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Department of Microbiology and Immunology and Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40536
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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activation, were similar in B cell Ag receptor (BCR)-
and CD72-stimulated B cells and that BCR- but not CD72-mediated early
signaling events were blocked by protein kinase A activation. The
present report shows that CD72 ligation induces a variety of
tyrosine-phosphorylated proteins, most of which were of the same
molecular mass as those seen in anti-IgM-treated B cells, except
for a 72-kDa protein. Further analysis showed that the tyrosine kinases
lyn and blk were activated in CD72-ligated B cells. Interestingly, the
non-src kinase syk was not activated in CD72-stimulated cells whereas
the tec family kinase btk was activated in both CD72- and
BCR-stimulated B cells. Furthermore, B cells from xid mice were
unresponsive to CD72-induced proliferation, indicating an essential
role for btk in CD72-induced signaling events. Surprisingly, tyrosine
phosphorylation of phospholipase C-2 was normal in CD72-stimulated
cells in spite of a lack of activation of syk. Furthermore, B cell
proliferation through CD72 was blocked by the immunosuppressive agents
cyclosporin A and FK506, indicating the important role for
Ca2+-regulated activation events similar to
BCR-stimulated cells. We propose that btk can substitute for syk in
inducing phospholipase C-2 tyrosine phosphorylation and initiating
calcium mobilization in CD72-stimulated B lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2
(PLC-2) (8), phosphatidylinositol 3-kinase (9), and ras-GAP (10)
undergo tyrosine phosphorylation leading to generation and
amplification of diverse signals into the cell. For BCR-induced
signaling events, initial activation of the src-kinase lyn is essential
for activation of syk and btk (11, 12), which in turn control tyrosine
phosphorylation and activation of PLC-2 (13, 14). However, recent
evidence demonstrates a lyn-independent activation of syk (15) in
BCR-stimulated B lymphocytes. Thus coordinate activation and
interaction of different signaling molecules tightly regulate cellular
responses to various signals. The B cell specific coreceptor CD72 is a 45-kDa type II transmembrane glycoprotein expressed in all stages of B cells except plasma cells. Stimulation through CD72 induces B cell proliferation (16, 17, 18), an increase in levels of surface MHC class II (19, 20), and augmentation of IL-4-dependent CD23 expression (21). CD72 has an important role in differentiation of B cells into Ab-secreting cells in responses to both thymic-independent and thymic-dependent Ags (20, 22, 23). Thus, activation of Ag-specific B lymphocytes by the thymic independent Ag TNP-Ficoll was enhanced by signaling through CD72 (22).
Although the exact mechanisms involved in CD72-mediated B cell
activation are unknown, ligation of CD72 induces hydrolysis of inositol
phospholipids (18, 24) and elevation of intracellular calcium (18, 19).
Previously we showed that both these signaling events were similar in B
cells stimulated through CD72 and BCR (18). However, pathways used by
CD72 and BCR were differentially regulated by PKA. Thus, cAMP-elevating
agents like forskolin (18) or PGE2 (25) inhibited
BCR-mediated but not CD72-mediated B cell proliferation. cAMP inhibited
BCR-induced activation of phospholipase C- and
[Ca2+]i mobilization indicating a
"cross-talk" between cAMP and phosphatidylinositol pathways for
BCR- but not CD72-mediated signaling pathways (18). These results
suggested that the potential targets of PKA may be, at least in part,
at the level of PTK activation and/or PLC activation. CD72-mediated
signals partially overcome apoptosis in B lymphocytes caused by
hyper-cross-linking the BCR in vitro (26). The PKA resistance of CD72
signaling, together with its ability to deliver antiapoptotic signals,
strongly suggests that CD72 signals may have a bystander role to
antagonize negative effects on B cell activation during immune
responses.
