Major histocompatibility complex class II
molecules and the B cell antigen receptor (BCR) transduce similar
signals when cross-linked by ligand. Therefore, studies were conducted
to determine whether the protein tyrosine phosphatase CD45 regulates
signaling via these transmembrane receptors in an analogous manner.
Cross-linking of either class II molecules or the BCR on CD45-positive
K46-17µm
B lymphoma cells was observed to induce activation of the
Src family protein- tyrosine kinase Lyn, tyrosine phosphorylation of
Syk and phospholipase C
, and the production of inositol
1,4,5-trisphosphate leading to intracellular mobilization as well as
extracellular influx of Ca2+. In the absence of CD45,
cross-linking of either class II molecules or the BCR failed to induce
activation of Lyn. Syk was inducibly phosphorylated on tyrosine in a
normal manner, whereas phospholipase C
exhibited a high basal level
of tyrosine phosphorylation that was not significantly increased upon
stimulation. Nevertheless, phospholipase C
appeared to be functional
because CD45-negative cells produced elevated levels of inositol
1,4,5-trisphosphate following stimulation through class II or the BCR.
Regardless of this, CD45-negative cells exhibited Ca2+
mobilization responses that were greatly diminished and transient in
nature. Whereas little or no mobilization of Ca2+ was
observed in response to class II cross-linking, CD45-deficient cells
mobilized Ca2+ from intracellular stores but not the
extracellular environment in response to BCR cross-linking. These
results demonstrate that CD45 regulates both Src family kinase
activation and Ca2+ mobilization associated with class II-
and BCR-mediated signal transduction.
 |
INTRODUCTION |
Major histocompatibility complex
(MHC)1 class II molecules
expressed by B cells play an important role as restricting elements in
the generation of thymus-dependent immune responses (1-4). There is now a significant amount of evidence indicating that class II
molecules also deliver signals to the B cell that affect several
parameters related to their immune function. MHC class II-mediated
signals have been implicated in regulation of antigen presentation,
up-regulation of accessory molecule expression (i.e. B7.2),
cell-cell adhesion, apoptosis, proliferation, and differentiation (5-15). The signals transduced in response to cross-linking of class
II molecules have been partially characterized and are complex in
nature. Ligation of class II can elicit either the production of cAMP
and the subsequent translocation of protein kinase C to the nucleus
(16-20) or the activation of protein-tyrosine kinases (PTK),
hydrolysis of phosphatidylinositol, and mobilization of Ca2+ (21-25). The exact molecular mechanism(s) by which
class II molecules are coupled to these distinct signaling pathways is
at present unclear but appears to be regulated in part by the
activation state of the B cell. In both the human and mouse,
cross-linking of MHC class II molecules on unstimulated B cells elicits
the production of cAMP and promotes translocation of protein kinase C
(23, 26). In contrast, differences in coupling of MHC class II
molecules to the PTK-dependent signaling pathway in human
versus mouse appear to exist. Whereas cross-linking of class
II molecules in human tonsillar B cells and B cell lines leads to PTK
activation (21, 22), studies using quiescent mouse splenic B cells have demonstrated that class II-mediated PTK activation is dependent on the
receipt of a priming signal delivered via the B cell antigen receptor
(BCR) in conjunction with IL-4 (24). The apparent differences in
coupling of class II to the PTK-dependent pathway in human versus mouse have been attributed to inherent differences in
the activation state of the cells being examined.
Structure/function studies have demonstrated that the ability of MHC
class II to mediate PTK activation resides in the
transmembrane/extracellular region of the
and/or
chains
(25-28). This finding suggests that class II is coupled to PTK
activation through its interaction with an intermediate transducer
protein(s) in much the same way that membrane immunoglobulin is coupled
to the Src PTKs through an interaction with the Ig
/Ig
heterodimer. Further analogies between class II-mediated signaling and
that of the BCR can be drawn from a range of studies. Cross-linking of
MHC class II molecules on B cells leads to the activation of the Src
family PTKs Lyn and Fgr (29). Additionally, class II has been shown to
regulate the activation of the PTK Syk in both tonsillar B cells and B leukemia cell lines (30). Downstream PTK-dependent
processes including activation of phospholipase C
(PLC
),
phosphatidylinositol turnover, and increases in the concentration of
intracellular Ca2+ have also been documented following
cross-linking of class II molecules in human tonsillar B cells (21, 22,
24, 25). These findings suggest that cross-linking of MHC class II
molecules activates a signaling pathway that is similar to the one
associated with the BCR.
Because the signaling pathway activated by cross-linking of MHC class
II molecules is similar to that triggered by ligand binding to the BCR,
it was of interest to determine whether signal transduction via MHC
class II is regulated by the protein-tyrosine phosphatase (PTP) CD45.
Numerous studies have demonstrated that optimal BCR-mediated signaling
requires CD45 expression on the surface of the cell (31, 32). In the
absence of CD45, BCR cross-linking does not mediate recruitment and
activation of Src family PTKs (33-35), whereas the PTK Syk is
recruited to the BCR complex and is activated (34, 35). Although Syk
function appears to be intact in cells lacking CD45, the absence of
this PTP is manifest by the fact that BCR-mediated mobilization of
Ca2+ is abnormal, and activation of Ras is abrogated
(36-38). Thus, it is clear that the loss of CD45 may affect not only
Src PTK activation but downstream processes as well.
To determine whether CD45 regulates class II-mediated signaling,
experiments were conducted using the K46 B cell lymphoma in conjunction
with two CD45-deficient variants. The K46 lymphoma is ideal for these
studies because it exhibits a primed MHC class II signaling phenotype
(25). Studies demonstrate that MHC class II-mediated activation of the
Src family protein tyrosine kinase Lyn is abrogated, and
Ca2+ mobilization is drastically reduced in the absence of
CD45. These findings indicate that CD45 plays an analogous role in
regulating both MHC class II- and BCR-mediated signal transduction.
 |
EXPERIMENTAL PROCEDURES |
Biological Reagents--
Monoclonal Abs used in these studies
included the following: D3.137.5.7 (rat IgG2a, anti-mouse
MHC class II, Iad,b haplotype), B76 (rat IgG1,
anti-mouse µ heavy chain), RG7/9.1 (mouse IgG2b anti-rat
light chain), I3/2.3 (rat IgG2b, anti-mouse CD45), MB23G2 (rat
IgG2a, anti-mouse CD45 Exon B), C363.16A (rat IgG2a,
anti-mouse CD45 Exon B), M1/42.3.9.8 (rat IgG2a, anti-mouse class I MHC, H-2K), and 187.1 (rat IgG1 anti-mouse
light chain). Monoclonal Abs were purified using protein G-Sepharose 4B
fast flow (Amersham Pharmacia Biotech). The mAbs were biotinylated using N-hydroxysuccinimidobiotin (Sigma) as described
previously (39). 4G10 (mouse IgG2b, anti-phosphotyrosine
(Tyr(P)), Upstate Biotechnology, Inc., Lake Placid, NY) and mouse
anti-bovine phospholipase C
-1 (mixed monoclonal IgG antibodies,
Upstate Biotechnology, Inc.) were purchased for these studies. The
anti-Lck BP1 polyclonal Ab (murine HS1) was generously provided by Dr.
