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Integrated Department of Immunology, University of Colorado Health Sciences Center, and National Jewish Medical and Research Center, Denver, CO 80206
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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/Ig-
signal-transducing complex. To begin
to explore the possibility that anergy is the consequence of receptor
destabilization, we analyzed a panel of B lymphoma transfectants
expressing constant amounts of signal-competent Ag receptors and varied
amounts of a receptor with identical specificity, but bearing mutations
that render it incapable of interacting with Ig-/Ig-. This
analysis revealed that coaggregation of signal-incompetent receptors
prevented Ag-induced Ig- and Syk phosphorylation, mobilization of
Ca2+, and the up-regulation of CD69 mediated by competent
receptors. In contrast, Ag-induced Cbl and Erk phosphorylation were
unaffected. Data indicate that coaggregation of destabilized receptors
(as few as 
15% of total) with signal-competent receptors
significantly affects the ability of competent receptors to transduce
signals. Thus, BCR destabilization may underlie the Ag unresponsiveness
of anergic B cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/Ig- dimer that is responsible for signal transduction.
Receptor aggregation by multivalent Ags induces phosphorylation of the
immunoreceptor tyrosine-based activation (ITAM) motifs within Ig-
and Ig- by receptor-associated Src-family tyrosine kinases
(1). This creates high-affinity docking sites that recruit
kinases and linker molecules that propagate divergent signaling
cascades involving Ras/mitogen-activated protein kinase,
phospholipase C
/Ca2+, and phosphatidylinositol
3-kinase. The minimum number of receptors required within an aggregate
for signal transduction, as well as requirements for spatial
distribution of signal transducing components, remains unclear. Studies
using Ags of varying valence have suggested that at least 12 receptors
must be cross-linked to induce signal transduction (2).
Signal transduction is dependent on optimal receptor aggregation,
implying dependence on receptor density, Ag affinity, and Ag valence
(3, 4).
Signal transduction through the BCR determines the fate of B
lymphocytes. Under conditions where cognate T cell help is provided,
BCR signal transduction leads to differentiation and Ab secretion. In
the absence of secondary T cell-mediated stimulation, signals
transduced through the BCR lead to receptor desensitization and
cellular unresponsiveness (5, 6). Cells bearing
desensitized receptors are unable to respond to Ag stimulation and are
termed anergic. Desensitized receptors continue to bind Ag but fail to
transduce signals that lead to tyrosine phosphorylation of
Ig-/Ig-, as well as other substrates, and to
Ca2+ mobilization. In the anti-hen egg
lysosome (HEL) Ig transgenic mouse model of B cell anergy, ligation of
as few as 5% of receptors on naive cells initiates sufficient receptor
aggregation and signal transduction to induce anergy. Anergic cells
fail to undergo tyrosine kinase activation upon further Ag-induced
receptor aggregation, yet show sustained Ca2+
oscillation and ERK activation. Although in the anti-HEL model
anergic cells exhibit selective and marked decreases in surface IgM
levels, which could result in the unresponsiveness, in other
low-affinity Ig transgenic models of anergy, such as the Ars/A1,
anti-Smith, and anti-ssDNA systems, relatively little
receptor down-modulation is seen, but available receptors are
desensitized
(7, 8).5
We have recently used relatively low-affinity lymphoma and Ig
transgenic models to define biochemical changes that render cells
unresponsive to subsequent receptor aggregation. Analysis of kinase
activation in cells bearing desensitized receptors has shown that
Ag-induced phosphorylation of receptor-associated kinases such as Lyn,
Blk, Shc, and Syk is impaired. Most interestingly, these cells exhibit
normal levels of inactive receptor-associated Lyn kinase, and this
kinase can be activated by exposure to a doubly phosphorylated ITAM
substrate in vitro (5). Finally, desensitized receptors
remain responsive to Abs against Ig/Ig (9). These
data suggest that the failure of desensitized receptors to activate
signaling pathways is not due to a defect intrinsic to kinases or
kinase association with resting receptors, but rather reflects a defect
at the level of transmission of signal through the receptor. Coincident
with BCR desensitization, Ag stimulation leads to destabilization of
the receptor complex in these cells (9). Destabilization
occurs within 15 min of receptor stimulation and typically results in a
50–80% loss of coprecipitable Ig-/Ig- in anti-µ
immunoprecipitates. Receptor destabilization occurs on the cell surface
and the dissociated Ig-/Ig- complexes remain responsive to
anti-Ig- ligation, suggesting that destabilization contributes
to the desensitized phenotype.