As an initial step toward understanding the key differences in early
signals generated through CD72 and BCR, we investigated the role of
tyrosine phosphorylation and activation of different PTKs following
stimulation through these receptors. Ligation of CD72 resulted in
activation of PTKs, lyn, and blk. Unlike BCR-ligated B cells, the
non-src family kinase syk was not activated upon signaling through
CD72. Surprisingly, tyrosine phosphorylation of PLC-2 was apparently
normal in CD72-stimulated B cells without activation of syk. Instead,
btk, the tec family member, was found to be critical for CD72-mediated
B cell activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female DBA/2, BALB/c, CBA/Ca (non-xid), and CBA/N (xid) mice were obtained from the National Cancer Institute, Frederick, MD.
Reagents
Details have been previously described of monoclonal
anti-CD72 Abs K10.6 (27, 28) and 10.1.D2 (17) and of
isotype-matched control Ab (CAb) MOPC195 (M195) (18) used in this
study. Goat anti-mouse IgM F(ab')2 was purchased from
Organon Teknika (Durham, NC). Genistein and herbimycin A were from
Calbiochem-Behring (San Diego, CA). The stock solutions of these PTK
inhibitors were prepared in DMSO and stored at -20°C until further
use. Cyclosporin A was from Sandoz Research Institute (East Hanover,
NJ). FK506 was a kind gift from Fujisawa GmbH (Munich, Germany). Abs to
lyn, blk, syk, btk, and PLC-2 were purchased from Santa Cruz Biotech
(Santa Cruz, CA). IL-4 was purchased from Genzyme (Cambridge, MA) and
recombinant horseradish peroxidase (HRP)-conjugated
anti-phosphotyrosine Ab (RC20H) was obtained from Transduction
Laboratories (Lexington, KY).
B cell stimulation and Western blotting
Splenic B cells were prepared after T-depletion as described previously (18), and their purity exceeded 95% in most experiments. Splenic B cells in serum-free Iscove/F-12 (IF-12) medium were prewarmed at 37°C for 45 min. B cells were stimulated at 37°C with 10 µg/ml of anti-IgM, 30 µg/ml of anti-CD72, or M195. In some experiments, cells were pre-incubated with PTK inhibitors, genistein (100 µM), or herbimycin A (7 µg/ml) for 30 min at 37°C and later stimulated with the stimulatory Abs. Reactions were stopped with the addition of 1x cold PBS, pH 7.2, containing phosphatase inhibitors as described earlier (29, 30). After two to three washes with PBS, cells were lysed with 1% Triton-X 100 lysis buffer containing 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 1 mM EDTA, 1 mM PMSF, 2 µg/ml aprotinin, and 2 µg/ml of leupeptin for 30 min on ice. Lysates were cleared of nuclear debris by centrifugation at 12,000 x g for 30 min at 4°C.
For immunoprecipitation studies, lysates prepared from 25 x
106 B cells were precleared with protein A beads (Repligen,
Cambridge, MA) for 1 h at 4°C. Abs to lyn, blk, syk, PLC-2,
or btk were added and incubated at 4°C for 6 h. Immune complexes
were collected by incubating with protein A beads for 2 h.
Proteins were eluted by boiling with sample buffer for 3 to 5 min and
were separated on 10% SDS-PAGE. Resolved proteins were transferred
onto Immobilon-P membranes (Millipore, Bedford, MA), then blocked for
30 min with 1.5% BSA. Membranes were incubated with
anti-phosphotyrosine Ab RC20 for 1 h at room temperature.
After extensive washing, the blots were developed with an enhanced
chemiluminescence kit (DuPont/NEN, Boston, MA) according to the
manufacturer’s instructions. For Western blotting of the individual
enzymes, blots were incubated with their respective Abs for 1 h at
room temperature followed by horseradish peroxidase-conjugated
anti-rabbit secondary Ab (Santa Cruz Biotech, Santa Cruz, CA). The
proteins were visualized as described above.
