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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1382-1392
CD22 Cross-Linking Generates B-Cell Antigen Receptor-Independent
Signals That Activate the JNK/SAPK Signaling Cascade
By
Joseph M. Tuscano,
Agostino Riva,
Salvador N. Toscano,
Thomas F. Tedder, and
John H. Kehrl
From the Department of Medicine, UC Davis Cancer Center, University
of California, Davis, Sacramento, CA; and the Department of Immunology,
Duke University Medical Center, Durham, NC.
 |
ABSTRACT |
CD22 is a B-cell-specific adhesion molecule that modulates
BCR-mediated signal transduction. Ligation of human CD22 with
monoclonal antibodies (MoAbs) that block the ligand binding site
triggers rapid tyrosine phosphorylation of CD22 and primary B-cell
proliferation. Because extracellular signal-regulated kinases (ERKs)
couple upstream signaling pathways to gene activation and are activated
by B-cell antigen receptor (BCR) signaling, we examined whether CD22
ligation also activated ERKs and/or modified BCR-induced ERK
activation. Ligation of CD22 on either primary B cells or B-cell lines
failed to significantly activate the mitogen activated protein kinase (MAPK) ERK-2, but did activate the stress-activated protein kinases (SAPKs; c-jun NH2-terminal kinases or JNKs). In contrast, BCR ligation
resulted in ERK-2 activation without significant SAPK activation.
Concurrent ligation of CD22 and BCR enhanced BCR-mediated ERK-2
activation without appreciably modulating CD22-induced SAPK activation.
Consistent with its induction of SAPK activity, there was a marked
increase in nuclear extracts of activator protein-1 (AP-1) and c-jun
levels within 2 hours of exposure of primary B cells to the CD22 MoAb.
Despite their differences in ERK activation, both CD22 and BCR ligation
triggered several Burkitt lymphoma cell lines to undergo apoptosis, and
the 2 stimuli together induced greater cell death than either signal
alone. The pro-apoptotic effects were CD22-blocking MoAb-specific and
dose-dependent. Examination of expression levels of Bcl-2 protoncogene
family members (Bcl-2, Bcl-xL, Mcl-1, and Bax) showed a
downregulation of Bcl-xL and Mcl-1 after CD22 ligation.
This study provides a plausible mechanism to explain how CD22 and BCR
signaling can costimulate B-cell proliferation and induce apoptosis in
Burkitt lymphoma cell lines.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
CD22 IS A MEMBER OF the sialoadhesin
subclass of the Ig superfamily whose members function as mammalian
lectins.1-3 Although the physiologic significance of the
adhesive properties of CD22 remains unknown, the extracellular portion
of CD22 mediates interactions with sialic acid-bearing ligands
expressed by lymphocytes, neutrophils, monocytes, erythrocytes, and
some nonhematopoietic cell types.2-6 In vitro and in vivo
studies have demonstrated that CD22 ligation regulates B-cell function,
and CD22 binding to its ligand on T cells provides a costimulatory
signal (reviewed in Tedder et al3).
The intracellular portion of CD22 contains 6 tyrosines that are
potential targets for tyrosine phosphorylation. Two pairs of tyrosines
are within regions that are structurally homologous to the
tyrosine-based activation motif (TAM).7 TAMs are found in
many hematopoietic signaling receptors and are thought to serve as
platforms for docking of src family tyrosine kinases.8-10
The physiologic relevance of these motifs in CD22 had been demonstrated by showing that the intracellular portion of CD22 physically associates with several effector proteins, including the src kinase Lyn, the
src-related kinase Syk, PI-3 kinase, and phospholipase C (PLC).7,11-13 The TAM motifs and the effector proteins
with which they associate are implicated in the delivery of potent mitogenic signals. Indeed, CD22 cross-linking triggers B-cell proliferation and augments the proliferative response induced by
lipopolysaccharide, CD40, or anti-Ig.12-16 The
intracellular portion of CD22 also contains 4 tyrosine-based inhibitory
motifs (TIMs), which provide docking sites for the SH2 domains of SHP1 protein tyrosine phosphatase.17 TIMs have been found in
transmembrane proteins involved in the inhibition of signals through
other receptors and include the FcRIIB and the natural killer
(NK) cell-inhibitory receptors p58 and p70.17
CD22 has been shown to associate with SHP1 and the recruitment of CD22
into the antigen receptor complex likely inhibits antigen receptor
signaling.11,18-20
Recently, 4 groups independently generated targeted deletions of the
mouse CD22 gene.21-24 Although the observed
CD22 / phenotype resolved some of the
questions related to the role of CD22 in B-cell function, many
questions remained unanswered. Each of the groups demonstrated a
downregulation of surface IgM expression on peripheral
CD22/ B cells and an augmented calcium flux
after BCR cross-linking, results consistent with an inhibitory role for
CD22 in BCR signaling. However, some significant differences in the
observed CD22/ phenotype were also reported.