The observation that BCR destabilization occurs coincident with BCR
desensitization suggests that separation of Ig-/Ig- from mIg may
mediate cellular unresponsiveness. However, if only 50–80% of
receptors dissociate from the Ag-binding µ-H chain, one would predict
that the remaining 20–50% of receptors sheathed by Ig-/Ig-
should be sufficient to initiate signal transduction upon a second
encounter to Ag. This prediction is supported by the fact that
concentrations of ligand that occupy only 10–20% of BCR on naive
cells induce robust B cell activation. In view of these findings, we
hypothesized that destabilized receptors may modulate signaling by
competent receptors, but only when the receptors are coaggregated. To
investigate whether destabilized receptors can modulate competent
receptors from transducing signals, we created a panel of B cell
lymphomas expressing both signal-competent (mIgD) and
signal-incompetent (mIgM(i)) receptors to mimic the B cell surface
following BCR destabilization. Analysis of this lymphoma panel
indicates that the spatial organization of the signal-transducing
molecules within the receptor aggregate plays a key role in determining
signal transduction. Coligation of mIgD and mIgM(i) prevented Ig-
and Syk phosphorylation, mobilization of Ca2+,
and the up-regulation of the activation marker, CD69. In contrast, Erk
and Cbl phosphorylation were unaffected by coaggregation of
signal-incompetent with signal-competent receptors. These data indicate
that inclusion of receptors lacking Ig-/Ig- in aggregates
containing functional BCR leads to disruption of the spatial
organization within the aggregate, thereby inhibiting the activation of
key signaling molecules. Based on these findings, we hypothesize that
receptor destabilization plays a key role in reducing the BCR response
in anergic cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mAbs AC38 (anti-idiotype), B.7-6 (anti-µ), JA12.5
(anti-
), and HM-79 (anti-Ig-) were purified from culture
supernatants using protein A or protein G Sepharose. Other reagents
include FITC-labeled anti-CD69 (BD PharMingen, San Diego, CA);
anti-phosphotyrosine-specific Ab, Ab-2 (Oncogene Science,
Manhasset, NY); anti-Cbl (Santa Cruz Biotechnology, Santa Cruz,
CA); and anti-extracellular signal-related kinase (Erk) 1/Erk 2
(Santa Cruz Biotechnology, Santa Cruz, CA). HRP-conjugated secondary
Abs include rat anti-mouse IgG1 (BioSource International,
Camarillo, CA) and protein A (Zymed Laboratories, San Francisco, CA).
The rabbit polyclonal anti-Syk and anti-Ig- have been
previously described (5). The Abs used for staining in
fluorescence microscopy were FITC-labeled b-7-6 (anti-µ) and rat
anti-mouse IgD-RPE (Southern Biotechnology Associates,
Birmingham, AL). The Abs used for flow cytometric analysis were
FITC-labeled b-7-6 (anti-µ) and FITC-labeled JA12.5
(anti-µ), along with biotinylated AC38 (anti-idiotype) and
streptavidin-FITC (Caltag, Burlingame, CA).
Cell culture and selection
The K46 B lymphoma cells (2a+,
+) were transfected individually with plasmids
encoding 3-nitro-4-hydroxy-5-iodophenylacetyl (NIP)-specific
IgD, and/or a Y
L-mutated Ig-/IgM chimera. Cells were selected in
medium containing mycophenolic acid (1 µg/ml) and
hypoxanthine/xanthine (15 µg/ml:250 µg/ml). Cells were cultured in
IMDM supplemented with 5% FCS (HyClone, Logan, UT), 1 mM sodium
pyruvate, 50 µg/ml gentamicin, 100 U/ml penicillin, 100 µg/ml
streptomycin, 2 mM glutamine, and 50 µM 2-ME at 37°C in 7.5%
CO2.
