By
§
§
From the * Department of Immunology, The cytosolic SHP-1 and transmembrane CD45 protein tyrosine phosphatases (PTP) play critical roles in regulating signal transduction via the B cell antigen receptor (BCR). These PTPs
differ, however, in their effects on BCR function. For example, BCR-mediated mitogenesis is
essentially ablated in mice lacking CD45 (CD45
The pivotal role for B cell antigen receptor (BCR)1
stimulation in driving B lymphocyte differentiation
and activation is realized through a complex intracellular
signaling network that biochemically translates BCR engagement to nuclear response. Transmission of ligand binding signals via this biochemical network is dependent upon
reversible protein tyrosine phosphorylation and mediated by the relative effects of protein tyrosine kinases (PTKs) and phosphatases (PTPs; 1, 2). As the BCR lacks intrinsic tyrosine kinase activity, tyrosine phosphorylation of its Ig At present, the regulatory roles for PTPs in BCR signaling are not as well defined as those of PTKs. However, two
PTPs that have been identified as key elements in modulating the outcome of BCR engagement are the CD45 transmembrane and SHP-1 cytosolic proteins, enzymes that are
both expressed in hemopoietic cell lineages (4, 5). Analyses
of CD45-deficient mutant cell lines as well as B cells from
mice genetically deficient for CD45 have indicated that
CD45 activity is used to couple BCR stimulation to cell
proliferation (6). The involvement of CD45 in B cell differentiation has also been revealed by the recent findings that CD45-deficient mice manifest a reduction in splenic
B cells with phenotypic markings of the mature B cell pool
(8). Together, these data suggest a critical role for CD45 in
promoting the coupling of BCR stimulation to both B cell
mitogenesis and transit from the immature to mature stage
of differentiation. Similarly, multiple lines of evidence indicate that the SH2 domain-containing SHP-1 tyrosine
phosphatase plays a major role in the regulation of BCR signaling capacity. These data include, for example, the demonstration that loss of function mutations in the SHP-1
gene are responsible for the severe haemopoietic abnormalities found in motheaten (me) and viable motheaten (mev)
mice (9, 10). These animals, which express no (me) or catalytically compromised (mev) SHP-1, manifest high levels of
serum immunoglobulins and autoantibodies. In addition,
they exhibit a profound reduction in conventional B-2
cells, but an overexpansion of CD5+ B-1 cells in the periphery (11, 12). B cells from me and mev mice have also
been shown to be hyperresponsive to BCR stimulation, the mutant cells proliferating in response to normally submitogenic concentrations of F(ab Although the available data indicate opposing effects of
CD45 and SHP-1 on the signaling events triggered by BCR
engagement, it is currently unclear whether these PTPs exert their antagonistic effects by coordinate regulation of a
single signaling pathway or by the modulation of distinct,
parallel signaling cascades involving disparate downstream
signaling effectors. It is also unclear whether the effects of
these individual PTPs on B cell maturation are realized via
the modulation of BCR signaling capacity and, in particular, through the alteration of BCR thresholds for signal
propagation to the nucleus. To address these issues, we have
examined the ontogeny and signaling properties of the B
lineage population that develops in mice lacking both the
CD45 and SHP-1 tyrosine phosphatases. Analysis of these
mice has revealed their expression of a peripheral B cell
population comprised largely of mature, conventional B-2
cells that proliferate in response to BCR engagement. As
described herein, these data strongly suggest that SHP-1
and CD45 act in concert to modulate the coupling of BCR
stimulation to mitogenesis and maturation.
Cell Preparation and Proliferation Analysis.
Single cell suspensions of splenic B cells were obtained from the PTP-deficient
control mice after previously published procedures (8, 13). For
FACS® analysis, the spleen cells were subjected to erythrocyte lysis in ammonium chloride. Cells (1-5 × 105/sample) were
stained by standard procedures using FITC-anti-IgM (hybridoma
33-60), PE-anti-IgD (Southern Biotechnology Assoc., Birmingham, AL), and PE-anti-CD5 (PharMingen, San Diego, CA) antibodies and 104 cells/sample were analyzed on a FACScan® flow
cytometer (Becton Dickinson, San Jose, CA). For proliferation assays, single cell suspensions of splenic cells were subjected to
erythrocyte lysis in ammonium chloride, T cell lysis by anti-CD4/CD8/Thyl.2 antibody and complement treatment, and
separation over Percoll gradients as previously described (8, 13).
