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Rheumatologische Universitätsklinik Basel, Felix Platter-Spital, CH-4055 Basel, Switzerland; and
* Unité d’Immunopathologie Humaine, Hôpital Broussais, INSERM U 430, 75674 Paris Cedex 14, France
1Correspondence: Hôpital Broussais, Unité d’Immunopathologie Humaine, INSERM U 430, 96, rue Didot, 75674 Paris Cedex 14, France. E-mail: moncef.zouali{at}wanadoo.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONBCR: A PLATFORM FOR...KINASES, PHOSPHATASES,...B CELL SELECTION AND...SETTING THE BALANCE OF...IMPLICATIONS FOR AUTOIMMUNITY IN...ANALOGIES IN BCR AND...CONCLUSIONSREFERENCES |
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Key Words: T cell • BCR • gene expression • SLE
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONBCR: A PLATFORM FOR...KINASES, PHOSPHATASES,...B CELL SELECTION AND...SETTING THE BALANCE OF...IMPLICATIONS FOR AUTOIMMUNITY IN...ANALOGIES IN BCR AND...CONCLUSIONSREFERENCES |
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BCR: A PLATFORM FOR TRANSDUCING SIGNALS |
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TOPABSTRACTINTRODUCTIONBCR: A PLATFORM FOR...KINASES, PHOSPHATASES,...B CELL SELECTION AND...SETTING THE BALANCE OF...IMPLICATIONS FOR AUTOIMMUNITY IN...ANALOGIES IN BCR AND...CONCLUSIONSREFERENCES |
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/Igß (CD79 /ß) subunits (1
, 2)
.
The mIg is a tetramer consisting of a heavy (H) chain homodimer and two
or two 
light (L) chains. H chains possess three or four
constant region domains, CH
1-CH 3/CH 4 and one
variable (VH) region, whereas the L chains
contain one CL (C or
C) and one VL
(V or V). The H chain
homodimer is linked by a disulfide bridge between the
CH 1 and CH 2 domains, and
C or C are bound to
each CH 1. The CH 2 to
CH 3/CH 4 domains represent
the Fc region of the antibody, whereas the CH 1
and the VH domains together with the
CL and the VL form the Fab
portion. Within the variable regions are three hypervariable regions
that encode for the complementarity-determining regions CDR1, CDR2, and
CDR3. CDR3 is responsible for the highest variability, being generated
by the rearrangement of germline V, D, and J sequences of the H chain
and V and J sequences of the L chain. The rearrangements can be limited
or extensive, and several mechanisms ensure that only B cells
expressing functionally intact Igs are viable (3
, 4)
.
In contrast, the constant regions are encoded for by germline
sequences, which are post-translationally modified, e.g., by
glycosylation. Nine H chain isotypes distinguish the IgG subclasses
IgG1–IgG4; IgM, IgA1, and IgA 2; and IgD and IgE. mIg are primarily of
the IgM or IgD isotype. Whereas the former is secreted as a pentamer in
substantial amounts, minimal concentrations of the latter are found in
serum. The Fc region mediates complement and Fc receptor binding
depending on the Ig isotype; in the case of mIg, insertion into the
plasma membrane. The membrane insertion region of Ig is essential for
the expression of a functional BCR, without which B cell development
cannot proceed (5)
. Heavy chain isotype expression is
amenable to switching by recombination at switch regions, allowing the
expression of one
VHDHJH
rearrangement together with different constant exons (6)
,
depending on the nature of antigen and stage of response
(7)
.
The other component of the BCR, the disulfide-linked CD79 /ß
heterodimer, is noncovalantly bound to mIg. Although there is no direct
evidence that mIg has the capacity to mediate signals on binding of
antigen to the variable regions, CD79 and ß possess immune
receptor tyrosine-based activation motifs (ITAMs) in their
intracellular domains (8)
. Cross-linking of the BCR is
rapidly followed by phosphorylation of the CD79 /ß ITAMs
(9)
. This in turn enables the binding of SH-2
domain-containing proteins, including protein tyrosine kinases (PTKs)
and protein tyrosine phosphatases (PTPases), which propagate and
modulate further signaling as discussed below.
In addition to the association of phosphorylated ITAMs with SH-2
domain-containing proteins, interaction of surface molecules with the
cytoskeleton provides a structural basis for signaling. mIg has long
been known to associate with cytoskeletal elements, especially actin,
during capping (10
, 11)
. The association is not uniformly
induced by cross-linking of mIg (12)
, and the specific
nature of the cross-linking may allow differential activation of signal
transduction pathways (13
, 14)
. The interaction of the BCR
with actin has been shown to locate several surface and signal
molecules in proximity to the BCR. These include p21ras
(15)
, the src family PTK Lyn and the non-src PTK Syk
(16)
, GTP binding proteins (17)
, and the
adapter protein SH3P7 (18)
. A different link to the
cytoskeleton is provided by Vav, a GTPase exchange factor
constitutively associated with the cytoskeletal membrane anchors talin
and vinculin (13
, 19)
.
