Regulation of mast cell signaling through high-affinity IgE receptor by CD45 protein tyrosine phosphatase

Kiichi Murakami, Shintaro Sato, Shigeharu Nagasawa and Toshiyuki Yamashita

Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan

Correspondence to: T. Yamashita


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The transmembrane tyrosine phosphatase CD45 regulates the activity of src family protein tyrosine kinases (PTK) and thereby influences the signaling via such receptors as T and B cell antigen receptors associated with these PTK. However, its implication in signaling through the mast cell receptor with high affinity for IgE (Fc{epsilon}RI) is less clear, although Lyn, a member of the src family, plays an important role in Fc{epsilon}RI-mediated signaling. To define a role for CD45 in Fc{epsilon}RI signal transduction, we established CD45 high expressing rat basophilic leukemia cell lines (RBL-CD45H) and cell lines expressing trace amounts of CD45 (RBL-CD45L). We demonstrate that although all RBL-CD45L cell lines degranulate following IgE- and antigen-induced Fc{epsilon}RI aggregation, the response is significantly reduced at a low dose of antigen. The cells show a delayed and slowed Ca2+ mobilization even though at a higher dose where the cells degranulate to a similar extent as RBL-CD45H. This diminished Ca2+ response is restored by reconstitution of RBL-CD45L with a chimeric molecule containing the cytoplasmic phosphatase domains of rat CD45. Furthermore, tyrosine phosphorylation of Fc{epsilon}RI, association of Fc{epsilon}RI with Lyn and PTK activity associated with Fc{epsilon}RI, all of which are enhanced upon Fc{epsilon}RI aggregation in RBL-CD45H, are impaired in RBL-CD45L. Finally, we show that Fc{epsilon}RI is physically associated with CD45 in RBL-CD45H prior to receptor aggregation. Thus, we propose that, although not indispensable in mast cell degranulation, CD45 positively regulates the signaling through Fc{epsilon}RI by promoting the activation of Fc{epsilon}RI-associated Lyn.

Keywords: Fc{varepsilon}RI, Lyn, tyrosine phosphorylation, rat basophilic leukemia


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The high-affinity IgE receptor on mast cells and basophils (Fc{varepsilon}RI) plays a central role in allergic responses (1,2). Fc{varepsilon}RI is a multisubunit receptor comprising an IgE-binding {alpha} chain, a ß chain and two {gamma} chains (3). Aggregation of Fc{varepsilon}RI initiates a series of intracellular biochemical cascades that lead to the secretion of inflammatory mediators. Phosphorylation of a select set of cellular proteins on tyrosine residues is an early event in Fc{varepsilon}RI-mediated signal transduction (4). The rapid tyrosine phosphorylation on ß and {gamma} subunits of Fc{varepsilon}RI upon receptor aggregation suggests an aggregation-dependent activation of protein tyrosine kinase (PTK) that is closely associated with Fc{varepsilon}RI. Several lines of evidence suggest that the src family kinase Lyn, which is constitutively associated with the ß chain of Fc{varepsilon}RI and activated following receptor aggregation, is responsible for the tyrosine phosphorylation of Fc{varepsilon}RI subunits (510).

The cytoplasmic domains of the ß and {gamma} chains each contain a sequence termed the immunoreceptor tyrosine-based activation motif (ITAM) that is found in other multisubunit immune recognition receptors such as the B cell antigen receptor (BCR) and TCR (1114). The phosphorylation of two conserved tyrosine residues within the ITAM consensus sequences plays a critical role for receptor-mediated signal transduction. Current evidence suggests that tyrosine- phosphorylated ITAM of the ß chain leads to reorientation of receptor-associated Lyn and also recruitment of additional Lyn possibly due to src homology 2 (SH2) domain-mediated binding (7,8,10,15). Such binding is known to increase the specific activity of Lyn (16,17). Thus, the Fc{varepsilon}RI-associated kinase activity derived from Lyn is strongly enhanced by receptor aggregation. On the other hand, tyrosine phosphorylation of ITAM of {gamma} chain recruits another PTK, Syk, a member of the Syk/ZAP-70 family of PTK. The Syk thus recruited becomes tyrosine phosphorylated by receptor-associated Lyn and/or autophosphorylation, leading to its full activation and propagation of downstream signals (7,1820).

CD45 is a transmembrane tyrosine phosphatase containing the two tandem phosphatase domains in its intracellular region (21). CD45 modulates the activity of src family PTK by dephosphorylating the tyrosine residue located in the C-terminal regulatory domain. When phosphorylated, this tyrosine appears to bind the kinase's own SH2 domain and suppress PTK activity (22). Many studies have shown that CD45 regulates BCR- and TCR-mediated signaling. However, it is unclear whether CD45 is required for initiating Fc{varepsilon}RI-mediated activation. For example, Berger et al. reported that CD45 was required for Fc{varepsilon}RI-mediated degranulation using primary mast cell cultures derived from CD45-deficient mice (23). In contrast, some CD45-deficient rat basophilic leukemia (RBL) cells were reported to degranulate upon Fc{varepsilon}RI aggregation (24,25).

