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Keywords:

  • B lymphocytes;
  • Ca2+ signaling;
  • Siglec;
  • B cell maturation;
  • SHP-1

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

CD22 is an inhibitory coreceptor for B cell receptor (BCR) signaling. The inhibition is most likely mediated by activation of SHP-1. We found that SLP65/BLNK reaches maximal tyrosine-phosphorylation at earlier time points in CD22–/– than in wild type B cells upon BCR cross-linking, suggesting that SLP65/BLNK is a substrate of SHP-1. However, in contrast to the defective Ca2+ mobilization of SLP65/BLNK–/– B cells, there was a clear Ca2+ response in SLP65/BLNK×CD22 double-deficient B cells. This implies that SLP65/BLNK is not the sole target of SHP-1 in the regulation of the Ca2+ signaling strength. While SLP65–/– mice show several blocks of B cell differentiation, in SLP65/BLNK×CD22 double-deficient mice the maturationblock of B cells in the spleen was partially rescued. However, the proliferative responses of B cells from both SLP65/BLNK–/– and double-deficient mice were defective after IgM- or CD40-stimulation. These results show that SLP65/BLNK is not absolutely essential for Ca2+ induction in B cells, because the deficiency of this adapter can be by-passed by the additional deletion of an inhibitory receptor. Furthermore, these experiments suggest that B cell maturation in the spleen is directly dependent on the strength of BCR-derived Ca2+ signals.

Abbreviations:
BCR:

B cell receptor

ITIM:

Immunoreceptor tyrosine-based inhibition motif

Siglec:

Sialic acid-binding immunoglobulin-like lectin

2,6 Sia:

α2–6 linked sialic acids

T1:

Transitional type 1

T2:

transitional type 2

MZ:

Marginal Zone

TI:

Thymus independent

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

B cell maturation and B cell activation is controlled by B-cell receptor (BCR)-derived signals. The strength and quality of the signals, triggered by antigen binding to the BCR, determines thesurvival, maturation and immune response of the B cells. BCR signaling is modulated by accessory transmembrane molecules or coreceptors. The accessory molecule CD22 is an important negative regulator on B lymphocytes. This has been clearly demonstrated by the generation of CD22-deficient mice 14. B cells of these mice showed strongly increased Ca2+ mobilization upon BCR cross-linking. CD22 is associated with IgM and IgD on the B cell surface. After anti-IgM treatment CD22 is quickly tyrosine phophorylated on its cytoplasmic tail. The tyrosine kinase mainly responsible for CD22 phosphorylation is Lyn, as was demonstrated by reduced CD22 phosphorylation in Lyn-deficient mice 5, 6. The cytoplasmic tail of CD22 contains six tyrosines, three of which belong to the ITIM (immunoreceptor tyrosine-based inhibition motif) consensus sequence. The phosphorylated ITIM motifs of CD22 recruit the tyrosine phosphataseSHP-1, an important negative regulator of many signaling pathways in hematopoietic cells 7. Since SHP-1-deficient motheaten mice display a similar increased Ca2+ mobilization as CD22-deficient mice 8, it is generally assumed that SHP-1 is the crucial signaling molecule which mediates the inhibitory function of CD22 9. However, the mechanism by which CD22-activated SHP-1 regulates the strength of the Ca2+ flux is still unsolved. Surprisingly, the negative regulation of Ca2+ signaling by CD22 does not takeplace in a cell line expressing an IgG receptor 10.

