparation, treatment and flow cytometry
For the analysis of splenic lymphocytes, single-cell suspensions of total splenic tissue were prepared. Peritoneal cells were isolated by peritoneal lavage with 5 ml of RPMI medium containing 5% FCS. Cells from bone marrow were obtained by flushing both femurs with 1 ml of RPMI medium using a syringe. Prior to FACS analysis, all cells were depleted of erythrocytes by lysis with hypotonic Geys solution. Sialidase treatment was performed with 0.2 U/ml neuraminidase from A. ureafaciens (Roche) (for 1 h at 37°C in PBS/0.1% BSA). Then 15 x 105 erythrocyte-depleted cells were stained with various combinations of antibodies (PharMingen and Becton Dickinson) as indicated. Staining with NeuGc
2,6GalSAAPFITC or linkerSAAPFITC was performed by incubating the cells with the probe for 45 min on ice. For three-color staining using biotinylated antibodies, as well as the sialosideSAAPFITC, the bound biotinylated antibody was well saturated with streptavidinCyChrome, before the NeuGc
2,6GalSAAPFITC was added. Various control stainings were performed to exclude that free streptavidin binding sites on the sialosideSAAPFITC may cross-react with the biotin on biotinylated antibodies. Flow cytometry was performed on a FACSCalibur cytometer (Becton Dickinson). The data were analyzed using CellQuest software (Becton Dickinson).
In vitro stimulation
Mouse splenocytes were prepared as described above and plated in 24-well plates at 1 x 106 cells/ml in PRMI medium with 5% FCS. The cells were stimulated with goat F(ab)2 anti-IgM (10 µg/ml) (Jackson and Dianova) plus 100 U/ml IL-4, lipopolysaccharide (20 µg/ml) (Calbiochem) or anti-CD40 (5 µg/ml) (PharMingen and Becton Dickinson) for different time points. After the stimulation, cells were harvested, washed, stained with labeled antibodies and analyzed by flow cytometry, as described above.
 |
Results
|
---|
Previous studies using fluorescence microscopy with mouse cells had indicated that B cell subsets exist with unmasked CD22 (14). These studies were conducted with a sialosidePAA-based probe containing the NeuAc
2,6Gal linkage. Yet, when the same probe was used in FACS analysis, no unmasked cells were observed (not shown). However, while both murine and human CD22 have a strong preference for the sialoside sequence Sia
2,6Galß1,4GlcNAc, they differ in their preference for the N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). Human CD22 binds both NeuAc as well as NeuGc, while murine CD22 strongly prefers the NeuGc form (28,29). We have previously developed a synthetic probe with NeuGc
2,6Galß1,4GlcNAc coupled to PAA (NeuGc
2,6GalPAA) that was highly specific for murine CD22 (15). However, this probe also did not detect unmasked cells on FACS analysis. In contrast, using a new probe format containing the NeuGc
2,6Galß1,4GlcNAc sequence, unmasked cells were detected (B. E. Collins et al., unpublished). As described in Methods, this probe format is comprised of biotinylated sialiside adsorbed to commercial SAAP, consisting of streptavidin coupled to alkaline phosphatase in a molar ratio of 2:1. While initially developed for ELISA assays for Siglec specificity, by coupling FITC to SAAP prior to binding of the sialoside, the probe was found to be suitable for flow cytometry. Figure 1 shows the binding of this probe to mouse B cells from the spleen. NeuGc
2,6GalSAAP stained B cells specifically when they were pre-treated with sialidase. In contrast, most of B220+ B cells are not stained without sialidase treatment. There is, however, a significant fraction of untreated B cells of C57BL/6 mice (
10%) which can be stained with the sialosideSAAP probe. The specificity of these stainings are demonstrated by two controls. First, when B cells from CD22-deficient mice were used, the NeuGc
2,6GalSAAP conjugate stained only a minor fraction of both untreated and sialidase-treated cells (
23%). Secondly, the linker without sialoside coupled to SAAP (linkerSAAP) gives a similar background staining of
23% of all B220+ B cells, both before and after sialidase treatment (Fig. 1). This demonstrates that the NeuGc
2,6Gal sialoside has a high specificity for CD22. In contrast to the previously used NeuGc
2,6GalPAA probe, this synthetic ligand readily detects a population with unmasked CD22 in C57BL/6 mice.