For example, the response to thymus-independent antigen was found to be
impaired by 1 group,22 but unaffected by
another.23 One group found that the lifespan of B cells in the periphery of CD22/ mice was
shortened,22 perhaps secondary to enhanced apoptosis. Another group found expansion of long-lived CD5+ B cells in
the peritoneum.23 Two groups reported impaired
proliferation after BCR cross-linking,22,23 whereas a third
group found the converse.21 Although the basis of these
differences remains elusive, the impaired response to T-independent
antigen, reduced number of long-lived cells, and impaired proliferative
response after BCR cross-linking would all be consistent with a
positive role for CD22 in B-cell physiology. A recent comparison of
BCR-mediated signaling events in CD19/ and
CD22/ mice showed a reciprocal change in Vav
phosphorylation.25 The CD19/ B
cells had decreased and the CD22/ B cells had
increased Vav phosphorylation. However, this study also showed that,
after BCR cross-linking, CD22/ B cells had
reduced levels of several tyrosine phosphorylated proteins, including
Ig- , Ig- , SH2-containing inositol phosphatase (SHIP),
and PLC, a result perhaps inconsistent with CD22 solely being an
inhibitor of BCR-mediated signal transduction. Furthermore, this study
failed to see an alteration in BCR-mediated mitogen-activated protein
kinase (MAPK) activation in the CD22/ B
cells. This contrasts with a recent study in which the use of a
CD22 immobilization technique suggested that the recruitment of CD22
into the BCR complex inhibited BCR-mediated MAPK
activation.26
Thus, although CD22 can inhibit BCR-mediated signaling events, it may
also play a positive role in B-cell function. Dissection of the
intermediate and late signaling events that occur after CD22 ligation
may help to better define the role of CD22 in normal B-cell physiology.
In this study, we used monoclonal antibodies (MoAbs) that are highly
efficient at initiating early CD22-mediated signaling events to
identify the downstream signaling cascades used by this receptor. Many
recent reports have identified the importance of stress-activated
protein kinase (SAPK; also JUN kinase) and MAPK in the propagation of
proliferative signals in B cells. Specifically, ERK-2 has been
identified as the principal MAPK involved in BCR-mediated signals in
primary B cells,27 whereas the SAPK signaling cascade has
been implicated in mediating the mitogenic effects after CD40
ligation.28 We show here that CD22 ligation activates the
SAPK pathway and amplifies the ERK-2 activation observed after BCR
cross-linking.
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MATERIALS AND METHODS |
Reagents.
The blocking (HB22.7, HB22.23, and HB22.33) and nonblocking (HB22.27)
MoAbs were purified from ascites and previously
characterized.12 The goat antihuman IgM
F(ab')2 was purchased from Jackson Immunoresearch (West Grove, PA). The biotinylated goat antirabbit MoAb and horseradish peroxidase (HRP)-conjugated streptavidin used for immunoblotting were
purchased from Dako (Carpinteria, CA). Antimouse IgG-coated magnetic
beads were used for immobilization and cross-linking CD22, as well as
for immunoprecipitation for kinase assays (Dynal, Oslo, Norway). The
anti-c-Jun antisera used for the mobility shift assay (MSA) and
immunoblotting was a kind gift of Kevin Johnson (National Cancer
Institute [NCI], National Institutes of Health [NIH], Bethesda,
MD). The anti-JNK antisera used for the JNK/SAPK kinase
assay was purchased from Pharmacia (Piscataway, NJ). The anti-ERK-2
antiserum used for the ERK-2 kinase assay was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). The GST-c-Jun and myelin basic protein
(MBP) used as a substrate for the in vitro kinase assays were purchased
from Santa Cruz Biotechnology and Sigma (St Louis, MO), respectively.
The anti-Bcl-xL, anti-Bcl-2, anti-Mcl-1, and anti-Bax
used for intracytoplasmic flow cytometery were purchased from Santa
Cruz Biotechnology. The p38/JNK2 inhibitor SB203580 was purchased from
Calbiochem (San Diego, CA).
B-cell preparations, cell culture, and cell lines.
Human tonsillar B cells were isolated as described.12 These
cell preparations are routinely greater than 98% CD20+ as
assessed by flow cytometry. The Burkitt lymphoma cell line Ramos and
the plasmacytoma cell line HS-Sultan were obtained from the American
Type Culture Collection (Rockville, MD). The Burkitt lymphoma cell
lines ST486 and BL41 were kind gifts of E. McGrath (NCI, NIH). The cell
lines were maintained in RPMI 1640 supplemented with 10% fetal calf
serum at 0.5 × 106/mL. The anti-CD22 MoAbs were
immobilized by incubating 20 µg of MoAb with 16 × 106 Dynabeads for 20 minutes at 4°C. The complex was
washed with phosphate-buffered saline (PBS) and used to stimulate 2 × 107 tonsillar, 107 Ramos, or
107 HS-Sultan B cells for various periods of time, as indicated.
In vitro kinase assays.
