Transmodulation of BCR Signaling by TransductionIncompetent Antigen Receptors: Implications for Impaired Signaling in Anergic B Cells1 Barbara J. Vilen,2 Kathy M. Burke, Michelle Sleater, and John C. Cambier3 B cell tolerance can be maintained by functional inactivation, or anergy, wherein B cell Ag receptors (BCR) remain capable of binding Ag, but are unable to transduce signals. Although the molecular mechanisms underlying this unresponsiveness are unknown, some models of B cell anergy are characterized by disruption of proximal BCR signaling events, and by destabilization of the BCR complex. Receptor destabilization is manifest by a reduced ability to coimmunoprecipitate membrane Ig with the Ig-␣/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 Ca2ⴙ, 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. The Journal of Immunology, 2002, 168: 4344 – 4351.
T
he B cell Ag receptor (BCR)4 is a multimeric complex composed of membrane Ig (mIg) noncovalently associated with an Ig-␣/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 Srcfamily 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). Integrated Department of Immunology, University of Colorado Health Sciences Center, and National Jewish Medical and Research Center, Denver, CO 80206 Received for publication October 16, 2001. Accepted for publication March 1, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grants AG13983, AI26519, and AI22295. J.C.C. is an Idan and Cecil Green Professor of Cell Biology. B.J.V. was supported by a National Research Service Award from the National Institute of Allergy and Infectious Disease. 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:
[email protected] 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.
Copyright © 2002 by The American Association of Immunologists
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 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.
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The Journal of Immunology 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- 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 -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.
Materials and Methods Reagents The mAbs AC38 (anti-idiotype), B.7-6 (anti-), 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) 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-) and FITC-labeled JA12.5 (anti-), 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 Y3 L-mutated Ig-/IgM chimera. Cells were selected in medium containing mycophenolic acid (1 g/ml) and hypoxanthine/xanthine (15 g/ml:250 g/ml). Cells were cultured in IMDM supplemented with 5% FCS (HyClone, Logan, UT), 1 mM sodium pyruvate, 50 g/ml
4345 gentamicin, 100 U/ml penicillin, 100 g/ml streptomycin, 2 mM glutamine, and 50 M 2-ME at 37°C in 7.5% CO2.
DNA constructs The constructs encoding wild-type mIgD and signal-incompetent mIgM (IgM-Mut/Y3 L) 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 Y3 L ITAM-mutated Ig- cytoplasmic tail. This construct has previously been referred to as IgM-Mut/Y3 L, and is referred hereto as mIgM(i). It is noteworthy that an alternative construct composed of a 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 ⫻ 106 cells/ml. Cells were resuspended at 5 ⫻ 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. CD69 up-regulation was analyzed following exposure of cells to NP7BSA for 0, 4, 6, or 10 h. At the indicated time points, cells were washed in ice-cold staining medium (balanced salt solution containing 2% FCS and 0.2% sodium azide) then resuspended at 10 ⫻ 106/ml. Cells were subsequently incubated with the appropriate primary Ab for 30 min, washed extensively, then incubated with fluorochrome-conjugated secondary Ab. Finally, the samples were washed and resuspended in staining medium at 10 ⫻ 106/ml. Analysis was performed on FACScan (BD Biosciences, Mountain View, CA) and data was analyzed using CellQuest (BD Biosciences).
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 g/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 ⫻ 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 g of precipitating Ab was incubated with 1 ⫻ 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 ⫻ 106/ml in buffer A. Indo-1/AM in DMSO was added to the cell suspensions to a final concentration of 10 M. 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 ⫻ 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 ⫻ 106/ml unstimulated and Ag-stimulated cells (1 g NP7BSA/5 ⫻ 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
4346 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- (b7-6), 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- 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 (-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 -specific stain)(-normalization factor) ⫽ total number of receptors. The proportion of mIgM(i) receptors on the mixed cell lines was then calculated by: (MF -specific stain)(-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.
Results Preparation of NIP-specific B lymphoma lines expressing signalcompetent and signal-incompetent receptors 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
FIGURE 1. Schematic representation of the signal-competent mIgD and the signal-incompetent mIgM(i) constructs. The mIgD construct contains an NIP-specific extracellular domain linked to the ␦-transmembrane domain and the ␦-cytoplasmic tail. The mIgM(i) construct contains the NIP-specific extracellular domain linked to the H-2Kb transmembrane domain and an ITAM-mutated Ig- cytoplasmic tail.
BCR SIGNAL TRANSMODULATION 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. The constructs encoding the wild-type mIgD and the signalincompetent mIgM(i) receptors were transfected into the K46 B cell lymphoma, and cell lines expressing either mIgD, mIgM(i), or both receptors were isolated (Fig. 2). 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). Both mIgD and mIgM(i) receptors participate in Ag-induced receptor aggregates 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 Agbinding 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 in subsequent studies, we surface-stained naive and Ag (saturating concentrations) stimulated cell lines with anti- 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- 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- 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- 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 signalincompetent 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,
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FIGURE 2. Relative expression of mIgD and mIgM(i) on the panel of B lymphomas. Cell lines expressing mIgD, mIgM(i), or a mixture of mIgD ⫹ mIgM(i) were isolated by cell sorting. Upper left panel, The relative levels of mIgM(i). Upper right panel, The relative levels of mIgD. Lower left panel, The level of idiotype reactive receptors. The MF values for the different stains are compiled in the table (lower right). These data are representative of four independent experiments.