To investigate the precise role of CD45 in Fc{varepsilon}RI-mediated signaling, we established multiple RBL variants that were CD45 on FACS analysis but expressed trace amounts of CD45 as revealed by immunoblotting. Since parental RBL-2H3 cells are heterogenous in CD45 expression, we also established RBL cell lines that stably expressing high levels of CD45. We determined their ability of degranulation and analyzed their early intracellular events initiated by receptor aggregation. The results argue strongly that, although not required for Fc{varepsilon}RI-mediated degranulation, CD45 positively regulates the signaling by enhancing the recruitment of Lyn to Fc{varepsilon}RI and thereby activating the receptor-associated Lyn.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell culture
RBL-2H3 cells and various RBL cell lines were grown in a humidified atmosphere containing 5% CO2 in Eagle's MEM (Sigma, St Louis, MO) supplemented with 10% FBS (ICN, Irvine, CA), penicillin (100 U/ml), streptomycin (100 µg/ml) and Fungizone (Life Technologies, Grand Island, NY; 0.25 µg/ml). Cells were harvested following exposure to 1 mM EDTA in Dulbecco's PBS for 10 min and assayed in suspension.

Antibodies
Monoclonal anti-DNP IgE was prepared from the ascites of mice bearing the H1 DNP-{varepsilon}-26.82 hybridoma (26). [125I]IgE was prepared by the chloramine T method (27). Mouse mAb directed against rat CD45 (OX-1) was purchased from Serotec (Kidlington, UK) and used for FACS analysis. Polyclonal anti-CD45 antibody was obtained by immunizing rabbits with bacterially expressed 6xHis-tag fusion proteins containing the extracellular domain (residues 1–229) or C-terminal (1008–1142) of rat CD45RO, and used for immunoblot analysis and immunoprecipitation respectively. Anti-rat MHC class I (RT1.A) mAb was from Cedarlane (Hornby, Ontario, Canada). Anti-guinea pig Fc{gamma}RIIB mAb was prepared as described (28). Anti-mouse IgE was raised in guinea pigs and its affinity-purified IgG2 fraction was covalently bound to Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden) for immunoprecipitation of IgE-bound Fc{varepsilon}RI. FITC-conjugated sheep F(ab')2 fragment of anti-mouse IgG was from Cappel (West Chester, PA). The anti-phosphotyrosine mAb 4G10 and rabbit anti-Lyn were purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit anti-Csk was from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP) conjugates of donkey F(ab')2 anti-rabbit Ig and streptavidin were obtained from Amersham (Little Chalfont, UK).

cDNA and generation of CD45 chimeras
The cDNA encoding rat CD45RO (29,30) was obtained by using total RNA from RBL-2H3 cells as the template and RT-PCR with the RNA LA PCR Kit (Takara Shuzo, Otsu, Japan) with primers bearing appropriate restriction sites. The PCR products were cut with restriction enzymes and cloned into the pBluescript II SK+ or KS+ vector (Stratagene, La Jolla, CA). A phosphatase-inactive mutant was generated by mutating cysteines 690 and 1005 to serine with PCR as described (31). For the construction of a chimeric CD45, the cytoplasmic region of rat CD45 or its phosphatase-negative form was attached to the sixth amino acid of the cytoplasmic domain of the guinea pig Fc{gamma}RIIB (32) by PCR using primer 5'-AAG CAG CCT CCA GCC GAT GAA CAG CAG GA-3'. Underlined nucleotides are from 5' end of CD45 cytoplasmic domain. After confirming their structure by DNA sequencing, the constructs were subcloned into pcDNA3 (Invitrogen, San Diego, CA) or pcXN2 expression vector (33) kindly provided by Dr J. Miyazaki (Osaka University, Osaka, Japan).

Transfections
RBL cells expressing chimeric CD45 were produced according to protocols provided by the supplier of the Lipofect Amine reagents (Life Technologies). Stably transfected clones were selected with 600 µg/ml of G418 (Life Technologies). Surface expression was analyzed on FACSort (Becton Dickinson, San Jose, CA) using anti-guinea pig Fc{gamma}RIIB and FITC–sheep anti-mouse IgG (32). Several monoclonal transfectants were obtained and cells stably expressing the highest levels of chimeras were used for experiments.

Analysis of mediator release
DNP-conjugated BSA (11 mol of DNP/mol of protein) was prepared as described (34). RBL cells (107 /ml) were incubated with 10 µg/ml mouse anti-DNP IgE for 1 h at room temperature. Cells were washed and resuspended in assay buffer containing 25 mM PIPES (pH 7.2), 119 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.4 mM MgSO4, 5.4 mM glucose and 0.1% BSA at 1x107 /ml. Cells (106) were stimulated with various amounts of DNP-BSA for 1 h at 37°C. Cells were also stimulated simultaneously with 50 nM phorbol myristate acetate (PMA) and 5 µM calcium ionophore A23187 to determine their maximal ability to degranulate. The culture supernatant was collected and the cells were lysed with assay buffer containing 0.2% Triton X-100. For determination of hexosaminidase release, 10 µl of supernatant or cell lysate was incubated with 50 µl of 4 mM p-nitrophenyl-N-acetyl-ß-D-glucosaminide in 0.1 M citrate buffer, pH 4.5, for 20 min at 37°C. At the end of the incubation, 150 µl of 0.2 M glycine buffer, pH 10.4, was added and the absorbance at 405 nm was determined.

Calcium analysis
Measurements of intracellular free calcium concentration ([Ca2+]i) were performed using Fura-PE3 (35) as a Ca2+ indicator. Cells were incubated with 10 µg/ml mouse anti-DNP IgE and 3 µM Fura-PE3/AM (Texas Fluorescence, Austin, TX) for 1 h at room temperature. Cells were washed and resuspended in assay buffer at 5x106/ml. Fluorescence of the stirred cell suspension was continuously monitored with a fluorescence spectrophotometer Shimadzu RF-5300PC. Fluorescence was excited alternatively at 340 and 380 nm, and the signal (emission at 510 nm) at each excitation wavelength was plotted against time. [Ca2+]i was calibrated and computed as described (35).