Tonsillar, Ramos, or HS-Sultan B cells were stimulated with immobilized
MoAb or media for the indicated periods of time. Cells were then lysed
in kinase lysis buffer (20 mmol/L HEPES, pH 7.4, 2 mmol/L EGTA, 50 mmol/L -glycerol phosphate, 1 mmol/L dithiothreitol [DTT], 1 mmol/L sodium orthovanidate, 1% Triton X-100, 10% glycerol, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 1 mmol/L
phenylmethyl sulfonyl fluoride [PMSF]) for 1 hour at
4°C. Cleared lysates were incubated with (5 µL) anti-JNK or
anti-ERK-2 MoAb for 1 hour at 4°C. Immunoprecipitates were
captured with antimouse IgG Dyna beads for 2 to 3 hours at 4°C. The
immunoprecipitates were washed twice with lysis buffer, once with LiCl
wash buffer (500 mmol/L LiCl, 100 mmol/L Tris-HCl, pH 7.6, 0.1% Triton
X-100, and 1 mmol/L DTT), and twice with kinase assay buffer (20 mmol/L
MOPS, pH 7.2, 2 mmol/L EGTA, 10 mmol/L MgCl2, 1 mmol/L DTT,
0.1% Triton X-100, and 100 µmol/L sodium orthovanidate). GST-c-Jun
(1 µg) and MBP (10 µg) were used as substrates for the SAPK and
ERK-2 kinase assays, respectively. Immunoprecipitates were resuspended
in 80 µL of kinase assay buffer (50 mmol/L MgCl2, 500 µmol/L ATP, 5 µCi [32P]ATP), with either GST-c-Jun
or MBP as a substrate. Assays were incubated at 30°C for 20 minutes
and the reactions were stopped by adding 25 µL of 4× sodium
dodecyl sulfate (SDS)-sample buffer. Subsequently, the
immunoprecipitates were boiled, size-fractionated on a 10%
SDS-polyacrylamide gel, and visualized by autoradiography. The amount
of 32P incorporation into MBP or GST-c-Jun was determined
by excising the appropriate band and liquid scintillation counting.
Mobility shift assays and immunoblot analysis.
Nuclear extracts were prepared using a standard protocol.
Oligonucleotides containing an AP-1 binding site and its complementary strand were annealed and gel-purified. A 32P-labeled AP-1
probe was incubated with 5 µg of nuclear extracts at room temperature
for 20 minutes in a binding reaction followed by electrophoresis on
4.5% polyacrylamide gels in 0.25% TBE buffer.29 For the
analysis of c-Jun, expression nuclear extracts (50 µg) were
size-fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE),
and transferred to nitrocellulose, and the membrane was blocked with
TTBS (150 mmol/L NaCl, 50 mmol/L Tris-Cl, pH 7.4, and 0.05% Tween)
plus 10% milk. The membranes were then incubated with anti-c-Jun or
anti-p52 NF- B MoAb (1:1,000) in TTBS plus 5% milk overnight at
4°C. Subsequently, they were then incubated with biotinylated goat
antimouse antibodies (1:5,000) in 5% bovine serum albumin (BSA) and
HRP-Streptavidin (1:10,000). Detection was performed by enhanced
chemiluminescence (ECL; Amersham, Little Chalfont, UK).
Flow cytometry, cell cycle, and apoptotic analysis.
Ramos cells were stimulated with media, blocking CD22 MoAbs (HB22.7,
HB22.23, and HB22.33), nonblocking MoAb HB22.27, or antihuman IgM (all
at 20 µg/mL) for 24 or 48 hours. Cells were examined for viability by
their ability to exclude trypan blue. Apoptotic analysis was performed
by propidium iodide (PI) uptake, as previously described,30
and also by using the APO-BRDU kit (Pharmagen, San Diego,
CA) and following the manufacturer's recommendations. Briefly,
106 cells were fixed in 1% paraformadehyde (wt/vol) and
stored in 70% ethanol. Fragmented DNA was labeled with BR-dUTP and
TdT, followed by detection with anti-BRDU-fluorescein isothiocyanate (FITC) (Becton Dickinson, San Jose, CA). Data from 50,000 events were acquired using a Becton Dickinson FACstar flow cytometer and analyzed using Multicycle cell cycle analysis software (Phoenix Systems, San Diego, CA). Intracytoplasmic florescence-activated cell
sorting (FACS) analysis was performed as previously
described.31,32 Briefly, after appropriate stimulation,
106 Ramos cells were fixed with 1:1 4%
paraformaldehyde/lysing solution (Becton Dickinson). After 2 washes
with PBS/0.5% Tween 20, cells were blocked with 10% heat-inactivated
human serum/PBS for 30 minutes, stained with indicated antisera at
1:1,000, and detected with goat-antirabbit-FITC (Becton Dickinson).
Fifty thousand nonapoptotic events were analyzed by gating out PI
staining cells.
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RESULTS |
CD22 cross-linking with the blocking MoAb HB22.7 activates the SAPK
pathway in tonsillar B cells and in several B-cell lymphoma cell lines.