Yoshihiro Takemoto (Institute of Immunology, Chiba, Japan). Rabbit
polyclonal anti-Syk Ab was obtained either from Dr. Joseph Bolen (DNAX,
Palo Alto, CA) or was purchased from Upstate Biotechnology Inc.
Polyclonal goat anti-mouse IgG coupled to horseradish peroxidase (HRPO)
and goat anti-rabbit IgG coupled to HRPO were purchased from Biosource International (Camarillo, CA). Streptavidin was purchased from Pierce.
Cell Lines--
The B lymphoma cell line K46-17µm
(K46)
was generously provided by Dr. Michael Reth (Max-Planck Institut fur
Immunobiologie, Freiburg, Germany). K46 cells were cultured in
Iscove's modified Dulbecco's medium (IMDM) supplemented with 5%
fetal calf serum (HyClone Laboratories, Logan, UT), 2 mM
L-glutamine, 50 µM
-mercaptoethanol, 100 µg/ml streptomycin-penicillin, and 50 µg/ml gentamycin (Sigma) at
37 °C under 7% CO2. CD45-negative variants of the K46
line were isolated following chemical mutagenesis with methanesulfonic acid ethyl ester (EMS, Sigma). K46 cells (2 × 107)
were incubated in 25 ml of IMDM supplemented with 5% fetal calf serum
containing EMS at a final concentration of 480-760 µg/ml. The cells
were cultured in the presence of EMS overnight at 37 °C after which
they were washed and resuspended in IMDM with 5% fetal calf serum.
Within 1 to 2 days the CD45-negative cell population was enriched by
negative selection with anti-CD45 mAbs and rabbit complement.
EMS-treated cells were resuspended in IMDM at a concentration of 5 × 106 cells/ml. A mixture of anti-CD45 mAbs containing
I3/2.3, MB23G2, and C363.16A each at a final concentration of 20 µg/ml was added, and the cells were incubated for 20 min on ice.
Subsequently, mAb-treated cells were washed and were incubated with
rabbit complement for 2-3 h at 37 °C. Additional complement was
then added, and cells were incubated for an additional 2 h after
which they were washed and resuspended in IMDM with 5% fetal calf
serum. Complement-mediated negative selection of CD45-positive cells
was performed 2-3 times. CD45-negative cells were then positively
selected using fluorescence-activated cell sorting. CD45-negative cells
isolated by fluorescence-activated cell sorting were then subjected to
limiting dilution cloning to isolate several CD45-negative clones, two
of which (3S5 and 35S5) are described below. K46 cells and the
CD45-negative clones were examined using immunofluorescence staining as
described previously (40) to ensure comparable expression of key
surface markers.
Metabolic Labeling--
K46 cells or CD45-negative variants were
washed two times in PBS and were resuspended at a concentration of
5 × 106/ml in cysteine/methionine-free IMDM (Sigma)
supplemented with 5% dialyzed fetal calf serum. The cells were
incubated for 1 h at 37 °C prior to the addition of 0.1 mCi of
Tran35S-label (specific activity >1000 Ci/mmol, ICN
Pharmaceuticals, Costa Mesa, CA) per 1 × 107 cells.
Cells were subsequently incubated at 37 °C for 3 h in the
presence of [35S]cysteine/methionine. Following the 3-h
incubation, cells were washed three times with PBS and were used for
immunoprecipitation experiments (2 × 107/sample).
Metabolically labeled cells that were not used immediately were frozen
as a pellet at
20 °C.
Northern Blot Analysis--
Total RNA was isolated from 1 × 108 cells using the TRIzol extraction technique (Life
Technologies, Inc.) as described by the manufacturer. Thirty-five µg
of RNA from each cell line was electrophoresed on a 1%
agarose-formaldehyde denaturing gel and then transferred to
nitrocellulose membrane (Amersham Pharmacia Biotech) by capillary blotting using 20× SSC. The membrane was hybridized with a full-length CD45 cDNA obtained from Dr. M. L. Thomas (Washington
University, St. Louis, MO). The cDNA was labeled using nick
translation to a specific activity of 1.8 × 108
cpm/µg using [32P]dCTP (NEN Life Science Products). The
membrane was probed overnight at 42 °C and was subsequently washed 2 times with 0.2× SSC at a temperature of 55 °C. The membrane was
then exposed to XAR-5 x-ray film (Kodak) to visualize the mRNA band
encoding CD45.
Immunoprecipitation and Immunoblotting--
MHC class II- and
BCR-mediated signaling events were analyzed following stimulation of
cells with the respective mAbs as described below. To analyze MHC class
II-mediated signaling CD45-positive K46 cells or the CD45-negative
variants (3S5 or 35S5) were harvested, resuspended in IMDM with 5%
fetal calf serum (2.5 × 107/sample), and incubated in
the presence of biotinylated anti-class II mAb (D3.137.5.7, 10 µg/ml)
for 15 min at RT. Cells were then washed once with PBS and were
resuspended in IMDM with 5% fetal calf serum (1 ml/sample). The cells
were equilibrated for 20 min at 37 °C and were stimulated for
various times with streptavidin at a final concentration of 10 µg/ml.
Stimulation of cells with either anti-class II mAb or streptavidin
alone did not elicit a signaling response as determined by measurement
of calcium mobilization (data not shown). For consistency cells
stimulated through the BCR were handled in the same manner as those
stimulated with anti-class II mAb and streptavidin. They were
harvested, incubated at RT for 15 min, and then equilibrated at
37 °C in medium alone prior to being stimulated for varied times by
the addition of the mAb B76 (anti-µ heavy chain, 10 µg/ml).
Following stimulation, cell samples were washed twice in PBS and were
lysed in 0.5 ml of lysis buffer (25 mM Hepes, 150 mM NaCl, pH 7.8, 10 mM EDTA, 1 mM
EGTA, 0.1 mM Na3VO4, and 1%
Nonidet P-40). Cells were lysed for 1 h on ice, and the lysates
were centrifuged at 12,000 × g for 15 min at 4 °C.