DNA constructs
The constructs encoding wild-type mIgD and signal-incompetent
mIgM (IgM-Mut/YL) have been previously
described (10). These constructs encode Ag receptors
specific for the hapten NIP using the H chain B-1-8 and 
1 L chain.
The IgD construct contains the wild-type transmembrane domain and
cytoplasmic tail of the -H chain, and hence, sheaths Ig-/Ig-
normally. The mIgM construct contains the transmembrane domain of MHC
class I H-2Kb fused to a YL ITAM-mutated
Ig- cytoplasmic tail. This construct has previously been referred to
as IgM-Mut/YL, and is referred hereto as
mIgM(i). It is noteworthy that an alternative construct composed of a
µ chain containing only transmembrane region YS to VV mutations also
does not associate with Ig-/Ig-, but modulates responses when
coaggregated with mIgD (data not shown).
B cell stimulation, CD69 up-regulation, and surface staining
Preparations of hapten conjugates have been previously described
(5). Cell stimulation was performed using cultures grown
to a density of 1 x 106 cells/ml. Cells
were resuspended at 5 x 106/ml, warmed to
37°C, then stimulated with 500 ng nitrophenol
(NP)7BSA/ml (7 mol of NP-hapten per mole of BSA
carrier) for 1 min. This Ag concentration was shown in preliminary
experiments and in Fig. 3
to saturate receptors. Following the
stimulation, the cells were pelleted by brief centrifugation.
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Cell lysates, immunoprecipitation, and immunoblot analysis
Lysates were prepared by the addition of lysis buffer containing
1% Nonidet P-40, 150 mM NaCl, 10 mM Tris (pH 7.5), 2 mM sodium
orthovanadate, 1 mM PMSF, 0.4 mM EDTA, 10 mM NaF, and 1 µg/ml each of
aprotinin, leupeptin, and 1-antitrypsin to cell pellets. Lysates
were held on ice for 10 min followed by the removal of particulate
material by centrifugation at 12,000 x g for 10 min at
4°C.
Abs used in the immunoprecipitations were conjugated to cyanogen bromide-activated Sepharose 4B according to manufacturer’s instruction (Amersham Pharmacia Biotech, Uppsala, Sweden). Approximately 0.5–1 µg of precipitating Ab was incubated with 1 x 106 cell equivalents of cleared lysate for 30 min at 4°C. Immunoprecipitates were washed twice with lysis buffer, resuspended in reducing SDS-PAGE sample buffer, and then fractionated by 10% SDS-PAGE. Separated proteins were transferred to polyvinylidene difluoride membranes using a semidry blotting apparatus following the conditions recommended by the manufacturer (Millipore, Bedford, MA). Polyvinylidene difluoride membranes were blocked in TBS containing 4% BSA, then incubated with the various immunoblotting Abs followed by the appropriate HRP-conjugated secondary Abs. Immunoreactive proteins were detected based on ECL (NEN, Boston, MA).
Calcium mobilization
To determine intracellular free calcium concentrations ([Ca2+]i), cells were washed once in buffer A (10 mM HEPES in HBSS, pH 6.9), then resuspended at 5 x 106/ml in buffer A. Indo-1/AM in DMSO was added to the cell suspensions to a final concentration of 10 µM. Following a 30 min incubation at 37°C, the cells were diluted 1/2 with buffer B (10 mM HEPES containing 3% FCS in HBSS, pH 7.4) and incubated an additional 30 min. The excess Indo-1/AM was removed by two washings in buffer C (IMDM containing 3% FCS). Finally, the cells were resuspended in buffer C at a concentration of 5 x 106/ml and maintained at room temperature until analysis. Flow cytometry analysis of [Ca2+]i was performed using the MoFlo (Cytomation, Fort Collins, CO). Data were analyzed using MTime (Phoenix Flow Systems, San Diego, CA).