Remarkably, the aggregation of BCR molecules is selective, since no
evidence for cytoskeletal attachment of MHC class I or II has been
found on BCR ligation (12)
. This observation was recently
extended by the finding that BCR cross-linking and CD79 /ß ITAM
phosphorylation are followed by association of CD79 /ß with MHC
II, which is likely related to internalization of capped mIg
(20)
. Until now, the structural basis for cytoskeletal
reorganization and the interaction of signaling elements have not been
precisely defined, though the finding that mIg capping and
internalization can occur independent of CD79 /ß translocation to
the insoluble fraction indicates that the cytoplasmic portion of mIg
may play a role in signal transduction.
Recent experiments have led to the identification of glycosphingolipid-
and cholesterol-rich plasma membrane microdomains, termed lipid rafts.
These lipid microdomains, which sequester glycosylphosphatidylinositol
(GPI) -linked proteins, are thought to function as platforms for both
signal transduction and membrane trafficking (21)
. After
cross-linking, the BCR is rapidly translocated into lipid rafts that
contain Lyn and CD79 and exclude the PTPase CD45R
(22)
. Remarkably, lipid rafts, which can be viewed as
floating membrane domains, seem to be involved in various phenomena. In
contrast to mature lymphocytes, the BCR does not translocate into lipid
rafts after cross-linking in immature B cell lines (23)
.
This may account in part for the differences in signaling outcomes in
mature and immature B cells. Furthermore, in tolerant B cells, the BCR
is not efficiently translocated into lipid rafts after cross-linking
(24)
.
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KINASES, PHOSPHATASES, ACCESSORY, AND ADAPTER MOLECULES: SETTING THE THRESHOLD OF BCR SIGNALING |
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TOPABSTRACTINTRODUCTIONBCR: A PLATFORM FOR...KINASES, PHOSPHATASES,...B CELL SELECTION AND...SETTING THE BALANCE OF...IMPLICATIONS FOR AUTOIMMUNITY IN...ANALOGIES IN BCR AND...CONCLUSIONSREFERENCES |
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). The ITAMs on the cytoplasmic domains of CD79 /ß (8)
associate noncovalently with Src family PTKs via their SH2 domains
(1
, 25)
. A prerequisite for this binding is the
phosphorylation of CD79 /ß by the Src family PTKs Lyn, Fyn, Lck,
and Blk upon activation of the BCR (26)
. In addition to
enabling the binding to the ITAMs, these PTKs (27)
and the
PTKs Syk (28
29
30)
and ZAP-70 (31)
are
activated and not only autophosphorylate, but also phosphorylate each
other as well as other downstream substrates. The phosphorylation of
these ITAMs allows the recruitment of other SH2 domain-containing
elements.
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Differential responses can be evoked depending on the individual
tyrosines phosphorylated. For example, the association of Lyn with the
CD79 /ß ITAMs and its activation upon BCR cross-linking or antigen
binding were markedly reduced by increased constitutive phosphorylation
at Y508 in cells lacking the transmembrane PTPase CD45
(32)
. In a different system, Lyn was hyperphosphorylated
and constitutively activated in the absence of CD45 (33)
.
Syk also associates with the CD79 /ß ITAM (28
29
30)
.
This requires two functional Syk SH2 domains and the Syk
autophosphorylation site (34)
. Syk activation has been
reported to be dependent on Lyn (35)
. Syk
autophosphorylation within the catalytic and inter-SH2 domain enhances
Syk binding to the CD79 ITAMs and kinase activity more efficiently
in cells that express Lyn (36
, 37)
. Syk recruitment and
activation may also occur independent of Lyn, since in CD45-deficient
cells, Lyn recruitment to the CD79 ITAMs was practically abolished
whereas Syk phosphorylation, activation, and association with the BCR
were unaffected (32)
. A regulatory role for Lyn is
suggested by inhibition of Syk activity due to phosphorylation of Syk
linker region tyrosines by Lyn and the hyperactivation of Syk by
mutation of one of these sites (36
, 38)
. The association
of Syk with phospholipase C (PLC) through its SH2 domain has been
reported both to mediate and not to mediate inositol
1,4,5-trisphosphate (IP3) generation, depending on the experimental
system (39
, 40)
. A slower, more persistent intracellular
Ca2+ release is mediated by Lyn in a manner
independent of Syk and PLC-IP3 generation, which are necessary for
rapid, higher level Ca2+ influx
(39)
. These findings indicate that Lyn negatively
regulates Syk and that Syk and Lyn possess distinct distal signaling
pathways. Syk is a negative regulator of BCR signaling (36
, 37)
that could participate in the transition from the immature
to the mature, recirculating B cell stage (41
, 42)
.
In addition to Lyn and Syk, the PTKs Blk, Fyn, and Lck are activated by
ligation of mIg. Anti-IgM activated B cells show binding of
phosphoproteins to SH2 domains of Blk and Fyn, whereas a fraction of
pre-B cells show such binding spontaneously (43)
. Fyn is
not a prerequisite for BCR signaling, since
Fyn-/- mice show no discernible alterations in
proliferation, PTK activity, IP3, or Ca2+
response (44)
. Fyn specifically binds to CD19 via its SH2
domain and possibly activates phosphatidylinositol 3 (PI-3)-kinase
recruited to CD19 via its SH3 domain (45)
. In addition,
Btk, Itk, and Tec bind to the SH3 domain of Fyn, which possibly
prevents phosphorylation of Btk at inhibitory sites (46)
.