View larger version (60K):
[in this window]
[in a new window]
|
Fig. 1. A subpopulation of splenic B cells carries unmasked CD22. Splenocytes of C57BL/6 or CD22/ mice were either treated with sialidase at 37°C, or left untreated at 37°C, and then stained with B220 and NeuGc 2,6GalSAAP (upper four panels) or the linkerSAAP control conjugate (lower two panels). The numbers give the percentage of B220+ cells positive for SAAP binding (± SD from four experiments).
|
|
We next wanted to analyze the subpopulation of splenic B cells with unmasked CD22 in more detail. Since B cells with CD22 molecules which are not ligand bound may show altered signaling, it was interesting to see whether these cells are enriched in certain developmental B cell stages, because B cell maturation depends on the BCR signaling capacity. When immature B cells enter the spleen they have the transitional phenotype 1 (T1 B cells) which is characterized by the expression pattern IgMhiIgDloCD21lo. They then enter the T2 stage (IgMhiIgDhiCD21hi). Finally, a maturation to follicular, mature B cells (IgMloIgDhiCD21int) takes place (30). Figures 2 and 3 show three-color staining of C57BL/6 mice with antibodies which separate these developmental stages. Populations corresponding to T1, T2 or mature cells were gated and the NeuGc
2,6GalSAAP binding analyzed. Cells with unmasked CD22 were found in all three populations. However, while within the mature B cell population there was a subpopulation of
89% with unmasked CD22, the population with unmasked CD22 was increased to
14% in transitional T1 and 20% in T2 cells (Figures 2 and 3). Also included in these gated populations are marginal zone B cells, which have overlapping surface markers with both T1 and T2 cells, and are characterized in more detail in Fig. 5. Negative controls using the linkerSAAP for staining of various B cell populations always gave a similar background staining of
2% of B cells as in Fig. 1 (not shown).
It was important to exclude that the higher NeuGc
2,6GalSAAP binding in T1 and T2 cells was due to higher CD22 surface expression. Figure 4(A) shows that T2/marginal zone cells express a similar level of CD22 as mature B cells, while T1 cells express less. Thus probe binding to a higher fraction of T1 or T2 cells cannot be attributed to higher CD22 surface expression. One might expect that unmasking of CD22 correlates directly with down-modulation of 2,6Sia on the surface. This was analyzed by staining with CD22Fc protein and detection with anti-human Ig antibody. The 2,6Sia expression level was 2-fold lower on T1 than on T2 and mature B cells (Fig. 4B). Thus the level of 2,6Sia on the B cell surface did not clearly correlate with unmasked CD22. The population of marginal zone B cells was analyzed separately with CD23/CD21 staining. In addition, marginal zone B cells (CD21hi CD23lo/) contain a subpopulation with increased binding of the NeuGc
2,6GalSAAP probe when compared to the CD21loCD23hi cells, which are mainly mature B cells (Fig. 5).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4. The expression level of CD22 and 2,6Sia in various splenic B cell compartments. Three-color staining was done with anti-CD21 and anti-IgM, and B cell populations gated as shown in Fig. 2. The third color was CD22 (A) or CD22Fc stain for 2,6Sia on the surface (B). m: mean fluorescence intensity of the indicated stainings.
|
|
The masking status of CD22 on B cells was also analyzed in other organs. In the bone marrow, the B cells which were stained with the NeuGc
2,6GalSAAP probe were mature IgDhi B cells. Similar to splenic mature B cells,
10% mature bone marrow B cells carried unmasked CD22 (Fig. 6A). The other B cell population which had unmasked CD22 were transitional B cells of the bone marrow; however, no significant staining of immature B or pre-B cells was detected (not shown). B cells of the blood also contain a population with unmasked CD22 (Fig. 6B). When compared to a parallel staining of splenic B cells (as shown in Fig. 1), there was a higher subpopulation with unmasked CD22 in the blood (16.6% of B220+ cells in the blood versus 10% B220+ cells in the spleen). Finally, peritoneal B cells were analyzed. The peritoneum contains a population of CD5+ B1a cells in mice. A higher fraction of B cells with unmasked CD22 was detected on these B1a cells, when compared to the B2 population (Fig. 7).