The purified splenic B cells (>80% sIg+ by FACS®) were cultured (5 × 104 cells/well) for 48 h in culture media alone or in
the presence of 5 µg/ml B76 (rat IgG1 anti-mouse IgM) or 15 µg/ml LPS, and proliferation was evaluated after a 6-h pulse with
1 µCi/well [3H]thymidine (Dupont/New England Nuclear, Boston, MA). Data are expressed as cpm and values represent means
(± SEM) of triplicate experiments.
Mouse Strain Typing.
Genotyping for the mev mutation was
carried out by PCR amplifying a 69-bp fragment encompassing
the site of the T Antitopoisomerase Assay.
Serial dilutions of sera in PBS/3%
FCS were added to topoisomerase coated wells (Scl-70; Advanced Biological Products, Brampton, Canada) and incubated
for 1 h at room temperature. Wells were washed, incubated with
goat anti-mouse Ig coupled to horseradish peroxidase (HRP;
Sigma Chemical Co., St. Louis, MO) for 1 h, washed again, incubated with substrate (2,2 Histological Analysis.
After fixation in formalin and paraffin
embedding, 3-micron sections of renal tissues harvested from normal and mutant mice were successively incubated at room temperature with rabbit anti-mouse IgM (1.5 h) and biotinylated goat
anti-rabbit (30 min) antibodies (Zymed Labs., Inc., S. San Francisco, CA), peroxidase-conjugated streptavidin (30 min), and aminoethylcarbazole in 0.2 M sodium acetate (15 min; Sigma Chemical
Co.). Sections were then counterstained in hematoxylin and mounted
with Crystal/Mount (Biomeda Corp., Foster City, CA). Original
magnification was at 250. Positive staining is indicated by the red-brown deposition seen most prominently in the lower left panel
(Fig. 2 C).
Immunoprecipitation and Immunoblotting.
Before preparation of
cell lysates, purified splenic B cells were resuspended in culture
media at 3 × 107/ml and left untreated or, alternatively, stimulated for 5 min at 37°C with 5 µg/ml B76 anti-Ig antibody. For
biotinylation, 5-6 × 107 splenic B cells were resuspended in ice-cold 1.5 ml PBS and mixed with 15 µl sulfo-NHS-Biotin solution (Pierce Chem. Co., Rockford, IL). After 30 min of incubation at room temperature, the reaction was quenched by 5 min of
incubation with 500 µg/ml lysine in PBS. Cells were then stimulated as above. For preparation of cell lysates from nonbiotinylated or biotinylated cells, the cells were pelleted by 0.5 min centrifugation and lysed for 15 min in 400 µl cold buffer containing
150 mM NaCl, 50 mM Tris-HCl, pH 8, 2 mM EDTA, 50 mM
NaF, 1 mM PMSF, 1 mM sodium orthovanadate, 0.05% NaN3,
and 1% NP-40. Lysates were then centrifuged at 4°C for 10 min
at 14,000 g, electrophoresed through SDS-polyacrylamide gels,
transferred to nitrocellulose, and incubated at 4°C for at least 1 h
in Tris-buffered saline with Tween solution (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.05% Tween 20) plus 3% gelatin. Filters
were then incubated for 2 h at room temperature with antiphosphotyrosine antibody (4G10; Upstate Biotechnology Inc., Lake
Placid, NY) in Tris-buffered saline with Tween followed by the
space addition of goat anti-mouse antiserum labeled with peroxidase (Amersham Corp., Arlington Heights, IL) and HRP conjugate (Bio Rad Labs., Hercules, CA). Filters derived using protein
lysates from biotinylated cells were also probed with HRP-avidin
(Pierce Chem. Co.). Immune complexes were detected using an
enhanced chemiluminescence system (Amersham Corp.). For immunoprecipitations, lysate proteins (1 mg) were precleared by incubation with protein A-Sepharose (Pharmacia, Baie d'Urfe, Quebec; 40 µl lysate in 1 ml volume beads) for 1 h at 4°C and for an
additional hour with 40 µl beads and 5 µl rabbit preimmune serum. Lysates were then incubated for 3 h at 4°C with 5 µl anti-
SHP-1 (rabbit polyclonal produced in our lab) or anti-CD19 (rat
monoclonal produced by the ID3 hybridoma) antibodies or rabbit
preimmune serum and 10 µl packed protein A-Sepharose beads
and the immune complexes then collected by centrifugation, washed
in lysis buffer, and resuspended in SDS sample buffer. Samples
were then boiled for 5 min, electrophoresed through 8% SDS-PAGE, and subjected to immunoblotting analysis as described
above. Stripping and reprobing of the blots were performed according to Amersham Corp.'s recommended protocol. Antibody
to mb-1 (rabbit anti-mb-1 cytoplasmic tail) was provided by Dr.