In vitro kinase assay
B cells (1 x 107) were stimulated for
indicated time points, and immunoprecipitates from lysates were
prepared as above. Protein A beads were incubated in 500 µl of kinase
buffer (20 mM HEPES, pH 7.5, 10 mM MnCl2, 5 mM DDT, 1 mM
Na3VO4, and 1 mM PMSF) with 5 µCi
[-32P]ATP for 30 min at 30°C. Reactions were stopped
by adding kinase buffer containing 5 mM EDTA (stop buffer). Beads were
extensively washed with stop buffer and proteins were eluted as
described above. The proteins were resolved by SDS-PAGE and visualized
by autoradiography. The autoradiograms were scanned with a UMAX
flat-bed scanner (UMAX Data System, Taiwan, R.O.C.), and relative
intensities expressed in arbritary units (OD) of bands were
quantified using the National Institutes of Health image program.
Radioactivity of bands for syk experiments were quantified with a
Molecular Dynamics (Sunnyvale, CA) computing densitometer using a
volume-integrated mode, and the results were expressed as fold
increase.
Proliferation assay
DBA/2 B cells (2 x 105) were cultured in the presence or absence of 50 µg/ml of Abs (anti-IgM or anti-CD72) or inhibitors (CsA or FK506) in a final volume of 200 µl in IF-12 medium containing 10% FBS (Atlanta Biologics, Norcross, GA). Bacterial LPS was used at a concentration of 10 µg/ml. For studying proliferation of xid and non-xid B cells, 2 x 105 cells were cultured with increasing concentrations of anti-CD72 Abs in the presence or absence of 50 U/ml of IL-4. B cells were also stimulated with 50 µg/ml of anti-IgM (in the presence or absence of IL-4) to make comparisons on the proliferative ability of cells to BCR engagement. Cells were cultured for 48 h in 5% CO2 at 37°C and pulsed with 1 µCi of tritriated thymidine (sp. act. 2 Ci/mmol, New England Nuclear, Boston, MA) during the last 4 h. Cultures were harvested on glass fiber filters using a Packard automatic cell harvester, and thymidine incorporation was determined by a Matrix-96 ß counter (Packard, Downers Grove, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To analyze the initial signaling events after engaging CD72, B
lymphocytes were stimulated with Abs to the CD72 receptor for different
time periods. Following stimulation, lysates were prepared and analyzed
using the anti-phosphotyrosine Ab RC20. As a control, cells were
stimulated with an isotype-matched control Ab. Lysates from
anti-IgM-stimulated cells were used to compare differences or
similarities in the profile of induction of various proteins after BCR
or CD72 engagement. Stimulation through CD72, but not the control Ab,
resulted in tyrosine phosphorylation of several proteins of molecular
masses (indicated by arrows in Fig. 1
A) of 40, 45, 52 to
56, 60, 90 to 95, 110 to 120, and a prominent protein at 140 kDa.
Proteins at 30 to 32 and 35 kDa were also phosphorylated but with
lesser intensity. Induction was detected as early as 1 min (52-, 56-,
and 140-kDa bands), reaching peak levels at 10 to 15 min and returned
to basal levels of phosphorylation by 45 min, although the
phosphorylation reached peak levels at 30 min for a 40-kDa protein.
Similar results were obtained with another anti-CD72 Ab, 10.1.D2
(data not shown). When compared with anti-IgM-stimulated cells,
there was an absence of tyrosine phosphorylation of a 72-kDa protein in
CD72-stimulated cells. The reappearance of tyrosine-phosphorylated
proteins at molecular mass < 28 kDa and 43 kDa at 30 min in
CD72-stimulated cells was not seen in other similar experiments. Two
PTK-specific inhibitors, genistein (Fig. 1B) and
herbimycin A (data not shown) were used to determine whether the
appearance of tyrosine-phosphorylated substrates required activation of
tyrosine kinases. As expected, there was very little increase in
protein tyrosine phosphorylation in both CD72- and BCR-stimulated cells
pretreated with genistein (Fig. 1B). These results
indicate that CD72 stimulation resulted in the appearance of several
tyrosine-phosphorylated proteins due to PTK activation similar to
BCR-stimulated cells.