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 7. More peritoneal B1a than B2 cells carry unmasked CD22. Peritoneal cells were stained with anti-CD5 and anti-IgM, the typical B1a or B2 population gated, and then analyzed for binding of NeuGc 2,6GalSAAP. B1a cells have a higher proportion of unmasked CD22, when compared to B2 cells. **P < 0.01, Students t-test; tested for significant difference between B1a and B2 cells.
|
|
Since unmasking of CD22 may be influenced by cellular activation processes, we studied whether those splenic B cells with unmasked CD22 have a different expression level of B cell activation markers. Figure 8(A) shows that B cells which could bind the NeuGc
2,6GalSAAP probe have an increased expression of the activation markers B7-2 (CD86), CD69 (not significantly increased) or MHC class II. Experiments were performed in which splenic B cells were stimulated in vitro with various stimuli to analyze whether this treatment leads to unmasking. One example of these experiments is shown in Fig. 8(B). Stimulation of splenic B cells with the shown stimuli for 48 h did not lead to any stronger CD22 unmasking, although the B cells were stimulated well, as demonstrated by B7-2 (CD86) up-regulation (Fig. 8B). Neither were significant changes in CD22 masking status detected at earlier (30 min, and 1, 2, 12 and 24 h) or later (72 h) time points during the cellular stimulation (not shown). This result is in contrast to a similar study on human peripheral blood B cells which demonstrated CD22 unmasking after in vitro stimulation (13).
 |
Discussion
|
---|
CD22 is an important negative regulator of B cell signaling. The binding of CD22 to sialylated ligands in cis controls the efficiency of the inhibition (25,26). Therefore it is important to determine to which degree CD22 is bound to endogenous ligands on B cells. This was analyzed in this study by use of a new probe which contained the natural ligand for CD22: NeuGc
2,6Galß1,4GlcNAc (NeuGc
2,6Gal), coupled via a biotinylated linker to a dimeric SAAP conjugate. This oligomeric ligand stained specifically CD22 on B cells, if the B cells were pre-treated with sialidase. The specificity was demonstrated by the use of B cells of CD22/ mice as a negative control. While most untreated B cells were not stained with the NeuGc
2,6GalSAAP probe, there was a subpopulation of
10% of splenic B cells that could be stained. This staining was interpreted to result from a B cell population with unmasked CD22. The subpopulation with unmasked CD22 was enriched in immature transitional B cells (particularly in T2 cells) and on marginal zone B cells in the spleen. We could also detect subpopulations of B cells with unmasked CD22 in the bone marrow, blood and peritoneal cavity. Finally, the B cell subpopulation which bound the probe showed an activated phenotype.
We have previously used a synthetic CD22 ligand where the sialoside NeuGc
2,6Gal was coupled to a PAA platform (15). In contrast to the probe used in this study, the NeuGc
2,6GalPAA could not readily detect a substantial B cell population with unmasked CD22, although after sialidase treatment binding to almost 100% of the B cells was detected, similar to with the NeuGc
2,6GalSAAP probe. We think that these differences could be simply explained by a higher avidity of the NeuGc
2,6GalSAAP probe. In this case, the probe can better compete with cis glycoprotein ligands. Nevertheless, it is important to discuss whether the staining of a subpopulation of B cells with NeuGc
2,6GalSAAP really detects CD22. We think this is the case, because the probe does not stain CD22-deficient B cells, either before or after sialidase treatment. The weak background binding of CD22-deficient B cells is the same as the background staining of the linkerSAAP probe of wild-type B cells and can thus be attributed to unspecific binding of the SAAP backbone. It is relevant that another study also detected unmasked CD22 in murine B cell subpopulations from the spleen, lymph node and bone marrow by use of a NeuAc
2,6GalPAA probe and fluorescence microscopy (14).