Fluorescence microscopy
A total of 1.5 x 106/ml unstimulated and Ag-stimulated cells (1 µg NP7BSA/5 x 106 cells/ml, 37°C, 45 min) were gently bound to poly(D)-lysine (3 mg/ml) coated coverslips by brief centrifugation then fixed for 10 min with 3% paraformaldehyde/3% sucrose. Cells stained with the indicated Abs were observed using a Leica DMXRA epifluorescence microscope (Intelligent Imaging, Denver, CO). Visual data were acquired using a Cooke Corporation SensiCam CCD camera (Intelligent Imaging) and were digitally deconvolved using a nearest neighbor algorithm with SlideBook software (Intelligent Imaging) as described previously (11).
Calculation of receptor level
Cell surface staining was performed as described above using
anti-µ (b-7-6), anti- (JA12.5), and an
anti-idiotype-specific mAb that recognizes the B-1-8/1 receptor
(AC38). The density of mIgM and mIgD was calculated by dividing the
mean fluorescence (MF) of the anti-idiotype staining by the MF of
the anti-µ or anti- stain on the control cell lines that
contain only mIgM(i) or mIgD to obtain a ratio for normalization of
anti-idiotype and the H chain-specific Ab stains (µ-normalization
factor and -normalization factor, respectively). In any cell line
containing both receptors (IgM(i) and mIgD) the following calculation
was made: (MF -specific stain)(-normalization factor) + (MF
µ-specific stain)(µ-normalization factor) = total number of
receptors. The proportion of mIgM(i) receptors on the mixed cell lines
was then calculated by: (MF µ-specific stain)(µ-normalization
factor)/total receptor number = percentage of mIgM. The proportion
of receptors on the mixed cell lines that are mIgD was calculated by:
(MF -specific stain)(-normalization factor)/total receptor
number = percentage of mIgD.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously it has been reported that aggregation of 5–25% of
surface Ig leads to transduction of signals and cellular activation
(5, 12). We have recently shown that Ag aggregation of
similar proportions of surface Ig leads to destabilization of 50–80%
of Ig-/Ig- from mIg, and is coincident with subsequent Ag
unresponsiveness (9). Interestingly, the 20–50% of
receptors that remain coupled to Ig-/Ig- are unable to transduce
signals when cells are restimulated. One explanation for this lack of
response is that destabilized receptors modulate signaling of the
coaggregated functional receptors. To explore this possibility, we
prepared a panel of cell lines expressing nearly constant levels of
signal-competent IgD receptors (mIgD) and variable levels of
signal-incompetent IgM receptors (mIgM(i)). We chose distinct Ig
isotypes to prevent mispairing of functional and nonfunctional receptor
H chains, to enable independent stimulation of each receptor and to
allow quantification of relative expression of functional and
nonfunctional receptors. Although both receptor constructs
encoded a NIP-specific mIg (Fig. 1), only the competent receptors
associated with endogenous Ig-/Ig-. The signal-incompetent
construct encoded the extracellular domain of mIgM, the transmembrane
domain of H-2Kb, and the cytoplasmic tail of
Ig-. The Ig- cytoplasmic tail contained tyrosine to phenylalanine
mutations within the ITAM tyrosines that rendered the construct unable
to transduce signals upon BCR ligation (Ref. 10 ; B.
J. Vilen and J. C. Cambier, unpublished observations). This
chimeric mIg did not associate with Ig-/Ig-, and therefore,
mimicked the 50–80% of surface Ig that are destabilized following Ag
stimulation.
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). Lines bearing mIgM(i)
were prepared by transfecting aliquots of a single line previously
transfected with, and therefore, expressing mIgD. Three cells lines
were established that expressed nearly constant mIgD with varying
levels of mIgM(i) (Fig. 2, upper left panel; see
Materials and Methods for calculations of receptor
expression). In the mIgD + 0.13 mIgM(i) line, 13% of total receptors
were mIgM(i) and 87% were mIgD. In the mIgD + 0.32 mIgM(i) line, 32%
of total receptors were mIgM(i) and 68% were mIgD. In the IgD + 0.53
mIgM(i) line, 53% of total receptors were mIgM(i) and 47% were mIgD.