Detergent-soluble lysates were precleared by incubation with protein
G-Sepharose (for Lyn, HS1, PLC-
, and Syk experiments) or RG7/9.1
bound to Sepharose 4B (for CD22 experiments) for 1 h at 4 °C to
minimize nonspecific protein binding. Proteins of interest were
immunoprecipitated from precleared lysates either by the addition of
soluble Abs followed by the addition of protein G-Sepharose (Lyn, Syk,
HS1, and PLC
) or by the addition of Ab coupled directly to Sepharose
4B beads (CD22). Each immunoprecipitation step was performed for 1 h at 4 °C with rotation. Immune complex-coated beads were collected
and washed four times with lysis buffer containing 0.2% Nonidet P-40.
The beads were resuspended in 50 µl of reducing SDS-PAGE sample
buffer, boiled for 4 min, and centrifuged at 12,000 × g for 5 min. The proteins contained in 15 µl of
supernatant from each sample were separated on 8% acrylamide gels
using SDS-PAGE and were transferred to Hybond-ECL nitrocellulose
membranes (Amersham Pharmacia Biotech). The membranes were blocked with
10% nonfat dry milk in TBST for 1 h at room temperature, washed
five times in TBST, and incubated with the appropriate primary Ab for
1 h at RT. The membranes were then washed five times with TBST,
incubated with the appropriate HRPO-conjugated secondary Ab for 1 h at RT, washed five times with TBST, and visualized using enhanced
chemiluminescence (ECL, Pierce) according to the manufacturer's
instructions. The same blots were stripped of Abs by incubation in
stripping buffer (10 mM Tris, pH 2.3, 150 mM
NaCl) at 60 °C for 1 h and then were washed extensively as
above. The membranes were then blocked with 3% blot qualified bovine
serum albumin (Promega, Madison, WI) in PBS, washed extensively, and
incubated with anti-Tyr(P) mAb (4G10 at a 1/2000 dilution, Upstate
Biotechnology Inc.) for 1 h. The membranes were washed and
developed as above using ECL.
In Vitro Kinase Assay--
Lyn was recovered from cells that
were stimulated with mAbs directed against class II or the BCR as
described previously by immunoprecipitation with rabbit anti-Lyn Ab and
protein G-Sepharose. To perform in vitro kinase assays, Lyn
bound to protein G-Sepharose 4B beads was incubated in 50 µl of
reaction buffer containing 25 mM Imidazole HCl, pH 7.2, 5 mM MnCl2, 0.1% gelatin, 0.5% Nonidet P-40,
and 10 µCi of [
-32P]ATP (specific activity, 4500 Ci/mM, ICN Biochemicals, Inc., Irvine, CA) for 15 min at
37 °C. The catalytic activity of Lyn was monitored by addition of
the exogenous substrate acid-denatured enolase (5 µg per reaction,
Sigma). Phosphorylation of enolase was allowed to proceed at room
temperature for 5 min. All reactions were terminated by the addition of
50 µl of reducing SDS-PAGE sample buffer. Radiolabeled proteins were
boiled and then separated using SDS-PAGE. Incorporation of
32P into the exogenous substrate enolase was visualized by
autoradiography using Kodak XAR-5 x-ray film. In selected experiments
radiolabeled proteins were transferred to nitrocellulose followed by
immunoblotting to detect Lyn. Densitometric analysis was used to
confirm equivalent recovery and loading of the respective PTK.
Membranes were then exposed to x-ray film to visualize radiolabeled
proteins including enolase. Following excision of the enolase band from
the membrane, incorporation of 32P was quantitated using
scintillation counting.
Measurement of IP3 Production--
Experiments to
measure the production of inositol 1,4,5-trisphosphate
(IP3) were performed using [3H]inositol
1,4,5-trisphosphate Radioreceptor Assay Kit (NEN Life Science Products)
following the manufacturer's protocol. The samples were prepared for
assay by stimulating K46 cells or CD45-negative variants (1 × 107/sample) with mAbs directed against either class II or
the BCR as described previously. Following stimulation the cells were washed in 0.5 ml of PBS. The cells were lysed on ice with 100 µl of
100% trichloroacetic acid, and the trichloroacetic acid was extracted
with a 3:1 solution of 1,1,2-trichloro-1,2,2-trifluoroethane to
trioctylamine based on the manufacturer's recommendations. The assays
were performed as indicated in the protocol; however, the lyophilized
IP3 standard was reconstituted in PBS instead of distilled
H2O. Each condition was prepared in duplicate with each
sample also being assayed in duplicate. The results depicted are
representative of three separate experiments.
Measurement of Calcium Mobilization--
Analysis of
Ca2+ flux in K46, 3S5, and 35S5 cells was performed as
described previously (40). Cells (1 × 106/ml) were
loaded with Indo-1 AM (Molecular Probes, Eugene, OR) at a final
concentration of 5 µM. Indo-1-loaded cells were analyzed using a Becton Dickinson FacsVantage (San Jose, CA) equipped with an
Enterprise laser from Coherent (Santa Clara, CA) set for excitation at
~364 nm at a power setting of 60 milliwatts. Fluorescence emissions were separated by a 505-nm short pass beam splitter into two component emissions by passage through 405- and 485-nm centered 10-nm band pass
filters to detect violet and blue, respectively. The ratio of emissions
for 405/485 was determined, and a plot was constructed of fluorescence
ratio versus time. Analysis of class II-mediated Ca2+ mobilization was performed as described previously
(40). Just prior to analysis cells were incubated with biotinylated
anti-class II mAb (D3.137.5.7, 5 µg/ml, 15 min at RT), were washed,
and the base-line level of intracellular free Ca2+ was
established. Once the base line was measured, streptavidin (5 µg/ml)
was added to the mAb-treated cells and the analysis resumed.
BCR-mediated calcium flux was measured in response to anti-µ (B76, 5 µg/ml) added to cells once an initial base line had been established.
Measurement of intracellular mobilization versus
extracellular influx of Ca2+ was performed as described
previously (40). Briefly, cells were resuspended in
Ca2+/Mg2+-free PBS just prior to analysis. A
base line was established, and the cells were stimulated as described
above. Following the resolution of the intracellular mobilization
response, the analysis was stopped and CaCl2 was added to
the sample to a final concentration of 4 mM. Measurement of
intracellular free Ca2+ was resumed immediately.
 |
RESULTS |
Analysis of CD45-negative K46 Mutants--
The K46 cell line was
chosen for studies to examine the role of CD45 in MHC class II-mediated
signaling because it is functionally equivalent to a primed murine B
cell in which class II is coupled to the PTK-dependent
signal transduction pathway associated with calcium mobilization.