Fyn and Blk activation and, to a lesser extent, Btk activation were
dependent on CD45 expression (47)
. Btk interacts with SH3
domains of Fyn, Lyn, and Hck that are mediated by two 10-aa motifs in
Btk. An analogous site with the same specificity is also present in
Itk, the T cell-specific homologue of Btk (48)
.
Only recently was it realized that signaling molecules are coupled to
their target substrates by adapter proteins that are able to interact
with multiple transducing proteins and to act as molecular scaffolds
required for formation of potent signaling complexes. As an example of
adapter proteins, BASH, whose expression is restricted to B cells, is
rapidly phosphorylated by the PTK Syk after BCR ligation. Not only does
this molecule play a role in peripheral B cell maturation and in
activation/proliferation of B cells, but is also critical for pre-B
receptor signaling (49)
.
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B CELL SELECTION AND TOLERANCE |
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, 51)
. In contrast to
adult bone marrow-derived B cells, B cells arising from the fetal liver
are able to differentiate into both conventional B cells and B cells
expressing the pan-T cell marker CD5, designated B-2 and B-1 cells,
respectively (52)
. In the fetus, both conventional and
CD5-positive B cells fail to express MHC class II antigens. This
precludes antenatal antigen presentation to CD4+
T cells by B cells, allowing for tolerance and cell elimination due to
BCR signaling (53)
. Most fetal liver B cells have been
reported to express CD5 (52)
. In the adult, bone marrow
stem cells differentiate into B-2 cells, whereas the B-1 cells are
largely self-replenishing and are preferentially located in the
peritoneal and other serosal cavities (54)
. In addition to
its negative effect in B-1 cells (55)
, recent evidence
indicates that CD5 signaling is linked to maintaining tolerance in B-2
cells (56)
.
In early ontogeny, pro-B cells express the stem cell marker AA4.1, high
levels of germline H chain genes, the pan B cell marker CD45 (B220)
(57)
, and Rag-1 and Rag-2 gene products that mediate the
joining of H chain gene segments (58)
. Thereafter, Rag-1
and -2 mRNA and protein are down-regulated until they are re-expressed
in pre-B cells, coincident with L chain rearrangement
(59)
. BCR surface expression depends on successful gene
rearrangement, without which cells cannot survive and proceed in their
development (60)
. This involves surrogate templates,
against which rearranged H chains are tested for their ability to
associate with L chains (61)
and other molecules that
possibly test for functional signal transduction, such as CD79 and
ß (62)
. If rearrangements are unsuccessful or result in
autoreactivity, secondary rearrangement can be induced, which is also a
mechanism for alterations in specificity (60
, 63)
. When
successfully rearranged and functional BCR is expressed on the cell
surface, signaling induced by specific antigen encounter may lead to
deletion or anergy of developing autoreactive B cells
(64)
.
Ligand-mediated selection mechanisms involving B cell surface receptors
also operate at later stages of B cell ontogeny. At least two signaling
molecules, Syk and CD45, are necessary for B cell maturation beyond the
peripheral immature B cell stage (41
, 65)
. That the number
of newly generated immature B cells greatly exceeds that of mature B
cells suggests that a vast majority of these cells undergo negative
selection. It is estimated that in the periphery, 70% of peripheral
immature B cells expressing functional surface IgM that reach the
spleen mature into the long-lived pool of recirculating mature B cells
(66)
. A comparison of BCR repertoires of sorted immature
and mature B cell populations of an individual mouse revealed that
receptor-based selection governs the transition from immature to mature
B cells in the periphery (67)
.
In addition to negative selection, B cells expressing certain variable
gene combinations undergo positive selection depending on the
specificity of mIgs. B cells with receptors for self-molecules would be
selected over others and dominate the repertoire of mature B cells.
This view is supported by recent evidence indicating that self-antigen
can positively influence the fate of CD5+ B-1
cells and generate a B cell pool with autoreactivity (68)
.
Although it is not known whether there is an endogenous ligand that
modulates the signaling, self-reactivity may be responsible in part for
the dominance of certain Ig variable region genes in normal circulating
peripheral B cells. It is also possible that environmental antigens and
nonpathogenic flora may be ‘preselecting’ a naive recirculating B
cell repertoire that is predisposed to recognize pathogen-related
antigens it may later confront. It is unclear, however, whether B-2
cells also require positive selection to mature and persist. Recently,
autoreactive peripheral B cells have been observed to differentiate
into B-1 or B-2 cells with identical specificity, possibly due to a
shift in BCR signaling (69)
. Other mechanisms ensure that
after acquiring the potential for activation in response to antigen
binding, B cells maintain their propensity for tolerization or cell
death (64)
. Nonautoreactive mature B cells require
continuous BCR signaling in the form of a steady tickle by endogenous
antigens, reflecting a role for low-affinity antigen in shaping the B
cell repertoire and continued expression of the BCR to survive
(70)
.