Staining with an anti-idiotype-specific Ab indicated that the total
number of receptors remained relatively constant, regardless of the
ratio of mIgD to mIgM(i) (Fig. 2, lower left panel). In
addition, the level of mIgD, although relatively high on all cell lines
(>70% of control), decreased slightly as the level of mIgM(i)
increased (Fig. 2, upper right panel and table).
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The structure of surface Ig varies between the mIgM and mIgD
isotypes. Murine mIgM contains four constant-region domains, while mIgD
contains two such domains. Consequently, the Ag-binding domain of mIgM
extends further from the membrane than IgD. Given this difference, it
seemed possible that mIgD might be unable to compete for Ag when the
level of mIgM(i) increased. To determine whether both mIgD and mIgM(i)
receptors bind Ag and are coaggregated under the conditions
of stimulation used insubsequent studies, we surface-stained naive
and Ag (saturating concentrations) stimulated cell lines with
anti-µ and anti- Abs, and measured receptor cocapping
(Fig. 3
: mIgD, panels 1 and
2; mIgD + 0.53 mIgM(i), panels 3 and
4; and mIgM(i), panels 5 and 6).
Diffuse plasma membrane staining with anti-µ was apparent in both
the unstimulated mIgD + 0.53 mIgM(i) cell line and the mIgM(i)
line (Fig. 3, top row, panels 3 and
5). Upon Ag-induced receptor aggregation, anti-µ
staining became punctuate, indicating aggregation of mIgM(i) (Fig. 3, top row, panels 4 and 6). Anti-
staining of naive and Ag-stimulated cells showed a similar fluorescence
pattern with the mIgD cell line and the mIgD + 0.53% mIgM(i) line
(Fig. 3, middle row, panels 1–4). An overlay of
the anti-µ and anti- staining showed that both mIgM and
mIgD receptors were present on the surface of the mIgD + 0.53% mIgM(i)
line and most importantly, receptors colocalized in the Ag-induced
receptor aggregates (Fig. 3, bottom row, panels 3
and 4). These data, which are representative of four
independent experiments, indicate that despite the difference in size,
both the mIgD and mIgM(i) receptors participate in Ag-induced receptor
aggregates in Ag-stimulated mIgD/mIgM(i)-expressing cells. They also
indicate that despite the fact that mIgM and mIgD may occur in distinct
receptor oligomers on unstimulated cells (13), they can be
coaggregated into caps where they cooperate in transduction of
signals.
Coaggregation of signal-competent mIgD and signal-incompetent
mIgM(i) fails to induce tyrosine phosphorylation of Ig- and Syk
To examine whether the signal-incompetent mIgM(i) receptors affect
transduction of signals through the competent mIgD receptors, we
analyzed the level of phosphotyrosine in lysates of mIgD, mIgM(i), and
mIgD + 0.53 mIgM(i) cell lines following stimulation with saturating
concentrations of Ag or anti- Abs. Aggregation of mIgD with an
anti- Ab-induced robust whole-cell tyrosine phosphorylation in
both the mIgD and the mIgD + 0.53 mIgM(i) cell line (Fig. 4A, middle panel,
lanes 1 and 2), indicating that the receptor
density of mIgD on the mIgD + 0.53 mIgM(i) line was sufficiently high
to initiate signal transduction. These data also showed that in the
absence of coaggregation, expression of mIgM(i) does not affect signal
transduction through mIgD. However, Ag stimulation of the mIgD + 0.53
mIgM(i) lead to a diminished response compared with the mIgD cell line
(Fig. 4A, right panel, lane 1 compared
with 2). To examine tyrosine phosphorylation of proteins
that have previously been defined as proximal signaling intermediates
in the BCR signaling pathway, we immunoprecipitated the Ig-/Ig-
complex and Syk. As shown in Fig. 4, B and C,
aggregation of mIgD on either the mIgD or the mIgD + 0.53 mIgM(i) cell
line with anti--induced robust tyrosine phosphorylation of both
Ig-/ and Syk (Fig. 4, B and C, middle
panel, compare lane 1 with 2). In contrast,
Ag-induced coaggregation of mIgD and mIgM(i) on the mixed
receptor-expressing cell line led to diminished tyrosine
phosphorylation of both Ig- and Syk (Fig. 4, B and
C; right panel, compare lane 1 with
2). Importantly, anti- stimulation of the mIgD cell
line led to robust tyrosine phosphorylation of Ig-, Ig-, and Syk.