Cross-linking of class II expressed on the K46 cell line with
biotinylated anti-class II mAb and a secondary reagent (i.e.
streptavidin) will elicit a calcium mobilization response without the
need to first prime the cell with anti-Ig and IL-4 (25). This
characteristic was essential for studies to evaluate the role of CD45
in MHC class II-mediated signaling because it may not be possible to
prime cells via the BCR and/or IL-4 receptor in the absence of
CD45.
CD45-negative mutants of the K46 parental cell line were generated by
subjecting cells to chemical mutagenesis and negative selection as
described under "Experimental Procedures." Two clones were selected for further analysis that expressed less than 8% of the
parental level of CD45 as determined by immunofluorescence staining and
flow cytometry. Clones 3S5 and 35S5 were examined using a panel of mAbs
directed against several transmembrane proteins expressed on B cells
including CD45, membrane immunoglobulin M, Ig
light chain, MHC class
II, MHC class I, and CD22. Depicted in Fig.
1 are the immunofluorescence staining
results for the phenotypic analysis of the CD45-negative clone 35S5. As
can be seen, with the exception of CD45 these cells express normal
levels of surface receptors when compared with the parental K46 cells. The 3S5 clone exhibits a similar phenotype (data not shown). Additional immunofluorescence analysis demonstrated that the expression of CD19
was comparable between the parental K46 cells and the CD45-negative clones, whereas the level of CD21 and CD81 expression was elevated (~2-fold) on the 3S5 and 35S5 clones (data not shown). It can be
concluded from these analyses that the CD45-negative clones exhibit a
relatively selective deficiency in their expression of CD45.

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Fig. 1.
The CD45-deficient clone 35S5 exhibits
comparable expression of surface receptors other than CD45 when
compared with parental K46 cells. Cells (1 × 106) were stained with optimal dilutions of fluorescein
isothiocyanate-conjugated mAbs directed against the surface markers
indicated. Isotype-matched control mAbs were used to gate out
nonspecific fluorescence. The histograms depict surface receptor
expression of CD45-positive K46 cells as well as the CD45-deficient
35S5 clone. The surface receptor phenotype of the 35S5 clone is
representative of the 3S5 clone (data not shown). mIgM,
membrane immunoglobulin M.
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Further examination of the 3S5 and 35S5 clones to detect CD45 message
revealed that there is little or no detectable message based on
Northern blot analysis of total RNA (Fig.
2A). In agreement with the
immunofluorescence and Northern blot analyses, metabolic labeling of
3S5 and 35S5 with [35S]methionine/cysteine followed by
immunoprecipitation of CD45 with the I3/2.3 mAB failed to detect
intracellular CD45 (Fig. 2B). It should be noted, however,
that when the 3S5 and 35S5 clones were maintained in culture for
extended periods (>6 months), CD45-positive revertants were observed.
Thus, it is likely that chemical mutagenesis did not result in specific
mutations within the CD45 gene locus in the 3S5 and 35S5
clones but instead affected the ability of the cells to transcribe the
CD45 gene.

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Fig. 2.
The 35S5 and 3S5 clones do not express
intracellular CD45 or message based on metabolic labeling and Northern
blot analysis. A, the 35S5 and 3S5 clones do not contain
detectable levels of mRNA encoding CD45. Thirty-five µg of total
RNA from the K46 parental cells and each of the CD45-deficient clones
(3S5 and 35S5) was separated on a 1% formaldehyde agarose gel and was
transferred to a nitrocellulose membrane. The membrane was probed with
a 32P-labeled full-length CD45 cDNA. The membrane was
then exposed to x-ray film, and autoradiography was used to visualize
hybridizing bands corresponding to CD45. B, the 35S5 and 3S5
clones do not express intracellular CD45 based on metabolic labeling.
Cells (2 × 107) labeled with
[35S]methionine/cysteine were washed and lysed in buffer
containing 1% Nonidet P-40. Detergent-soluble lysates were precleared
by the addition of irrelevant mAb coupled to Sepharose 4B beads. CD45
was then immunoprecipitated using the anti-CD45 mAb I3/2.3 coupled to
Sepharose 4B, and the immune complex material was separated by
SDS-PAGE. Radiolabeled bands were visualized by autoradiography.
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Loss of CD45 Expression Abrogates Src Family PTK Activation in
Response to MHC Class II Cross-linking--
The loss of CD45
expression has been demonstrated to inhibit antigen receptor-mediated
activation of Src family PTKs in T and B cells (32). Additionally, it
has been shown that class II molecules mediate the activation of the
Src family PTKs Lyn and Fgr in response to ligand binding (29).
Therefore, experiments were performed to evaluate whether cross-linking
of class II leads to activation of the PTK Lyn in the absence of CD45.
Whereas Lyn exhibited comparable basal levels of tyrosine
phosphorylation in the presence or absence of CD45, there was a marked
inhibition of inductive phosphorylation in response to cross-linking of
class II in the 35S5 clone when compared with parental K46 cells (Fig. 3, upper panel). When the
membrane was stripped and reprobed with antibody directed against Lyn
(Fig. 3, middle panel), it was noted that the amount of Lyn
in each lane was comparable, although it did exhibit an
activation-dependent change in its electrophoretic mobility
in response to class II cross-linking in the CD45-positive K46 cells
but not the CD45-negative 35S5 clone. This finding suggests that
cross-linking of MHC class II may promote the inducible phosphorylation of Lyn on serine/threonine residues thereby decreasing its
electrophoretic mobility in a manner similar to that previously
reported for Lck (41) and that this process is regulated by CD45.

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Fig. 3.
CD45 expression is required for activation of
the Src family PTK Lyn. Parental cells and CD45-negative (35S5)
cells (1 × 107/sample) were incubated in medium alone
or in the presence of anti-class II mAb (anti-Ia, D3.137, 10 µg/ml)
on ice for 10 min. Cells were washed twice in ice-cold PBS and were
resuspended in 1 ml of IMDM containing 10% fetal bovine serum.