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In B lymphocytes, two src-kinases, lyn and blk, were activated
within seconds of BCR engagement (31), which is usually accompanied by
an increase in the tyrosine phosphorylation status of PTKs. Hence it
was interesting to identify candidate PTKs that are activated on CD72
stimulation. B cells were stimulated with anti-IgM Ab or
anti-CD72 Ab for different time periods, and the phosphotyrosine
content of immunoprecipitates of these src-kinases was analyzed. As
shown in Figure 2A,
stimulation through BCR for 10 min resulted in increased tyrosine
phosphorylation of lyn (sixfold). An increase in the tyrosine
phosphorylation of lyn was seen as early as 1 min of CD72 ligation,
which was two- to threefold over control after 30 to 45 min of
stimulation. Immunoblot (IB) analysis of samples probed with
anti-lyn Ab accounted for equivalent loading of proteins. On the
other hand, there was a modest but highly reproducible increase in the
tyrosine phosphorylation of blk in both BCR- and CD72-stimulated cells
(Fig. 2B). Similar to lyn, maximal levels of tyrosine
phosphorylation of blk were seen at 30 min of CD72 ligation. This
pattern was highly reproducible in two other experiments.
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C). On the other hand, there was no increase in the
tyrosine phosphorylation of syk in CD72-stimulated B cells at any time
point tested (Fig. 2C). Immunoblot analysis of syk
immunoprecipitates showed that the changes were not due to differences
in relative amounts of the protein loaded in each lane. These data
suggest that PTKs lyn and blk, but not syk, may be activated in
CD72-ligated B cells. Kinase activities of PTKs lyn and blk, but not syk, are increased in CD72-ligated B cells
The lack of tyrosine phosphorylation of syk may be due to a
failure by CD72 to stimulate an increase in kinase activities of the
src kinases although increased tyrosine phosphorylation of these
enzymes was evident. To confirm that the increase in phosphotyrosine
content of lyn and blk in CD72-stimulated cells in fact reflected an
increase in their kinase activities, in vitro autophosphorylation
assays on these enzymes were performed. Stimulation through either CD72
or BCR increased kinase activities of both lyn (Fig. 3A) and blk (Fig. 3B). As expected, BCR ligation resulted in enhanced
syk activity (Fig. 4A),
in agreement with the previously reported data on syk
autophosphorylation activity in such kinase assays (11, 14, 31).
However, there was no detectable change in syk activity in
CD72-stimulated cells (Fig. 4A). Longer exposures of
autoradiograms did not reveal any increase in syk activity in
CD72-ligated B cells (data not shown). To be certain that the failure
to detect syk activity upon CD72 ligation is not due to limitations in
the sensitivity of detection, we performed kinase assays with
immunoprecipitates from five times more the number of cells (Fig. 4B) compared with that used in the experiment of
Figure 4A. Phosphorimager analysis of the gel further
confirmed the inability of CD72 to stimulate syk activity. Under the
same conditions, BCR ligation induced a 2.8-fold increase in syk
activity (Fig. 4B). These data clearly indicate that
the src family kinases lyn and blk, but not the non-src kinase syk,
were activated in CD72-stimulated cells, accounting for the first major
difference in the initial signaling pathways between CD72 and
BCR.
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Previous studies have indicated that the tec family kinase btk,
whose function was defective in xid mice, was activated after src
kinase(s) activation in cells triggered through the BCR (32, 33, 34).
Furthermore, absence of wild-type btk function resulted in apoptosis of
anti-IgM-activated splenic B cells (35), indicating its essential
role in B cell activation. Btk was activated in xid B cells following
BCR engagement, suggesting that a single mutation in the pleckstrin
homology (PH) domain of the enzyme does not alter its catalytic
activity (34). Since src kinases were activated upon CD72 triggering,
we tested the possible involvement of btk in CD72-stimulated B cells.
