Published online before print May 10, 2004
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receptors FcRI/ and FcRIIA differ in their interaction with Syk and with Src-related tyrosine kinases
University of Pennsylvania School of Medicine, Hematology and Oncology Division, Philadelphia
1Correspondence: University of Pennsylvania School of Medicine, Hematology and Oncology Division, Biomedical Research Building II/III, Room 705, 421 Curie Blvd., Philadelphia, PA 19104. E-mail: schreibr{at}mail.med.upenn.edu
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RIIA, which uses the Ig tyrosine-activating motif (ITAM) within its own cytoplasmic domain, and FcRI, which transmits signals by means of an ITAM located within the cytoplasmic domain of its associated -chain. For example, in transfected epithelial cells and COS-1 cells, FcRIIA mediates phagocytosis of IgG-coated red blood cells more efficiently than does FcRI/, and enhancement of phagocytosis by Syk kinase is more pronounced for FcRI/ than for FcRIIA. In addition, structure/function studies indicate that the -chain ITAM and the FcRIIA ITAM have different requirements for mediating the phagocytic signal. To study the differences between FcRIIA and FcRI/, we examined the interaction of FcRIIA and the FcRI/ chimera FcRI-- (extracellular domain–transmembrane domain–cytoplasmic domain) with Syk kinase and with the Src-related tyrosine kinases (SRTKs) Hck and Lyn in transfected COS-1 cells. Our data indicate that FcRIIA interacts more readily with Syk than does FcRI-- and suggest that one consequence may be the greater phagocytic efficiency of FcRIIA compared with FcRI/. Furthermore, individual SRTKs affect the efficiency of phagocytosis differently for FcRI-- and FcRIIA and also influence the ability of these receptors to interact with Syk kinase. Taken together, the data suggest that differences in signaling by FcRIIA and FcRI-- are related in part to interaction with Syk and Src kinases and that individual SRTKs play different roles in FcR-mediated phagocytosis.
Key Words: ITAM • phagocytosis • Hck
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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R) expressed in hematopoietic cells [1
, 2
]. Members of each of the three classes of human FcR, FcRI, FcRII, and FcRIII, are able to induce aphagocytic signal. FcRIIA mediates phagocytosis as a single chain, whereas FcRI and FcRIIIA transmit a phagocytic signal by association with the subunit (FcRI/ and FcRIIIA/) [3
4
5
]. For FcRIIA-, FcRI/- and FcRIIIA/-mediated phagocytosis, a cytoplasmic amino acid motif termed the immunoreceptor tyrosine-based activation motif (ITAM), also present in the B cell, T cell, and Fc
RI receptor complexes, is essential [6
, 7
]. The typical ITAM, such as that found in the FcRI- and FcRIIIA-associated subunit, contains two YXXL sequences separated by seven nonconserved amino acids (Fig. 1
). In FcRIIA, 12 amino acid residues separate the two YXXLs.
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Figure 1. Sequences of the ITAM regions in the cytoplasmic domains of FcRIIA and the -chain. ITAM sequences are underlined, and tyrosine residues (Y) are highlighted in bold. In structure/function studies, tyrosines in the -chain and in FcRIIA were replaced by phenylalanine. The cytoplasmic domain of FcRIIA contains a tyrosine residue (Y1), seven amino acids upstream of the ITAM, which is not in a typical YXXL context. Similarly, human (but not murine ) contains a Y1 tyrosine seven residues upstream of the first ITAM YXXL [3
].
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R cross-linking (e.g., refs. [10
11
12
]). These doubly phosphorylated ITAMs recruit Src homology 2-containing molecules such as the protein tyrosine kinases Syk or 
-associated protein-70, as well as tyrosine phosphatases. The binding of Syk to the phosphorylated ITAM initiates a cascade of downstream signaling events that eventually leads to functional responses such as phagocytosis. Using cells derived from knockout mice deficient in individual Src kinases as well as other approaches, we and others have demonstrated that deficiencies in specific SRTKs (such as Src and Fyn) reduce the efficiency of FcR-mediated phagocytosis (e.g., refs. [13
14
15
]).
The essential role of the tyrosine kinase Syk in the phagocytic process has also been established. We have demonstrated that antisense oligonucleotides which ablate the expression of Syk significantly reduce FcR-mediated phagocytosis in cultured human monocytes [16
], and we and others have demonstrated that Syk-deficient murine macrophages do not signal for FcR-mediated phagocytosis [11
, 17
, 18
]. In addition, overexpression of Syk kinase in FcR-expressing fibroblasts significantly enhances FcR-mediated phagocytosis [19
].