Streptavidin (10 µg/ml) was added to the cells, and they were
incubated at 37 °C for varied periods. Control cells did not receive
streptavidin but were incubated for 10 min at 37 °C. Reactions were
terminated by the addition of ice-cold PBS containing 1% Nonidet P-40,
and Lyn was immunoprecipitated using a polyclonal rabbit anti-Lyn
antibody. Immune complex material was separated by SDS-PAGE and
transferred to nitrocellulose. The membrane was probed with anti-Tyr(P)
mAb (4G10) coupled to HRPO. Phosphorylation of Lyn was visualized using
enhanced chemiluminescence (upper panel). The same membrane
was stripped and reprobed with polyclonal antibody directed against Lyn
followed by a secondary goat anti-rabbit antibody coupled to HRPO. ECL
was then used to monitor loading of Lyn (middle panel). The
activation of Lyn was assessed based on phosphorylation of the
exogenous substrate enolase (lower panel). Cells were
stimulated as described above. Lyn was immunoprecipitated from cell
lysates and was incubated with denatured enolase in 50 µl of reaction
buffer containing 10 µCi of 32P-labeled ATP. Reactions
were allowed to proceed for 5 min at RT. Radiolabeled enolase was
isolated by SDS-PAGE and was visualized by autoradiography.
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Additional studies were performed to measure the enzymatic activity of
Lyn in the presence or absence of CD45 following class II ligation. K46
and 35S5 cells were stimulated for varied periods as indicated in Fig.
3 (bottom panel). Subsequently, Lyn was recovered by
immunoprecipitation, and its catalytic activity was assayed using an
in vitro kinase assay in the presence of the exogenous substrate enolase. Cross-linking of MHC class II molecules in the
CD45-positive K46 cells, but not the CD45-negative 35S5 clone, resulted
in the rapid activation of Lyn within 1 min. The composite results for
both BCR- and class II-mediated Lyn activation in the presence or
absence of CD45 are depicted in Fig. 4.
Ligation of either the BCR or class II molecules on CD45-positive K46
cells caused a 2-3-fold increase in the activity of Lyn based on
phosphorylation of enolase. In contrast, antibody binding to either the
BCR or MHC class II failed to activate Lyn in the 3S5 and 35S5 clones (Fig. 4). These data indicate that expression of CD45 is required for
activation of Lyn in response to stimulation via either the BCR or MHC
class II.

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Fig. 4.
Loss of CD45 expression abrogates Lyn
activation in response to BCR or class II cross-linking. K46,
35S5, and 3S5 cell lines (1 × 107 cells/sample) were
incubated in medium alone or in the presence of either anti-Ig mAb
(B76, 10 µg/ml) or anti-class II mAb (biotinylated D3.137, 10 µg/ml) and streptavidin (10 µg/ml) for 1 min at 37 °C. Cells
were then resuspended in ice-cold PBS, washed, and lysed in buffer with
1% Nonidet P-40. Lyn was immunoprecipitated from cell lysates and
incubated with enolase and [ -32P]ATP at RT for 5 min.
Radiolabeled proteins were separated by 10% SDS-PAGE and were
transferred to nitrocellulose. The recovery of Lyn was monitored by
immunoblotting with anti-Lyn polyclonal Ab followed by goat anti-rabbit
Ig coupled to HRPO. Lyn was visualized using ECL and autoradiography.
Incorporation of 32P into enolase was quantitated using
scintillation counting. Each stimulation condition was assayed in
triplicate, and the data are depicted as the mean stimulation index.
The stimulation index is defined as the fold increase in
32P incorporation into enolase mediated by Lyn isolated
from stimulated cells divided by that for Lyn isolated from
unstimulated cells. The basal kinase activity (in cpm of
32P incorporated into enolase) for Lyn isolated from K46
cells was 29,116, 27,657, and 31,217; clone 35S5 was 27,981, 28,533, and 30,701; and clone 3S5 was 26,897, 29,911, and 28,679. The results
depicted are representative of two independent experiments.
mIg, membrane immunoglobulin.
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The PTK Syk Is Inducibly Phosphorylated on Tyrosine in the Absence
of CD45--
Previous studies have demonstrated that the non-Src
family PTK Syk is recruited to the BCR complex and becomes activated
following cross-linking of the BCR in the absence of CD45 (34, 35). To
examine whether class II-mediated Syk activation requires CD45, experiments were conducted in which K46 and 35S5 cells were stimulated with either anti-IgM (B76) mAb or biotinylated anti-class II (D3.137) mAb and streptavidin. Subsequently, Syk was immunoprecipitated from
cell lysates and subjected to immunoblot analysis to measure tyrosine
phosphorylation. As depicted in Fig. 5
Syk was inducibly phosphorylated in response to either anti-IgM or
anti-class II, regardless of whether CD45 was expressed on the surface
of the cell. These findings suggest that Syk is inducibly activated
following MHC class II cross-linking and are in agreement with previous results related to Syk activation via the BCR. That Syk is
catalytically active in the absence of CD45 is supported by experiments
in which tyrosine phosphorylation of putative Syk substrates was
examined in the K46 and 35S5 cell lines. Cells were stimulated via the BCR or class II molecules, and inducible tyrosine phosphorylation of
HS1 and CD22 was measured. HS1 exhibited a constitutively elevated level of tyrosine phosphorylation that was not appreciably increased upon stimulation via the BCR or MHC class II (Fig.
6, A and B). In
contrast, CD22 exhibited a slight increase in its basal phosphorylation in the absence of CD45 and was hyperphosphorylated in response to
cross-linking of either the BCR (Fig.
7A) or MHC class II (Fig. 7B) when compared with K46 cells. Thus, in the absence of
CD45 expression or detectable Src PTK activation, these data suggest that the PTK Syk is able to function resulting in increased basal and/or inducible tyrosine phosphorylation of multiple downstream substrates.

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Fig. 5.
Inducible tyrosine phosphorylation of Syk
occurs in the absence of CD45 in response to BCR or class II
cross-linking. Experiments were performed with K46 cells and the
35S5 clone (1 × 107 cells/sample) in which the cells
were incubated for varied periods in medium alone or in the presence of
mAbs directed against the BCR (anti-Ig, B76, 10 µg/ml) or class II
(anti-Ia, biotinylated D3.137, 10 µg/ml + streptavidin, 10 µg/ml).
At the appropriate time points cells were resuspended in ice-cold PBS,
washed, and lysed in buffer with 1% Nonidet P-40. Lysates were
precleared, and Syk was immunoprecipitated with polyclonal rabbit
anti-Syk Ab. Immune complex proteins were separated by 10% SDS-PAGE
and transferred to nitrocellulose. The membranes were probed with
anti-Tyr(P) mAb (4G10 coupled to HRPO), and tyrosine phosphorylation of
Syk was visualized with ECL. The membranes were then stripped and
reprobed with anti-Syk Ab to monitor loading of Syk in each well.
A, analysis of Syk phosphorylation in response to
cross-linking of the BCR. B, analysis of Syk phosphorylation
in response to cross-linking of class II molecules.
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Fig. 6.