Recently, SAPK has been implicated as a critical mediator of c-jun
phosphorylation after CD40 ligation in B cells28 as well as
a pathway used for BCR-mediated apoptosis in neoplastic B-cell lines.33 To test whether CD22 cross-linking might also
activate SAPK, we immobilized the CD22 MoAbs HB22.7, HB22.23, and
HB22.33, which are known to block the binding of CD22 to its ligand, on magnetic beads and used them to cross-link CD22 on primary tonsillar B cells. SAPK activity was measured by performing immune
complex kinase assays with GST-c-jun as a substrate.28 CD22
cross-linking induced a consistent 3-fold increase in SAPK activity
after 20 minutes of ligation (Fig 1A). In
contrast, cross-linking of the BCR with F(ab')2
fragments of anti-IgM failed to significantly induce SAPK. Concurrent
cross-linking of CD22 and the BCR resulted in a similar, albeit
slightly less pronounced increase in SAPK activity (Fig 1A). We
extended these studies to analyze the effects of CD22 cross-linking on
2 B-lymphocyte cell lines, Ramos and HS-Sultan. The Ramos cell line is
derived from a Burkitt lymphoma, has a germinal center phenotype, and
is CD77+, CD22+, and sIgM+.
HS-Sultan cells have features of a plasmacytoma cell line and, although
the cells are CD22+, they are surface
Ig. We observed a similar induction of SAPK activity
in both cell lines after CD22 ligation with the blocking MoAbs (Fig 1B
and C). Similar levels of SAPK activation were seen after cross-linking with either of the blocking MoAbs, HB22.7 or HB22.23 (Fig 1C). Also,
there was no significant difference in SAPK induction observed when
CD22 and sIgM were cross-linked concurrently on the surface of Ramos
cells (data not shown). By way of comparison, CD40 cross-linking induced a 6- and 9-fold induction of SAPK activity in Ramos and HS-Sultan cells, respectively (Fig 1B and C). No significant SAPK induction was observed with the control anti-CD3 MoAb in either cell
line. All studies were performed in at least triplicate, with those
shown being representative.
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| Fig 1.
Increased SAPK activity after CD22 ligation on primary B
cells and several B-cell lines. (A) SAPK kinase assays performed with
lysates obtained after stimulating primary human tonsillar B cells (2 × 107/lane) with media, immobilized CD22 MoAb HB22.7 (20 µg/mL), F(ab')2 fragments of goat MoAb to BCR (20 µg/mL), or both. Immune complex kinase assays were performed using
GST-c-Jun as a substrate. (B) SAPK kinase assays performed as described
in (A) after stimulation of the B-cell line Ramos
(107/lane) with HB22.7 (20 µg/mL), CD40 (2 µg/mL), or
CD3 (10 µg/mL). (C) SAPK kinase assays performed as described in (A)
after stimulation of the B-cell line HS Sultan (107/lane)
with the CD22 MoAbs HB22.7 and HB22.23 (20 µg/mL) or CD40 (2 µg/mL).
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Cross-linking CD22 with the blocking MoAb HB22.7 fails to
significantly activate ERK-2 but prolongs BCR-mediated ERK-2
activation.
We next examined the effects of CD22 cross-linking on MAPK activation.
The best characterized MAPKs are the extracellular signal-regulated
protein kinases (ERKs) 1 and 2.34 Previous studies have demonstrated that BCR cross-linking activates
MAPKs27,35 and increases c-fos expression and AP-1
activity.36 Because ERK-2 is the principal MAPK isoform
activated after BCR cross-linking,27 we measured ERK-2
kinase activity after cross-linking CD22 on human tonsillar B cells.
However, the HB22.7 MoAb failed to significantly induce ERK-2 activity
(Fig 2A). As expected, anti-IgM
cross-linking resulted in a 3- to 4-fold increase in ERK-2 activity.
Maximal activity was seen after ligating for 5 minutes, with a
significant decrease observed after 30 minutes and a return to near
baseline after 45 minutes. Concurrent cross-linking of CD22 and IgM
resulted in a slight increase in ERK-2 activity above that seen with
anti-IgM alone. Similar experiments using Ramos cells again showed an
additive effect on ERK-2 activation after concurrent CD22 and anti-IgM cross-linking; however, more strikingly, ERK-2 activity failed to
decline at the rate seen with anti-IgM alone (Fig 2B). All studies were
performed in at least triplicate, with those shown being
representative. Significant heterogeneity of baseline ERK activity was
seen in tonsilar B cells, which likely represents the nonhomogenous
activation state of tonsilar B cells, which may explain the difference
in ERK-2 kinase induction between tonsilar and Ramos B cells.
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| Fig 2.
CD22 and BCR coligation prolongs BCR-mediated ERK-2
activation. (A) ERK-2 kinase assays were performed on lysates obtained
after stimulating primary human tonsil B cells (2 × 107/lane) with media, immobilized CD22 MoAb HB22.7 (20 µg/mL), F(ab')2 fragments of goat antihuman IgM (20 µg/mL), both, or CD40 (2 µg/mL). Immune complex kinase assays were
performed using MBP as a substrate. (B) ERK-2 kinase assays of lysates
obtained from Ramos cells (107/lane) stimulated as
described in (A).