These data indicate that coaggregation of unsheathed mIgM with mIgD
inhibits mIgD-mediated phosphorylation of Ig-, Ig-, and Syk (Fig. 4, B and C, middle panel).
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isolated from the lines differed
slightly in mass distribution (Fig. 4B). This finding is
consistent with previous reports that mIgD-associated Ig- is more
heavily glycosylated than mIgM-associated Ig- (14).
Regardless, densitometric analysis revealed that very similar amounts
of Ig- were precipitated from the three cell lines. Coaggregation of mIgM(i) does not affect Ag-induced phosphorylation of (Casitas B lineage lymphoma) Cbl and Erk
To further investigate the extent to which coaggregated mIgM(i)
impairs signal transduction through mIgD, we assessed the
phosphorylation of the downstream signaling intermediates Cbl and the
mitogen-activated protein kinases, Erk1 and Erk2 (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27).
As shown in Fig. 5, the basal levels of
tyrosine phosphorylation of both Cbl and Erk1/2 were low in naive cells
expressing only mIgD. Not surprisingly since they were derived from the
same mIgD+ parent line, basal Cbl and Erk1/2
expression and phosphorylation were equivalent in mIgM(i) lines (data
not shown). Upon Ag stimulation, a marked increase in both Cbl and
Erk1/2 phosphorylation was observed while no phosphorylation was seen
in the mIgM(i) cell line (Fig. 5, lanes 1, 2, and
7). Analysis of cell lines expressing varying levels of
mIgM(i) and a constant amount of mIgD revealed that coaggregation of
mIgM(i) with mIgD did not affect the ability of the competent mIgD
receptor to transduce signals leading to phosphorylation of Cbl and
Erk1/2, regardless of the level of coexpressed mIgM(i). Taken together,
these data show that coaggregation of incompetent with signal-competent
receptors does not affect Ag-induced Cbl and Erk tyrosine
phosphorylation.
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To assess the effect of coaggregation of mIgM(i) on mIgD-mediated
mobilization of calcium, we stimulated cell lines with saturating
concentrations of anti-, anti-idiotype or Ag, and monitored
calcium levels. As depicted in Fig. 6A, row 1,
aggregation of the mIgDT in the absence of mIgM(i) by anti-,
anti-idiotype, or Ag resulted in calcium increases following
receptor ligation. The ability of anti- to induce mobilization
of calcium was unaffected by coexpression of mIgM(i) with mIgD (Fig. 6A, column 1). However, coaggregation of even
small amounts of mIgM(i) (13%) reduced the mIgD-mediated calcium
mobilization following anti-idiotype or Ag stimulation (Fig. 6A, row 2). In this cell line, mIgM(i) expression
was quite variable. The ability of a 13% contamination of functional
receptors with nonfunctional receptors to modulate signaling
should be viewed as a rough approximation. As the level of
signal-incompetent mIgM(i) increased to 32%, the ability of
coaggregated mIgD to mobilize calcium in response to Ag or
anti-idiotype was almost completely ablated (Fig. 6A,
rows 3 and 4). Finally, when mIgM(i) on the mIgD
+ 0.53 mIgM(i) cell line was aggregated independently using monoclonal
anti-µ (b-7-6) Abs, there was no effect on the concurrent
anti--induced response (Fig. 6B).