Immunoprecipitation and immunoblotting
Cells were solubilized in lysis buffer containing 0.5% Triton X-100, 50 mM Tris–HCl (pH 7.6), 50 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 5 mM Na4P2O7, 50 mM NaF, 1 mM PMSF, and 10 µg/ml each of aprotinin, leupeptin and pepstatin A. When Fc{varepsilon}RI was immunoprecipitated, Triton X-100 was substituted for 10 mM CHAPS. Postnuclear supernatants were precleared with Protein A–Sepharose 4 Fast Flow beads (Pharmacia) and then immunoprecipitated with various antibodies, followed by incubation with the beads. To determine the association of Fc{varepsilon}RI with Lyn, IgE-bound Fc{varepsilon}RI was directly immunoprecipitated with Sepharose 4B bead-bound anti-mouse IgE. After immunoprecipitation, the immune complexes were washed in lysis buffer, resolved by SDS–PAGE and transferred to 0.2 µm nitrocellulose (BA83; Schleicher & Schuell, Keene, NH). Non-specific binding sites were blocked by incubation with blocking buffer (50 mM Tris–HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20, 4% protease-free BSA, 0.01% human IgG and 0.01% thimerosal). Blots were then incubated with optimal concentrations of various primary antibody, washed 3 times with the blocking buffer and incubated with HRP-conjugated secondary antibody or streptavidin–HRP complex (Amersham). After a further wash with the blocking buffer, the immunoblots were developed by enhanced chemiluminescence (Amersham). For some experiments, membranes were stripped of primary antibodies according to the protocol of the reagents and reprobed.

In vitro kinase assay
Immunoprecipitates were washed twice with kinase buffer (20 mM Tris–HCl, pH 7.5, 0.15 M NaCl, 20 mM MgCl2 and 1 mM Na3VO4) and resuspended in 28 µl kinase buffer containing exogenous substrates (cdc-2-derived peptide; KVEKIGEGTYGVVKK; final concentration 1 mM) (3638). Kinase reactions were initiated by the addition of 2 µl ATP (final 0.1 mM cold ATP and 10 µCi [{gamma}-32P]ATP (3000 Ci/mmol; NEN, Boston, MA). After incubation for 1 h at 30°C, kinase reaction products were separated by SDS–PAGE on 16% tricine gels, fixed in 7% acetic acid and analyzed by autoradiography.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of CD45 in RBL-2H3 cell and its variants
We first examined which isoform(s) of CD45 is expressed in RBL-2H3 because potentially eight isoforms can be generated by the alternative splicing of three exons (21). Using RT-PCR with internal sense and antisense primers for the N-terminal of CD45, where mRNA splicing occurs, we confirmed that the cells expressed only CD45RO isoforms (data not shown). Because we were not able to find a commercially available antibody that recognizes CD45 on immunoblots, we immunized rabbit with a recombinant protein containing the extracellular domain of rat CD45RO. Immunoblotting of RBL-2H3 lysates with the obtained anti-CD45 showed a single band (180 kDa) corresponding to the CD45RO isotype. This antibody did not react with native CD45 either on FACS analysis or by immunoprecipitation.

Consistent with the previous report (25), FACS analysis using a mouse anti-rat CD45 mAb OX-1 demonstrated that RBL-2H3 were heterogenous in expression levels of CD45 (Fig. 1Go). We attempted to obtain RBL cell lines expressing different amounts of CD45 by limiting dilution. Individual colonies were screened by FACS analysis using OX-1 and several cloned RBL variants expressing different levels of CD45 were obtained. Two clones were found to stably express high revels of CD45 and one of these clones (RBL-CD45H) was used for further experiments. On the other hand, three clones (RBL-CD45L1, L2 and L3) were essentially devoid of CD45 when assessed by FACS. However, immunoblotting showed that these clones expressed trace amounts of CD45 (Fig. 2Go). By densitometry of the blot, it was estimated that RBL-CD45H expressed ~100 times as much CD45 as three RBL-CD45L clones. These clones expressed comparable amounts of Fc{varepsilon}RI, when assessed by their IgE bindings. We also checked amounts of Lyn, Csk and Syk, all of which are PTK involved in early events following Fc{varepsilon}RI aggregation, with immunoblotting and failed to find any notable difference among them.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1. FACS analysis of Fc{varepsilon}RI and CD45 expression in the parental RBL-2H3 and the cloned RBL variants. Cells were stained with saturating amounts of anti-CD45 mAb (OX-1) (solid line) or mouse IgE (broken line), followed by F(ab')2 FITC–sheep anti-mouse Ig. The negative controls (dotted line) were stained with the secondary antibody alone.

 


View larger version (79K):
[in this window]
[in a new window]
 
Fig. 2. Immunoblot analysis of CD45, Lyn, Csk and Syk expression in RBL-CD45H (H) and -CD45L1, L2 and L3 (L1–L3).

 
Effect of CD45 on degranulation
To determine the involvement of CD45 in Fc{varepsilon}RI-mediated signaling, we tested for the ability of RBL-CD45L cell lines to degranulate. The Fc{varepsilon}RI on cells were saturated with anti-DNP IgE and aggregated with 1 or 1000 ng/ml of DNP-BSA. Cells were also stimulated simultaneously with PMA and calcium ionophore A23187 to estimate their maximal ability to degranulate. The degree of degranulation after 1 h incubation was determined by measuring the release of the granule enzyme ß-hexosaminidase. As shown in Fig. 3Go, all three RBL-CD45L cells were capable of degranulating in response to Fc{varepsilon}RI aggregation with 1000 ng/ml of antigen. However, compared to the responses of RBL-CD45H cells, their responses at low concentrations of antigen (1 ng/ml) were significantly reduced. It should be noted that the other CD45 high expressing cell line and two more CD45L clones showed the same trend (data not shown).