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CD22 cross-linking results in increased AP-1 activity.
Signals that lead to B-cell activation are often initiated by receptor
ligation and propagated by signaling cascades that eventually modify
the transcriptional activity of AP-1 and NF-B. AP-1 is a heterodimer
that consists of c-jun paired with a member of either the Fos or ATF
family of transcription factors. Full AP-1 activation requires
phosphorylation of residues Ser 63 and Ser 73 in c-jun (reviewed in
Karin and Hunter37), which is catalyzed by the SAPKs.
Because the ligation of CD22 with the blocking MoAbs resulted in
increased SAPK activity, we examined AP-1 activity in B cells after
CD22 cross-linking using MSAs. Human tonsillar B cells were stimulated
with either beads alone or the MoAb HB22.7 immobilized on beads.
Nuclear extracts were examined by MSA with an oligonucleotide probe
containing an AP-1 site. There was a substantial induction of AP-1
binding activity after 2 hours of stimulation
(Fig 3). This activity diminished
significantly after 12 hours of stimulation. Detection of the AP-1
complex was eliminated when the binding reactions for the gel shift
assay were performed in the presence of a c-jun antisera (data not
shown), indicating that the AP-1 complex contained c-jun. Preincubating
the reaction mixture with 100-fold excess of cold probe also abolished
the AP-1 band, demonstrating that the pattern is AP-1-specific (Fig 3). Replicate MSAs were performed in triplicate, with those shown being
representative. To confirm these results, we also examined the nuclear
extracts for the presence of c-jun by immunoblotting. Consistent with
the results of the MSAs, 2 hours of CD22 ligation resulted in a
significant increase in c-jun levels in the nucleus (Fig 4A). The immunoblot was stripped and
reprobed with antibody that recognizes the transcription factor p52
NF-B. The levels of p52 NF-B were found to be uniform at all time
points examined, suggesting equal loading of the nuclear extracts (Fig
4B).
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| Fig 3.
AP-1 activity increases after stimulating primary human
tonsil B cells with the CD22 blocking MoAb HB22.23. Tonsillar B cells
were stimulated with media alone or with immobilized HB22.23 (20 µg/mL) for 2, 6, and 12 hours. Nuclear extracts were prepared and
AP-1 activity was assayed by MSA.
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| Fig 4.
c-Jun protein levels increase after stimulating primary
human tonsillar B cells with the CD22 MoAb HB22.23. (A) Tonsil B cells
were stimulated as described in Fig 3. Nuclear extracts were prepared,
analyzed by immunoblotting with anti-c-Jun antisera, and detected by
ECL. (B) The immunoblot described above was stripped and reprobed with
anti-p52 NF-B MoAb and detected by ECL.
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CD22 ligation with HB22.7 causes cell cycle arrest and apoptosis in
the cell line Ramos.
Immune development and function are dependent on eupeptic elimination
of dysfunctional or autoreactive lymphocytes. Suicide signals initiated
by the CD95/Fas receptor, tumor necrosis factor (TNF)
receptor, and, in specific cases, the BCR are propagated via the
SAPK/JNK signaling cascade.33,38,39 Whereas BCR
cross-linking in primary B cells generates a proliferative signal,
cross-linking the BCR on several B-cell lymphoma cell lines initiates
the apoptotic process.40-42 Whether this represents
standard elimination of germinal center B cells or a dysfunction
specific for these malignant clones remains unclear, but it does
provide a functional endpoint to examine CD22 and anti-Ig signaling.
One study has demonstrated that, upon CD22 ligation and cross-linking,
apoptosis could be induced in the Burkitt B-cell lymphoma cell line,
Ramos.42 However, this was dependent on extensive
cross-linking, and the amount of B-cell apoptosis was modest. The
effect of CD22 MoAbs, which block the interaction with its ligands and
induce SAPK activation, on Ramos cells has not previously been
examined. We found that addition of the blocking anti-CD22 MoAbs,
HB22.7 and HB22.33, to Ramos cell cultures for 48 hours resulted in
apoptosis in 48% and 82% of the cells, respectively
(Fig 5). A more complete analysis of the
effects of HB22.7 alone and in combination with anti-IgM after 24 and
48 hours of incubation yielded similar results
(Fig 6). In contrast, the nonblocking CD22
MoAb HB22.27 induced the apoptosis of only 7% to 10% of the cells
(Fig 6). Ramos cell cultures treated the control MoAb CD3 contained a
similar number of apoptotic cells as did those cultures treated with
HB22.27 (Fig 6). After 48 hours of treatment with HB22.7, nearly 505 to
60% of the cells had undergone apoptosis, compared with 10% with
HB22.27 (Fig 6). The extent of apoptosis after ligation with HB22.7 was
similar to that observed with anti-IgM (Fig 6). Ligation of CD22 with the blocking MoAb resulted in a modest increase in G1 cell cycle arrest, which was not seen with the nonblocking MoAb or anti-Ig (data
not shown). The further addition of a cross-linking MoAb did not affect
the changes in cell cycle or degree of apoptosis observed with CD22
alone (data not shown). Recently, a pyridinyl imidizole (SB203580) has
been identified that specifically and effectively blocks p38 and JNK2
kinases, but not JNK3, JNK4, or ERK kinases.43,44 To
elucidate the role of these kinases on CD22-mediated apoptosis in Ramos
cells, we preincubated Ramos cells with 25 and 50 µmol/L SB203580
before stimulation with HB22.7 and anti-IgM. No change in the extent of
apoptosis was observed (data not shown), which is consistent with JNK1
and ERK kinases mediating the effects of CD22 and sIgM ligation,
respectively.