L. Matsuuchi (University of British Columbia, Vancouver, Canada).
Assay of Mitogen-activated Protein Kinase Activity.
Cell lysates
were prepared from unstimulated and anti-Ig antibody (5 µg/ml
B76)-treated splenic B cells (3 × 107) from the various PTP-deficient mice and the lysate proteins then immunoprecipitated with
anti-Erk2 antibody (Santa Cruz Labs., Santa Cruz, CA). Equal aliquots of the anti-Erk2 immune complexes were then resuspended in 25 µl reaction buffer (30 mM Tris-HCl, pH 8.0, 20 mM MgCl2, 2 mM MnCl2 containing 5 µg myelin basic protein (Upstate Biotechnology Inc.) and 10 µg [ To address the relative contribution of the CD45 and
SHP-1 enzymes to B lymphocyte activation and development, mice lacking both SHP-1 and CD45 activities were
derived by breeding the mev mutation, which engenders
expression of catalytically compromised SHP-1, into mice
homozygous for a CD45-exon 6 null mutation (9, 19).
Double mutant (CD45 As shown by the representative example in Fig. 1, flow
cytometric analysis of splenic cells obtained from double
mutant mice (CD45
To investigate the physiologic basis for restoration of
B cells displaying a normal phenotype in the CD45 CD45 We next addressed the biochemical basis for the disparities observed in BCR-mediated activation events that distinguished CD45
In addition to induction of tyrosine phosphorylation,
anti-Ig elicited activation of the MAP kinase cascade, a more
downstream signaling event that couples BCR-induced
Ras activation to nuclear response, was also examined in
the PTP-deficient cells (34). As shown in Fig. 3 D, analysis
of ERK2-immunoprecipitable kinase activities in these
cells revealed that the induction of MAP kinase activity after BCR engagement was no different between CD45 In summary, our results indicate that SHP-1 deficiency
rescues antigen-receptor-elicited proliferation in CD45-deficient mice. The relatively normal splenic B cell phenotype observed in the double mutant mice is most likely a
consequence of the restoration of the selective forces that
promote normal B cell maturation in the spleen. It remains
to be determined whether the effects observed are directly
or indirectly determined by the interactions of the PTPs. It
is possible that altered selection at various levels of B cell
development results in quite distinct populations of splenic
B cells. The signaling defects observed in CD45-deficient B cells suggest that BCR-mediated activation is normally restricted to conditions that allow the engagement of CD45.
This restriction appears to be required during the final stages
of B cell maturation as transitional cells are recruited into
the mature B cell pool, and may serve to restrain the amplitude of the immune response by setting a threshold for B cell
activation. A key role for SHP-1 in this regulatory circuit is
also apparent as the absence of SHP-1 circumvents the requirement for CD45 in B cell maturation/activation. Thus,
the data reported here, which suggest that the balance of SHP-1 and CD45 activities substantially influence the outcome of BCR engagement, identify the biochemical mechanisms whereby these PTPs coordinately modulate BCR
activation as being critical to the maintenance of normal B cell
responses and the prevention of autoreactivity.
Address correspondence to Dr. C. Paige, The Wellesley Hospital Research Institute, Wellesley Hospital, 160 Wellesley St. East, Toronto, Ontario M4Y 1J3, Canada. Phone: 416-926-7751; FAX: 416-926-5109;
E-mail: Paige{at}whri.on.ca Received for publication 24 January 1997 and in revised form 6 June 1997.