Prominent substrates for SHP-1 are expected to show a higher tyrosine-phosphorylation level in CD22-deficient B cells. However, the general level of tyrosine phosphorylation does not reveal changes in CD22-deficient B cells, when compared to control B cells, both before and after BCR stimulation 1, 11. Various groups searched for candidate tyrosine-phosphatase substrates of the proximal BCR pathway. So far only Vav-1 and CD19 have been found to be stronger tyrosine phosphorylated in CD22–/– B cells 11, 12. In addition to SHP-1, other proteins are recruited via their SH2 domains to the tyrosine-phosphorylated tail of CD22, which are normally positively involved in BCR signaling. These include Syk, PLCγ2, PI3K, Grb-2 and Shc 1315. Grb-2 binds to another phosphorylated tyrosine of CD22, distinct from the ITIM tyrosines which are bound by SHP-1 12, 15. The other proteins have partially overlapping binding sites with SHP-1. One group has shown that the lipid phosphatase SHIP can bind indirectly to the CD22 tail by interaction with Grb-2 and Shc 14. SHIP is the crucial phophatase activated by the inhibitory receptor FcγRII in B cells 16. However, B cells of SHIP-deficient mice do not show increased Ca2+ mobilization when their BCR is stimulated alone (without co-crosslinking to the FcγRII) 17. Thus, there is so far no evidence showing that SHIP is the crucial phosphatase regulating Ca2+ signaling downstream of CD22.

In vivo, CD22 regulates early B cell activation and B cell maturation. CD22-deficient mice do not show defects on early B-cell differentiation in the bone marrow, but characteristic changes in later B cell maturation and migration. A preferential development to follicular, mature B cells, but defective development to marginal zone B cells can be attributed to the higher activation status of CD22–/– B cells, resulting from increased Ca2+ fluxing 1, 18. A defect of recirculating B cells in homing to the bone marrow can be explained by the function of CD22 as an adhesion receptor 19. B1 cells of CD22–/– mice do not show a change in numbers. This can be explained by the fact that CD22 does not inhibit Ca2+ mobilization in B1 cells 20.

Apart from its function as inhibitory receptor, CD22 has properties of an adhesion molecule and belongs to the Siglec (sialic acid-binding immunoglobulin-like lectin) family 21. This family of inhibitory adhesion receptors is characterized by binding specifically to sialic acids. CD22 has a high specificity for Neu5Acα2–6Galβ1–4Glc(NAc) or α2–6-linked sialic acid (2,6 Sia). 2,6 Sia is a common structure on N-linked glycans and abundantly expressed on the surface of lymphocytes or other cells. We and others have recently shown that the 2,6 Sia-binding function of CD22 directly regulates signaling 22, 23. CD22 is bound to ligands on the B cell surface in cis, on most B cells. When this interaction is destroyed, less CD22 tyrosine phosphorylation, less SHP-1 recruitment and a higher B cell Ca2+ flux is induced.

Here we demonstrate that the B cell-specific adapter molecule SLP65/BLNK (also called Bash) 2426 is tyrosine-phosphorylated after BCR cross-linking at earlier time points in CD22–/– B cells than in control B cells. SLP65/BLNK is a crucial intracellular adapter molecule that acts as a scaffold by assembling a macro-molecular complex that includes important enzymes for signaling (PLCγ2, Btk and Vav1), as well as further linker proteins (Grb-2 and Nck) 24, 25. In mice and a chicken B-cell line it has been demonstrated that SLP65/BLNK-deficiency leads to impaired Ca2+ signaling 2729. To test whether the inhibitory functions of CD22 and SHP-1 on Ca2+ are mediated via dephosphorylation of SLP65, we produced SLP65/BLNK×CD22 double-deficient mice.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