We have examined the requirements for FcR-mediated signaling using transfected COS-1 cells as a model system and the phagocytosis of IgG-coated cells as a downstream signaling event (e.g., refs. [3
4
5
, 19
20
21
22
23
]). Because COS-1 cells do not express endogenous FcR, this model allows investigation of phagocytosis by individual FcR in the absence of other FcR. Using this model system, we determined that although FcRIIA and the -chain use several signaling molecules in common (e.g., Syk kinase; ref. [24
]), there are important differences between FcRIIA and the -chain in mediating phagocytosis. For example, in both transient and stable transfectants of COS-1 cells, FcRIIA is consistently more efficient in the phagocytosis of antibody-coated cells than the -chain-dependent receptors FcRI/ and FcRIIIA/ [3
]. Overexpression of Syk kinase in COS-1 cells significantly enhances phagocytosis mediated by FcR, but this enhancement is notably more pronounced for FcRI/ and FcRIIIA/ than for FcRIIA [19
]. The structural requirements of the cytoplasmic ITAM sequences involved in phagocytosis also differ for the human FcRI -chain and FcRIIA. Extensive structure/function studies have shown that substitution of the ITAM tyrosines (see Fig. 1
) with phenylalanine has different effects on FcRI/- and FcRIIA-mediated phagocytosis [3
, 5
, 21
].
In this report, we present further evidence for differences in the signaling properties of the two receptors. Our data suggest that FcRIIA associates more readily with Syk kinase than do the -chain-dependent FcR. In addition, we demonstrate that although SRTK activity is essential to mediate FcR phagocytosis in phagocytic cells, individual Src family members, such as Hck, Fgr, and Lyn, interact differently with FcRIIA than with FcRI/. These individual SRTKs affect not only the efficiency of phagocytosis but also the ability of the receptors to interact with Syk kinase. Taken together, the data suggest that the differences observed between signaling by FcRIIA and FcRI/ may be related to their ability to interact with Syk and Src kinases.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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and CD8/FcRIIA {CD8-- [extracellular domain–transmembrane domain–cytoplasmic domain (EC–TM–CY)] and CD8-IIA-IIA (EC–TM–CY) wild-type (WT) and mutant in the CY} were constructed using the two-step extension overlap polymerase chain reaction. All CD8 chimeras and the FcRIIA and FcRI-- (EC–TM–CY) cDNAs used for transient expression in COS-1 cells were subcloned into the pCDNA3.1Myc/His vector (Invitrogen, San Diego, CA).
Cell culture and transfection
COS-1 cells were cultured and maintained in Dulbecco’s modified Eagle’s medium containing glucose (4.5 mg/mL), glutamine (2 mM), streptomycin (100 U/mL), penicillin (100 µg/mL), and 10% heat-inactivated fetal bovine serum (FBS). COS-1 cells were transiently transfected with cDNAs using the calcium phosphate mammalian transfection kit (5'Prime 3'Prime, Boulder, CO), according to the manufacturer’s instructions. Following transfection, cells were incubated for 48 h before analysis.
COS-1 cell lines stably expressing the FcR FcRIIA or the chimeric receptor FcRI-- were constructed by selection for neomycin resistance using the neomycin analog G418 (500 µg/mL). G418-resistant cells were sorted for FcR expression by flow cytometry, and single-cell clones were obtained from anti-FcR antibody-positive cells by the limiting dilution method. We also prepared cells stably expressing FcRIIA or FcRIIIA-- in the human lung epithelial cell line HS24 (kindly provided by Dr. Ronald Crystal, Columbia University, New York, NY).
THP-1 cells were maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 10% heat-inactivated FBS, glutamine (2 mM), streptomycin (100 U/mL), and penicillin (100 µg/mL). Peripheral blood mononuclear cells from healthy individuals were isolated as described previously [25 ]. Briefly, the heparinized blood was centrifuged on Ficoll-Hypaque (lymphocyte separation medium, Organon Teknika, Durham, NC), and interface cells were washed twice in phosphate-buffered saline (PBS). Mononuclear cells were resuspended and maintained in RPMI-1640 medium.
Binding and phagocytosis of IgG-sensitized red blood cells [RBC; (EA)]
Antibody-coated sheep RBCs (Rockland, Gilbertsville, PA) were prepared in magnesium- and calcium-free PBS by incubating 109 sheep RBC per ml with an equal volume of the highest subagglutinating concentration of IgG rabbit anti-sheep RBC antibody (Cappel Laboratories, West Chester, PA), as described previously [4
]. The medium was removed from COS-1 transfectants, and the cells were overlaid with EA (108 cells/ml) and incubated at 37°C for 30 min. Unbound EA were removed by washing with PBS. Externally bound EA were removed by a short (40 s) hypotonic wash. The cells were stained with Wright-Geimsa, and the number of COS-1 cells with more than one internalized EA was determined in a blinded menner by light microscopy. Approximately 300 cells were counted for each determination. For the experiments with the Syk kinase inhibitor piceatannol, COS-1 cells (106 cells) stably expressing FcRIIA or FcRI-- were preincubated for 30 min at 37°C with 1 ml piceatannol (Sigma Chemical Co., St. Louis, MO) at the indicated concentrations. Following incubation, the cells were washed twice with PBS, and EA was added as described above to the assay for phagocytosis, expressed as the phagocytic index (PI), the number of EA internalized per 100 COS-1 cells. The PI was corrected for variations in cell-surface receptor expression, as determined by flow cytometry.