We could not detect any reproducible increase in the tyrosine
phosphorylation status of btk on CD72 or BCR ligation. Hence, in vitro
kinase assays were performed on btk immunoprecipitates prepared from B
cell lysates from xid and non-xid control mice after stimulation
through CD72 or BCR. Btk activity was measured at 1, 3, and 5 min of
stimulation with anti-CD72 or anti-IgM Ab. Maximal kinase
activity was demonstrated after 3 min of stimulation for both CD72- and
BCR-ligated cells. As shown in Figures 5A (non-xid) and 5B
(xid), btk activity was detectable in unstimulated cells, which was
elevated in both CD72- and BCR-ligated B lymphocytes. Although the
exaggerated increase in btk activity at 3 min of CD72 stimulation in
CBA/N mice (Fig. 5B) was not reproducible in other
experiments, there was a consistent increase in btk activity in all
experiments. Btk activity increased to comparable levels in both CBA/N
and CBA/Ca B cells on BCR ligation (Fig. 5C).
Similarly, btk activity induced by CD72 was comparable in CBA/N and
CBA/Ca B cells after 1 and 3 min of stimulation (Fig. 5C). Btk activity returned to basal levels by 5 min
of BCR or CD72 stimulation (data not shown).
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, A and B).
An allele-specific anti-CD72 Ab (10.1.D2) that does not recognize
the CD72b allele in CBA/N and CBA/Ca mice was used as a
control for these experiments. IL-4 had minimal effects in enhancing
CD72-induced B cell proliferation in CBA/Ca mice (Fig. 6A). In xid mice, addition of IL-4 increased the
level of CD72-induced B cell proliferation to two- to threefold when
compared with cells stimulated with anti-CD72 alone (Fig. 6B). However, the magnitude of response of xid B
cells to CD72 plus IL-4 was only 20% of the response of CBA/Ca B
lymphocytes. As expected, xid B cells were unresponsive to
anti-IgM-induced B cell proliferation (Fig. 6C).
IL-4 enhanced the anti-IgM response almost sevenfold in xid mice as
opposed to less than threefold in CBA/Ca mice. However, the
absolute magnitude of the xid response to anti-IgM plus IL-4 was
only 25% of the response of the wild-type mice. These results clearly
show that btk is activated by CD72 ligation and that its intact PH
domain-related function is essential for CD72-mediated B cell
activation.
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2 is inducibly phosphorylated in CD72-stimulated cells
We have previously shown that CD72-mediated B cell activation
resulted in PI hydrolysis (18, 24). Previous reports using the chicken
DT40 B cell lymphoma showed the importance of both syk and btk in
tyrosine phosphorylation and activation of PLC-2 (13). Hence it was
interesting to verify whether PLC-2 was tyrosine phosphorylated in
CD72-stimulated cells in which btk, but not syk, was activated. As
shown in Figure 7, PLC-2 was inducibly
tyrosine phosphorylated to a similar extent in both CD72- and
BCR-ligated cells.
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Since we previously showed that stimulation through CD72 resulted
in activation of phospholipase- and mobilization of
[Ca2+]i, we asked whether
Ca2+-dependent activation events were similar in CD72- and
BCR-ligated cells. Two immunosuppressive agents CsA and FK506 have been
previously shown to block Ca2+-dependent activation of the
ser/thr phosphatase, calcineurin, which is required for the
translocation of NF-AT to the nucleus (36). B cells were cultured with
anti-IgM or two different anti-CD72 Abs (K10.6 and 10.1.D2) in
the presence of various concentrations of CsA. There was a
dose-dependent inhibition of CD72-stimulated proliferation in the
presence of CsA similar to that of BCR-ligated cells (Fig. 8A). Similar results
were obtained in experiments conducted in the presence of FK506 (Fig. 8B). The growth response of B cells to bacterial LPS
was minimally inhibited by both these agents.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several reports have suggested that tyrosine phosphorylation of protein
substrates was mediated by the src-family PTKs. Activation of PTKs
occur in a temporal fashion following BCR engagement in a murine B cell
lymphoma (31). Src family kinases lyn and blk were activated as early
as 5 sec, followed by activation of btk and syk at 5 to 10 min after
receptor ligation. Stimulation through CD72 caused a time-dependent
increase in the phosphotyrosine content of src kinases lyn and blk, but
to a lesser extent when compared with BCR-stimulated cells. The
increase in phosphorylation closely correlated with the activation of
these enzymes. The lesser strength of activation may be due to the
absence of a canonical immunoreceptor-based activation motif in the
cytoplasmic tail of CD72. However, the cytoplasmic domain of CD72
contains three tyrosine residues that could be potential
phosphorylation sites (39). We could not detect any evidence of
tyrosine phosphorylation of CD72, based on a detailed kinetic analysis
of anti-CD72, anti-IgM, or pervanadate-treated cells (C.