In order to clarify the mechanism of the CD22-mediated inhibition of the Ca2+ signal, we compared the tyrosine phosphorylation level of different intracellular signaling proteins in CD22-deficient and wild-type B cells. We expected some proteins to be stronger tyrosine phosphorylated, which are known to trigger the signaling cascade leading to Ca2+ mobilization. Therefore, B cells of control and CD22-deficient mice were stimulated with anti-IgM and specific immunoprecipitations of tyrosine kinases, adapter proteins and PLCγ2 were done. We could not detect any stronger phophorylation pattern in CD22–/– B cells when we immunoprecipitated the tyrosine kinases Lyn and Syk or the phospholipase PLCγ2 (not shown). These findings are in accordance with results from other groups 11, 12. However, when we compared the activation of the adapter protein SLP65/BLNK, a different tyrosine phosphorylation pattern was detectable. SLP65/BLNK was tyrosine phophorylated at earlier time points in CD22-deficient B cells upon anti-IgM treatment, as was demonstrated by two different methods (Fig. 1A and B). In CD22–/– B cells full tyrosine phosphorylation of SLP65/BLNK was reached after 10 s (Fig. 1A), or after 30 s (Fig. 1B), which was much faster than in control B cells. In these, full activation of SLP65/BLNK took 1 to 3 min. The adapter protein SLP65/BLNK is crucial for triggering Ca2+ responses in B cells 2729.

The earlier SLP65/BLNK tyrosine phosphorylation in CD22–/– B cells could indicate that in normal wild-type B cells the adapter is a direct substrate of SHP-1, which is activated via CD22. If SLP65/BLNK is the exclusive substrate for SHP-1 in the regulation of Ca2+ mobilization, then the SLP65/BLNK deficiency should have a dominant Ca2+ signaling defect, even in the presence of an additional CD22 deficiency. To test this, we crossed the CD22–/– mice with SLP65/BLNK-deficient mice. B cells of single-deficient or double-deficient mice were stimulated with anti-IgM and the Ca2+ mobilization was measured. SLP65/BLNK–/– B cells show the expected impaired and CD22–/– B cells the described higher Ca2+ mobilization (Fig. 2). Surprisingly, in double-deficient B cells a Ca2+ response was measured which was clearly higher than in SLP65/BLNK–/– B cells. This partial rescue of Ca2+ demonstrated that SLP65/BLNK cannot be the only CD22/SHP-1 substrate in the regulation of Ca2+ signaling.

SLP65/BLNK-deficient mice display several blocks in B cell differentiation. One block is at the preB cell stage in the bone marrow. The second block is in the maturation of transitional B cells to mature B cells in the spleen. Additionally, B1a cells are not present in the usual frequency 2628. Since SLP65/BLNK×CD22 double-deficient mice show a rescued Ca2+ mobilization, we analyzed which of these developmental blocks could be overcome by the increased signaling. Analysis of bone marrow cells revealed that there was no rescue of early B-cell differentiation in double-deficient mice (Fig. 3). Both SLP65–/– and double-deficient mice showed increased numbers of pro/preB cells (B220loIgM) and a relative decrease of immature (B220loIgMlo), transitional (B220lo-hiIgMhi) and mature B cells (B220hiIgMlo), when compared to control mice. SLP65 is a tumor suppressor and its deficiency leads to spontaneous preB cell tumors in SLP65–/– mice 30, 31. We did not observe a changed rate of these spontaneous tumors in SLP65/BLNK×CD22 double-deficient mice.

In the spleen, however, the maturation block of SLP65/ BLNK–/– B cells at the transitional type 2 (T2) stage was partially rescued in double-deficient mice. Fig. 4A and B illustrates a decrease in the relative number of T2 cells and a corresponding relative increase in mature (M) B cells in the double-deficient mice. As has previously been shown, CD22–/– mice have a relatively decreased T1 and T2 compartment and more mature B cells, probably due to increased BCR signaling 1. The total splenocyte numbers of SLP65/BLNK- and double-deficient mice were similarly 25 to 30% reduced (average from five exp.: control: 8.7×107, CD22–/–: 9×107, SLP65/ BLNK–/–: 6.4× 107, SLP65/BLNK×CD22 double-deficient mice: 7×107 splenocytes). Therefore, there was also an improvement of the absolute number of mature B cells in double-deficient mice. However, the mature B cells numbers of wild type mice were not reached.