Monocytes and THP-1 cells (2.5x105 cells) in 150 µL PBS were incubated with EA (108 cells/ml) for 30 min at 37°C. The cells were centrifuged and subjected to a 40-s hypotonic wash to remove unbound and externally bound erythrocytes. Following cytospin onto glass slides and staining of cells with Wright-Geimsa, the PI was evaluated by light microscopy. For treatment with the SRTK inhibitor 4-amino-5-[-4-chloro-phenyl]-7-[-t-butyl]pyrazola[3,4-d]pyrimidine (PP2), 106 freshly isolated peripheral blood monocytes or THP-1 cells were incubated for 30 min at 37°C with 2 ml PP2 at concentrations varying from 1 to 10 µM. Following treatment, the cells were washed twice with PBS, and EA was added to the assay for phagocytosis.
Flow cytometry analysis
Cells were incubated on ice for 30 min with anti-FcRI monoclonal anbitody (mAb) 32.2 (for FcRI--), anti-FcRII mAb IV.3 (for FcRIIA), or anti-CD8 mAb RPA-T8 (BD PharMingen, San Diego, CA; for chimeras containing the CD8 extracellular domain). The cells were washed and labeled with fluorescein isothiocyanate-conjugated F(ab')2 goat anti-mouse IgG (Tago Inc., Burlingame, PA) for 30 min on ice. Following washing, the cells were fixed in a solution of 2% paraformaldehyde.
Immunoprecipitation and Western blotting
COS-1 cell transfectants were stimulated with EA (108 cells/ml) at 37°C for 20 min or 10 µg/ml anti-CD8 mAb (RPA-T8, BD PharMingen) for 30 min at 0°C. EA-stimulated cells were then placed on ice to inhibit further phagocytosis, and externally bound EA were removed by hypotonic lysis. Anti-CD8-stimulated cells were washed twice with ice-cold PBS and incubated for 15 min at 37°C with F(ab')2 goat anti-mouse IgG (25 µg/ml; ICN Pharmaceuticals Inc., Irvine, CA). Cells were lysed by incubation on ice for 30 min with 1.0 ml 1% Triton X-100 or 1.0 ml 1% BRIJ lysis buffer (150 mM NaCl, 25 mM Tris HCl, 1 mM EGTA). The cell lysis solutions contained the following protease inhibitors: 1 mM NaVO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 50 µg/ml leupeptin, and 100 µg/ml soybean trypsin inhibitor. Cleared lysates from stimulated cells were obtained by centrifugation at 12,000 rpm at 4°C. Cleared lysates were immunoprecipitated with anti-Syk antibody, 4 µg/ml (Upstate Biotechnology, Lake Placid, NY) or 2 µg/ml anti-Myc mAb 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Alternatively, whole cell lysates were separated directly by SDS-PAGE. Proteins transferred to nitrocellulose were immunoblotted with 0.3 µg/ml anti-Syk antibody 4D10 (Santa Cruz Biotechnology), 1 µg/ml antiphosphotyrosine mAb 4G10 (Upstate Biotechnology), or 1 µg/ml anti-Myc mAb 9E10. Immunoblots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (BioRad, Richmond, VA) and were visualized by enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, Piscataway, NJ).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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RIIA and FcRI-- with Syk kinase: competition for Syk kinaseRIIA and FcR signaling through the -chain differ in the efficiency of phagocytic signaling in transfected COS-1 cells and that there are pronounced differences in the enhancement of phagocytosis by Syk kinase for these FcR [3
, 19
, 23
]. Our studies with FcRIIA/-chain chimeras further highlight these differences. We have observed that phagocytosis is increased significantly (about twofold) in cells bearing a modified -chain in which -chain YXXL ITAM sequences were replaced by the FcRIIA YXXL ITAM sequences (Fig. 2A
). In contrast, phagocytosis in cells expressing a FcR chimera in which the -chain CY was substituted for the FcRIIA CY (IIA–IIA-) was significantly decreased compared with WT FcRIIA (16±2% that of WT IIA; Fig. 2B
). Furthermore, although accurate counting is not possible due to the degradation of ingested EA, our general observation is that even when cells are incubated for longer periods (beyond 30 min) with EA, the -chain does not achieve the phagocytic efficiency of FcRIIA in the presence or absence of Syk kinase.