We thank Caren Furlonger for superb technical assistance with this work, Fengao Xu for scientific contributions, Lori Mason for her assistance with the immunochemical studies, and Drs. John Cambier, Doug
Fearon, and Tak Mak for their generous contribution of anti-CD22 antibody, anti-CD19 antibody, and
CD45 This work was supported by grants from the Medical Research Council of Canada and the National Cancer
Institute of Canada. G. Pani is a recipient of a Leukemia Foundation of Canada Fellowship award and a fellowship from the Italian Ministry of Public Education, and K.A. Siminovitch is a Career Scientist of the Ontario Ministry of Health and an Arthritis Society of Canada Research Scientist.
Department of Medicine and Molecular and Medical Genetics,
The
Wellesley Hospital Research Institute,
), but is enhanced in SHP-1-deficient motheaten (me) and viable motheaten (mev) mice. To determine whether these PTPs act independently or coordinately in modulating the physiologic outcome of BCR engagement, we assessed B cell development and signaling in CD45-deficient mev (CD45
/SHP-1
) mice. Here
we report that the CD45
/SHP-1
cells undergo appropriate induction of protein kinase
activity, mitogen-activated protein kinase activation, and proliferative responses after BCR aggregation. However, BCR-elicited increases in the tyrosine phosphorylation of several SHP-1-associated phosphoproteins, including CD19, were substantially enhanced in CD45
/SHP-1
,
compared to wild-type and CD45
cells. In addition, we observed that the patterns of cell surface expression of µ,
, and CD5, which distinguish the PTP-deficient from normal mice, are
largely restored to normal levels in the double mutant animals. These findings indicate a critical
role for the balance of SHP-1 and CD45 activities in determining the outcome of BCR stimulation and suggest that these PTPs act in a coordinate fashion to couple antigen receptor engagement to B cell activation and maturation.
and
chains after ligand engagement is achieved through
recruitment of cytosolic PTKs, the activities of which create phosphotyrosine sites for recruitment and activation of
SH2 domain containing PTKs and other secondary signaling molecules (3). PTK-induced phosphorylation thus provides the framework for the sequential protein activation and amalgamation that ultimately serves to couple BCR
stimulation to lymphocyte response.
)2 anti-Ig antibody, but
responding normally to other mitogenic stimuli such as
LPS (13). Developing B cells from mev mice bearing hen
egg lysozyme (HEL) and anti-HEL transgenes have also
been shown to be hyperresponsive to HEL stimulation, the anti-HEL-bearing SHP-1-deficient cells undergoing deletion when exposed to a level of antigen below that normally required to induce deletion in this system (14). Together, these data indicate a major role for SHP-1 in
modulating B cell development and in regulating the signaling events linking the BCR to both proliferation and
clonal deletion/negative selection. In contrast to CD45,
however, SHP-1 effects on BCR signaling appear largely
inhibitory, a contention also consistent with recent data
indicating that SHP-1 interacts with and modulates the
signaling functions of both the Fc
RIIB1 and CD22 receptors, two transmembrane molecules also implicated in
the downregulation of BCR-elicited signaling cascades
(15).
A transversion in the mev SHP-1 gene using the
primer pair 5
-CGTGTCATCGTCATGACT-3
(forward) and
5
-AGGAAGTTGGGGCTTTGCCGT-3
(back primer that introduces an RsaI site in the vicinity of the mutation) followed by
RsaI digestion of the PCR products. The wild-type SHP-1 allele
is detected as 48- and 21-bp RsaI restriction fragments, whereas
the mev allele shows as an intact 69-bp fragment. The genotype
with respect to CD45 was determined by cell surface analysis of
the B220 isoform of CD45 since, as previously described, there is
complete correlation between the presence of B220 and the presence of the wild-type CD45 gene (19). The CD45
mice used in
these studies were from the eighth backcross generation to
C57BL/6.
-azino-bis; Sigma Chemical Co.), and
absorbance read at 405/630 nm. To quantify the amount of antitopoisomerase, serially diluted purified mouse IgG was added to
ELISA plates (Costar Corp., Cambridge, MA) coated with goat
anti-mouse IgG (Sigma Chemical Co.), and the plates were then
washed, blocked with PBS/3% FCS, and developed as described above. Based on the standard, OD values of 70, 50, and 30 were estimated to correspond to ~30, 15, and 1 ng/ml, respectively.