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 3. Degranulation of RBL variants in response to Fc{varepsilon}RI aggregation. RBL-CD45H or three RBL-CD45L cell lines were sensitized with anti-DNP IgE and stimulated with 1 or 1000 ng/ml DNP-BSA. Cells were also stimulated with PMA (50 nM) plus A23187 (5 µM). After 1 h, ß-hexosaminidase activity was measured from cell lysates and supernatants, and release is expressed as the percentage of total. The data shown represent the mean ± SE for at least three separate experiments.

 
Effect of CD45 on Fc{varepsilon}RI-mediated calcium mobilization
We next addressed whether calcium mobilization is affected by CD45, because calcium flux is an early event induced upon Fc{varepsilon}RI aggregation. The IgE-sensitized and Fura-PE3-loaded cells were stimulated with 1 or 100 ng/ml DNP-BSA, and their [Ca2+]i levels were determined by spectrofluorometry. In RBL-CD45H cells, [Ca2+]i reached the maximum level within 20 s after stimulation (Fig. 4AGo). The second peak at 1 min is perhaps derived from influx of extracellular Ca2+, because it disappeared by chelation of extracellular Ca2+ with EGTA (Fig. 4BGo). In contrast, the calcium level in three RBL-CD45L cell lines increased slowly after stimulation and reached the maximum level at ~70 s post-stimulation. This impaired calcium response was more prominent when the extracellular Ca2+ was depleted. With 1 ng/ml DNP-BSA, which induced degranulation of RBL-CD45H but hardly that of RBL-CD45L cells, a delayed and slow [Ca2+]i increase was observed in RBL-CD45H, whereas three RBL-CD45L cell lines showed almost no response (Fig. 4CGo).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 4. Fc{varepsilon}RI-induced calcium flux of RBL variants. Cells were loaded with Fura-PE3/AM and anti-DNP IgE, and stimulated at the indicated time point (arrow) with 100 ng/ml DNP-BSA in the absence (A) or presence of 1 mM EGTA (B). The cells were also stimulated with 1 ng/ml DNP-BSA in the absence of EGTA (C). The fluorescence response at 340 and 380 nm excitation was measured. The traces show calculated [Ca2+]i (A and C) or the 340/380 nm fluorescence ratio (B).

 
To confirm that these diminished Ca2+ responses are due to a poor expression of CD45, we made a chimeric construct in which intracellular sequences of rat CD45 are fused to the extracellular and transmembrane sequence of guinea pig type IIB Fc{gamma}R and transfected it into RBL-CD45L1. Stably transfected clones were selected with G418 and surface expression was analyzed on using anti-guinea pig Fc{gamma}RIIB mAb. One clone thus obtained expressed high levels of chimeric CD45 (Fig. 5AGo) and scarcely expressed CD45RO as is the case with parental RBL-CD45L1, when assessed by immunoblotting (data not shown). As shown in Fig. 5(B)Go, the chimeric CD45 completely restored the Fc{varepsilon}RI-mediated calcium response. These results strongly suggest that CD45 is involved in Fc{varepsilon}RI-mediated signaling and positively regulates calcium response.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5. (A) FACS analysis of chimeric CD45 expression in a RBL-CD45L variant (FcR/CD45). The expression was determined by staining cells with anti-guinea pig Fc{gamma}RIIB mAb. (B) Fc{varepsilon}RI-induced calcium flux of the RBL variant expressing chimeric CD45 (FcR/CD45). Cells were loaded with Fura-PE3/AM and anti-DNP IgE, and stimulated at the indicated time point (arrow) with 100 ng/ml DNP-BSA. [Ca2+]i was measured as described in Fig. 3Go.

 
Effect of CD45 on tyrosine phosphorylation of Fc{varepsilon}RI and its association with Lyn
We next examined whether the tyrosine phosphorylation of receptor itself upon aggregation is abolished in RBL-CD45L, because several lines of evidence suggest that Lyn is responsible for the tyrosine phosphorylation of Fc{varepsilon}RI subunits (79). RBL-CD45H and -CD45L1 were stimulated with various amounts of DNP-BSA for 2 min, and their Fc{varepsilon}RI were immunoprecipitated. Immunoblotting with the anti-phosphotyrosine mAb 4G10 showed that aggregation of Fc{varepsilon}RI induced tyrosine phosphorylation of receptor subunits in RBL-CD45L1 (Fig. 6AGo). However, the ß and {gamma} chain in RBL-CD45L were less tyrosine phosphorylated than those in RBL-CD45H even at 1000 ng/ml DNP-BSA where RBL-CD45L1 were fully capable of degranulation. This impaired tyrosine phosphorylation was also observed in RBL-CD45L2 and L3 cells (data not shown). Introduction of CD45 chimeras into RBL-CD45L1 cells restored the aggregation-induced tyrosine phosphorylation of Fc{varepsilon}RI (Fig. 6BGo). These results suggest that CD45 is required for full activation of Lyn, although the basal activity of Lyn is sufficient to induce the tyrosine phosphorylation of Fc{varepsilon}RI.