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 mIgDmediated phosphorylation of Ig-␣, Ig-, and Syk (Fig. 4, B and C, middle panel). It is curious that Ig-␣ isolated from the lines differed slightly in mass distribution (Fig. 4B). This finding is consistent with previous
FIGURE 3. Fluorescence microscopy images of mIgM(i) and mIgD receptors. Panels 1 and 2, Anti- and anti-␦ staining (rows 1 and 2, respectively) of either unstimulated or NP7BSA-stimulated mIgD-expressing cells. Panels 3 and 4, Anti- and anti-␦ staining (rows 1 and 2, respectively) of unstimulated or NP7BSA-stimulated mIgD ⫹ 0.53 mIgM(i)-expressing cells. Panels 5 and 6, Anti- and anti-␦ staining (rows 1 and 2, respectively) of unstimulated or NP7BSA-stimulated mIgM(i)-expressing cells. Row 3, The overlay of the anti- and anti-␦ staining of each cell line. These data are representative of three replicate experiments.
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FIGURE 5. Coaggregation of mIgD and mIgM(i) induces normal tyrosine phosphorylation of Cbl and Erk. Anti-phosphotyrosine (rows 1 and 3), anti-Cbl (row 2), or anti-Erk (row 4) immunoblots of either anti-Cbl immunoprecipitates (rows 1 and 2) or anti Erk1/2 immunoprecipitates (rows 3 and 4). Lane 1, Unstimulated mIgD cell line; lane 2, NP7BSAstimulated mIgD-expressing cells; lane 3, NP7BSA-stimulated mIgD ⫹ 0.25 mIgM(i); lane 4, NP7BSA-stimulated mIgD ⫹ 0.45 mIgM(i); lane 5, NP7BSA-stimulated mIgD ⫹ 0.65 mIgM(i); lane 6, NP7BSA-stimulated mIgD ⫹ 0.70 mIgM(i); and lane 7, NP7BSA-stimulated mIgM(i). These data are representative of findings in three independent experiments. FIGURE 4. Coaggregation of IgD and IgM(i) fails to induce tyrosine phosphorylation of several effectors, including Ig-␣ and Syk. A, Anti-phosphotyrosine immunoblots of unstimulated, anti-␦-stimulated, or NP7BSAstimulated mIgD, mIgD ⫹ 0.53 mIgM(i), or mIgM(i) cell lines. B, Antiphosphotyrosine (upper panel) and anti-Ig-␣ (lower panel) immunoblots of an anti-Ig- immunoprecipitation of the cell treatments described above. C, Anti-phosphotyrosine (upper panel) and anti-Syk (lower panel) immunoblots of a Syk immunoprecipitation of the cell treatments described above. This experiment is representative of three independent replicates.
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 (15–27). 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.
Coaggregation of signal-incompetent mIgM(i) inhibits signalcompetent mIgD-mediated mobilization of Ca2⫹ 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 antiidiotype 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- (b-7-6) Abs, there was no effect on the concurrent anti-␦-induced response (Fig. 6B). These data show that coaggregation of signal-incompetent receptors drastically affects the signal-transducing ability of the competent receptor. They also show that the difference between Ag and anti-␦ 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
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FIGURE 7. Coaggregation of mIgD and mIgM(i) blocks up-regulation of CD69. Cell surface staining of CD69 following Ag (NP7BSA) or anti-␦ stimulation. Panel 1, mIgD-expressing cell line. Panel 2, mIgD ⫹ 0.53 mIgM(i) cell line; panel 3, mIgM(i)-expressing cell line. Panel 4, Anti-␦ stimulation of the mIgD, mIgM(i), and mIgD ⫹ 0.53 mIgM(i) cell lines at 6 h. Data are representative of three experiments.
FIGURE 6. A, Coaggregated mIgM(i) modulates mIgD-mediated Ca2⫹ mobilization. Each profile reflects the nanometers of [Ca2⫹]i as a function of time. The intracellular calcium concentration of resting cells was determined before stimulation. Cell lines expressing varied proportions of mIgM(i) as indicated (left panels) were stimulated with anti-␦ (left panels), anti-idiotype (middle panels), or NP7BSA (right panels). B, The mIgD and mIgD ⫹ 0.53% mIgM(i) cell lines were stimulated with NP7BSA, anti-␦, or anti-␦ combined with anti-. The results shown are representative of more than four independent experiments.
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.
Discussion We have previously shown that Ag stimulation leads to destabilization of a large proportion of BCR as manifest by a decreased ability to coimmunoprecipitate Ig-␣/Ig- signal-transducing sub-
units 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
4350 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 (33–36). 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.
BCR SIGNAL TRANSMODULATION
Acknowledgments We thank Michael Neuberger for generously providing the IgM and IgD constructs, and Sandy Duran for assistance in preparation of the manuscript.
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