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Figure 2. Effect of ITAM substitutions on phagocytosis by FcR. WT or mutant FcR were transfected into COS-1 cells. Phagocytosis of IgG-coated RBC was determined as described in Materials and Methods. (A) The -chain Y2XXL ITAM sequence YTGL was replaced with the FcRIIA Y2XXL sequence YMTL, and the -chain Y3XXL ITAM sequence YETL was replaced with the FcRIIA Y3XXL ITAM sequence YLTL in the FcRIIIA/ chimera RIIIA-- (EC–TM–CY). (B) The FcRIIA cytoplasmic domain was replaced with the -chain cytoplasmic domain. Phagocytosis by FcRIIIA-- is increased when the -chain YXXL sequences are replaced with the FcRIIA YXXL sequences, and FcRIIA phagocytosis is decreased when the FcRIIA cytoplasmic domain is replaced by the -chain cytoplasmic domain.
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R to interact with Syk kinase. To examine the possibility that Syk may interact more efficiently with FcRIIA than with the -chain, we further examined the in vitro association of Syk with these receptors. For these studies, we compared FcRIIA and the chimeric FcR/ FcRI--.
One approach was to investigate whether coexpression of FcRIIA affects the association of FcRI-- with Syk kinase (Fig. 3A
). Syk kinase was cotransfected with 1) Myc-tagged FcRIIA in COS-1 cells stably expressing untagged FcRI-- or with 2) Myc-tagged FcRI-- in COS-1 cells stably expressing untagged FcRIIA. Controls were parental COS-1 cells cotransfected with Syk and either FcRIIA or FcRI--. FcR on the cell surface were cross-linked with EA.
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Figure 3. Coimmunoprecipitation of FcRIIA (IIA) and FcRI-- (I--) with Syk kinase: competition for Syk kinase. FcR on the cell surface were cross-linked with EA. Cleared cell lysates were immunoprecipitated with anti-Syk antibody before SDS-PAGE (A) or directly subjected to SDS-PAGE (C). (A) Anti-Myc immunoblot of anti-Syk immunoprecipitates. Lane 1, Cotransfection of Syk and Myc-tagged FcRI-- in COS-1 cells stably expressing FcRIIA (FcRIIA not tagged with Myc); lane 2, cotransfection of Syk and Myc-tagged FcRIIA in COS-1 cells stably expressing FcRI-- (FcRI-- not tagged with Myc); lane 3, cotransfection of Syk and Myc-tagged FcRIIA in control COS-1 cells; lane 4, cotransfection of Syk and Myc-tagged FcRI-- in control COS-1 cells. (B) Anti-Syk immunoblot of (A) demonstrating equal amounts of Syk kinase protein in each lane. (C) Anti-Myc immunoblot of whole cell lysates. Lane 1, Cotransfection of Syk and Myc-tagged FcRI-- in COS-1 cells stably expressing FcRIIA (FcRIIA not tagged with Myc); lane 2, cotransfection of Syk and Myc-tagged FcRIIA in COS-1 cells stably expressing FcRI-- (FcRI-- not tagged with Myc); lane 3, cotransfection of Syk and Myc-tagged FcRIIA in control COS-1 cells; lane 4, cotransfection of Syk and Myc-tagged FcRI-- in control COS-1 cells. Expression of each transfected Myc-tagged FcR is comparable in control cells and in the reciprocal, stable cell line. (D) Anti-Syk immunoblot of (C) demonstrating comparable expression of Syk in each lane. (E) Analysis by flow cytometry of FcRI-- (I--) expression in COS-1 cells stably expressing FcRI-- (left) and of FcRIIA (IIA) expression in COS-1 cells stably expressing FcRIIA (right). Our data indicate that FcRI-- does not associate efficiently with Syk in COS-1 cells stably expressing FcRIIA (A, lane 1), but FcRIIA readily associates with Syk kinase in COS-1 cells stably expressing FcRI-- (A, lane 2). These data suggest that Syk interacts more efficiently with FcRIIA than with the -chain.
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RI-- readily coimmunoprecipitates with Syk kinase in the absence of FcRIIA (Fig. 3A
, lane 4), it did not coimmunoprecipitate with Syk when FcRI-- and Syk kinase were cotransfected into COS-1 cells stably expressing FcRIIA (Fig. 3A
, lane 1). In contrast, transfected FcRIIA readily associated with Syk kinase both in parental COS-1 cells (absence of FcRI--, Fig. 3A
, lane 3) and in COS-1 cells stably expressing FcRI-- (Fig. 3A
, lane 2).