Fig. 2.
B cell proliferative responses and autoantibody production in
PTP-deficient mice. (A) Effects of anti-Ig and LPS on proliferation ([3H]thymidine incorporation) of CD45/SHP-1
, CD45
, and CD45+/
SHP-1+ (wild-type) B cells (all LPS-treated groups as well as anti-µ-treated groups (CD45+/SHP-1+) and (CD45
/SHP-1
) differed significantly
from their counterparts in the No stimulation group based on analysis by a
two-tailed t test (P <0.05 in all cases). (B) Comparisons of antitopoisomerase antibody titers in 4-wk-old CD45
/SHP-1
, CD45
, CD45+/
SHP-1+ and SHP-1
mice. (C) Immunostaining of renal tissues from
4-wk-old CD45+/SHP-1+, CD45
, CD45
/SHP-1
, and SHP-1
mice.
[View Larger Versions of these Images (30 + 166K GIF file)]
32P]ATP (Dupont/
New England Nuclear). After a 15 min incubation at 30°C, reactions were terminated by addition of 6 µl 5× loading buffer and the
samples were then boiled, electrophoresed through 15% polyacrylamide gels, and the phosphorylated myelin basic protein bands
were visualized by phosphoimaging. Equal aliquots of the remaining portions of the anti-Erk2 immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose, and the filters
were then probed as described above with anti-Erk 2 antibody.
/SHP-1
) mice derived from these
matings developed the wasting disease normally associated
with the lack of SHP-1, and consequently manifested severe runting and early mortality. However, in contrast to
the splenomegaly characteristically found in the mev homozygous (mev or CD45+/SHP-1
) mice (11, 12), splenic
size was markedly reduced in the double mutant animals As
a consequence of these differences, double mutant littermate and age-matched wild-type control mice differed significantly with respect to splenic cellularity and composition. Thus, assessment of peripheral B cell populations in
these animals requires consideration of not only absolute
cell numbers, which depend highly on individual animal
size, and particularly spleen size, but also evaluation of the
relative frequencies of the cell populations being studied.
Accordingly, to examine the effects of combined CD45/
SHP-1 deficiency on B cell development, the presence of
mature splenic B cells was assessed in the double mutant
mice derived from these matings by evaluating the proportion as well as absolute numbers of four splenic cell populations distinguished by relative levels of cell surface µ and
.
/SHP-1
) revealed the proportion of
hiµlo B cells to be similar to that detected in wild-type
(CD45+/SHP-1+) spleens (48 versus 45%). This result suggests that progression to the mature and active stage of B
cell differentiation occurs despite the absence of these two
PTP activities. By contrast, the proportion of
hiµlo cells in
the splenic B cell compartment of CD45
mice is markedly
reduced relative to that detected in wild-type mice (7 compared to 45%). In absolute terms, the CD45
mice have
threefold fewer
hiµlo splenic B cells than do normal mice,
despite having twice as many total splenic B cells. Mev mice
also have fewer
hiµlo B cells in absolute terms, but this reduction parallels the reduced size of these animals and the
reduced number of B lineage cells present. Fig. 1 also
shows that two populations which emerge in mev spleens
(
µ+ and
+µ
populations) are eliminated in the normal
double mutant mice. A further manifestation of impaired B
cell differentiation in mev mice is the marked over expression of CD5-expressing B-1 cells. These cells constitute the
majority of splenic B cells in SHP-1
mice (see legend to
Fig. 1), but represent only a minor population in the B cell
compartments of either wild-type or CD45
mice. The
proportion of CD5+ splenic B cells is significantly reduced
in double mutant CD45
/SHP-1
mice, although their
levels remain higher than normal indicating a failure to
completely restore normal B cell development. It is possible, for example, that the B cell development in the double mutant mice is still subjected to trans effects mediated by
abnormal monocytes. Nonetheless, these observations reveal the development of significant numbers of conventional B-2 cells in CD45
/SHP-1
mice, a population that
fails to develop in CD45+ mice deficient for SHP-1. They
also reveal that CD45
/SHP-1
mice do not suffer the
block in progression of B cells from the
hiµhi to the
hiµlo
stage of differentiation that is typical of CD45
mice. The
number of
hiµlo cells that develop is appropriate for the
small spleens of the double mutant mice. Together these
results strongly suggest that these PTPs act in concert to
promote B cell maturation and activation and specifically
the differentiation events yielding B-1 or B-2 cells.