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| Fig 5.
Ligation of CD22 on the Burkitt lymphoma cell line Ramos
results in apoptosis. Ramos B cells were incubated with media (Null) or
with 20 µg/mL of the CD22 blocking MoAbs HB22.7 or HB22.33. Apoptosis
was assessed by BRDU incoporation by DNA fragments and detecting
apoptotic cells with anti-BRDU-FITC via FACS.
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| Fig 6.
CD22-mediated apoptosis induction was CD22 blocking
MoAb-specifc and equivalent to ligation of sIgM. Ramos B cells were
incubated with media, the CD22 MoAb HB22.7 (20 µg/mL), the
nonblocking CD22 MoAb HB22.27 (20 µg/mL), F(ab')2
fragments of goat MoAb to BCR (20 µg/mL), or CD3 (10 µg/mL) for 24 and 48 hours. Apoptosis was assayed by trypan blue exclusion and
confirmed via PI staining and BRDU incorporation as described in Fig 5.
The results shown are an average of 4 replicate experiments, with the
error bars representing the standard deviation.
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We next examined the dose-dependency of the pro-apoptotic effects of
the 2 MoAbs HB22.7 and HB22.33. Forty-eight hours of incubation with 5, 10, 20, 40, and 80 µg/mL of each MoAb induced a similar degree of
apoptosis, but at significantly different concentrations
(Fig 7). Eighty-two percent of Ramos cells
were apoptotic with 80 µg/mL of HB22.7, with a similar degree
observed with only 10 µg/mL of HB22.33 (Fig 7). The effects of these
MoAb were also examined in the Burkitt lymphoma cell lines BL41 and ST486. Both BL41 and ST486 are CD22+ and sIgM+
(J. Tuscano, unpublished observation) and have been
previously described.45,46 Incubating these cell lines with
the blocking (HB22.7 and HB22.33) and nonblocking (HB22.27) MoAb
induced 72% to 80% and 6% to 10% apoptosis, respectively
(Fig 8).
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| Fig 7.
Titration of apoptotic effects of HB22.7 and HB22.33 in
the Burkitt lymphoma cell line Ramos. Ramos B cells were incubated with
5, 10, 20, 40, or 80 µg/mL of MoAb for 48 hours. Apoptosis was
assessed by PI staining or BRDU incororation as described in Fig 5.
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| Fig 8.
Ligation of CD22 with blocking MoAbs induces apoptosis in
several Burkitt lymphoma cell lines. Twenty micrograms per milliliter
of the blocking (HB22.7 and HB22.33) or nonblocking (HB22.27) MoAbs
were incubated with the Burkitt lymphoma cell lines Ramos, ST486, and
BL41 for 48 hours. Apoptosis was assessed by propidium iodide staining
and BRDU incorporation as described in Fig 5.
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Ligation of CD22 results in downregulation of Bcl-xL and
Mcl-1.
The Bcl-2 protoncogene family of proteins has been found to be
important in regulating apoptosis in B cells. These proteins include
the anti-apoptotic proteins Bcl-2, Mcl-1, and Bcl-xL and the pro-apoptotic protein Bax.47-51 Using intracytoplasmic
flow cytometry (FACS), we examined the levels of these proteins in Ramos cells before and after stimulation with HB22.33 for 24 and 48 hours. Use of intracytoplasmic FACS and gating out PI-staining or
apoptotic cells allowed for examination of protein levels in pre-apoptotic cells only. After 48 hours of stimulation, only Bcl-xL and Mcl-1 were significantly downregulated
(Fig 9). Bcl-2 levels did not change
appreciably, and Bax levels increased slightly (Fig 9).
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| Fig 9.
Bcl-xL and Mcl-1 levels are downregulated in
Ramos cells after CD22 ligation. Ramos B cells were stimulated with 20 µg/mL of HB22.33 for 0, 24, and 48 hours. Cells were assessed for
Bcl-2, Bcl-xL, Mcl-1, and Bax levels via intracytoplasmic
FACS. Only nonapoptotic cells were assessed by gating out PI staining
cells.