View larger version (43K):
[in this window]
[in a new window]
 
Fig. 6. (A) Tyrosine phosphorylation of Fc{varepsilon}RI upon receptor aggregation. Cells were sensitized with anti-DNP IgE and stimulated with various concentrations of DNP-BSA for 2 min. Then Fc{varepsilon}RI was immunoprecipitated and subjected to immunoblotting with anti-phosphotyrosine mAb 4G10. SDS–PAGE (12% gel) was performed under non-reducing conditions. (B) Restored tyrosine phosphorylation of Fc{varepsilon}RI by introduction of chimeric CD45 into RBL-CD45L1 cells. Cells were unstimulated or stimulated with 1 µg/ml DNP-BSA for 2 min. (C) Association of Fc{varepsilon}RI with Lyn. Cells were either unstimulated or stimulated with 1 µg/ml DNP-BSA for 2 min. Then Fc{varepsilon}RI were immunoprecipitated and analyzed by immunoblotting with anti-Lyn.

 
It has been well documented that Fc{varepsilon}RI is pre-associated with Lyn and the association is enhanced following receptor aggregation (58). The receptors were immunoprecipitated from unstimulated or stimulated cells and analyzed by immunoblotting with anti-Lyn. As shown in Fig. 6(C)Go, the level of pre-association was similar between two cell lines. However, the receptor aggregation in RBL-CD45L1 did not induce an increase in association of Lyn with the receptors, which was seen in RBL-CD45H. At this antigen dose (1000 ng/ml), the receptors in RBL-CD45L1 were tyrosine phosphorylated although much less so than in RBL-CD45H (Fig. 6AGo). Previous reports have described that tyrosine-phosphorylated ITAM of ß chain leads to recruitment of additional Lyn via SH2 domain-mediated binding (7,8,10,15). Perhaps, as described in BCR signaling (39), the dephosphorylation of a regulatory tyrosine on Lyn may be required for this binding. Thus, in RBL-CD45L1 cells the regulatory tyrosine may not be dephosphorylated due to low-level expression of CD45, which prevents recruitment of additional Lyn proteins.

Effect of CD45 on Fc{varepsilon}RI-associated PTK activity
It is well known that aggregation of Fc{varepsilon}RI results in an enhanced PTK activity associated with the receptor. To determine whether CD45 affects the Fc{varepsilon}RI-associated PTK activity, Fc{varepsilon}RI was immunoprecipitated from RBL-CD45H or -CD45L1 cells and subjected to in vitro kinase assay. We confirmed that the PTK activity of receptors from RBL- CD45H was markedly enhanced by receptor aggregation with 1000 ng/ml antigen for 2 min, as judged by the PTK activity toward exogenous substrates that is known to be a good substrate for the src family kinases (Fig. 7Go) (3638). In contrast, although the basal activity was indistinguishable between two cell lines, the Fc{varepsilon}RI from RBL-CD45L1 did not show an enhanced PTK activity upon receptor aggregation. The aggregation-induced PTK activation was again restored by introduction of CD45 chimeras into RBL-CD45L1 cells. These data clearly indicate that CD45 is required for the enhancement of Fc{varepsilon}RI-associated PTK activity by receptor aggregation.



View larger version (58K):
[in this window]
[in a new window]
 
Fig. 7. Fc{varepsilon}RI-associated PTK activity toward exogenous substrates. Anti-IgE immunoprecipitates were prepared from RBL-CD45H, -CD45L1 and -FcR/CD45 cells, and their in vitro kinase activities were assessed by incubating with 1 mM exogenous peptide substrates (cdc-2 peptide) and 10 µCi [{gamma}-32P]ATP in a kinase assay. Incorporation of radioactivity into the substrate peptides was visualized by SDS–PAGE on 16% tricine gels and autoradiography.

 
Association of Fc{varepsilon}RI with CD45
To address the potential interaction of Fc{varepsilon}RI with CD45, we immunoprecipitated Fc{varepsilon}RI from RBL-CD45H cell lysates and immunoblotted with the rabbit anti-CD45. [125I]IgE-sensitized RBL-CD45H were unstimulated or stimulated with 1000 ng/ml DNP-BSA for 2 min, solubilized with 10 mM CHAPS, incubated with or without anti-IgE antibody and immunoprecipitated with Protein A–Sepharose beads. As shown in Fig. 8(A)Go, CD45 was co-immunoprecipitated with Fc{varepsilon}RI. Aggregation of receptors did not affect or slightly enhanced the extent of association. Comparing the intensity of the bands on immunoblots of the unfractionated detergent extract and the specific precipitate of receptor, we estimated that ~0.2% of the total cellular CD45 was co-immunoprecipitated with Fc{varepsilon}RI. Conversely, Fc{varepsilon}RI was co-immunoprecipitated when CD45 was immunoprecipitated with polyclonal anti-CD45 antibody that recognizes the C-terminal region of rat CD45 (Fig. 8BGo). A small but substantial fraction of Fc{varepsilon}RI was co-immunoprecipitated with CD45 comparing with fractions of co-precipitated Fc{varepsilon}RI when anti-rat MHC class I (RT1.A) mAb as well as normal rabbit IgG and mouse IgG1 mAb (anti-guinea pig Fc{gamma}RIIB) were used for immunoprecipitation.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 8. Association of Fc{varepsilon}RI with CD45. (A) IgE-bound Fc{varepsilon}RI was immunoprecipitated from lysates of unstimulated or stimulated (1 µg/ml DNP-BSA for 2 min) RBL-CD45H and analyzed by immunoblotting with rabbit anti-CD45 antibody. As a control, lysates were incubated without anti-IgE and precipitated with Protein A–Sepharose beads. (B) RBL-CD45H cells sensitized with [125I]IgE were unstimulated or stimulated with antigen, solubilized and immunoprecipitated with affinity-purified rabbit IgG anti-CD45 (15 µg) or mouse IgG1 mAb anti-RT1.A (15 µg). As a control, cell lysates were immunoprecipitated with the same amounts of normal rabbit IgG or mouse IgG1 mAb directed against guinea pig Fc{gamma}RIIB. Percentages of co-precipitated Fc{varepsilon}RI were calculated from precipitated radioactivities. The data shown represent the mean ± SE for at least three separate experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
It is controversial whether CD45 is required for signal transduction through mast cell Fc{varepsilon}RI. An early study suggested the involvement of CD45 in IgE-induced histamine release by demonstrating the inhibitory effects of anti-CD45 antibody (40). A recent report using primary mast cell cultures derived from CD45-deficient mice (23) and a transfection study using a CD45-deficient T cell line (41) further indicated the importance of CD45 in Fc{varepsilon}RI-mediated signaling. On the other hand, some RBL variants that lack CD45 have been shown to degranulate upon Fc{varepsilon}RI aggregation (24,25). There is also a report indicating that the functional Fc{varepsilon}RI can be reconstituted in CD45 fibroblasts (42).