Fig. 1.
Representative flow cytometric analyses of splenic B cells
from PTP-deficient mice. Expression of IgM and IgD was evaluated on splenic cells from age-matched
4-wk-old mice: CD45+/SHP-1+
(C57B1/6 wild type); CD45/
SHP-1+ (C57B1/6 CD45 exon 6
/
;
originally derived by K. Kishihara;
reference 8; eighth backcross generation), CD45+/SHP-1
(C57B1/6
mev/mev); CD45
/SHP-1
mice
(derived by mating C57B1/6 CD45
exon 6
/
[eighth backcross generation] with C57B1/6 mev/+ mice). The percentages of cells within the boxed regions correspond to the following pattern: I, percent
+µ
in the lymphoid gate; II, percent
hiµlo of III; III, percent
+µ+ in the lymphoid gate; IV, percent
µ+ in the lymphoid gate. The lymphoid gate was set by standard procedures relying on the forward and side scatter properties of the splenic cells. The absolute numbers of various subsets of cells found in the
spleens of the same mice (× 106) for CD45+/SHP-1+, CD45
/SHP-1+, CD45+/SHP-1
, CD45
/SHP-1
), total splenic cells were 79, 120, 140, and
56, respectively. Lymphoid gate: 68, 95, 95, 41; µ+
+: 33, 76, 7.2, 9.1; µlo
lo: 15, 5.2, 3.6, 4.4; µ+
, 1.6, 3.4, 8.2, 1.1; µ
+: 0.5, 0.06, 1.6, 0.05;
µ+CD5+: 5.7 (representing 16% of µ+ cells), 4.5 (6%), 11 (71%), 3.6 (37%). Data are representative of four independent experiments carried out as described in Materials and Methods.
[View Larger Versions of these Images (37 + 9K GIF file)]
/SHP-1
mice, we examined the proliferative response of splenic B
cells stimulated by BCR ligation. As assessed by [3H]thymidine incorporation, anti-Ig induced proliferative responses of CD45
/SHP-1
B cells were lower in magnitude than
those observed in wild-type cells, but were substantially increased relative to the negligible response detected in similarly treated B cells from CD45-deficient mice (Fig. 2 A).
By contrast, LPS-induced proliferation that has previously
been shown to be unaffected by SHP-1 deficiency (13) was
marginally enhanced in the context of CD45 deficiency,
but markedly diminished in CD45
/SHP-1
cells. These
findings suggest a complex interaction between CD45 and
SHP-1 in relation to the coupling of LPS stimulation to B cell
proliferation, and also reveal the capacity of the mev mutation to rescue the BCR signaling defect engendered by
CD45 deficiency. Previous data have indicated that BCR-evoked proliferation is augmented in the absence of SHP-1
(13, 14). Coupled with this observation, the additional data
presented here support the postulate of antigen receptor
signaling thresholds (20). The threshold level required for
transducing a physiologic, BCR-mediated response is presumably attained in CD45
/SHP-1
B cells in which the
inhibitory effects of SHP-1 on the BCR are eliminated,
but not in CD45
B cells in which SHP-1 function is intact. These observations also suggest that the positive selection of B lymphocytes, as assessed by the progression from
loµhi to
hiµhi to the
hiµlo stage of B cell development,
depends upon coordinate effects of SHP-1 and CD45 on
BCR signaling capacity. The reduction of CD5+ B-1 cells
in CD45
/SHP-1
mice further suggests that BCR-driven
positive selection of not only mature B-2, but also B-1,
cells depends on the interplay between CD45 and SHP-1.
These observations are consistent with other evidence linking B-1 cell development to (auto)antigenic stimulation of
the BCR (21, 22). From this perspective, the expansion of
B-1 cells in SHP-1-deficient mice might be attributable to
the loss of SHP-1 inhibitory effects on BCR activation and the subsequent indiscriminate triggering of unengaged BCRs.