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induced responses in the mIgD + 0.53 mIgM(i) cell line is
not a function of differences in ligand affinity/avidity because the
high-affinity anti-idiotype Abs that coaggregate mIgD and mIgM(i)
are also unable to induce a calcium response. Taken together, our data
suggest that if as few as 13% of receptors in an aggregate are signal
incompetent, they can affect the ability of the signal-competent
receptors to function. Coaggregation of incompetent receptors blocks BCR-mediated up-regulation of CD69
The data reveal that low levels of coaggregated signal-incompetent
receptors drastically affect Ag-induced Ig- and Syk tyrosine
phosphorylation and calcium mobilization. In contrast, phosphorylation
of Erk and Cbl are unaffected by signal-incompetent receptors. To
evaluate how coaggregation of signal-incompetent and signal-competent
receptors affect downstream biological responses, we assessed
up-regulation of CD69 expression in cell lines responding to BCR
aggregation. Ag aggregation of the mIgD in the absence of mIgM(i) lead
to a modest CD69 up-regulation (Fig. 7, top panel). In contrast, Ag aggregation of the
signal-incompetent BCR on the mIgM(i) cell line did not induce CD69
up-regulation. On the mIgD + 0.53 mIgM(i) cell line, Ag coaggregation
of the BCR failed to induce CD69 up-regulation. To ensure that failure
to up-regulate CD69 was not due to some intrinsic defect within the
mIgD + 0.53% mIgM(i) cell line, we aggregated the signal-competent IgD
and observed CD69 up-regulation comparable to that seen in control mIgD
cells. These data show that coaggregation of signal-incompetent and
signal-competent receptors affects Ag induction of downstream biologic
responses despite inhibiting only selected indicators of signal
transduction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/Ig- signal-transducing
subunits with mIg (9). More recently, we have confirmed
these findings using fluorescence resonance energy transfer (S. Gauld
and J. C. Cambier, manuscript in preparation). Destabilization
occurs on the cell surface, leaving receptors capable of binding Ag but
apparently incapable of transducing normal signals. Following initial
documentation of this phenomenon, it was unclear how destabilization
could cause cell unresponsiveness to Ag because sufficient receptors
appeared to remain "stable" to transduce signals that would lead to
cell activation. In the present study, we investigated whether
destabilized (incompetent) Ag receptors can modulate signal
transduction by coaggregated stable (competent) receptors. We
demonstrate that analogs of destabilized receptors inhibit signaling by
competent receptors, but only when receptors are coaggregated. These
effects are evident when as few as 13% of receptors within the
aggregates are incompetent. Two major questions arise from these studies: 1) how can occupancy of a relatively small proportion of receptors cause destabilization of most of the cell’s BCR, and 2) how do destabilized BCR affect signaling by coaggregated competent receptors? Regarding the former question, one possibility is that stimulation of cells by subsaturating Ag doses destabilizes both occupied and nonoccupied (bystander) receptors by homologous/heterologous mechanisms. This possibility is consistent with findings in a T cell model where cross-antagonism of a T cell clone expressing two distinct TCR has been shown (28). In this model, antagonist engagement of TCR desensitized distinct receptors binding agonist peptide. This was correlated with recruitment of the SH2-containing tyrosine phosphatase-1 to bystander TCR. However, others have been unsuccessful in showing cross "antagonism" of the TCR (29, 30). It is noteworthy that, to date, destabilization has only been documented in B cell models in which affinity for Ag is low/moderate, i.e., in 3–83 and Ars/A1 (Ref. 9 ; R. J. Benschop and J. C. Cambier, manuscript in preparation). These findings suggest a second mechanistic possibility involving serial engagement of receptors. In this scenario, Ag may be capable of serial receptor engagement due to its high off-rate. Ag would trigger destabilization of only occupied receptors; however, these receptors would remain destabilized for some period of time following dissociation of Ag. This would result in accumulation of more destabilized receptors than are occupied at a single point in time. Clearly, either mechanism could result in the generation of the observed proportion of occupied and destabilized receptors.
A serial occupancy mechanism could probably be operative only in cells with low to moderate affinity to Ag. Consistent with this possibility, we have been unable to detect receptor destabilization in Ag-stimulated MD4 anti-HEL B cells where receptor affinity is very high. In B cells with high Ag affinity, serial occupancy would be limited by low rates of disassociation and more efficient Ag-induced receptor endocytosis. In high affinity cells, which are probably rare in the primary repertoire, unresponsiveness may result from reduced numbers of receptors on the cell surface or from some undefined mechanism.