The binding of Siglecs to sialic acid-carrying cellular ligands in cis seems to be a general phenomenon within this family. The only exception may be sialoadhesin (Siglec-1), which due to its size (17 Ig-like domains) extends out of the cellular surface, away from sialylated transmembrane glycoproteins (8). Accordingly, B cells from the ST6GalI-deficient mouse line, which do not express 2,6Sia on the cellular surface, show completely unmasked CD22 (15,31). It is interesting to speculate how the level of cis binding controls the function of the Siglecs as inhibitory signaling receptors. The only example of Siglecs where this has been clearly demonstrated so far is CD22. When cis ligand binding of CD22 is disturbed, tyrosine phosphorylation of CD22 and SHP-1 recruitment are decreased, and inhibition of Ca2+ signaling is subsequently affected (25,26). This means that B cells with unmasked CD22 could potentially show less CD22-mediated inhibition and hence stronger BCR signaling. It has previously been shown that small changes of expression levels of CD22 can already cause a signaling phenotype. Even heterozygous loss of the CD22 gene (and resulting lower surface expression) can contribute to an autoimmune phenotype in BCR transgenic mice (32). Also, after in vitro stimulation of B cells with IL-4, lipopolysaccharide or anti-IgM, CD22 expression levels are characteristically up- or down-regulated respectively (33,34). This suggests that B cell signaling can be regulated by a modulation of the expression level of CD22.
We would like to suggest another model by which the B cell can regulate the strength of inhibition of BCR signaling through CD22. The level of inhibition can also be controlled by
2,6-linked sialylation on the B cell surface. Down-regulation of 2,6Sia on crucial protein ligands by up-regulation of a sialidase or down-regulation of a sialyltransferase may lead to unmasking of CD22, and hence a released inhibition and stronger BCR signaling. We would have expected to detect lower 2,6Sia by staining with CD22Fc protein on those populations (such as T2 cells) which show a higher degree of unmasking. However, our analysis showed that T2 cells not only express a similar amount of CD22 protein on the surface as mature B cells, but are also stained similarly well with CD22Fc. One way to explain this is that CD22Fc may only bind well to accessible 2,6Sia moieties on the cellular surface, but may not detect subtle changes which may affect only certain crucial CD22 ligands. Alternatively, unmasking of CD22 may be the movement into another compartment on the plasma membrane, away from the compartment of the predominant ligand. Exclusion of lipid raft localization may be such a mechanism.
It is very intriguing that we have detected a significantly increased population of transitional T2 B cells with unmasked CD22 in the spleen. Transitional T2 cells are a crucial population as a developmental checkpoint for selection into the pool of mature, follicular or marginal zone B cells (30,35). Numerous knockout mice, deficient for various signaling proteins required for BCR signaling, which have a block in the transition from T2 to mature B cells, have exemplified that this transition is a process dependent on the BCR signal (3639). Since B cells with unmasked CD22 would be expected to show stronger Ca2+ signaling, our findings suggest that these B cells are enriched in the pool of transitional B cells in order to allow a stronger B cell response. The blood contains a relatively high proportion of transitional T1 B cells, which are on their way from the bone marrow to the spleen (30). These may contribute to the higher population of B cells with unmasked CD22 in the blood. Marginal zone B cells are a specialized B cell population in the spleen. Marginal zone B cells are involved in specific immune responses to blood-borne particulate antigens (40). Also, for these cells, less inhibition by CD22 through unmasking would potentially lead to stronger signaling, which would distinguish them from follicular B cells. In future experiments we will address the question whether unmasking of CD22 truly correlates with lower SHP-1 recruitment and stronger Ca2+ signaling. Attempts to study this by comparing Ca2+ mobilization of NeuGc
2,6GalSAAP bound and unbound B cells have not revealed differences so far, but it is not clear how the bound sialoside conjugate affects CD22 function.