Several mutually exclusive distinct responses are probably important in
preventing immature, self-reactive B cells from progressing to a mature
stage. Besides the concentration and affinity of antigen, negative
selection is also affected by signaling through molecules such as CD19,
SHP-1, and CD45 (64)
, leading to anergy and apoptosis in
immature B cells and activation of mature B cells. During their passage
through the lymph node germinal centers, mature B cells undergo
apoptosis if they encounter self-antigen or if they are not positively
selected by an antigenic signal. The opposite consequences of BCR
triggering, tolerance vs. response, could be explained by
differential regulation of other accessory surface molecules according
to the developmental stage and environment of the cells. Thus, BlyS
(BAFF, TALL-1, THANK), a TNF family B cell activation factor, allows
the survival and differentiation of splenic transitional type 2 B
cells, a process likely to occur in the lymph node marginal zone
(71)
. The surface receptors for zTNF4 are B cell
maturation antigen (BCMA) and TACI (transmembrane activator and CAML
interactor), which are also the receptors for APRIL (a
proliferation-inducing ligand), a molecule that partially emulates
zTNF4 effects. BCMA, which is expressed only by B cells, mediates cell
survival signaling via NF-B (72
73
74)
. Mature B cells
that have survived censoring for autoreactivity can up-regulate CD86, a
ligand for CD28, and MHC class II, a ligand for TCR, and thus engage in
immune responses. Increases in
[Ca2+]i are thought to
mediate the antigen-induced changes in CD86 seen in mature B cells and
RAG-2 seen in immature B cells (75)
.
These observations demonstrate that B cell development and survival are severely constrained. BCR signaling and its modulation ensure that only cells that fulfill rigorous selection requirements of both a positive and negative nature are given the opportunity to participate in antibody production and regulation of the immune response. The data raise the question of whether altered signaling may interfere with development, survival, and autoreactivity of B cells.
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SETTING THE BALANCE OF BCR SIGNALING |
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TOPABSTRACTINTRODUCTIONBCR: A PLATFORM FOR...KINASES, PHOSPHATASES,...B CELL SELECTION AND...SETTING THE BALANCE OF...IMPLICATIONS FOR AUTOIMMUNITY IN...ANALOGIES IN BCR AND...CONCLUSIONSREFERENCES |
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), which
mediate changes in gene expression required for differentiation. Their
contribution to B cell function has been studied by transgene
expression or disruption of individual signaling elements. Several of
these alterations have resulted in abnormal B cell development in mice
(Table 1
). Apart from indicating that these molecules set thresholds for BCR
signaling at different developmental states of B cells, they have also
revealed novel potential mechanisms for the induction of autoimmunity.
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An important insight came from inactivating the CD19 gene,
which codes for a receptor expressed at high levels on human B lineage
cells. Multiple signaling pathways converge on CD19 to amplify signals
generated through the BCR. B cells from CD19-deficient mice are
hyporesponsive to transmembrane signals (76)
. In the
absence of CD19, differentiation of peritoneal B-1 cells, including
those with anti-Sm reactivity, is markedly reduced in nonautoimmune
mice transgenic for an H chain specific for the autoantigen Sm
(69)
. By contrast, mice that overexpress CD19 produce
significantly higher levels of antibodies reactive with DNA and other
autoantigens (76
, 77)
. A double transgenic mouse model, in
which simultaneous expression of the antigen hen egg lysozyme and the
corresponding specific IgM and IgD surface receptors renders peripheral
B cells anergic to the antigen, was also used to evaluate the effect of
CD19 on B cell reactivity. In this model of peripheral tolerance,
overexpression of CD19 resulted in a breakdown of peripheral tolerance
and the production of autoantibodies (78)
. Thus, altered
signaling thresholds due to CD19 overexpression may result in the
breakdown of peripheral tolerance, shifting the balance from tolerance
to immunity by augmenting BCR signaling.
CD22, another B cell-specific surface receptor, is a member of the Ig
superfamily. It is associated with the BCR at low stoichiometry and is
present at low density on mIgM+ immature B cells
and at high density on mature B cells. Initially, CD22 was thought to
be a positive regulator of cell activation. However, further
experiments revealed that CD22 serves primarily as an inhibitor
(79)
. Upon BCR cross-linking, immunoreceptor inhibitory
motifs (ITIMs) in its cytoplasmic tail are tyrosine phosphorylated by
Lyn. The phosphorylated ITIMs bind SHP-1, a negative regulator of BCR
signaling, which suppresses BCR signaling (80)
. Targeted
disruption of the cd22 gene results in a hyperactive B cell phenotype,
an augmented thymus-independent immune response, an expanded B-1 cell
population, and increased titers of serum autoantibodies
(81
82
83
84)
. It is interesting that as the CD22-deficient
mice age, a large proportion develops high titers of high-affinity
serum IgG directed against dsDNA (85)
. Thus, a single gene
defect, exclusive to B cells, suffices to trigger autoantibody
production in mice. Remarkably, one of the loci contributing to
autoimmunity in New Zealand mixed (NZM) mice (Sle3) has been
mapped to a region of chromosome 7 near cd22
(86)
. These autoimmune mice produce aberrant CD22 mRNA
species-containing insertions that result from alternative splicing due
to an insertion in one intron of the cd22 gene
(87)
. Heterozygous expression of this allele, called
cd22a, promoted autoantibody production in
mice bearing the Y chromosome-linked autoimmune acceleration gene
Yaa (87)
, implying that even a partial CD22
deficiency may contribute to lupus susceptibility. In parallel with
overexpression of CD19 in mice expressing an H chain specific for Sm,
CD22 deficiency caused a marked increase in anti-Sm levels. There was
also a marked increase in anti-Sm peritoneal B cells (69)
.