In CD45
/SHP-1
mice, however, the absence of CD45-positive effects on BCR activation appears to counteract
the heightened "excitability" of mev BCRs so as to mitigate
the preferential selection of B-1 cells. This interpretation of
the data is supported by the findings of reduced splenic B-1
cell numbers in not only CD45
animals, but also Btk-deficient xid mice (23) as well as mice rendered genetically
deficient for other signaling effectors implicated in the coupling of the BCR to cell response, such as CD19 (24) and
Vav (25).
/SHP-1
mice also exhibited reduced levels of
serum immunoglobulins (data not shown) and autoantibodies, as evaluated by antitopoisomerase antibody titers
(Fig. 2 B). Similarly, renal immune complex deposition was
less evident in CD45
/SHP-1
than in SHP-1-deficient
mev mice (Fig. 2 C). However, in contrast to these findings,
overexpansion and tissue accumulation of myelomonocytic
cells were of comparable severity in CD45
/SHP-1
and
SHP-1
mice (data not shown) and, as a result, these animals were indistinguishable in terms of their physical appearance and survival. These observations concur with previous data showing that elimination of B-1 cells by addition
of the xid gene has no effect on mev phenotype and mortality (26). Together, these data suggest that the impact of
SHP-1 and CD45 on the signaling events that promote activation and differentiation are different in myeloid and
monocytic cells than in lymphoid cells.
/SHP-1
B cells from those found in
the single PTP-deficient mice. Comparison of biochemical
differences between populations of different developmental profiles should always be undertaken with caution, since
the observed differences may result from a combination of
direct effects due to the loss of the PTPs in question and
indirect consequences that appear due to the absence of
these PTPs during B cell development. Nonetheless, we
compared these B cell populations by first determining their
profiles of protein tyrosine phosphorylation. As is consistent with previous data linking SHP-1 to the inhibition of
BCR-driven signaling cascades (13, 14) anti-Ig-induced
tyrosine phosphorylation was found to be somewhat increased in the SHP-1
compared to wild-type cells (Fig. 3
A). By contrast, although CD45 has been implicated in
PTK activation after antigen receptor engagement (6, 27),
little difference was detected between stimulated CD45
/
and wild-type, and the CD45
/SHP-1
cells with respect
to the pattern or degree of total protein tyrosine phosphorylation. The capacity of SHP-1 deficiency to rescue BCR-induced proliferative responsiveness in B cells lacking CD45
suggests a role for altered tyrosine phosphorylation of proteins that normally would be dephosphorylated by SHP-1.
We therefore analyzed the tyrosine phosphorylation of proteins coprecipitated with SHP-1 from these B cell populations after BCR ligation. The results of this analysis revealed
the tyrosine phosphorylation of several SHP-1-associated
phosphoproteins (of ~85-90, 115, and 120 kD) to be
strikingly enhanced in both SHP-1
and CD45
/SHP-1
cells relative to wild-type B cells. By contrast, the CD45
/
cells exhibited relatively reduced tyrosine phosphorylation
of SHP-1 as well as the various phosphoproteins coprecipitated with this PTP, including a 140-kD species identified
by immunoblotting analysis (data not shown) as CD22, a B
lineage-specific transmembrane glycoprotein implicated in
the negative regulation of BCR signaling (Fig. 3 B) (16,
28). In view of preliminary data from our group revealing
the capacity of SHP-1 to associate with CD19, a 115-120-kD cell-specific transmembrane glycoprotein that is rapidly tyrosine phosphorylated after BCR stimulation (29, 30), the
possibility that CD19 is differentially phosphorylated in the
context of CD45 deficiency versus CD45/SHP-1 deficiency was directly investigated. As shown by antiphosphotyrosine immunoblotting analysis of CD19 immunoprecipitates from the various PTP-deficient B cells (Fig. 3 C),
CD19 phosphorylation was dramatically reduced in the
CD45
cells, but increased both constitutively and after BCR
ligation in the CD45
/SHP-1
cells compared to wild-type
B cells. The observation of reduced CD19 phosphorylation
in CD45