In this study, we have established multiple RBL cell lines expressing trace amounts of CD45. These variants show a reduced degranulation when stimulated with relatively low concentrations of antigen, even though it fully degranulates in response to higher concentrations. The IgE-induced calcium response is also significantly impaired in these cell lines. Transfection of a chimeric CD45 into RBL-CD45L1 restored the IgE-induced calcium response. Thus, CD45 is critical for facilitating signal transduction through Fc{varepsilon}RI. It still remains unclear whether even such small amounts of CD45 as those detected by immunoblot analysis might be sufficient to induce degranulation upon Fc{varepsilon}RI aggregation. To address the issue, we transfected a phosphatase-negative version of chimeric CD45 into RBL-CD45L1 and determined whether a dominant negative effect was observed. The calcium response of the transfectant did not further deteriorate even when stimulated with 100 ng/ml DNP-BSA (data not shown). This suggests that the CD45 expressed in RBL-CD45L1 are not functional or the level of CD45 expression is too low to affect the signaling. Thus, it is likely that CD45 expression is not required for IgE-induced degranulation.

The finding that RBL-CD45L1 failed to enhance the PTK activity associated with Fc{varepsilon}RI following receptor aggregation suggests that CD45 is critical to activate the receptorassociated kinase, probably Lyn. However, CD45 is not required for initiating tyrosine phosphorylation of Fc{varepsilon}RI subunits upon receptor aggregation. The basal activity of receptor-associated kinase may be sufficient to phosphorylate receptor subunits by transphosphorylation and recruit Syk to the phosphorylated {gamma} ITAM. We observed that the extent of tyrosine phosphorylation and PTK activity of Syk after receptor aggregation in RBL-CD45L1 was less than in -CD45H (data not shown). Even not fully activated, it is likely that the recruited Syk can propagate signals leading to degranulation.

We also found that the association of Fc{varepsilon}RI with Lyn was not enhanced by receptor aggregation in RBL-CD45L1. A previous report using chemical cross-linking indicated that the recruitment of additional Lyn to aggregated receptors was dependent on tyrosine phosphorylation (8). Other in vitro studies suggested that Lyn SH2 domain can bind to the tyrosine-phosphorylated ITAM of the receptor's ß subunit (15). Recently, it was demonstrated that Lyn from CD45-deficient B cells failed to bind the tyrosine-phosphorylated ITAM peptides (39). This suggests that the phosphorylation of Lyn C-terminal regulatory tyrosine prevents its association with the phosphorylated ITAM, perhaps due to its preferential binding to the Lyn's own SH2 domain. Thus, CD45 is a prerequisite for Lyn binding to phosphorylated ß ITAM via its SH2 domain.

To our knowledge, this is the first study to demonstrate the association of Fc{varepsilon}RI with CD45. This association is very weak and detectable only when cells are solubilized with 10 mM CHAPS as far as we tested to date. Only a small fraction of CD45 (0.2%) is associated with receptor in these conditions. We speculate that the majority of the association is disrupted during solubilization and the actual percent of CD45 associated with Fc{varepsilon}RI may be higher, as is the case with the receptor–Lyn association (8). Unlike Fc{varepsilon}RI–Lyn association, the association is hardly enhanced by receptor aggregation. The importance of this association is unknown. However, it seems probable that only CD45 constitutively associated with Fc{varepsilon}RI is involved in signaling. The action of CD45 may be restricted to the receptor-associated Lyn and the Lyn that is located in the proximity of aggregated receptors, which becomes dephosphorylated and is then recruited to aggregated receptors.

The present findings demonstrate that, although not indispensable in mast cell degranulation, CD45 is involved in the signaling through Fc{varepsilon}RI and facilitates the signal transduction by promoting the activation of Fc{varepsilon}RI-associated Lyn following receptor aggregation. Based on our results and other reports, we propose the following model for the activation of receptor-associated Lyn. In resting cells, only a fraction of Fc{varepsilon}RI is associated with Lyn (8). When aggregated, the receptor-associated Lyn has access to adjacent receptors and thereby transphosphorylates tyrosines of the ß and {gamma} ITAM (38). We suggest that the basal activity of receptor-associated Lyn is sufficient to phosphorylate them. The constitutively associated Lyn, which binds to the ß ITAM in a tyrosine phosphorylation-independent manner prior to aggregation (7,10), now switches to bind to tyrosine-phosphorylated ß ITAM via its SH2 domain. The phosphorylated ß ITAM also promotes the recruitment of additional Lyn (8). In both cases, the dephosphorylation of Lyn C-terminal negative regulatory tyrosine by CD45 is required for the binding via SH2 domain. This binding increases the specific activity of Lyn (13,17). The receptor-associated kinase activity is now strongly enhanced, resulting in a large increase in phosphorylation of the receptors and associated molecules (8). In this scenario, CD45 is a key molecule that modulates the signaling by regulating Lyn activity. Thus, the expression level of CD45 may play an important role in determining the magnitude of IgE-induced mast cell activation.