HS1 is constitutively hyperphosphorylated on
tyrosine in the absence of CD45 expression. Analysis of putative
Syk substrate phosphorylation was performed in K46 cells and the 35S5
clone. Cells (1 × 107/sample) were incubated in the
presence of either anti-Ig (B76, 10 µg/ml, A) or
anti-class II ( -Ia, biotinylated D3.137 10 µg/ml + streptavidin,
10 µg/ml, B) for 1-10 min at 37 °C. As a control,
cells were incubated for 10 min in medium alone (NT). At the
appropriate time points cells were resuspended in ice-cold PBS, washed,
and lysed in buffer with 1% Nonidet P-40. HS1 was immunoprecipitated
with polyclonal rabbit anti-HS1 and protein G-Sepharose. Proteins were
separated by 10% SDS-PAGE and were transferred to nitrocellulose.
Tyrosine phosphorylation of HS1 was visualized by probing the membranes
with anti-Tyr(P) mAb coupled to HRPO (4G10) followed by development of
the blot with ECL (upper immunoblots). The membranes were
stripped and were reprobed with anti-HS1 Ab followed by goat
anti-rabbit IgG coupled to HRPO. Loading of HS1 on the gel was
visualized using ECL (lower immunoblots).
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Fig. 7.
CD22 exhibits both basal and inducible
hyperphosphorylation after BCR or class II cross-linking in cells that
lack CD45. Analysis of CD22 tyrosine phosphorylation was performed
using K46 cells and the 35S5 clone as described previously. Cells
(1 × 107/sample) were incubated in the presence of
either anti-Ig (B76, 10 µg/ml, A) or anti-class II
( -Ia, biotinylated D3.137 10 µg/ml + streptavidin, 10 µg/ml,
B) for 1-10 min at 37 °C. As a control, cells were
incubated for 10 min in medium alone (NT) or in the presence
of the PTP inhibitor pervanadate (PV). At the appropriate
time points cells were resuspended in ice-cold PBS, washed, and lysed
in buffer with 1% Nonidet P-40. CD22 was immunoprecipitated with the
mAb NIMR-6 coupled to Sepharose 4B. Proteins were separated by 10%
SDS-PAGE and were transferred to nitrocellulose. Tyrosine
phosphorylation of CD22 was visualized by probing the membranes with
anti-Tyr(P) mAb coupled to HRPO (4G10) followed by development of the
blot with ECL.
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MHC Class II-mediated Phosphatidylinositol Hydrolysis Does Not
Require CD45 Expression--
Studies have demonstrated that Syk
regulates tyrosine phosphorylation and activation of PLC
leading to
the hydrolysis of phosphatidylinositol to produce the second messengers
diacylglycerol and IP3 that mediate protein kinase C
activation and calcium mobilization, respectively (42). If Syk is
functional in the absence of CD45 expression, then tyrosine
phosphorylation of PLC
and hydrolysis of phosphatidylinositol should
occur. Experiments were conducted to assess whether class II
cross-linking on 35S5 cells mediates tyrosine phosphorylation of PLC
and the production of IP3. As seen in Fig.
8 PLC
is constitutively phosphorylated
on tyrosine in the absence of CD45 and exhibits only a minor increase
in phosphorylation after cross-linking of either the BCR or MHC class
II. This finding, together with the data concerning tyrosine
phosphorylation of CD22 and HS1, suggests that Syk may be
constitutively active in the absence of CD45.

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Fig. 8.
PLC is constitutively phosphorylated on
tyrosine in cells that lack CD45 expression. PLC
phosphorylation on tyrosine was examined in K46 parental cells and the
35S5 clone as described previously. Cells (1 × 107/sample) were incubated in the presence of either
anti-Ig (B76, 10 µg/ml, A) or anti-class II (anti-Ia,
biotinylated D3.137 10 µg/ml + streptavidin, 10 µg/ml,
B) for 1-30 min at 37 °C. As a control, cells were
incubated for 30 min in medium alone (NT). At the
appropriate time points cells were resuspended in ice-cold PBS, washed,
and lysed in buffer with 1% Nonidet P-40. PLC was
immunoprecipitated with mouse anti-bovine phospholipase C -1 (mixed
mAb) and protein G-Sepharose. Proteins were separated by 10% SDS-PAGE
and were transferred to nitrocellulose. Tyrosine phosphorylation of
PLC was visualized by probing the membranes with anti-Tyr(P) mAb
coupled to HRPO (4G10, upper immunoblot in each panel)
followed by development of the blot with ECL. The membranes were
stripped and then reprobed with anti-bovine PLC -1 mAb followed by
goat anti-mouse IgG coupled to HRPO. PLC was visualized using ECL
(bottom immunoblot in each panel).
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To determine whether PLC
is functional in the CD45-negative 35S5
cell line, experiments were conducted to measure the production of
IP3 in response to BCR or class II cross-linking. In
agreement with the possibility that PLC
may be constitutively active
based on an analysis of tyrosine phosphorylation (Fig. 8),
IP3 levels were constitutively elevated (~1.5-fold) in
the 35S5 cells when compared with the CD45-positive K46 parental cells.
Both CD45-positive and CD45-negative cell lines exhibited an increase
in the level of IP3 when stimulated via the BCR or MHC
class II (Fig. 9). Whereas the increase
in IP3 production was relatively transient in the CD45-positive K46 cells, decreasing almost to basal levels within 10 min, the 35S5 cells exhibited a prolonged elevation in the level of
IP3 after stimulation. Moreover, levels of IP3
produced in the CD45-negative cells were consistently 1.5-2-fold
higher than those in the parental cells at all time points examined. Thus, these data indicate that PLC
is functional in the absence of
CD45 and that it can be inducibly activated.

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Fig. 9.
BCR- and class II-mediated hydrolysis of
phosphatidylinositol occurs in cells that lack CD45.
IP3 production was measured in K46 cells and the 35S5 clone
treated either with anti-Ig mAb (B76, 10 µg/ml, A) or
anti-class II mAb (biotinylated D3.137, 10 µg/ml + streptavidin, 10 µg/ml, B) for varied periods using an IP3
radioreceptor binding assay. Cell samples were set up in triplicate,
and each sample was then assayed in triplicate with the IP3
receptor binding assay. The data depicted are representative of three
independent experiments.