cells suggests a role for CD45 activity in promoting
the tyrosine phosphorylation of this membrane glycoprotein. This may well be due to the effect of CD45 on the activation of the Lyn PTK, an enzyme previously shown to associate with CD19 (31). Conversely, the enhanced tyrosine
phosphorylation of CD19 observed in SHP-1-deficient B
cells (Fig. 3 B) suggests that SHP-1 exerts an inhibitory influence over CD19 tyrosine phosphorylation state either by
direct dephosphorylation of this protein or by negative regulation of a PTK involved in CD19 phosphorylation. This
interpretation is supported by the detection of a phosphoprotein that co-migrates with CD19 in SHP-1 immunoprecipitates, suggesting a capacity for SHP-1 to associate with CD19 or a CD19-bound protein. Thus, augmented
phosphorylation of CD19 in the CD45
/SHP-1
cells likely
reflects the loss of both SHP-1-driven CD19 dephosphorylation and CD45
dependent, PTK-mediated CD19 phosphorylation. Since CD19 has been shown to serve as a coreceptor that positively modulates signal transducing capacity of
the BCR (32), these data suggest that the counterbalance of
CD45 and SHP-1 effects on CD19 recruitment to the
BCR signaling cascade represents one biochemical mechanism whereby these PTPs exert their coordinate regulation
of BCR signaling capacity. This hypothesis, as well as the
identity of the other SHP-1-associated phosphoprotein species appearing hyperphosphorylated in the CD45
/SHP-1
cells, require further investigation. Nonetheless, the suggestion that CD45 and SHP-1 coordinate effects on BCR
function are realized at the level of CD19 phosphorylation
is consistent with the data indicating a pivotal role for
CD19 in modulating the threshold for coupling BCR
stimulation to not only proliferation, but also to the development of B-1 cells (24, 33).
Fig. 3.
Analysis of SHP-1 phosphoprotein binding and MAP kinase
activation in stimulated B cells from PTP-deficient mice. (A) Comparison of protein tyrosine phosphorylation in resting and anti-Ig-treated B cells
from wild-type (CD45+/SHP-1+), CD45CD45
/SHP-1
, and CD45+/
SHP-1
mice. Loading of equivalent amounts of lysate proteins was confirmed by reblotting with anti-mb-1 antibody (bottom). (B) Antiphosphotyrosine (anti pTyr) immunoblots (top) showing the tyrosine-phosphorylated species coprecipitated with SHP-1 from resting and anti-Ig-treated B cells from wild-type (CD45+/SHP-1+), CD45
, and CD45
/SHP-1
mice (left) and from CD45+/SHP-1
mice (right). Arrows indicate the
positions of SHP-1 and three associated phosphoproteins that appear differentially phosphorylated in the CD45
compared to CD45
/SHP-1
and SHP-1
cells. Mobilities of molecular mass standards are shown on
the left. Loading of equivalent amounts of lysate protein was confirmed by reblotting with anti-SHP-1 antibody (bottom). Data are representative of three independent experiments on nine mice. (C) Antiphosphotyrosine immunoblot (top) showing the tyrosine phosphorylation status of CD19
immunoprecipitates derived from biotinylated resting and anti-Ig-treated
wild-type, CD45
, and CD45
/SHP-1
cells was carried out as described in Materials and Methods. The position of CD19 is indicated by
the arrow on the right. Loading of equivalent amounts of CD19 was confirmed by reblotting with HRP-avidin (bottom). (D) Representative example showing the levels of MAP kinase activities before and 5 min after
BCR ligation in wild-type, CD45
, CD45
/SHP-1
, and CD45+/SHP-1
cells (top). Analysis of equivalent amounts of Erk-2 was confirmed by
anti-Erk2 immunoblotting of equivalent aliquots of each Erk-2 immunoprecipitate (bottom).
[View Larger Versions of these Images (54 + 53K GIF file)]
/
SHP-1
and wild-type cells, but was relatively enhanced in
SHP-1
cells, and essentially abrogated in CD45
cells.
These results indicate a parallel between MAP kinase activation and the capacities of these PTP-deficient B cells to
proliferate in response to BCR ligation. Thus, the molecular
interactions which allow SHP-1 and CD45 to cooperatively
influence the outcome of BCR engagement appear to occur at or proximal to activation of the MAP kinase cascade.
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by The Rockefeller University Press.