Fig. 4C shows a specific staining for marginal zone (MZ) B cells. SLP65–/– mice have a normal number of MZ B cells, which has not been analyzed before. Also SLP65/ BLNK×CD22 double-deficient mice have normal MZ B cell numbers, whereas CD22–/– mice have the described MZ B cell defect 18. While the CD22-deficiency could apparently correct developmental blocks in the spleen, it could not rescue the defective development of B1a cells in SLP65/BLNK–/– mice. Fig. 5 shows the analysis of cells of the peritoneal cavity. The relative number of B1a cells of control mice is unchanged in CD22–/– mice and drops about tenfold both in SLP65/BLNK–/– and double-deficient mice.

SLP65/BLNK–/– B cells do not proliferate when stimulated with anti-IgM or anti-CD40. These defects are not rescued in SLP65/BLNK×CD22 double-deficient B cells (Fig. 6A). In contrast to published results 27, our experiments revealed repeatedly normal proliferation of SLP65/BLNK–/– B cells when stimulated with both anti-CD40 plus IL-4. While SLP65/BLNK–/– B cells have a defective LPS response, the LPS response of double-deficient B cells was increased (Fig. 6B). LPS-induced proliferation of CD22–/– B cells is even higher than that of normal B cells. The mechanism for this higher response is not known, but it is apparently independent of the SLP65 defect, because both effects can compensate each other (Fig. 6B). Other immunological defects of SLP65/BLNK–/– mice, such as reduced serum levels of IgM and IgG3 or impaired thymus-independent immune responses, could not be rescued in SLP65/BLNK×CD22 double-deficient mice (not shown).

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Figure 1. Earlier tyrosine phosphorylation of SLP65/BLNK in CD22–/– mice. (A) Splenic B cells of control or CD22–/– mice were stimulated with anti-IgM (20 μg/ml) for indicated time points. The cells were lysed and proteins precipitated with a Grb2(SH3 SH2)-GST fusion protein. SLP65/BLNK is bound by the SH3 domain of Grb-2, therefore an equal amount of SLP65/BLNK was precipitated before and after stimulation (lower blot). The upper blot shows kinetics of tyrosine phosphorylation of SLP65/BLNK. This is quantified below by densitometric analysis (Signals of anti-P-Tyr divided by signals of anti-SLP65/BLNK, shown for first 3 min). (B) Splenic B cells were stimulated with anti-IgM (5 μg/ml), the cells were lysed and the proteins were immuno-precipitated with anti-SLP65/BLNK antiserum. The blotted proteins were stained with anti-phospho tyrosine antibodies, and after stripping reprobed with anti-SLP65/BLNK antiserum. Densitometric analysis was done as in (A). One out of three typical experiments (for both A and B) is shown.

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Figure 2. CD22 deficiency rescues Ca2+ mobilization in SLP65/BLNK–/– splenic B cells. Splenic cells of control, CD22–/–, SLP65/BLNK–/– or SLP65/BLNK×CD22 double-deficient mice were stimulated with 10 μg/ml B7–6 (anti-IgM antibody) at the time point indicated by arrows. B cells were gated and the ratio of bound to unbound Indo-1 was determined. Intracellular Ca2+ concentration is proportional to the ratio of bound to unbound indo-1. One out of three typical experiments is shown.

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Figure 3.  SLP65/BLNK×CD22 double-deficient mice show similar blocks of B cell development in the bone marrow as SLP65/BLNK–/– mice. Bone marrow cells were isolated from indicated mice and stained with anti-B220 and anti-IgM antiobodies. Percentages of non-B cells (B220 IgM), pro and preB cells (B220lo IgM), immature B cells (B220lo IgMlo), transitional B cells (B220lo-hi IgMhi) and mature, recirculating B cells (B220hi IgMlo) are given. One representative out of five experiments is shown.