The Src family PTK Lyn, a well-studied molecule, contributes to the
activation of Syk and the regulation of intracellular
Ca2+ concentrations through its association with
the CD79 /ß heterodimer. In Lyn-/- mice, B
cells fail to tyrosine phosphorylate upon BCR cross-linking and show no
synergy between BCR and CD40 ligand (CD154) signaling. They are
hyper-responsive to anti-IgM-induced proliferation, implying that Lyn
negatively regulates BCR signaling. Furthermore, stimulation with CD154
induces Fas expression in control but not in
Lyn-/- B cells, demonstrating that Lyn is
involved in the CD40-mediated induction of Fas (88)
.
Tyrosine phosphorylation of Fc
RIIB and CD22 coreceptors is also
impaired in Lyn-/- B cells (88
, 89)
. Since these receptors are important for feedback
suppression of BCR-induced signaling, Lyn may be critical for feedback
suppression of B cell activation. Inactivation of the lyn
gene gives rise to a hyperactive B cell phenotype, production of
autoantibodies, and severe immune complex-mediated lupus-like nephritis
(90
91
92)
. The knockout data suggest that Lyn participates
in clonal expansion and terminal differentiation of peripheral B cells
and in establishing and/or maintaining B cell tolerance.
In addition to kinases, negative signaling in B and T cells appears to
be due in part to the activity of SH2 motif-containing PTPases, SHP-1,
and SHP-2. SH2-containing protein tyrosine phosphatases are critical
for both positive and negative regulation of signaling by receptors
that use tyrosine phosphorylation to mediate their intracellular
effects. SHP-1 is capable of physically associating with the BCR
complex in resting B cells and to tyrosine dephosphorylate the CD79
unit (93)
. This phosphatase is a negative regulator of BCR
signaling that sets signaling thresholds for negative selection of B
cells (94)
. SHP-1 inhibitory effects on BCR activation are
mediated by interactions with various signaling effectors, including
CD22, as discussed above (80)
. SHP-1 also regulates the
signaling that mediates exclusion of mature B cells from lymphoid
follicles (95)
. The biological importance of SHP-1 is
exemplified by the naturally occurring mutations seen in the motheaten
(me) and motheaten viable
(mev) mouse strains. Viable motheaten
(mev/mev)
mice exhibit a spontaneous point mutation in the protein tyrosine
phosphatase SHP-1 that results in a protein with decreased phosphatase
activity (10–20% of normal activity) and immune defects, including
expansion of B-1 cells, a low threshold of membrane Ig signaling,
hypergammaglobulinemia, autoantibody production, and glomerulonephritis
(94
, 96
97
98)
. Their cells show increased proliferative
responses to in vitro stimulation with anti-Ig antibodies
(93)
. Surprisingly, the lower threshold for signaling
through mIgs coincides with an increased negative selection against
self-reactive B cells in these mice (94)
.
CD45 is protein tyrosine phosphatase involved in regulating B
lymphocyte selection and modulating signal thresholds
(65)
. Analysis of CD45-deficient mice revealed that CD45
couples BCR signaling to both cell proliferation and differentiation
from immature to mature cells (99)
. B lymphocytes lacking
CD45 show reduced activation of calcium and Erk signaling pathways and
have an elevated threshold for elimination of self-reactive B cells
(65)
. In ‘knock-in’ mice, a single E613R point mutation
in the putative inhibitory wedge of CD45 causes a lymphoproliferative
syndrome with polyclonal T and B lymphocyte activation and severe
autoimmune nephritis with autoantibody production (100)
.
The spontaneous autoimmunity seen in the mutant mice was limited to
production of anti-native DNA antibodies and lupus nephritis.
Endogenous or cross-reactive food antigens presumably were responsible
for triggering this autoimmune response. Since the lymphoproliferative
syndrome was present in both homozygous and heterozygous E613R knock-in
mice, this single alteration in a gene encoding a B and T lymphocyte
signaling molecule is able to cause a lupus syndrome in experimental
rodents in a dominant manner.
It is noteworthy that the autoimmune manifestations seen in Lyn- or SHP-1-deficient mice are more severe than the relatively modest production of autoantibodies seen in CD22-deficient mice. Whereas CD22 is expressed selectively in B cells, Lyn and SHP-1 are also expressed in other hematopoietic cells. It is possible that altered signaling in several cell types involved in the immune response is required for full expression of autoimmune phenomena. The more severe B cell phenotype in SHP-1-/- mice than that seen in CD22-/- or Lyn-/- mice suggests that SHP-1 may regulate B cells by additional pathways independent of CD22 and Lyn.