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| |
DISCUSSION |
In this report, we demonstrate that CD22 ligation with MoAbs that
specifically block the interaction of CD22 with its ligand lead to CD22
tyrosine phosphorylation and result in activation of the SAPK signaling
cascade. The induction of SAPK by CD22 ligation in the
surface Ig-negative B-cell line HS-Sultan implies a BCR-independent mechanism of activation of this pathway. Likely as a consequence of
SAPK activation, CD22 cross-linking leads to the translocation of AP-1
into the nucleus. Although CD22 cross-linking did not activate the ERK
pathway, concurrent ligation of CD22 and the BCR resulted in prolonged
ERK-2 activation.
The mechanism by which CD22 cross-linking results in SAPK activation
remains to be elucidated. Treatment of B cells with the CD22 MoAbs
induces TNF- production,52 a known inducer of SAPK activation; however, this is unlikely to account for the rapid induction of SAPK observed after CD22 cross-linking. Recently, a
subfamily of serine/threonine protein kinases has been identified that
activate the SAPK pathway. Each of the subfamily members triggers SAPK
activation without significant p38 or MAPK activation. Three of them,
germinal center kinase (GCK), germinal center kinase related (GCKR),
hematopoietic progenitor kinase (HPK), are known to be expressed by B
lymphocytes. However, the upstream signals that activate these kinases
are largely unknown. One signal known to activate them is TNF-.
TNF- increases the catalytic activity of GCK, GCKR, and that of
another GCK family member, germinal center like kinase
(GLK).53-55 Directly implicating GCKR in TNF--induced SAPK activation is the observation that a catalytically impaired form
of GCKR blocks TNF--induced SAPK activation.54 We are currently examining the effects of CD22 cross-linking on the activity of GCKR and GCK in B cells and have evidence that indeed GCKR is
involved in CD22-mediated SAPK activation (Tuscano and Kehrl, unpublished observations). In addition, we are testing
whether catalytically inactive forms of GCK and GCKR interfere with
CD40- and CD22-induced SAPK activation.
The adhesive properties of CD22 allow B cells to interact with other
hematopoietic cells and even some nonhematopoietic cells. Although the
importance of these interactions in modulating B-cell function remains
obscure, it seems unlikely that their only function would be to
immobilize CD22, thereby preventing its association with the BCR.
Several recent studies that manipulated the BCR/CD22 association using
either antibody-based immobilization or co-cross-linking techniques
have concluded that the function of CD22 is to inhibit BCR signaling
and that the immobilization of CD22 by its ligands impairs the
recruitment of CD22 into the BCR.26 It was argued that the
apparent positive signaling function observed with CD22 MoAbs was the
result of sequestration of SHP-1 from the BCR, leading to enhanced
signaling by the derepressed BCR. Although these studies offer a
compelling demonstration that co-cross-linking CD22 and the BCR
impairs BCR signaling, for several reasons we think that CD22 is an
independent signaling molecule capable of transmitting a positive
signal to the B cells rather than simply serving as a platform for the
recruitment of SHP-1. First, we have screened a panel of CD22 MoAbs and
found significant differences in their agonistic
activities.12,13 Several of these antibodies costimulate B-cell proliferation with anti-IgM and with CD40 and
cytokines.13 Although it can be argued that the CD22 MoAbs
recruit CD22 away from the BCR, thus increasing BCR
signaling, the results with CD22 MoAb and cytokines or CD40 cannot be
explained in that way. Also, other CD22 MoAbs that should recruit CD22
from the BCR complex as efficiently have little or no costimulatory
effect. Second, we find that the exposure of HS-Sultan cells, a cell
line that lacks surface Ig to agonistic CD22 MoAbs, activates SAPK, a
result consistent with a direct signaling role for CD22 independently of the BCR. Third, the agonistic CD22 MoAbs are as efficient as anti-IgM in inducing the apoptosis of Ramos cells, whereas nonagonistic CD22 MoAbs are no better than a control antibody. In addition, the
combination of the CD22 MoAbs and anti-IgM is better than either
antibody alone. Fourth, CD22 cross-linking not only triggers the
association of CD22 with Lyn and SHP-1, but also with Syk, PI-3 kinase,
and phospholipase C-.11,12,18-20 The last 3 effectors are usually regarded as positive effectors in signaling pathways.
So, if CD22 has a positive role in B-cell function, why is the
phenotype of the CD22/ mice most consistent
with CD22 downregulating BCR signaling. We would argue that, in fact,
the CD22/ B cells are only mildly compromised
compared with wild-type cells. Perhaps this is because losing CD22's
inhibitory effects is in part tempered by the loss of its stimulatory
effects. In fact, the CD22-deficient mice are fully capable of
generating an antibody response of normal magnitude to both
thymus-dependent and -independent antigens. Because significant BCR
cross-linking does not occur during the response to thymus-dependent
antigens, but rather the processing of antigen by B cells and direct
T-B-cell collaborations, it seems unlikely that the recruitment of
CD22 into the BCR complex would play a significant role in inhibiting
B-cell function. In this context, an interaction between CD22 ligands
on T cells and CD22 on B cells could promote B-cell activation.
Clearly, a more complete understanding of the consequences of the loss
of CD22 will require the identification of the ligands for CD22 and an examination of the consequences of their interactions with CD22 on
B-cell function.