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Figure 4. The maturation block of SLP65/BLNK–/– B cells in the spleen is partially rescued in SLP65/BLNK×CD22 double-deficient mice. (A) The staining with anti-IgM and anti-IgD antibodies separates developmental stages of the spleen: transitional type 1 (T1), transitional type 2 (T2) and mature (M) B cells. Marginal zone (MZ) B cells fall into the T1 gate. SLP65/BLNK–/– mice show a block at the T2 stage of development. In SLP65/BLNK×CD22 double-deficient mice this block is partially rescued, shown by decreased percentage of T2 and increased percentage of mature B cells. This partial rescue is also reflected in absolute numbers of B cells (see text). (B) The staining with anti-IgM and anti-CD21 separates T1, T2 and M B cells, as well. The same partial rescue as in (A) is observed in SLP65/BLNK×CD22 double-deficient mice. (C) Shown are anti-B220 gated cells, stained with anti-CD23 and anti-CD21. The indicated box are MZ B cells. MZ B cells are decreased in CD22–/– mice but found in normal numbers in SLP65/BLNK–/– and double-deficient mice. One representative out of four experiments is shown. The numbers give the percentage of cells. In (C) mean percentage ± SD is given.

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Figure 5. Both SLP65/BLNK–/– and SLP65/BLNK×CD22 double-deficient mice show decreased numbers of B1a cells in the peritoneum. Peritoneal lavage cells were stained with anti-CD5 and anti-IgM and analyzed by flow cytometry. One typical out of five experiments is shown.

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Figure 6. Impaired proliferative responses of SLP65/BLNK–/– B cells are not rescued by the CD22-deficiency. (A) and (B). Splenic B cells of the indicated mice were cultured for 48 h with the shown stimuli and [3H]thymidin incorporation was measured. Means of triplet measurements plus SD are shown. One typical out of three experiments is shown.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

In a search for substrates of the CD22/SHP-1 inhibitory pathway in B cells, we have identified the adapter protein SLP65/BLNK, which is earlier and stronger tyrosine-phosphorylated in CD22–/– B cells than in wild-type B cells upon BCR cross-linking. However, SLP65/BLNK×CD22 double-deficient B cells showed a clear Ca2+ response, in contrast to the practically absent Ca2+ response of SLP65/BLNK–/– B cells. This indicates that SLP65 cannot be the sole target of SHP-1 and CD22 in regulating the Ca2+ strength. In double-deficient mice maturation of B cells in the spleen was partially corrected, but differentiation blocks of preB cells in the bone marrow and of the peritoneal B1a cells were unaffected. Also, defects of SLP65/BLNK–/– mice in proliferative responses after IgM-or CD40-stimulation could not be overcome in double-deficient mice.

The main phenotype of CD22-deficient mice is the highly increased Ca2+ mobilization after anti-IgM stimulation of CD22–/– B cells 14. Themechanism of the negative regulation of Ca2+ signaling by CD22 has not been solved so far. Since SHP-1 is the prominent phosphatase binding to the ITIM motifs of CD22 7, 32 and since CD22 is associated with the BCR 33, 34, the general assumption is that intracellular substrates within the BCR signaling complex are dephosphorylated by the recruited SHP-1. Accordingly, in splenic B cells of CD22–/– mice one would expect to find prominent substrates of SHP-1 to be stronger tyrosine-phosphorylated. However, tyrosine phosphorylation of several candidates was not increased in CD22-deficient B cells, as was shown by us and other groups. Specifically, Igα, Igβ, Syk, Lyn, Blk, Fyn and PLCγ2 do not show a stronger tyrosine phosphorylation after activation with anti-IgM 11, 12. So far it has only been demonstrated that Vav1 and CD19 are higher tyrosine phosphorylated in CD22–/– B cells than in control B cells 11, 35. However, B cells from both Vav1- and CD19-deficient mice do not show strong defects in BCR-induced Ca2+ mobilization 35, 36.