It is notable that SHP-1 and CD45 are PTPases with opposite effects on
the signaling events triggered by BCR engagement. In contrast to CD45,
SHP-1 effects on BCR signaling are largely inhibitory. The balance of
CD45 and SHP-1 activities substantially influences the outcome of BCR
engagement (99)
, to maintain normal B cell responses and
prevent autoreactivity. A combined deficiency of two PTPases with
antagonistic effects would be anticipated to yield a phenotype
intermediate to that engendered by a deficiency of either of the two
enzymes. This is evident in mice lacking both CD45 and SHP-1
activities, which develop mature B cells, do not manifest aberrant
expansion of B-1 cells characteristic of
mev and me
mice, and lack autoimmune manifestations (93)
. These
findings indicate that the combination of certain defects in BCR
signaling may result in an equilibrium compatible with normal B cell
function. They also suggest that CD45 and SHP-1 are not absolutely
indispensable for signal delivery through the BCR and that their
influence is quantitative, rather than qualitative (65)
.
At least part of their effects could be coordinated through the
regulation of Lyn.
In a different system overexpression of zTNF4, a B cell activator,
caused an autoimmune disease with features of SLE (101)
.
Circulating zTNF4 was also elevated in strains in which spontaneous
lupus-like disease occurs. Successful inhibition of proteinuria and
delayed mortality by treatment with a TACI fusion protein in mice
overexpressing one of the receptors for zTNF4 and APRIL suggests that
this or similar strategies may be a therapeutic option in human
autoimmune disease (73)
.
Downstream signaling alterations identified in SHP-1-deficient mice
extend to activation of the transcription factor NF-B, which plays a
critical role in activation and regulation of multiple immune response
genes. NF-B consists of multiple subunits—p50 (NF-B1), p52
(NF-B2), RelA (p65), c-Rel, and Rel B—and is regulated by the
formation of heterodimers with the inhibitor protein IB. There were
reduced levels of RelA and RelB proteins and increased levels of p50
and c-Rel in B cells from mev mice, and
the level of the inhibitor IBa was significantly reduced
(102)
. Loss of SHP-1 and the resulting hypersensitivity of
the BCR and lowering of the signaling threshold could thus lead to
activation and nuclear translocation of NF-B. Consistent with an
intrinsic B cell defect leading to activation and hyperproliferation, B
cells from the BXSB/Yaa autoimmune mouse produce high levels
of RelA (103)
. The recent identification of the
NFB-pathway as a mediator of zTNF4 and APRIL signaling through BCMA
suggests that altered NFB signaling could be the mechanism by which
zTNF4 overexpression exerts its effects.
Downstream to the signaling molecules discussed above are transcription
factors. Pax5 encodes the B cell-specific activator protein
BSAP, which plays an essential role in B lineage commitment by
suppressing alternative lineage choices (104)
. Ets
proteins, including Fli-1, can function as transcription factors at
specific DNA binding sites in a broad range of cellular transcriptional
regulatory proteins. Fli-1 and Ets-1 are
expressed at high levels in B and T cells. Transgenic mice that
overexpress Fli-1 develop autoantibodies and immune complex-mediated
glomerulonephritis at a high incidence. In addition, B cells from these
transgenic mice are hyper-responsive to mitogens and exhibit
significant reduction in activation-induced cell death and prolonged
survival in culture (105)
.
Taken together, the observations on experimental animals reveal a novel mechanism for autoimmune disease susceptibility whereby alterations in BCR signaling result in persistence of high-affinity self-reactive B cells instead of their tolerization. The relevance of these recent contributions to the pathogenesis of autoimmune diseases in clinical settings is currently of great interest.
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IMPLICATIONS FOR AUTOIMMUNITY IN HUMANS |
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). In systemic sclerosis, overexpression of CD19 by B cells correlates
with autoantibody production (106)
, an observation
reminiscent of experimental studies suggesting that lowering B cell
signaling thresholds by increased CD19 expression may augment
susceptibility to the development of autoimmunity (77
, 78)
.
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As discussed above, B cells from mice deficient in Lyn
(90
91
92)
, CD22 (81
82
83
84)
, and SHP-1 (96
, 98)
exhibit a hyper-reactive phenotype and produce
autoantibodies. In human SLE, B cells exhibit increased
Ca2+ influx to antigen stimulation, exaggerated
phosphorylation, and spontaneous hyperactivity (107)
, a
pattern seen in Lyn-/- B cells
(90
91
92)
. Since Lyn-/- mice
develop circulating autoantibodies resulting in autoimmune
glomerulonephritis in a high proportion of cases, they provide a unique
model for autoimmune disorders that are largely due to the impaired
function of B cells. It will be interesting to see whether there is a
human counterpart of a similar autoimmune disorder with dysfunction of
Lyn. Since targeted disruption of cd22, which is exclusive to B cells,
results in the production of high titers of high-affinity serum IgG
directed against dsDNA in a large proportion of mice
(81
82
83
84
85)
, it was of interest to see whether a single gene
defect is also sufficient to trigger autoimmunity in humans. Variation
screening of the entire CD22 coding region was performed in 207 healthy
individuals and 68 SLE patients (108)
. An identified
variation (Q152E) seemed to accumulate in patients with SLE,
particularly those with central nervous system involvement. However,
variation screening of human SHP-1 revealed no variation within the
coding region in SLE patients (108)
; it will be of
interest to screen for Lyn.