Obviously, unmasking of CD22 from endogenous ligands on the same cellular surface potentially allows interactions with ligands on other cells. This may be directly relevant for the function of CD22 as a homing receptor, as has been demonstrated in the bone marrow (19,41). Ligands on other cells such as endothelial cells would have to compete with B cell cis ligands and the unmasked CD22 may favor these trans interactions. One recent study showed that 2,6Sia expression together with a specific cellular antigen on target cells could depress B cell activation in a CD22-specific fashion (20). Also, the level of unmasked CD22 on the B cell could be crucial for these types of interactions.
Another interesting finding of this study was that B cells which can bind the NeuGc
2,6GalSAAP probe have an activated phenotype. This suggests that during B cell activation in vivo, 2,6Sia on the surface is down-regulated and CD22 gets unmasked, allowing a stronger B cell signaling. It can only be speculated whether CD22 unmasking is the result of cellular activation or plays a physiological role in this process in vivo. There is evidence that surface sialylation is down-modulated after cellular activation of lymphocytes (42) and that sialidases are activated (43,44). However, we were unable to demonstrate an increased rate of unmasked CD22 after in vitro stimulation of murine B cells. Anti-IgM treatments even reduced the amount of detectable unmasked CD22, but this treatment also leads to down-regulation of total CD22 (33). These results are in contrast to a report of human B cells which showed partial unmasking of CD22 when stimulated in vitro with anti-IgM and anti-CD40 (13). In the study with human B cells, a PAA carrier was used for the 2,6Sia, whereas here we used a streptavidin-based carrier. In previous experiments, however, we also used NeuGc
2,6Gal coupled to PAA for murine B cells and could also not demonstrate specific binding to CD22 after in vitro stimulation (15). Whether these differences reflect real differences of B cell activation between human and mouse B cells, or technical difficulties in detecting CD22 unmasking under our in vitro conditions, remains to be analyzed in future experiments. In summary, we have shown that a novel sialoside probe can be used to detect unmasked CD22 on subpopulations of murine B cells. The enrichment of unmasked CD22 on B cells such as transitional B cells and B cells with activated phenotype could potentially release the BCR from CD22 inhibition, and thereby lead to stronger B cell signaling.
 |
Acknowledgements
|
---|
We thank Carolin Dix for expert technical help. This work was funded by the Deutsche Forschungsgemeinschaft through SFB 465 (L. N.), and the National Institutes of Health grants GM25042 (B. E. C.) and GM60938 (J. C. P.). We thank the Consortium for Functional Glycomics for providing reagents (NIGMS U54-GM62116).