Venkataraman and S. Bondada, unpublished observations). Also under
these detergent conditions, we could not find any CD72-associated
kinase activity (data not shown). It is also possible that CD72
physically associates with another protein that may recruit and
activate various PTKs. Use of a milder detergent to maintain
protein-protein interactions may help resolve this issue. Using 1%
Brij-96 lysis conditions, we could not demonstrate
coimmunoprecipitation of Ig
with CD72. However, under these
treatments, the physical association between membrane IgM and Ig
remained intact (Venkataraman et al., manuscript under
preparation). Several downstream substrates of lyn have been
identified, which include syk (11), HS1 (40, 41), and the CD19/CD21
complex (42, 43). Syk was neither inducibly tyrosine phosphorylated nor
activated in CD72-stimulated B cells. This is in contrast to other
surface molecules on B cells (e.g., BCR, CD40, and CD38) that activate
syk on receptor ligation (3, 5, 31).
Several receptors on B cells, including the BCR, IL-5R, and CD38,
involve activation of btk (6). B cells from xid mice do not respond to
signaling through these receptors, indicating the important role of btk
in various signal transduction pathways. Additionally, btk has been
shown to be essential for tyrosine phosphorylation of PLC-2 in a
chicken B cell lymphoma (14). We have identified btk as an important
PTK in CD72-mediated signal transduction. Btk was activated in both xid
and non-xid mice after CD72 or BCR ligation. There are very few reports
on the activation of btk in normal B cells in response to BCR
stimulation. Studies using the murine immature B cell line WEHI-231
(31), human tonsillar B cells, and Daudi B cells (44) showed a four- to
fivefold increase in btk kinase activity that remained elevated until
10 min of stimulation. However the extent of btk activation seen in our
studies in response to CD72 or BCR ligation is similar to that reported
in the chicken DT40 cell line (14) stimulated with anti-IgM. To our
knowledge, this is the first demonstration of an increase in btk
activity in response to BCR or CD72 signaling in normal mature splenic
B cells.
The importance of btk in CD72 signaling is underscored by the findings
that B lymphocytes from xid mice do not proliferate in response to CD72
signaling. This is similar to our earlier report, which showed that xid
mice heterozygous for CD72a expression did not respond
to stimulation with CD72a-specific mAb (10.1.D2) (17). This
is not due to expression of reduced levels of CD72 on xid B cells since
B lymphocytes from both xid and non-xid mice express similar levels of
CD72 as determined by flow cytometry (data not shown). These results
suggest that, similar to other receptors, interactions mediated through
the PH domain of btk were essential for complete signal transduction
through the CD72 receptor. Our preliminary data and an earlier report
(45) demonstrate normal levels of inducible protein tyrosine
phosphorylation in xid B cells in response to BCR ligation. Moreover,
levels of PLC-2 tyrosine phosphorylation and calcium mobilization in
response to anti-IgM stimulation are not altered in xid mice when
compared with wild-type control (data not shown). These results argue
that the xid B cells are not defective in inducing src tyrosine kinase
activity in response to BCR engagement.