SLP65/BLNK, on the other hand, is an important regulator of Ca2+ responses 29. Recently it has been demonstrated in DT40 cells (a chicken B cell line), that tyrosine phosphorylation of SLP65/BLNK is absolutely necessary for triggering BCR-mediated Ca2+ responses. SLP65/BLNK has three PLCγ2- and one BTK-binding site, all of which act in concert to control the strength of the Ca2+ signal 37. The main tyrosine kinase responsible for SLP65/BLNK phosphorylation is Syk 25. Our finding that SLP65/BLNK reaches the maximum of tyrosine phosphorylation at earlier time points in CD22–/– B cells could indicate that the tyrosines of SLP65/BLNK are phosphorylated and desphoshorylated by Syk and SHP-1, respectively, and it is the equilibrium of available activated kinases or phosphatases which regulates the activation status of SLP65/BLNK. It was previously shown that SLP65/BLNK is a direct substrate of SHP-1 in B cells 38, 39. However, when we analyzed SLP65/BLNK×CD22 double-deficient B cells we observed a partially rescued Ca2+ response. This suggests that the signaling defect of SLP65/BLNK–/– B cells is compensated by the higher Ca2+ mobilization, caused by the CD22 deficiency. If SLP65/BLNK is the sole effector of CD22 in regulation of Ca2+ signaling, then the double knockout should be as defective as the SLP65/BLNK knockout. This is not the case, therefore CD22 must have at least one other effector that regulates Ca2+, induced via a SLP65/BLNK-independent pathway. It is also important to note that the presence of SLP65/BLNK is not as essential for Ca2+ signaling in B cells, as had been previously suggested by studies in genetically deficient mice and B cell lines 2729. Ca2+ mobilization is still possible, even in absence of this adapter, as our SLP65/BLNK×CD22 double-deficient mice show.

If SLP65/BLNK is not the essential target of the CD22 and SHP-1 pathway, then there must be other substrates of SHP-1 which control the strength of the Ca2+ signal in B cells. Candidates are Vav1 and CD19 which both were found to be higher tyrosine-phosphorylated after BCR stimulation of CD22-deficient cells 11, 35. However, it is unlikely that these proteins are the crucial SHP-1 substrates, because both CD19–/– and Vav1–/– B cells show no strong defects in Ca2+ mobilization 35, 36, 40. A possible SHP-1 target is the Vav1 homologue Vav2. Vav2 can compensate for Vav1 function in B cells, and Vav1/ Vav2 double-deficient B cells have a clear Ca2+mobilization defect 36, 40. Another possibility to explain our findings is that SHP-1 targets only selected, specific tyrosines, in one of the crucial signaling enzymes or adapters. For instance, mouse SLP65/BLNK has seven different tyrosines that are bound by distinct SH2-domain containing signaling molecules 37. It is possible that a crucial binding site for PLCγ2 or BTK is dephosphorylated by SHP-1 without strongly affecting the overall tyrosine phosphorylation level of SLP65/BLNK. Also, SLP65/BLNK is recruited via a non-ITAM tyrosine of Igα to the membrane 41, 42. This tyrosine of Igα could be a selective target for SHP-1, without affecting the overall phosphorylation level of Igα significantly. These possibilities have to be studied in detail in the future. It has also been suggested that the crucial inhibitory downstream phosphatase of CD22 is SHIP, which binds indirectlytogether with Grb-2 and Shc to CD22 14. But so far no direct evidence supports the hypothesis that this binding regulates the Ca2+ signaling strength. An argument against SHIP being the important phophatase for inhibition of BCR-induced Ca2+ signaling comes from the analysis of SHIP–/– mice. These mice do not show impaired Ca2+ mobilization after BCR stimulation 17. SHIP is, however, crucial in FcγRII-mediated Ca2+ inhibition 16.