 |
Abbreviations
|
---|
2,6Siasialic acids in a
2,6 linkage
PAApolyacrylamide
NeuGc
2,6GalNeuGc
2,6Galß1,4GlcNAc
SAAPstreptavidinalkaline phosphatase
NeuAcN-acetylneuraminic acid
NeuGcN-glycolylneuraminic acid
PEphycoerythrin
T1 B celltransitional T1 B cell
T2 B celltransitional T2 B cell
 |
References
|
---|
- Campbell, M. A. and Klinman, N. R. 1995. Phosphotyrosine-dependent association between CD22 and protein tyrosine phosphatase 1C. Eur. J. Immunol. 25:1573.[ISI][Medline]
- Doody, G. M., Justement, L. B., Delibrias, C. C., Matthews, R. J., Lin, J., Thomas, M. L. and Fearon, D. T. 1995. A role in B cell activation for CD22 and the protein tyrosine phosphatase SHP. Science 269:242.[ISI][Medline]
- Law, C. L., Sidorenko, S. P., Chandran, K. A., Zhao, Z., Shen, S. H., Fischer, E. H. and Clark, E. A. 1996. CD22 associates with protein tyrosine phosphatase 1C, Syk, and phospholipase C-gamma(1) upon B cell activation. J. Exp. Med. 183:547.[Abstract]
- Nitschke, L., Carsetti, R., Ocker, B., Köhler, G. and Lamers, M. C. 1997. CD22 is a negative regulator of B-cell receptor signalling. Curr. Biol. 7:133.[ISI][Medline]
- OKeefe, T. L., Williams, G. T., Davies, S. L. and Neuberger, M. S. 1996. Hyperresponsive B cells in CD22-deficient mice. Science 274:798.[Abstract/Free Full Text]
- Otipoby, K. L., Andersson, K. B., Draves, K. E., Klaus, S. J., Farr, A. G., Kerner, J. D., Perlmutter, R. M., Law, C. L. and Clark, E. A. 1996. CD22 regulates thymus-independent responses and the lifespan of B cells. Nature 384:634.[CrossRef][ISI][Medline]
- Sato, S., Miller, A. S., Inaoki, M., Bock, C. B., Jansen, P. J., Tang, M. L. and Tedder, T. F. 1996. CD22 is both a positive and negative regulator of B lymphocyte antigen receptor signal transduction: altered signaling in CD22-deficient mice. Immunity 5:551.[ISI][Medline]
- Crocker, P. R. and Varki, A. 2001. Siglecs, sialic acids and innate immunity. Trends Immunol. 22:337.[CrossRef][ISI][Medline]
- Kelm, S., Pelz, A., Schauer, R., Filbin, M. T., Tang, S., de Bellard, M. E., Schnaar, R. L., Mahoney, J. A., Hartnell, A., Bradfield, P., et al. 1994. Sialoadhesin, myelin-associated glycoprotein and CD22 define a new family of sialic acid-dependent adhesion molecules of the immunoglobulin superfamily. Curr. Biol. 4:965.[ISI][Medline]
- Powell, L. D., Jain, R. K., Matta, K. L., Sabesan, S. and Varki, A. 1995. Characterization of sialyloligosaccharide binding by recombinant soluble and native cell-associated CD22. Evidence for a minimal structural recognition motif and the potential importance of multisite binding. J. Biol. Chem. 270:7523.[Abstract/Free Full Text]
- Engel, P., Nojima, Y., Rothstein, D., Zhou, L. J., Wilson, G. L., Kehrl, J. H. and Tedder, T. F. 1993. The same epitope on CD22 of B lymphocytes mediates the adhesion of erythrocytes, T and B lymphocytes, neutrophils, and monocytes. J. Immunol. 150:4719.[Abstract/Free Full Text]
- Hanasaki, K., Varki, A. and Powell, L. D. 1995. CD22-mediated cell adhesion to cytokine-activated human endothelial cells. Positive and negative regulation by alpha 26-sialylation of cellular glycoproteins. J. Biol. Chem. 270:7533.[Abstract/Free Full Text]
- Razi, N. and Varki, A. 1998. Masking and unmasking of the sialic acid-binding lectin activity of CD22 (Siglec-2) on B lymphocytes. Proc. Natl Acad. Sci. USA 95:7469.[Abstract/Free Full Text]
- Floyd, H., Nitschke, L. and Crocker, P. R. 2000. A novel subset of murine B cells that expresses unmasked forms of CD22 is enriched in the bone marrow: implications for B-cell homing to the bone marrow. Immunology 101:342.[CrossRef][ISI][Medline]