Previous studies have shown that tyrosine phosphorylation of PLC-2
was required for its activity (46). A study using chicken DT40 B cell
lymphoma showed an absolute requirement of syk activity for PLC-2
tyrosine phosphorylation after BCR stimulation (13). PLC-2 was
inducibly tyrosine phosphorylated to a lower extent in the
btk-deficient DT40 cell line, suggesting that syk can still
phosphorylate PLC-2 in the absence of btk involvement (14). In
contrast to these findings, normal levels of inducible phosphorylation
of PLC-2 were observed in CD72-stimulated cells, in spite of lack of
syk activation. The discrepancy in these results may be due to three
possible reasons: 1) There may be fundamental species differences in
the requirement of syk for PLC activation in murine and chicken B
cells. 2) There may also be differences based on the stage of B cell
development (immature vs mature); i.e., the chicken DT40 cell line is
an immature B cell lymphoma whereas our study used normal mature
splenic B cells. 3) Since btk has been shown to activate PLC-2 (14),
it might replace the need for syk in CD72-signaling pathway.
To our knowledge, this is the first demonstration of syk-independent
tyrosine phosphorylation of PLC-2 in mature B lymphocytes. The
absence of syk activation with normal PI hydrolysis (18, 24), calcium
mobilization (18, 19), and cell proliferation (16, 17, 18) in
CD72-stimulated B cells has at least two major implications. First,
this may explain the existence of "cross-talk" between cAMP and PI
pathways in BCR-, but not CD72-, mediated signaling (18, 25). Thus, in
neutrophils, the basis of "cross-talk" between the two pathways
lies in the inhibition of syk activity by cAMP-elevating agents (47).
Furthermore, the inhibition of syk is associated with serine
phosphorylation, supporting the involvement of cAMP-dependent PKA (47).
Whether such a mechanism can account for cAMP-mediated inhibition of
BCR signaling is currently being tested. Second, hyper-cross-linking of
surface Igon mature B cells induces apoptosis (48), which can be
rescued by the simultaneous addition of antibodies to CD40 (49) or CD72
(26) to B cell cultures. This led to a model suggesting the need for a
second costimulatory signal to prevent BCR-mediated death (26).
Although the exact regulatory mechanisms that rescue these cells are
unknown, syk-independent activation of the PLC-2 pathway and
subsequent signaling cascade by CD72 stimulation may have a role in
rescue from apoptosis and induction of growth in B cells.
Calcium-dependent activation events were required for BCR-induced proliferation that could be specifically blocked by immunosuppressive agents CsA and FK506 (50, 51). Previously we showed that CD72 engagement caused increased elevation of [Ca2+]i (18, 19). Consistent with this finding, both CsA and FK506 effectively inhibited CD72-induced B cell proliferation, indicating an important role for Ca2+-regulated activation pathways for CD72 signaling similar to that observed for BCR-stimulated cells.
In summary, our previous studies and this report have shown that CD72
stimulation results in differential activation of signaling events when
compared with BCR-stimulated cells. Similar to anti-IgM-stimulated
cells, CD72 induces activation of PTKs lyn, blk, and btk, activation of
phospholipase- (18, 24), calcium mobilization (18, 19), and
Ca2+-dependent signaling events required for growth
responses of B lymphocytes. Unlike BCR stimulation, CD72 ligation
induced tyrosine phosphorylation of PLC-2 in the apparent absence of
syk activation.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Pediatrics and Medical Microbiology and Immunology, Wexner Institute for Pediatric Research, Columbus, OH.
3 Current address: Division of Monoclonal Antibodies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD.
4 Address correspondence and reprint requests to Dr. Subbarao Bondada, 205 Sanders-Brown Center on Aging, 800 South Limestone St., University of Kentucky, Lexington, KY 40536. E-mail address:
5 Abbreviations used in this paper: BCR, B cell Ag receptor; PKA, protein kinase A; PTK, protein tyrosine kinase; PLC, phospholipase C; CAb, control Ab; IB, immunoblot; CsA, cyclosporin A; PH, pleckstrin homology; PI, phosphatidylinositol.