The generation of SLP65/BLNK×CD22 double-deficient mice described here allowed the analysis of B cell differentiation under the influence of these mutations. CD22 is expressed only weaklyon the surface of preB cells 1, 43 and it is not known whether it regulates preBCR signaling. CD22-deficient mice do not show any defects in early B-cell differentiation in the bone marrow. Therefore, it was not surprising that the CD22-deficiency could not rescue the developmental block found in SLP65/BLNK–/– mice at the proB/preB cell stage 27, 28. However, the second block in maturation of SLP65/BLNK-deficient B cells, in the differentiation to mature, follicular B cells in the spleen, was partially rescued in double-deficient mice. This rescue can most likely be explained by the higher signaling of double-deficient B cells. B cells need constant BCR signaling to survive and the strength of these signals controls the development to mature B cells, as has been shown in many genetically deficient mice 44. We cannot exclude that the CD22 and SLP65/BLNK mutations also shift the V(D)J repertoire of B cell surface immunoglobulins. This may affect B cell selection into the pool of mature B cells and also contribute to the observed changes in subpopulations. Another example of the rescue of B cell maturation by the CD22 deficiency was observed in mice deficient for the transcription factor Bob.1/OBF.1. These mice have also a defect in Ca2+ signaling and a block at theT2 stage of development. The crossing-in of the CD22-deficiency led to increased Ca2+ signals and a partial rescue of the differentiation to mature B cells 45.

In contrast, the B1a defect of SLP65/BLNK–/– mice could not be rescued by the CD22 deficiency. We have previously shown that CD22 does not inhibit Ca2+ signaling in B1a cells20. Therefore, the Ca2+ signaling capacity is expected to be unchanged in B1a cells of SLP65/BLNK×CD22 double-deficient mice compared to SLP65/BLNK–/– mice. Therefore, we did not expect to find a rescue of B1a cell numbers. Finally, we have shown for the first time, that SLP65/BLNK-deficient mice have a normal number of MZ B cells. There is accumulating evidence from a large number of gene-deficient mice that the signaling strength of the BCR controls differentiation into mature B cells versus MZ B cells. The knockout of genes, which lead to defects in various signaling pathways downstream of the BCR, show a defective development to mature B2 cells and to B1 cells. However, the MZ B cell compartment is usually not affected or even increased in size in the majority of knockout mice with BCR signaling defects 44, 46. In contrast, mice with a stronger BCR signaling, like CD22–/– mice, usually show an increased number of mature, but decreased number of MZ B cells 18, 44. Thus SLP65/BLNK-deficient mice, which have BCR signaling defects, show the pattern of developmental blocks in the periphery which fit to this model.

When analyzing the proliferative responses to anti-IgM stimuli of SLP65/BLNK–/– and SLP65/BLNK×CD22 double-deficient mice, we did not see any differences. CD22–/– mice show defects in anti-IgM-triggered proliferation 1, 3, 4, a phenotype which is not understood mechanistically, because MAP kinase pathways are notgrossly affected 12, 14. The defective proliferation of SLP65/BLNK-deficient B cells after CD40 stimulation was not rescued in double-deficient B cells. In contrast to our own previous experiments 1, CD22–/– B cells showed a higher proliferative anti-CD40 response than normal B cells. This was repeatedly seen and we can only attribute this to the other genetic background of the mice (here: C57BL/6×BALB/c F1). CD22–/– B cells mount stronger LPS responses than wild-type B cells. This finding has not been explained mechanistically so far. Possibly expression of TLR are up-regulated due to basal over-stimulation of CD22–/– B cells. It is also unclear why the LPS response is defective in SLP65/BLNK–/– B cells. Double-deficient mice showed a normal response, suggesting that LPS signaling is affected independently by both mutations and that both mutations seem to compensate each other.