    Acknowledgments
 
We gratefully acknowledge gifts of RBL-2H3 cells and H1 DNP-{varepsilon}-26.82 hybridomas from Drs. Henry Metzger and Juan Rivera, NIAMS/NIH. We also thank Dr Henry Metzger for critical review of the manuscript.


    Abbreviations
 
BCR B cell receptor
[Ca2+]i intracellular free Ca2+ concentration
Fc{varepsilon}RI high-affinity receptor for IgE
HRP horseradish peroxidase
ITAM immunoreceptor tyrosine-based activation motif
PMA phorbol myristate acetate
PTK protein tyrosine kinase
RBL rat basophilic leukemia
SH2 src homology 2

    Notes
 
Transmitting editor: D. Kitamura

Received 30 July 1999, accepted 14 October 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Metzger, H., Alcaraz, G., Hohman, R., Kinet, J. P., Pribluda, V. and Quarto, R. 1986. The receptor with high affinity for immunoglobulin E. Annu. Rev. Immunol. 4:419.[ISI][Medline]
  2. Ravetch, J. V. and Kinet, J. P. 1991. Fc receptors. Annu. Rev. Immunol. 9:457.[ISI][Medline]
  3. Blank, U., Ra, C., Miller, L., White, K., Metzger, H. and Kinet, J. P. 1989. Complete structure and expression in transfected cells of high affinity IgE receptor. Nature 337:187.[ISI][Medline]
  4. Benhamou, M. and Siraganian, R. P. 1992. Protein-tyrosine phosphorylation: an essential component of Fc{varepsilon}RI signaling. Immunol. Today 13:195.[ISI][Medline]
  5. Eiseman, E. and Bolen, J. B. 1992. Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinases. Nature 355:78.[ISI][Medline]
  6. Hutchcroft, J. E., Geahlen, R. L., Deanin, G. G. and Oliver, J. M. 1992. Fc{varepsilon}RI-mediated tyrosine phosphorylation and activation of the 72-kDa protein-tyrosine kinase, PTK72, in RBL-2H3 rat tumor mast cells. Proc. Natl Acad. Sci. USA 89:9107.[Abstract]
  7. Jouvin, M. H., Adamczewski, M., Numerof, R., Letourneur, O., Valle, A. and Kinet, J. P. 1994. Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor. J. Biol. Chem. 269:5918.[Abstract/Free Full Text]
  8. Yamashita, T., Mao, S. Y. and Metzger, H. 1994. Aggregation of the high-affinity IgE receptor and enhanced activity of p53/56lyn protein-tyrosine kinase. Proc. Natl Acad. Sci. USA 91:11251.[Abstract/Free Full Text]
  9. Oliver, J. M., Burg, D. L., Wilson, B. S., McLaughlin, J. L. and Geahlen, R. L. 1994. Inhibition of mast cell Fc{varepsilon}RI-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol. J. Biol. Chem. 269:29697.[Abstract/Free Full Text]
  10. Vonakis, B. M., Chen, H., Haleem-Smith, H. and Metzger, H. 1997. The unique domain as the site on Lyn kinase for its constitutive association with the high affinity receptor for IgE. J. Biol. Chem. 272:24072.[Abstract/Free Full Text]
  11. Reth, M. 1989. Antigen receptor tail clue. Nature 338:383.[ISI][Medline]
  12. Weiss, A. 1993. T cell antigen receptor signal transduction: a tale of tails and cytoplasmic protein-tyrosine kinases. Cell 73:209.[ISI][Medline]
  13. Cambier, J. C., Pleiman, C. M. and Clark, M. R. 1994. Signal transduction by the B cell antigen receptor and its coreceptors. Annu. Rev. Immunol. 12:457.[ISI][Medline]
  14. Cambier, J. C. 1995. New nomenclature for the Reth motif (or ARH1/TAM/ARAM/YXXL). Immunol. Today 16:110.[ISI][Medline]
  15. Kihara, H. and Siraganian, R. P. 1994. Src homology 2 domains of Syk and Lyn bind to tyrosine-phosphorylated subunits of the high affinity IgE receptor. J. Biol. Chem. 269:22427.[Abstract/Free Full Text]
  16. Johnson, S. A., Pleiman, C. M., Pao, L., Schneringer, J., Hippen, K. and Cambier, J. C. 1995. Phosphorylated immunoreceptor signaling motifs (ITAMs) exhibit unique abilities to bind and activate Lyn and Syk tyrosine kinases. J. Immunol. 155:4596.[Abstract]
  17. Clark, M. R., Johnson, S. A. and Cambier, J. C. 1994. Analysis of Ig-{alpha}-tyrosine kinase interaction reveals two levels of binding specificity and tyrosine phosphorylated Ig-{alpha} stimulation of Fyn activity. EMBO J. 13:1911.[Abstract]
  18. Benhamou, M., Ryba, N. J., Kihara, H., Nishikata, H. and Siraganian, R. P. 1993. Protein-tyrosine kinase p72syk in high affinity IgE receptor signaling. Identification as a component of pp72 and association with the receptor {gamma} chain after receptor aggregation. J. Biol. Chem. 268:23318.[Abstract/Free Full Text]
  19. Shiue, L., Zoller, M. J. and Brugge, J. S. 1995. Syk is activated by phosphotyrosine-containing peptides representing the tyrosine-based activation motifs of the high affinity receptor for IgE. J. Biol. Chem. 270:10498.[Abstract/Free Full Text]
  20. Kimura, T., Sakamoto, H., Appella, E. and Siraganian, R. P. 1996. Conformational changes induced in the protein tyrosine kinase p72syk by tyrosine phosphorylation or by binding of phosphorylated immunoreceptor tyrosine-based activation motif peptides. Mol. Cell. Biol. 16:1471.[Abstract]