These differences in FcR/Syk association cannot be attributed to how well transfected Syk is expressed in the pertinent cell lysates. Similar amounts of immunoprecipitated Syk were detected in each lane (Fig. 3B)
, and Syk expression was similar in all samples, as indicated in anti-Syk immunoblots of whole cell lysates (Fig. 3D)
. Furthermore, because we could visualize transfected receptor protein by blotting for the Myc epitope tag, we were able to demonstrate that similar amounts of transiently transfected Myc-tagged FcRIIA were expressed in parental cells (not expressing FcRI--) and cells stably expressing FcRI-- (Fig. 3C , compare lanes 3 and 2). Similar amounts of transiently transfected Myc-tagged FcRI-- were also expressed in parental cells (not expressing FcRIIA) and cells stably expressing FcRIIA (Fig. 3C
, compare lanes 4 and 1). The FcR (FcRIIA and FcRI--) stably expressed in transfected COS-1 cell lines were not epitope-tagged, and therefore, their expression levels could not be compared directly. However, analysis of FcR expression by flow cytometry indicated high levels of expression for each receptor in their respective stable cell lines (Fig. 3E)
. These observations suggest that FcRIIA can displace FcRI-- in a FcRI--/Syk complex and further suggest that FcRIIA associates with Syk more effectively than does FcRI--.
Effect of lysis conditions on the association of Syk kinase with FcRIIA and FcRI-- ITAM tyrosine mutants
Another approach to study FcR and Syk association is to compare the binding of Syk with the FcR under different buffer conditions. For these studies, we used chimeric FcR containing the EC of CD8 and the TM and ITAM-containing CY of either FcRIIA or the -chain (CD8-IIA-IIA and CD8--; Fig. 4
). The CD8 EC was used to eliminate the possibility of nonspecific binding by the Fc portion of the receptors. The Myc-tagged chimeric receptors cotransfected with Syk in COS-1 cells were cross-linked with anti-CD8/goat anti-mouse antibodies and lysed in buffers of different stringencies before precipitation with anti-Myc antibody.
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Figure 4. Effect of lysis detergent on the association of Syk kinase with the cytoplasmic domain of FcRIIA and the -chain. (A) Syk kinase cotransfected with WT or Y2F, Y3F mutants of the chimeric receptor CD8-IIA-IIA (EC–TM–CY). (B and C) Syk kinase cotransfected with WT or Y2F, Y3F mutants of the chimeric receptor CD8--. Y2F and Y3F are mutants in which the second or third tyrosine in the cytoplasmic domain is mutated to phenylalanine (see Materials and Methods). All receptors were tagged with Myc. (A and B) Immunoblots of transfected COS-1 cells lysed in Triton X-100 lysis buffer. (C) Immunoblot of transfected COS-1 cells lysed in the less-stringent BRIJ buffer. Cell lysates were precipitated with anti-Myc antibody and blotted with anti-Syk antibody. Cross-linking of the transfected FcR is indicated by + or –. The CD8 chimeras CD8-IIA-IIA and CD8-- were cross-linked with anti-CD8/F(ab')2 goat anti-mouse (G M) IgG. In support of the thesis that the cytoplasmic domain of FcRIIA binds to Syk more readily than does the cytoplasmic domain of the FcR -chain, the blots indicate that Syk coimmunoprecipitates with the Y2F mutant of CD8-IIA-IIA but not with the Y2F mutant of CD8-- in Triton buffer (stringent lysis conditions).
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- (Fig. 4B
, lanes 3 and 4) in Triton X-100 lysis buffer. Having previously established that differences exist in the requirements for ITAM tyrosines for FcRIIA and FcR/-mediated phagocytosis [20
21
22
], we examined Syk association with FcR chimeras in which the FcRIIA and -chain ITAM tyrosine residues, Y2 or Y3, were substituted with phenylalanine (F) (Y2F, Y3F CD8-IIA-IIA; Y2F, Y3F CD8--; see Fig. 1
for ITAM sequences). Syk coimmunoprecipitated with Y2F CD8-IIA-IIA (Fig. 4A , lanes 5 and 6) but not with the Y2F CD8-- (Fig. 4B
, lanes 5 and 6) in Triton X-100 lysis buffer. Under less-stringent lysis conditions (BRIJ lysis buffer, Fig. 4C
), there was indication of a weak association of Syk with Y2F CD8-- (Fig. 4C
, lane 4) when the film was overexposed. There was no evidence for Syk association with Y3F CD8-IIA-IIA in Triton X-100 (Fig. 4A
, lanes 7 and 8) or in BRIJ lysis buffer (not shown). Similarly, Syk did not associate with the Y3F CD8-- chimeras under either lysis condition (Fig. 4B
, lanes 7 and 8; Fig. 3C
, lane 3). These results, together with the data from the experiments in Figure 3
, support the thesis that FcRIIA binds to Syk more readily than does the FcR -chain. A corollary of this thesis is that the phagocytic efficiency of FcRIIA may be a reflection of the ability to use low levels of Syk kinase to mediate signaling pathways.