Received for publication September 18, 1997. Accepted for publication December 9, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2. J. Exp. Med. 179:673. and phosphatidylinositol 3-kinase. J. Immunol. 156:100.[Abstract]
2 in B lymphocytes. Proc. Natl. Acad. Sci. USA 89:5660. 2. J. Exp. Med. 184:31.) mechanisms of regulation of B lymphocyte activation. Int. Immunol. 5:949.1 by tyrosine phosphorylation. Science 250:1253.This article has been cited by other articles:
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  D. H. Li, J. W. Tung, I. H. Tarner, A. L. Snow, T. Yukinari, R. Ngernmaneepothong, O. M. Martinez, and J. R. Parnes CD72 Down-Modulates BCR-Induced Signal Transduction and Diminishes Survival in Primary Mature B Lymphocytes J. Immunol., May 1, 2006; 176(9): 5321 - 5328. [Abstract] [Full Text] [PDF]
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 M. Gururajan, R. Chui, A. K. Karuppannan, J. Ke, C. D. Jennings, and S. Bondada c-Jun N-terminal kinase (JNK) is required for survival and proliferation of B-lymphoma cells Blood, August 15, 2005; 106(4): 1382 - 1391. [Abstract] [Full Text] [PDF]
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 M. Ogimoto, G. Ichinowatari, N. Watanabe, N. Tada, K. Mizuno, and H. Yakura Impairment of B cell receptor-mediated Ca2+ influx, activation of mitogen-activated protein kinases and growth inhibition in CD72-deficient BAL-17 cells Int. Immunol., July 1, 2004; 16(7): 971 - 982. [Abstract] [Full Text] [PDF]
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I. Ishida, A. Kumanogoh, K. Suzuki, S. Akahani, K. Noda, and H. Kikutani Involvement of CD100, a lymphocyte semaphorin, in the activation of the human immune system via CD72: implications for the regulation of immune and inflammatory responses Int. Immunol., August 1, 2003; 15(8): 1027 - 1034. [Abstract] [Full Text] [PDF]
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H.-J. Wu, C. Venkataraman, S. Estus, C. Dong, R. J. Davis, R. A. Flavell, and S. Bondada Positive Signaling Through CD72 Induces Mitogen-Activated Protein Kinase Activation and Synergizes with B Cell Receptor Signals to Induce X-Linked Immunodeficiency B Cell Proliferation J. Immunol., August 1, 2001; 167(3): 1263 - 1273. [Abstract] [Full Text] [PDF]
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R. Goitsuka, H. Mamada, D. Kitamura, M. D. Cooper, and C.-l. H. Chen2 Genomic Structure and Transcriptional Regulation of the Early B Cell Gene chB1 J. Immunol., August 1, 2001; 167(3): 1454 - 1460. [Abstract] [Full Text] [PDF]
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T. Adachi, C. Wakabayashi, T. Nakayama, H. Yakura, and T. Tsubata CD72 Negatively Regulates Signaling Through the Antigen Receptor of B Cells J. Immunol., February 1, 2000; 164(3): 1223 - 1229. [Abstract] [Full Text] [PDF]
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A. M. Buhl and J. C. Cambier Phosphorylation of CD19 Y484 and Y515, and Linked Activation of Phosphatidylinositol 3-Kinase, Are Required for B Cell Antigen Receptor-Mediated Activation of Bruton's Tyrosine Kinase J. Immunol., April 15, 1999; 162(8): 4438 - 4446. [Abstract] [Full Text] [PDF]
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C. Venkataraman, S. Leung, A. Salvekar, H. Mano, and U. Schindler Repression of IL-4-Induced Gene Expression by IFN-{gamma} Requires Stat1 Activation J. Immunol., April 1, 1999; 162(7): 4053 - 4061. [Abstract] [Full Text] [PDF]
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