- Collins, B. E., Blixt, O., Bovin, N. V., Danzer, C. P., Chui, D., Marth, J. D., Nitschke, L. and Paulson, J. C. 2002. Constitutively unmasked CD22 on B cells of ST6Gal I knockout mice: novel sialoside probe for murine CD22. Glycobiology 12:563.[Abstract/Free Full Text]
- Kitagawa, H. and Paulson, J. C. 1994. Differential expression of five sialyltransferase genes in human tissues. J. Biol. Chem. 269:17872.[Abstract/Free Full Text]
- Lo, N. W. and Lau, J. T. 1996. Transcription of the beta-galactoside alpha 2,6-sialyltransferase gene in B lymphocytes is directed by a separate and distinct promoter. Glycobiology 6:271.[Abstract]
- Wang, X., Vertino, A., Eddy, R. L., Byers, M. G., Jani-Sait, S. N., Shows, T. B. and Lau, J. T. 1993. Chromosome mapping and organization of the human beta-galactoside alpha 2,6-sialyltransferase gene. Differential and cell-type specific usage of upstream exon sequences in B-lymphoblastoid cells. J. Biol. Chem. 268:4355.[Abstract/Free Full Text]
- Nitschke, L., Floyd, H., Ferguson, D. J. and Crocker, P. R. 1999. Identification of CD22 ligands on bone marrow sinusoidal endothelium implicated in CD22-dependent homing of recirculating B cells. J. Exp. Med. 189:1513.[Abstract/Free Full Text]
- Lanoue, A., Batista, F. D., Stewart, M. and Neuberger, M. S. 2002. Interaction of CD22 with alpha2,6-linked sialoglycoconjugates: innate recognition of self to dampen B cell autoreactivity? Eur. J. Immunol. 32:348.[CrossRef][ISI][Medline]
- May, A. P., Robinson, R. C., Vinson, M., Crocker, P. R. and Jones, E. Y. 1998. Crystal structure of the N-terminal domain of sialoadhesin in complex with 3' sialyllactose at 1.85 A resolution. Mol. Cell. 1:719.[ISI][Medline]
- van der Merwe, P. A., Crocker, P. R., Vinson, M., Barclay, A. N., Schauer, R. and Kelm, S. 1996. Localization of the putative sialic acid-binding site on the immunoglobulin superfamily cell-surface molecule CD22. J. Biol. Chem. 271:9273.[Abstract/Free Full Text]
- Bakker, T. R., Piperi, C., Davies, E. A. and Merwe, P. A. 2002. Comparison of CD22 binding to native CD45 and synthetic oligosaccharide. Eur. J. Immunol. 32:1924.[CrossRef][ISI][Medline]
- Sgroi, D. and Stamenkovic, I. 1994. The B-cell adhesion molecule CD22 is cross-species reactive and recognizes distinct sialoglycoproteins on different functional T-cell sub-populations. Scand. J. Immunol. 39:433.[ISI][Medline]
- Jin, L., McLean, P. A., Neel, B. G. and Wortis, H. H. 2002. Sialic acid binding domains of CD22 are required for negative regulation of B cell receptor signaling. J. Exp. Med. 195:1199.[Abstract/Free Full Text]
- Kelm, S., Gerlach, J., Brossmer, R., Danzer, C. P. and Nitschke, L. 2002. The ligand-binding domain of CD22 is needed for inhibition of the B cell receptor signal, as demonstrated by a novel human CD22-specific inhibitor compound. J. Exp. Med. 195:1207.[Abstract/Free Full Text]
- Blixt, O., Collins, B. E., Van Den Nieuwenhof, I. M., Crocker, P. R. and Paulson, J. C. 2003. Sialoside specificity of the Siglec family assessed using novel multivalent probes: identification of potent inhibitors of myelin associated glycoproteins. J. Biol. Chem. 278:31007.[Abstract/Free Full Text]
- Kelm, S., Schauer, R., Manuguerra, J. C., Gross, H. J. and Crocker, P. R. 1994. Modifications of cell surface sialic acids modulate cell adhesion mediated by sialoadhesin and CD22. Glycoconj. J. 11:576.[ISI][Medline]