Effect of piceatannol on FcR-mediated phagocytosis
Having observed that inhibition of Syk kinase expression by Syk antisense oligonucleotides abrogates FcR-mediated phagocytosis in monocytes and other cells [16
], we compared FcRIIA and FcR---mediated phagocytosis in the presence of the Syk kinase inhibitor piceatannol. We and others have demonstrated that inhibition of Syk kinase activity by piceatannol occurs through blocking of Syk phosphorylation [26
, 27
]. At concentrations known to be specific for inhibition of Syk kinase activity [26
], piceatannol consistently exerted a greater inhibitory effect on FcR-- than on FcRIIA (Fig. 5
). In COS-1 cells stably transfected with FcR, preincubation with 25 µg/ml (10 µM) piceatannol for 30 min inhibited FcRIIA phagocytosis by 54 ± 10% and FcRI-- phagocytosis by 77 ± 6%; preincubation with 50 µg/ml piceatannol inhibited FcRIIA phagocytosis by 66 ± 9% and FcRI-- phagocytosis by 86 ± 1% (Fig. 5A)
. Similar results were observed in HS24 cells stably transfected with FcRIIA or FcRIIIA-- (Fig. 5B) . FcRIIIA, like FcRI, requires the -chain for signaling. In HS24 cells stably transfected with FcR, preincubation with 25 µg/ml piceatannol for 30 min inhibited FcRIIA phagocytosis by 46 ± 7% and FcRIIIA-- phagocytosis by 68 ± 5%; preincubation with 50 µg/ml piceatannol inhibited FcRIIA phagocytosis by 65 ± 2% and FcRIIIA-- phagocytosis by 88 ± 7% (Fig. 5B)
. Thus, phagocytosis mediated by FcRI/ and FcRIIIA/ is more sensitive to inhibition by piceatannol than is FcRIIA.
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Figure 5. Effect of the Syk kinase inhibitor piceatannol on FcR-mediated phagocytosis. (A) Inhibition of phagocytosis by piceatannol in COS-1 cells stably expressing FcRIIA (RIIA) or FcRI-- (RI--). (B) Inhibition of phagocytosis by piceatannol in HS24 cells stably expressing FcRIIA (RIIA) or FcRIIIA-- (RIIIA--). Phagocytosis mediated by FcR/ is more susceptible to inhibition by piceatannol than is phagocytosis mediated by FcRIIA.
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R/ is more susceptible than FcRIIA to inhibition by piceatannol suggests that FcRIIA functions more effectively than FcR/ under conditions where Syk availability is compromised. This observation is consistent with the data from the competition and buffer stringency experiments. As a consequence of efficient binding to Syk, FcRIIA retains the ability to mediate phagocytosis when little Syk is available.
These data are also consistent with our observations from previous experiments. In Chinese hamster ovary cells or COS-1 cells, which express very low levels of Syk kinase, transfected FcRIIA, unlike transfected FcR/, efficiently mediates phagocytosis. In the absence of cotransfected Syk, the PI achieved by the -chain requiring FcR is generally minimal (
20), while the PI of FcRIIA is 
100 [3
, 19
, 23
]. With increased availability (cotransfection) of Syk in COS-1 cells, the FcR/ phagocytic signal generally increases three- to fivefold. However, even under these conditions, the PI of -chain requiring FcR rises to 60–80, still more modest than that of FcRIIA, which rises to a PI of 200.
The effect of SRTKs on FcRIIA and FcRI---mediated phagocytosis
Although the association of Syk with FcR is recognized as an early event in the signaling cascade for phagocytosis, the earliest detectable step following ligation of FcR at the cell surface is the activation of SRTKs [10
, 11
]. SRTKs thus activated, phosphorylate the cytoplasmic ITAM tyrosines of immunoreceptors, allowing them to become docking sites for activating molecules such as Syk kinase. We examined whether overexpression of individual SRTKs has the ability to modify the Syk/FcR association and the efficiency of FcR phagocytosis.
Previous work from this laboratory demonstrated that individual SRTKs can exert different effects on FcR-mediated phagocytosis [13
, 14
], and we and others have reported that mouse macrophages derived from knockout mice deficient in expression of the monocyte SRTKs Fgr, Hck, Lyn, and Src exhibit decreased FcR phagocytic efficiency [13
, 15
]. In these studies, the absence of an individual SRTK did not completely eliminate phagocytosis, suggesting that different Src family members can function in signaling for phagocytosis and that different SRTKs can functionally compensate for each other.
We confirmed the essential role of SRTKs in mediating an efficient FcR-mediated phagocytic signal using the PTK inhibitor PP2, which is selective for the Src family of tyrosine kinases [28
]. Our data indicate that PP2 inhibits FcR-mediated phagocytosis in freshly isolated peripheral blood monocytes (Table 1A
) and in the monocytic cell line THP1 (Table 1B)
. In human monocytes, phagocytic activity was virtually eliminated by 10 µM PP2 (97% reduction), and in THP1 cells, an 85–95% reduction in phagocytic efficiency was observed. Treatment with 10 µM PP2 did not disturb cell viability. Our results are thus consistent with other studies that report a decrease in FcR phagocytic efficiency in mouse macrophages derived from mice deficient in the expression of monocyte SRTKs [15
].