  21. Thomas, M. L. 1989. The leukocyte common antigen family. Annu. Rev. Immunol. 7:339.[ISI][Medline]
  22. Trowbridge, I. S. and Thomas, M. L. 1994. CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development. Annu. Rev. Immunol. 12:85.[ISI][Medline]
  23. Berger, S. A., Mak, T. W. and Paige, C. J. 1994. Leukocyte common antigen (CD45) is required for immunoglobulin E-mediated degranulation of mast cells. J. Exp. Med. 180:471.[Abstract]
  24. Schneider, H., Korn, M. and Haustein, D. 1993. CD45-deficient RBL-2H3 cells. Cellular response to Fc{varepsilon}R- and ionophore-induced stimulation. Immunol. Invest. 22:503.[ISI][Medline]
  25. Swieter, M., Berenstein, E. H. and Siraganian, R. P. 1995. Protein tyrosine phosphatase activity associates with the high affinity IgE receptor and dephosphorylates the receptor subunits, but not Lyn or Syk. J. Immunol. 155:5330.[Abstract]
  26. Liu, F. T., Bohn, J. W., Ferry, E. L., Yamamoto, H., Molinaro, C. A., Sherman, L. A., Klinman, N. R. and Katz, D. H. 1980. Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization. J. Immunol. 124:2728.[Free Full Text]
  27. McConahey, P. J. and Dixon, F. J. 1966. A method of trace iodination of proteins for immunologic studies. Int. Arch. Allergy Appl. Immunol. 29:185.[ISI][Medline]
  28. Shimamura, T., Nakamura, T. and Koyama, J. 1986. Analysis of Fc receptors for IgG on guinea pig peritoneal macrophages by the use of a monoclonal antibody to one of the Fc receptors. J. Biochem. 99:227.[Abstract]
  29. Thomas, M. L., Barclay, A. N., Gagnon, J. and Williams, A. F. 1985. Evidence from cDNA clones that the rat leukocyte-common antigen (T200) spans the lipid bilayer and contains a cytoplasmic domain of 80,000 Mr. Cell 41:83.[ISI][Medline]
  30. Barclay, A. N., Jackson, D. I., Willis, A. C. and Williams, A. F. 1987. Lymphocyte specific heterogeneity in the rat leucocyte common antigen (T200) is due to differences in polypeptide sequences near the NH2-terminus. EMBO J. 6:1259.[Abstract]
  31. Desai, D. M., Sap, J., Silvennoinen, O., Schlessinger, J. and Weiss, A. 1994. The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation. EMBO J. 13:4002.[Abstract]
  32. Yamashita, T., Shinohara, K. and Yamashita, Y. 1993. Expression cloning of complementary DNA encoding three distinct isoforms of guinea pig Fc receptor for IgG1 and IgG2. J. Immunol. 151:2014.[Abstract/Free Full Text]
  33. Niwa, H., Yamamura, K. and Miyazaki, J. 1991. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193.[ISI][Medline]
  34. Farah, F. S., Kern, M. and Eisen, H. N. 1960. The preparation and some properties of purified antibody specific for the 2,4-dinitrophenyl group. J. Exp. Med. 112:1195.[ISI]
  35. Vorndran, C., Minta, A. and Poenie, M. 1995. New fluorescent calcium indicators designed for cytosolic retention or measuring calcium near membranes. Biophys. J. 69:2112.[Abstract]
  36. Cheng, H. C., Litwin, C. M., Hwang, D. M. and Wang, J. H. 1991. Structural basis of specific and efficient phosphorylation of peptides derived from p34cdc2 by a pp60src-related protein tyrosine kinase. J. Biol. Chem. 266:17919.[Abstract/Free Full Text]
  37. Cheng, H. C., Nishio, H., Hatase, O., Ralph, S. and Wang, J. H. 1992. A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases. J. Biol. Chem. 267:9248.[Abstract/Free Full Text]
  38. Pribluda, V. S., Pribluda, C. and Metzger, H. 1994. Transphosphorylation as the mechanism by which the high-affinity receptor for IgE is phosphorylated upon aggregation. Proc. Natl Acad. Sci. USA 91:11246.[Abstract/Free Full Text]
  39. Pao, L. I. and Cambier, J. C. 1997. Syk, but not Lyn, recruitment to B cell antigen receptor and activation following stimulation of CD45 B cells. J. Immunol. 158:2663.[Abstract]
  40. Hook, W. A., Berenstein, E. H., Zinsser, F. U., Fischler, C. and Siraganian, R. P. 1991. Monoclonal antibodies to the leukocyte common antigen (CD45) inhibit IgE-mediated histamine release from human basophils. J. Immunol. 147:2670.[Abstract/Free Full Text]
  41. Adamczewski, M., Numerof, R. P., Koretzky, G. A. and Kinet, J. P. 1995. Regulation by CD45 of the tyrosine phosphorylation of high affinity IgE receptor ß- and {gamma}-chains. J. Immunol. 154:3047.[Abstract/Free Full Text]
  42. Scharenberg, A. M., Lin, S., Cuenod, B., Yamamura, H. and Kinet, J. P. 1995. Reconstitution of interactions between tyrosine kinases and the high affinity IgE receptor which are controlled by receptor clustering. EMBO J. 14:3385.[Abstract]