How do destabilized BCR affect signaling by coaggregated competent
receptors? The modulation of signal transduction in this model reflects
the proximal disruption of receptor coupling to a select set of signal
transduction pathways. Data indicate that both Ag-induced receptor
tyrosine phosphorylation of Ig-/ and Syk, and
Ca2+ mobilization are drastically reduced.
Surprisingly, tyrosine phosphorylation of Cbl and Erk are unaffected.
Analysis of downstream biological responses shows that coaggregation of
signal-competent and signal-incompetent receptors prevents CD69
up-regulation. Two obvious possibilities may explain the effect of
destabilized receptors on activation of select signaling pathways. By
disrupting the architecture of receptor aggregates, destabilized
receptors may simply reduce the efficiency of receptor phosphorylation.
As a consequence of this quantitative reduction, all "normal"
downstream signaling pathways may be activated, but with much reduced
efficiency. Activation of downstream events that are subject to more
enzymatic amplification (e.g., Cbl and Erk phosphorylation) might
appear normal, while activation of others (e.g.,
Ca2+ mobilization) might be inhibited. Thus, some
downstream biologic responses might be inhibited (e.g., CD69
up-regulation) while others are not.
An alternate possibility is that the observed differences in
Ig-/Ig- phosphorylation in anergic/desensitized vs naive cells
may reflect qualitatively distinct patterns of ITAM tyrosine
phosphorylation. Perhaps ITAM monophosphorylation is predominant
following stimulation of anergic cells. This would have the effect of
limiting activation of effectors whose function requires dual ITAM
phosphorylation, such as Syk (31), while sparing
activation of effectors whose function requires only ITAM
monophosphorylation, such as Lyn. Consistent with this possibility, we
have found in independent studies that only ITAM monophosphorylation is
required for maximal activation of the SH2-containing inositol-5
phosphatase SHIP and the adaptor Dok, which are downstream from
Lyn, and that these effectors are fully activated in anergic cells (I.
Tamir and J. C. Cambier, manuscript in
preparation).
Interestingly, several groups have recently suggested that in mature B
cells, phosphorylation of Ig- and Syk, as well as
Ca2+ mobilization, requires receptor movement to
the lipid raft microenvironment, while Erk activation occurs in the
absence of lipid rafts (33, 34, 35, 36). In addition, it has been
shown that in the MD4 anti-HEL model, Ag stimulation of anergic B
cells does not induce BCR movement to lipid rafts (33).
Based on these findings, it has been suggested that B cell anergy may
result from the inability of BCR to move to lipid rafts where the
microenvironment is optimally supportive of kinase activation and
signal propagation. It is important to note that receptor movement to
rafts has not been examined in models in which BCR destabilization has
been documented. Nonetheless, it is possible that destabilized
receptors exert their effect by preventing movement of receptor
aggregates to rafts. Future studies will address the effect of receptor
destabilization on Ag-induced movement of functional receptors to
rafts.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599.
3 Address correspondence and reprint requests to Dr. John C. Cambier, Integrated Department of Immunology, National Jewish Medical and Research Center, 1400 Jackson Street, K1001, Denver, CO 80206. E-mail address: cambierj{at}njc.org
4 Abbreviations used in this paper: BCR, B cell Ag receptor; ERK, extracellular signal regulated kinase; HEL, hen egg lysozyme; NP, nitrophenol; NIP, 1-hydro-5-iodo-3 nitrophenylacetyl; mIgM, membrane IgM; mIgD, membrane IgD; ITAM, immunoreceptor tyrosine-based activation motif; [Ca2+]i, intracellular free calcium concentration; MF, mean fluorescence.
5 M. Borrero, and S. H. Clarke. Low affinity anti-Sm B cells are regulated by anergy as opposed to developmental arrest or differentiation to B1. Submitted for publication.
Received for publication October 16, 2001. Accepted for publication March 1, 2002.
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