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Table 1. Effect of the SRTK Inhibitor PP2 on FcR-Mediated Phagocytosis
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R to associate with Syk kinase, we overexpressed individual SRTKs in COS-1 cells stably expressing FcR. Of special interest was the overexpression of the monocyte/macrophage-abundant SRTKs Hck, Lyn, and Src. Overexpression of Hck produced the greatest effect on FcRI---mediated phagocytosis (Fig. 6A
). We observed a 60% increase in the efficiency of FcRI---mediated phagocytosis in cells transfected with Hck compared with cells in which no SRTK was transfected. Transfection of the SRTKs Lyn and Src did not induce this effect. Transfection of Hck, Lyn, and Src kinases did not enhance the phagocytosis of EA in COS-1 cells stably expressing FcRIIA (Fig. 6B)
.
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Figure 6. Effect of individual SRTKs on FcR-mediated phagocytosis. Results are expressed as the percent change in PI compared with control cells not expressing transfected SRTK. (A) FcR-mediated phagocytosis evaluated in COS-1 cells stably expressing FcRI--. Cells were cotransfected with Hck, Lyn, or Src plus Syk kinase (N=10 for Hck; N=7 for Lyn). (B) Phagocytosis evaluated in COS-1 cells stably expressing FcRIIA. Cells were transfected with Hck, Lyn, or Src kinase (N=3). FcRIIA phagocytosis was determined for cells not cotransfected with Syk kinase, because addition of Syk increases phagocytosis to an extent that accurate determination of EA ingestion is difficult. Addition of Syk did not alter the effects of cotransfected SRTKs on FcRIIA-mediated phagocytosis. Overexpression of Hck produced the greatest positive effect on FcRI---mediated phagocytosis, illustrating that a specific SRTK can influence -chain-mediated phagocytic efficiency. Hck did not increase the efficiency of FcRIIA-mediated phagocytosis, illustrating that the effects of individual SRTKs may be receptor-specific.
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RI-mediated phagocytosis in transfected COS-1 cells. As demonstrated by Western blot, the expression of transfected Hck and Lyn (tagged with the Myc epitope) was similar in these experiments (Fig. 7A
). A direct, quantitative comparison of endogenous Hck and Lyn expression could not be determined, because different antibodies were required. However, it is evident that little or no Hck or Lyn is expressed in nontransfected COS-1 cells (Fig. 7B
and 7C)
. It is thus unlikely that the observed differences in phagocytosis are a result of large differences in the expression of the individual SRTKs.
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Figure 7. Expression of Hck and Lyn in COS-1 cells. (A) Expression of transfected Myc-tagged Lyn (lane 1) or Myc-tagged Hck (lane 2) in COS-1 cell lysates. Anti-actin blot (A, lower panel) demonstrates equality of protein loading. (B and C) Expression of total Hck and total Lyn in transfected COS-1 cells. Proteins in cell lysates were immunoblotted with anti-Hck (B, upper panel) or anti-Lyn (C, upper panel). (B and C, lane 1) Expression of endogenous Hck (B) or Lyn (C) in nontransfected COS-1 cells; lane 2, expression of transfected plus any endogenous SRTKs in COS-1 cells transfected with Hck (B) or Lyn (C). Anti-actin blots (B and C, lower panels) demonstrate the similarity of protein loading in lanes 1 and 2. The anti-Myc blots (A) indicate that the expression of transfected Hck and Lyn is similar in these experiments. In addition, although direct quantitative comparison of Hck and Lyn expression cannot be determined, it is evident that little or no endogenous Hck or Lyn is expressed in nontransfected COS-1 cells.
|
R-mediated phagocytosis are different for FcRI/ and FcRIIA, these data provide further evidence for the importance of individual SRTKs as upstream regulators of FcR-mediated phagocytosis [29
].
Interaction of Syk kinase with SRTKs Hck and Lyn
Because overexpression of Hck appeared to enhance the ability of FcRI-- to phagocytose EA in COS-1 cell transfectants, we considered the possibility that these SRTKs may boost phagocytic efficiency through interaction with Syk kinase. We therefore examined Syk/SRTK association as well as Syk tyrosine phosphorylation in cells coexpressing FcRI--, Syk, and 1) Hck kinase, which appears to have a positive effect on FcRI---mediated phagocytosis, or 2) Lyn kinase, which does not appear to enhance FcRI---mediated phagocytosis.