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Loss of CD45 Expression Attenuates Calcium Mobilization in Response
to Either BCR or MHC Class II Cross-linking--
Although class
II-mediated activation of the Src family PTK Lyn is abrogated in the
absence of CD45 expression, experiments suggest that both Syk and
PLC
are functional and that their activation results in the
production of IP3. Therefore, studies were conducted to
determine whether Ca2+ mobilization is normal in cells that
lack CD45 as one might predict based on the fact that the amount of
IP3 produced is equal to or greater than the level observed
in CD45-positive cells. Analysis of Ca2+ mobilization in
response to either anti-Ig or anti-class II mAbs, however, revealed
that there are profound alterations in the ability of cells to mobilize
Ca2+ in the absence of CD45. Whereas cross-linking of the
BCR elicited a transient and somewhat diminished Ca2+ flux
response (Fig. 10, top
panel), there was little or no detectable increase in the
intracellular concentration of free Ca2+
([Ca2+]i) after stimulation of the 35S5 clone
with anti-class II mAb and streptavidin (Fig. 10, middle
panel). The decreased Ca2+ flux in response to anti-Ig
or anti-class II stimulation of the CD45-negative 35S5 clone was not
due to unequal loading with the indicator dye Indo-1 as both K46 and
35S5 cells responded comparably when incubated with the
Ca2+ ionophore ionomycin (Fig. 10, bottom
panel).

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Fig. 10.
Calcium mobilization in response to
cross-linking of either the BCR or MHC class II is attenuated in the
absence of CD45 expression. Studies were performed with the K46
cells and 35S5 clone in which calcium mobilization was assayed in
response to either anti-Ig (B76, 10 µg/ml) or anti-class II ( -Ia,
biotinylated D3.137, 10 µg/ml + streptavidin, 10 µg/ml). Cells were
loaded with the calcium indicator dye Indo-1 as described under
"Experimental Procedures." To measure the response to cross-linking
of the BCR an unstimulated base line was established, the analysis was
stopped, and anti-Ig mAb was added. The analysis was resumed
immediately after addition of anti-Ig. For the analysis of class
II-mediated calcium mobilization, cells were first incubated with
biotinylated D3.137 for 10 min at RT. The cells were washed, and the
calcium base line was established. The analysis was then stopped and
streptavidin added, after which the analysis was resumed immediately.
The calcium ionophore ionomycin was used to stimulate cells to ensure
that they were loaded equivalently with Indo-1.
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Subsequently, experiments were performed to determine whether the
decreased Ca2+ mobilization response was due to lack of
intracellular mobilization and/or extracellular influx of
Ca2+. Because little or no detectable increase in the
[Ca2+]i could be observed after stimulation via
MHC class II, the separate analysis of intracellular mobilization
versus extracellular influx of Ca2+ was not
performed. However, experiments to monitor Ca2+
mobilization in response to anti-Ig revealed that CD45-negative cells
are able to mobilize Ca2+ from intracellular stores
(i.e. the endoplasmic reticulum) and that the response is
comparable to that seen in the parental cell line (Fig.
11, upper panel). In
contrast, the CD45-negative 35S5 clone did not exhibit the ability to
mobilize Ca2+ from the extracellular environment when
stimulated with anti-Ig, suggesting that plasma membrane
Ca2+ channels are not functional (Fig. 11). The small
increase in the [Ca2+]i seen in the 35S5 clone
after repletion of the medium with CaCl2 was due to normal
equilibration of the cytoplasmic compartment and does not reflect a
response to anti-Ig mAb because similar changes in the
[Ca2+]i are seen with the 35S5 clone in the
absence of stimulus (data not shown). The 3S5 clone exhibited
Ca2+ mobilization responses that were identical to those of
the 35S5 clone when stimulated with either anti-Ig or anti-class II and streptavidin (data not shown). These data indicate that there is a
significant defect in both BCR- and MHC class II-mediated Ca2+ mobilization in the absence of CD45 even though Syk
activation and the production of IP3 appear to occur.

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Fig. 11.
Loss of CD45 expression abrogates
extracellular influx of calcium in response to cross-linking of the
BCR. Analysis of the intracellular mobilization and extracellular
influx components of the calcium mobilization response mediated by
cross-linking of the BCR was performed. CD45-positive K46 cells and the
CD45-negative clone 35S5 were used for these experiments (1 × 106 cells/sample). Cells were loaded with Indo-1 as
described previously. Just prior to analysis the cells were washed and
resuspended in calcium-free PBS. The basal level of calcium in
unstimulated cells was established, and the analysis was stopped and
anti-Ig added (B76, 10 µg/ml). The analysis was resumed to monitor
the intracellular mobilization of calcium. After resolution of this
component of the response, the analysis was stopped a second time, and
CaCl2 was added to bring the final concentration of
Ca2+ to 4 mM. Analysis was then resumed to
monitor the influx of calcium from the extracellular environment.
Equivalent loading of cells was monitored using the calcium ionophore
ionomycin.
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DISCUSSION |
The K46 B lymphoma cell line in conjunction with derivative
CD45-negative variants constitutes a valuable model system for analyzing the role of CD45 in both BCR- and MHC class II-mediated signal transduction. The inherent advantage of utilizing the K46 cell
line is that signaling via both the BCR and MHC class II can be
analyzed in the same cell type without the need to prime the cell in
order to couple class II to its PTK-dependent signaling pathway. This facilitates the direct comparison of the effect that loss
of CD45 expression has on signal transduction via both receptors. The
results obtained from this study suggest that, with regard to the
requirement for CD45 expression, there are significant similarities
between the BCR-mediated signaling pathway and the
PTK-dependent pathway activated in response to
cross-linking of MHC class II.
Previous studies in both T cells and B cells have demonstrated that
CD45 physically associates with members of the Src family and that loss
of CD45 expression abrogates antigen receptor-dependent activation of Src family PTKs (32, 43, 44). In the B cell, BCR-mediated
activation of multiple Src family PTKs including Lyn, Fyn, and Blk is
attenuated in the absence of CD45 (33-35). The inability to activate
Src family PTKs in CD45-deficient cells results from
hyperphosphorylation of the carboxyl-terminal negative regulatory
tyrosine residues in the respective members of the family (32, 34, 35)
and the failure to properly recruit these PTKs to the BCR activation
complex (34). In agreement with previous work this study demonstrates
that, in the absence of CD45, cross-linking of either the BCR or MHC
class II fails to inducibly activate the PTK Lyn. Although Lyn did not
exhibit a significant increase in its basal level of tyrosine
phosphorylation in the CD45-negative 35S5 and 3S5 clones, it was
nevertheless clear that activation of this PTK is abrogated based on an
analysis of its ability to phosphorylate the exogenous substrate
enolase. Previous studies have implicated Fgr in addition to Lyn in
class II-mediated signaling in the B cell (29). However, because the K46 cell line does not express this PTK, it was not possible to determine whether CD45 expression is required for its inducible activation. It would be logical nonetheless to predict that the functional activation of this PTK would be inhibited as well because loss of CD45 expression has been shown to affect receptor-mediated activation of multiple Src family members in T and B cells (32).