Although the consequences of treating primary B cells with the
agonistic CD22 MoAbs have been examined, their effects on neoplastic B
cells have not. We have demonstrated here that these MoAbs are highly
efficient at inducing apoptosis in multiple Burkitt lymphoma cell
lines. The extent of this effect is similar to that observed after
surface Ig cross-linking and even to a greater extent when the IgM MoAb
(HB22.33) is used. This latter effect is likely to be secondary to the
pentameric structure of HB22.33 and a enhanced ability to cross-link
CD22. However, despite their similar effect on Burkitt lymphoma cells,
CD22 and surface Ig cross-linking result in distinctive patterns of ERK
activation. CD22 cross-linking predominantly induces SAPK activation,
whereas surface Ig cross-linking predominantly induces MAPK. Whereas
SAPK activation has been implicated in the induction of apoptosis in
certain cell types, CD40 is a potent SAPK activator yet prevents
apoptosis induced by anti-Ig.56 A potential explanation for
this dichotomy is that CD40 signaling also induces the translocation of
NF-B to the nucleus, which has been implicated in preventing
apoptosis.57 However, making this explanation less likely,
surface Ig cross-linking also induces the translocation of NF-B.
Alternatively, CD40 may induce a unique signaling pathway, which
results in rescue of the Burkitt cells. Whether these Burkitt cell
lines have relevance to normal B-cell physiology remains unclear;
however, they do provide a functional readout of surface Ig and CD22
signaling. The inability of the p38/JNK inhibitor SB203580 to block
CD22-mediated apoptosis in Ramos B cells is consistent with previous
reports that demonstrate that, whereas SB203580 effectively and
specifically inhibits p38 and JNK2, it does not inhibit JNK1, JNK3, or
JNK4.43,44
Examination of Bcl-2 protoncogene family member protein levels in
pre-apoptotic Ramos B cells before and after CD22 ligation showed a
significant decrease in Bcl-xL and Mcl-1 and suggests a
regulatory role for these proteins in CD22-mediated apoptosis in Ramos
B cells. However whether this pattern is CD22-specific or not, and its
relevance to normal B-cell physiology remains undefined, it is
consistent with previous reports that demonstrate a significant role
for these protein in regulating B-cell apoptosis.48,49,51
We also demonstrated that, although the engagement of CD22 does not
increase ERK-2 activity, it does enhance and prolong BCR-mediated ERK-2
induction. Although it may be hypothesized that this is secondary to
sequestration of SHP1 away from the BCR, the mechanism remains
undefined. Recently, the physiologic significance of the duration of
ERK activation has been explored in PC12 cells.58 These
studies have suggested that the duration of ERK activation may be
critical for determining cellular responses. Whereas a sustained
activation and nuclear accumulation of ERK induce cell differentiation,
a more transient activation leads to cell proliferation. The
significance of these effects in B cells has yet to be examined; however, they may begin to explain the ultimate physiologic consequence of the ability of CD22 to modulate BCR-mediated signaling events.
| |
ACKNOWLEDGMENT |
The authors thank Mary Rust for her editorial assistance and Dr Anthony
Fauci for his support.
| |
FOOTNOTES |
Submitted May 14, 1998; accepted April 21, 1999.
Supported by National Institutes of Health Grants No. CA-54464,
AI-26872, and HL50985.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to John H. Kehrl, MD, B Cell Molecular
Immunology Section, Laboratory of Immunoregulation, NIAID, NIH, Bldg
10, Room 11B08, Bethesda, MD 20892-1876.
| |
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K. L. Otipoby, K. E. Draves, and E. A. Clark
CD22 Regulates B Cell Receptor-mediated Signals via Two Domains That Independently Recruit Grb2 and SHP-1
J. Biol. Chem.,
November 16, 2001;
276(47):
44315 - 44322.
[Abstract]
[Full Text]
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S. Mathas, A. Rickers, K. Bommert, B. Dörken, and M. Y. Mapara
Anti-CD20- and B-cell Receptor-mediated Apoptosis: Evidence for Shared Intracellular Signaling Pathways
Cancer Res.,
December 1, 2000;
60(24):
7170 - 7176.
[Abstract]
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E. C. M. Brinkman-Van der Linden, E. R. Sjoberg, L. R. Juneja, P. R. Crocker, N. Varki, and A. Varki
Loss of N-Glycolylneuraminic Acid in Human Evolution. IMPLICATIONS FOR SIALIC ACID RECOGNITION BY SIGLECS
J. Biol. Chem.,
March 17, 2000;
275(12):
8633 - 8640.
[Abstract]
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J. C. Poe, M. Fujimoto, P. J. Jansen, A. S. Miller, and T. F. Tedder
CD22 Forms a Quaternary Complex with SHIP, Grb2, and Shc. A PATHWAY FOR REGULATION OF B LYMPHOCYTE ANTIGEN RECEPTOR-INDUCED CALCIUM FLUX
J. Biol. Chem.,
June 2, 2000;
275(23):
17420 - 17427.
[Abstract]
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ABSTRACT
INTRODUCTION