Figure 8
demonstrates that following cross-linking of FcRI-- with EA in cells expressing WT FcRI--, Syk coprecipitates with both Hck and Lyn and is highly phosphorylated on tyrosine (Fig. 8A and 8B
, lanes 1 and 2). There are, however, basic differences in Syk association with Hck and Lyn in cells expressing mutants of FcRI--. The association of Syk with Hck and the tyrosine phosphorylation of Hck-associated Syk were still vigorous in transfectants of Y2F FcRI-- (Fig. 8A
and 8B
, lane 4), and although reduced, Syk/Hck association and Hck phosphorylation were still significant in transfectants of Y3F FcRI-- (Fig. 8A and 8B
, lane 6). In contrast, the association of Syk with Lyn was severely inhibited, and the tyrosine phosphorylation of Lyn-associated Syk was minimal to nonexistent in Y2F FcRI-- (Fig. 8A
and 8B
, lane 3) and Y3F FcRI-- mutants (Fig. 8A
and 8B , lane 5). These data suggest that Hck may modify the requirements for Syk/FcR/-productive association.
 View larger version (39K): [in a new window] |
Figure 8. Association of Syk with Hck and Lyn in COS-1 cells cotransfected with WT or mutant FcRI--. Cells were cotransfected with Syk, either Hck or Lyn, and either WT or the Y2F or Y3F mutants of FcRI--, as indicated. Lysates were immunoprecipitated with anti-Lyn antibody (lanes 1, 3, and 5) or anti-Hck antibody (lanes 2, 4, and 6) and blotted with (A) antiphosphotyrosine (Anti-pTyr), (B) anti Syk antibody, (C) anti-Hck antibody, or (D) anti-Lyn antibody. pSyk indicates Phosphorylated Syk. Syk associates more readily with Hck than with Lyn, and tyrosine phosphorylation of Hck-associated Syk is greater than tyrosine phosphorylation of Lyn-associated Syk in cells expressing ITAM mutants of FcRI--.
|
RI--RI-- (Fig. 9
). The transfected FcR were immunoprecipitated with anti-Myc (receptor) antibody and blotted with anti-Syk (Fig. 9A)
or antiphosphotyrosine antibody (Fig. 9B)
. As expected, Syk associated with WT FcRI-- and was tyrosine-phosphorylated both in the absence and the presence of cotransfected SRTKs (Fig. 9A
, lanes 1–3). As an approach to determine whether Hck modifies the Syk/FcRI-- interaction, we examined the effect of Hck expression on the coimmunoprecipitation of Syk with mutants of FcRI--. Having previously demonstrated that the C-terminal ITAM tyrosine (Y3) is essential for -chain-mediated phagocytosis and FcR association with Syk [3
, 21
, 22
], we found of particular relevance the interaction of the -chain mutant Y3F with Syk in Hck-transfected cells. We observed that Syk coimmunoprecipitated with the Y3F mutant only in the presence of Hck (Fig. 9A
, lane 5) and not in the presence of Lyn (lane 6) or in the absence of these SRTKs (lane 4). Furthermore, the Syk associated with Y3F FcRI-- in cells expressing Hck was phosphorylated on tyrosine (Fig. 9B
, lane 5).
 View larger version (35K): [in a new window] |
Figure 9. Effect of Hck on association of Syk kinase with FcRI--. COS-1 cells were cotransfected with Syk kinase, Myc-tagged WT FcRI--, or Myc-tagged Y3F FcRI-- and Hck or Lyn kinases as indicated. Lysates were blotted with (A) anti-Syk or (B) antiphosphotyrosine (Anti-pTyr) antibody. The position of FcR-associated Syk kinase is indicated by an arrow. pSyk indicates phosphorylated Syk. These blots indicate that Syk coimmunoprecipitates with the Y3F FcRI-- mutant and undergoes a degree of phosphorylation on tyrosine(s) in the presence of cotransfected Hck but not in the presence of cotransfected Lyn.
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R//Syk kinase phagocytic signaling in professional phagocytes by promoting a more stable complex for Syk in a FcR//Syk/SRTK complex. However, the mechanism by which Hck enhances FcR function is still unclear. Our data suggest that Hck promotes association of Syk and FcRI/. Tyrosine phosphorylation of Syk by Hck may facilitate association of Syk with a -chain lacking an ITAM tyrosine; however, overexpression of Hck in cells transfected with Y3F FcRI-- and Syk was not in itself sufficient to set into motion the machinery for phagocytic signaling.
In summary, although signaling by FcRIIA and -chain-dependent receptors are similar in many ways, distinct differences exist in the manner in which the receptors interact with components of the phagocytic signaling cascade. Our data indicate that FcRIIA associates with Syk kinase more readily than does FcR/ and suggest that individual SRTKs play an important role in modulating the interaction of FcR with Syk kinase, essential for propagating the FcR phagocytic signal. Furthermore, these observations indicate that although different SRTKs may play a compensatory role under some conditions [13
, 14
], individual SRTKs are not completely interchangeable in their interaction in FcR/Syk kinase signaling.
Received November 13, 2003; revised March 3, 2004; accepted April 6, 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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