Although it was apparent from these studies that Src family PTK
function is abrogated in the absence of CD45, subsequent studies demonstrated that the non-Src family PTK Syk is inducibly
phosphorylated in response to cross-linking of both the BCR and MHC
class II molecules. Based on an analysis of putative Syk substrate
phosphorylation in response to cross-linking of either the BCR or class
II, it is likely that Syk is constitutively active in the 3S5 and 35S5 clones. HS1, CD22, and PLC
all exhibited increased basal levels of
tyrosine phosphorylation in the 35S5 and 3S5 clones when compared with
the CD45-positive K46 cells. Furthermore, CD22 was inducibly hyperphosphorylated suggesting that Syk activity is potentiated by BCR
or class II ligation in cells lacking CD45. Previous studies using the
J558L plasmacytoma and a CD45-positive transfectant of this cell line
have revealed that Syk is recruited to the BCR activation complex in
response to anti-Ig stimulation regardless of whether CD45 is expressed
on the surface of the cell (34). Activation of Syk in the absence of
CD45 has also been reported in studies using the chicken DT40 cell line
(35) as well as Syk-positive Jurkat T cells (45). Perhaps more
important in light of the observation that putative Syk substrates
exhibit basal hyperphosphorylation in the 35S5 and 3S5 clones is the
finding that Syk is constitutively activated in CD45-deficient B cells. Studies using the J558L plasmacytoma demonstrated that Syk was approximately 3-4-fold more active in unstimulated cells that lacked
CD45 when compared with CD45-positive transfectants (34). Therefore, it
is possible that the loss of CD45 expression may result in aberrant
constitutive activation of the PTK Syk.
Because it was apparent from these studies that Syk activity may be
constitutively elevated in the absence of CD45, it was of interest to
determine whether the downstream substrate PLC
is inducibly
phosphorylated and activated in response to BCR or MHC class II
cross-linking. Analysis of PLC
phosphorylation revealed that it is
constitutively phosphorylated on tyrosine in the 35S5 clone in
agreement with the possibility that Syk may be constitutively active in
these cells. This finding is in contrast to previous studies using B
cells isolated from CD45-deficient mice in which apparently normal
inducible tyrosine phosphorylation of PLC
was observed in response
to cross-linking of the BCR (46). Moreover, experiments with the
CD45-negative J558L cell line revealed inducible tyrosine
phosphorylation of PLC
1 and PLC
2 even though the basal activity
of Syk was elevated in these cells compared with CD45-positive J558L
transfectants (33).
Although PLC
exhibited high basal phosphorylation in the 35S5 clone,
subsequent studies to measure IP3 production demonstrated that PLC
is indeed functional and that its activity is increased upon cross-linking of the BCR or MHC class II. In agreement with the
observation that PLC
was constitutively hyperphosphorylated in the
CD45-negative clones, basal levels of IP3 were 1.5-2-fold higher in these cells when compared with parental K46 cells. Moreover, the level of IP3 present in CD45-deficient cells stimulated
via the BCR or MHC class II was elevated at all time points assayed. Previous studies with the CD45-deficient J558L plasmacytoma have demonstrated that cross-linking of the BCR mediates increased production of IP3 in the absence of CD45 (33). However,
because the basal level of IP3 in the J558L cells was only
a fraction of that in the CD45-positive transfectants, the maximum
level of IP3 produced in response to cross-linking of the
BCR was not observed to surpass the base-line level of IP3
present in the CD45-positive transfectants (33). Thus, although there
is general agreement that cross-linking of the BCR, as well as MHC
class II, leads to increased production of IP3 in the
absence of CD45, there is a discrepancy between the K46 and J558L cell
lines as to whether the net level of IP3 produced upon
stimulation is sufficient to mediate downstream signaling.
Consistent with previous studies of signal transduction via the BCR in
CD45-deficient B cell lines, experiments revealed that there is a
significant alteration in the Ca2+ mobilization response in
the 3S5 and 35S5 clones. Cross-linking of the BCR in the CD45-negative
clones leads to a transient rise in the [Ca2+]i
that was due to mobilization of Ca2+ from intracellular
stores. Little or no extracellular influx of calcium was observed
suggesting that the loss of CD45 expression either directly or
indirectly affects the function of plasma membrane Ca2+
channels. The loss of CD45 was observed to have an even more profound
effect on class II-mediated Ca2+ mobilization. Class II
ligation failed to induce a significant Ca2+ mobilization
response in the absence of CD45. The explanation for the difference
between BCR- and class II-mediated Ca2+ mobilization
responses is at present unclear but does not seem related to the
production of IP3.
The selective abrogation of the extracellular influx of
Ca2+ in the 35S5 and 3S5 clones in response to BCR
cross-linking is in agreement with previous results from studies with B
cells derived from CD45-deficient mice (46). The specific mechanism by
which influx of Ca2+ from the extracellular space is
inhibited in the absence of CD45 is not clear. Based on the results
presented in this study, it would appear that IP3
production leading to the depletion of intracellular Ca2+
stores occurs more or less normally in the absence of CD45. Recent studies have demonstrated that depletion of intracellular
Ca2+ stores leads to the generation of a signal that
activates Ca2+ influx across the plasma membrane through a
process termed capacitative Ca2+ entry (47). The specific
mechanism by which depletion-activated Ca2+ channels are
regulated is currently not well understood. However, there is evidence
to support the hypothesis that depletion of intracellular stores
initiates the production of a second messenger(s) from the microsomal
fraction of the cell that diffuses through the cytoplasm to the plasma
membrane where it opens the Ca2+ release-activated
Ca2+ (CRAC) channel (48). The identity of the calcium
influx factor is not well established at present. Nevertheless, the
direct involvement of CD45 in regulating the production of
Ca2+ influx factor might provide an explanation for the
lack of BCR-mediated Ca2+ influx in CD45-negative cells.
Alternatively, studies have suggested that depletion of intracellular
Ca2+ stores may regulate CRAC channel function via a
PTK-dependent mechanism (49, 50). Thus it is possible that
CD45 expression is required to enable a member(s) of the Src family, or
an as yet undefined PTK, to regulate CRAC channel function.
In summary, this study demonstrates that CD45 plays a comparable role
in regulating signaling via the BCR and MHC class II molecules in the B
cell. Loss of CD45 expression attenuates Src family PTK activation in
response to BCR or class II ligation. Additionally, the loss of CD45
expression has a profound effect on receptor-mediated Ca2+
mobilization. These findings confirm that CD45 plays an important role
in regulating B cell activation and further suggest that this PTP may
regulate clonal expansion and differentiation mediated by MHC class
II.