- Brinkman-Van der Linden, E. C., Sjoberg, E. R., Juneja, L. R., Crocker, P. R., Varki, N. and Varki, A. 2000. Loss of N-glycolylneuraminic acid in human evolution. Implications for sialic acid recognition by siglecs. J. Biol. Chem. 275:8633.[Abstract/Free Full Text]
- Loder, F., Mutschler, B., Ray, R. J., Paige, C. J., Sideras, P., Torres, R., Lamers, M. C. and Carsetti, R. 1999. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J. Exp. Med. 190:75.[Abstract/Free Full Text]
- Hennet, T., Chui, D., Paulson, J. C. and Marth, J. D. 1998. Immune regulation by the ST6Gal sialyltransferase. Proc. Natl Acad. Sci. USA 95:4504.[Abstract/Free Full Text]
- Cornall, R. J., Cyster, J. G., Hibbs, M. L., Dunn, A. R., Otipoby, K. L., Clark, E. A. and Goodnow, C. C. 1998. Polygenic autoimmune traits: Lyn, CD22, and SHP-1 are limiting elements of a biochemical pathway regulating BCR signaling and selection. Immunity 8:497.[ISI][Medline]
- Lajaunias, F., Nitschke, L., Moll, T., Martinez-Soria, E., Semac, I., Chicheportiche, Y., Parkhouse, R. M. and Izui, S. 2002. Differentially regulated expression and function of CD22 in activated B-1 and B-2 lymphocytes. J. Immunol. 168:6078.[Abstract/Free Full Text]
- Rudge, E. U., Cutler, A. J., Pritchard, N. R. and Smith, K. G. 2002. Interleukin 4 reduces expression of inhibitory receptors on B cells and abolishes CD22 and Fc gamma RII-mediated B cell suppression. J. Exp. Med. 195:1079.[Abstract/Free Full Text]
- Su, T. T. and Rawlings, D. J. 2002. Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development. J. Immunol. 168:2101.[Abstract/Free Full Text]
- Turner, M., Gulbranson-Judge, A., Quinn, M. E., Walters, A. E., MacLennan, I. C. and Tybulewicz, V. L. 1997. Syk tyrosine kinase is required for the positive selection of immature B cells into the recirculating B cell pool. J. Exp. Med. 186:2013.[Abstract/Free Full Text]
- Benatar, T., Carsetti, R., Furlonger, C., Kamalia, N., Mak, T. and Paige, C. J. 1996. Immunoglobulin-mediated signal transduction in B cells from CD45-deficient mice. J. Exp. Med. 183:329.[Abstract]
- Tedford, K., Nitschke, L., Girkontaite, I., Charlesworth, A., Chan, G., Sakk, V., Barbacid, M. and Fischer, K. D. 2001. Compensation between Vav-1 and Vav-2 in B cell development and antigen receptor signaling. Nat. Immunol. 2:548.[CrossRef][ISI][Medline]
- Jumaa, H., Wollscheid, B., Mitterer, M., Wienands, J., Reth, M. and Nielsen, P. J. 1999. Abnormal development and function of B lymphocytes in mice deficient for the signaling adaptor protein SLP. Immunity 11:547.[ISI][Medline]
- Martin, F., Oliver, A. M. and Kearney, J. F. 2001. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 14:617.[CrossRef][ISI][Medline]
- Nitschke, L., Floyd, H. and Crocker, P. R. 2001. New functions for the sialic acid-binding adhesion molecule CD22, a member of the growing family of Siglecs. Scand. J. Immunol. 53:227.[CrossRef][ISI][Medline]
- Bagriacik, E. U. and Miller, K. S. 1999. Cell surface sialic acid and the regulation of immune cell interactions: the neuraminidase effect reconsidered. Glycobiology 9:267.[Abstract/Free Full Text]
- Guthridge, J. M., Kaplan, A. M. and Cohen, D. A. 1994. Regulation of B cell:T cell interactions: potential involvement of an endogenous B cell sialidase. Immunol. Invest. 23:393.[ISI][Medline]
- Chen, X. P., Enioutina, E. Y. and Daynes, R. A. 1997. The control of IL-4 gene expression in activated murine T lymphocytes: a novel role for neu-1 sialidase. J. Immunol. 158:3070.[Abstract]