In summary, we have observed after activation of CD22–/– B cells that SLP65/BLNK is stronger phosphorylated at early time points. This suggests that SLP65/BLNK is a direct SHP-1 substrate. Nevertheless, CD22×SLP65/BLNK double-deficient B cells showed a higher Ca2+ mobilization than SLP65/BLNK-deficient B cells. Our results show that SLP65/BLNK is not as essential for Ca2+ induction in B cells as previously thought, because the deficiency of this adaptor can be by-passed by the additional deletion of an inhibitory receptor. Since a block in B cell differentiation was partially rescued in double-deficient mice, these experiments also suggest that B cell maturation in the spleen is directly dependent on the strength of BCR-derived Ca2+ signals. Both findings are important for understanding the regulation of BCR signaling and its influence on survival, selection and differentiation of B cells in the periphery.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Mice

For Fig. 1 C57BL/6 wild-type and CD22–/– mice (100% C57BL/6) were used. In all other experiments, control mice, CD22–/–, SLP65/BLNK–/– or SLP65/BLNK×CD22 double-deficient mice were on the F1 (BALB/c×C57BL/6) background. All mice were obtained from our breeding facility. Age-matched mice were analyzed 6–10 weeks after birth.

4.2 Immunoprecipitation and Western blot

Splenic B cells were purified by complement lysis of T cells 45. Before stimulation 1.2×107 B cells per time-point were preincubated on ice with FcγRII-blocking antibody (clone 2.4G2, our hybridoma collection). Cells were stimulated at 37°C with B7–6 (anti-IgM). Stimulation was stopped by lysing the pelleted cells in 1 ml ice-cold NP-40 buffer containing 1 mM Na3VO4, 5 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM PMSF. For immunoprecipitation 3 μl of rabbit anti-BLNK antiserum 29 was added to each lysate. Pull-down experiments with the glutathione S-transferase (GST)-Grb-2 fusion protein containing amino acid 1–168 (SH3-SH2) of human Grb-2 24 were performed by adding 5 μl GST-Grb2(SH3-SH2) coupled to glutathion-Sepharose beads (Amersham, Braunschweig, Germany) to each lysate. Precipitates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-phospho-tyrosine antibody (clone 4G10, Upstate, Lake Placid, NY) and anti-BLNK antiserum.

4.3 Flow cytometry analysis

Single-cell suspensions of bone marrow cells or splenocytes (after isotonic erythrocyte lysis) or directly isolated peritoneal lavage cells were incubated for 30 min at 4oC with different combinations of the following antibodies: anti-B220-biotin; anti-B220-FITC, anti-IgD-FITC, anti-CD21-FITC, anti-IgM-FITC; anti-IgM-PE, anti-CD23-PE, anti-CD5-PE (all from PharMingen, San Diego,CA). Stainings were performed in PBS containing 0.1% BSA, 0.1% Na-azide and saturating concentration of FcγRII-blocking antibody. Biotin-labeled antibody was revealed by Streptavidin-Cy-Chrome (PharMingen) in a second staining step. Cell surface marker expression was analyzed using a four-color flow cytometer (FACScalibur) and Cell Quest Software (Becton Dickinson, Heidelberg, Germany).

4.4 Measurement of Ca2+ movement

Splenocytes were Indo-1 loaded and stained extracellularly as described 45. Ca2+ movement was measured upon stimulation of splenocytes with 10 μg/ml B7–6 monoclonal anti-IgM antibody at 37°C. Increases in intracellular free Ca2+ of B cells was measured in real time with the use of a FACSvantage (Becton Dickinson), by gating on Mac.1- and Thy1.2-negative B cells.

4.5 Proliferation assay

Splenocytes from the indicated animals were T cell depleted by complement lysis as described 45. B cells (1×105) were stimulated for 48 h with LPS (Calbiochem,Novabiochem, Darmstadt, Germany), anti-IgM F(ab′)2 (Dianova, Jackson, Hamburg, Germany), B7–6, anti-CD40 (PharMingen, San Diego, CA) or IL-4 (200 U/ml). Proliferation was measured by[3H]thymidine incorporation (1 μCi/well) during the last 8 h of culture.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

We thank Astrid Heiter and Carolin Dix for technical help. This work was supported by grants through the Deutsche Forschungsgesellschaft SFB465/B8 (LN) and SFB549 (JW) and EU program QLG1-CT-2001-01536 (JW).

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