Recently it was realized that negative regulation of the BCR results
from a complex interaction between PTK Lyn, the coreceptor CD22, and
SHP-1 (109)
. Different expression patterns of these
factors, which seem to exhibit an incomplete penetrance, generate a
spectrum of BCR signaling. When the cumulative defects in these
molecules exceed a certain threshold, B cells are overtly hyperactive.
Combined heterozygous deletions of Lyn, CD22, and SHP-1 led to abnormal
B cell phenotypes quantitatively (109)
. The Lyn/CD22/SHP-1
and CD45 feedback pathway may be defective to different degrees in
human SLE B cells (110
, 111)
. This suggests an analogous
potential for alterations of BCR signaling in B cells in human SLE due
to disbalanced CD45 and SHP-1 activity. Since susceptibility to SLE
seems to be under polygenic control (86)
, disrupted BCR
signaling could drive self-reactive B cells to produce autoantibodies
after exposure to autoantigens not encountered during early
developmental stages. Abnormal postreceptor signaling could also alter
BCR-mediated lymphocyte death, anergy, or receptor editing. In
addition, dysregulation of BCR signaling could modify the threshold of
BCR triggering, leading to autoimmune phenomena, implying it is
critical for selection of the BCR repertoire and for prevention of
autoimmunity (Fig. 2
).
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ANALOGIES IN BCR AND TCR SIGNALING ALTERATIONS IN AUTOIMMUNITY |
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TOPABSTRACTINTRODUCTIONBCR: A PLATFORM FOR...KINASES, PHOSPHATASES,...B CELL SELECTION AND...SETTING THE BALANCE OF...IMPLICATIONS FOR AUTOIMMUNITY IN...ANALOGIES IN BCR AND...CONCLUSIONSREFERENCES |
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). First, Vav is a proto-oncogene expressed selectively in hematopoietic
cells and required for receptor clustering, proliferation and
differentiation of B and T cells. It is tyrosine phosphorylated upon
stimulation through several receptors, including the TCR and the BCR.
Studies of Vav-/- mice reveal that Vav adjusts
the threshold for antigen receptor-mediated B cell activation depending
on the nature of the antigen (13)
. With regard to
autoimmunity, differential regulation of Vav tyrosine phosphorylation
by CD19 and CD22 may provide a molecular mechanism for adjusting BCR
signaling thresholds (112)
. There is also
overphosphorylation of Vav tyrosine in T cells from lupus-prone
MRL/lpr mice (113)
. Conversely, Vav-deficient
mice have diminished numbers of B-1 and conventional B cells
(114
, 115)
.
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Second, Cbl-b is a molecular adapter that functions downstream from the
TCR and participates in signaling by receptor protein tyrosine kinases.
It has a positive role in T cell signaling, likely via direct
interaction with the upstream kinase Zap-70 (116)
. Because
Cbl-b is capable of degrading key intracellular proteins, it is thought
to be a negative regulator. Mice deficient in Cbl-b show
hyperproduction of lymphocytes and altered positive selection in the
thymus (117)
. Cbl-b plays a more subtle role in the
fine-tuning that regulates immune receptor signaling. Gene-targeted
mice lacking Cbl exhibit enhanced proliferation and interleukin 2
production in response to TCR ligation and expanded B cell
proliferation in response to CD40 or BCR cross-linking (118
, 119)
. In addition to lowering the threshold for immune receptor
triggering, inactivation of the cbl-b gene was associated
with an increased susceptibility to autoantibody production and tissue
damage (119)
as well as with experimental autoimmune
encephalomyelitis (118)
, a mouse model of multiple
sclerosis. These independent studies suggest that Cbl-b is important in
the series of events leading to autoimmune disease. Although the
molecular basis for this pathway remains unknown, it is possible that
Vav, a key guanine nucleotide exchange factor in lymphoid cells, is the
intracellular target for Cbl-b.
Third, the transmembrane protein PD-1 contains an ITIM that is induced
in lymphocytes and monocytic cells after activation. Mice deficient in
PD-1 develop SLE-like arthritis and glomerulonephritis, suggesting that
PD-1 serves as a negative regulator of immune responses and is involved
in maintenance of peripheral self-tolerance (120)
. Fourth,
there may be alterations of tyrosine kinases in both B and T cells.
Immature B cells lack two of the tyrosine kinases found in mature B
cells p59fyn and
p55frg1, indicating that expression of
the src family tyrosine kinases Fyn and Frg is regulated
developmentally in B lymphocytes and that their absence may contribute
to tolerization of the cells (121)
. At least three
cytoplasmic PTKs (p59fyn,
p56lck, and p70ZAP-70) are
involved in the initiation of early signal transduction via the TCR/CD3
complex. Initial studies of SLE T cells showed multiple cellular
abnormalities with decreased (122)
or accelerated and
sustained intracytoplasmic Ca2+ mobilization
(123)
. Abnormal free Ca2+ influx
suggested the existence of alterations in the biochemical events that
operate through the TCR/CD3 complex and that could result in abnormal
TCR transduction of the downstream signals. Other studies showed that
unstimulated T lymphocytes from SLE patients exhibit lower amounts of
p56lck and p59fyn kinases
compared with non-SLE patients and control individuals
(124)
. Another study showed that in PBL from active SLE
patients, p56lck activity was significantly
higher than inactive SLE patients and healthy controls
(125)
. Ligation through the TCR/CD3 complex induced a
significant decrease of p59fyn activity in T
lymphocytes from SLE patients, but not from controls
(124)
. Since it was reported that
p56lck activity was reduced in anergic T cell
clones (126
, 127)
, it is tempting to propose that there
are large numbers of anergic lymphocytes in SLE. Even though several
possibilities could account for the altered expression of Fyn protein,
there are indications that it may result indirectly from alterations in
other molecules involved in regulation of T cell signaling. A good
candidate was CD45, a PTPase that dephosphorylates both positive and
negative tyrosine residues (128)
. Not surprisingly,
reduced CD45 PTPase activity was described in T cells from SLE patients
and was inversely correlated with disease activity (129)
.
The importance of CD45 is also highlighted by a recent observation in a
patient with CD45 deficiency. The deficiency resulted in diminished T
cell numbers and unresponsiveness to mitogens. Despite normal B
lymphocyte numbers, serum Ig levels decreased with age
(130)
.
Since both T and B cells have been shown to be activated in SLE; since
Fyn is associated with signaling of immune receptors of these cells,
Fyn-deficient MRL/lpr mice were generated to investigate the
role of Fyn in the onset of the disease in MRL/lpr mice. The
mice developed markedly limited disease, with reduced production of
IgG3 anti-DNA autoantibodies and diminished glomerular deposits of IgG3
and C3, and lived longer than control mice (131)
. Thus,
modulation of a gene important in signal transduction through the B and
T cell receptors has been shown to affect disease outcome. Thus, the
Fyn PTK may be a potential therapeutic target in systemic autoimmune
diseases.
Fifth, further insight into TCR signaling in SLE comes from analysis of
the zeta (
) chain, also a CD45 substrate (132)
. A
defective tyrosine phosphorylation of the chain of peripheral blood
T cells from SLE patients was also noted in 67% of SLE patients of
different ethnic origins (133)
, but no correlation with
clinical activity was evident (134)
. Remarkably, a
defective TCR-mediated signaling and a decrease in chain expression
were also described in synovial T cells from rheumatoid arthritis
patients (135)
. Finally, the capping defect in human SLE T
cells has been attributed to markedly reduced cAMP-dependent kinase
(PKA) activity (136
, 137)
. It would be of great interest
to determine whether the PKA alterations are also present in B cells,
since the type I PKA isoenzyme colocalizes with the BCR
(138)
, and cAMP alters B cell responsiveness and
susceptibility to apoptosis (139)
.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONBCR: A PLATFORM FOR...KINASES, PHOSPHATASES,...B CELL SELECTION AND...SETTING THE BALANCE OF...IMPLICATIONS FOR AUTOIMMUNITY IN...ANALOGIES IN BCR AND...CONCLUSIONSREFERENCES |
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chain may result in
tolerance disruption, leading to autoimmune phenomena, which implies it
is critical for selection of the TCR repertoire and the prevention of
autoimmunity (140
141
142)
. This view is supported by
mounting evidence showing that lymphocyte receptor signaling molecules
play a role in lymphocyte selection (143
, 144)
. Thus,
alteration of TCR and BCR signaling thresholds, together with other
factors, may contribute to the disruption of self-tolerance in systemic
autoimmunity (145
, 146)
. The fact that reduced activity of
signaling molecules has been described in both T cells and B cells of
SLE patients strengthens the view that the abnormalities observed are
relevant to the pathogenesis (Fig. 2)
.
The mechanisms underlying the abnormal expression of these BCR and
TCR-associated molecules remain the focus of investigation. In a recent
study, there was a deletion—both heterozygous and homozygous—of exon
7 of the chain gene in some lupus patients (133)
.
Specifically, a chain lacking exon 7 was deficient in a tyrosine
residue in the third ITAM domain, presumably altering the TCR potential
to transduce signals (147)
. Variation screening of the
entire CD22 coding region performed in 68 SLE patients identified an
alteration that seemed to accumulate in patients with central nervous
system involvement (108)
. Since there is a tight
regulation of kinases and phosphatases, it is possible that further
studies will uncover other associated regulatory molecules or signaling
substrates in B and T cells from patients with systemic autoimmune
diseases. The results of experiments on Fyn-deficient MRL
lpr/lpr mice (131)
make it tempting to
speculate that such observations may open up a range of avenues to
design novel approaches for immunomodulation. One possibility is that
some of these molecules may serve as targets for immune intervention;
if it turns out that the disease is caused by a major signaling defect,
it is conceivable that the use of specific inhibitors of PTKs
(148)
or PTPases may modulate the autoimmune response and
alleviate the disease.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONBCR: A PLATFORM FOR...KINASES, PHOSPHATASES,...B CELL SELECTION AND...SETTING THE BALANCE OF...IMPLICATIONS FOR AUTOIMMUNITY IN...ANALOGIES IN BCR AND...CONCLUSIONSREFERENCES |
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