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Protein-tyrosine kinase Syk is required for pathogen engulfment in complement-mediated phagocytosis

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Protein-tyrosine kinase Syk is required for pathogen engulfment in complement-mediated phagocytosis
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  PHAGOCYTES Protein-tyrosinekinaseSykisrequiredforpathogenengulfmentincomplement-mediatedphagocytosis Yuhong Shi, Yumi Tohyama, Tomomi Kadono, Jinsong He, S. M. Shahjahan Miah, Ryoichi Hazama, Chisato Tanaka,Kaoru Tohyama, and Hirohei Yamamura The protein tyrosine kinase Syk plays acentral role in Fc   receptor–mediatedphagocytosis in the adaptive immunesystem. We show here that Syk alsoplays an essential role in complement-mediated phagocytosis in innate immu-nity. Macrophage-like differentiatedHL60 cells and C3bi-opsonized zymo-san comprised the pathogen-phagocytesystem. C3bi-opsonized zymosan par-ticles promptly attached to the cells andwere subsequently engulfed via comple-ment receptor 3. During this process,Syk became tyrosine phosphorylatedand accumulated around the nascentphagosomes. The transfer of Syk-siRNAor dominant-negative Syk (DN-Syk) intoHL60 cells resulted in impaired phagocy-tosis. Quenching assays using fluores-cent zymosan revealed that most of theattached zymosan particles were locatedinside parental HL60 cells, whereas fewwere ingested by the mutant cells. Thesedata indicated that Syk is required for theengulfment of C3bi-opsonized zymosan.During C3bi-zymosan–induced phagocy-tosis,actinaccumulationoccurredaroundphagosomesandwasfollowedbydepoly-merization, and further RhoA was acti-vatedtogetherwithtyrosinephosphoryla-tion of Vav. These responses includingthe actin remodeling were suppressed inSyk-siRNA– or DN-Syk–expressing cells.Our results demonstrated that Syk playsan indispensable role in complement-mediated phagocytosis by regulatingboth actin dynamics and the RhoA acti-vation pathway and that these functionsof Syk lead to phagosome formationand pathogen engulfment. (Blood. 2006;107:4554-4562) ©  2006 by TheAmerican Society of Hematology Introduction Phagocytosis is a central event in the innate immune responses thatare triggered by the association between ligands on the surface of pathogens and receptors on the membrane of phagocytes. Phago-cytes then engulf and eliminate the pathogens. Among the phago-cytic receptor types expressed on mammalian neutrophils andmacrophages, Fc   receptors and complement receptor 3 (CR3)have been characterized in detail. The clustering of these receptorsby their ligands bound to pathogens is followed by the activation of signaling pathways that trigger dynamic rearrangements of thecytoskeleton, which lead to the formation of phagosomes and theirfusion to the lysosome. 1,2 Some signaling molecules such asRho-GTPases, ADP-ribosylation factor 6 (ARF6), and PLC  regulate actin polymerization during phagosome formation. 3-5 The complement system comprises many distinct plasma pro-teins that react with one another to opsonize pathogens and inducea series of inflammatory responses that help to fight infection. 6,7 The most important action in this system is the acceleration of theuptake and destruction of pathogens by phagocytes. This comple-ment-mediatedphagocytosisbeginswiththespecificrecognitionof bound complement components by complement receptors (CRs).In these combinations, C3bi binding to CR3 is a highly effectivesignalbecausethisinteractionissufficienttostimulatephagocytosis.The protein tyrosine kinase Syk is expressed in a wide rangeof hematopoietic and nonhematopoietic cells and it plays a keyrole in immunoreceptor (B-cell receptor and Fc receptors)signaling including Fc  receptor–mediated phagocytosis. 8-14 Onthe other hand, the role of Syk in nonadaptive immunemechanisms is not well understood and the roles of Syk in innateimmunity have received considerable focus. 15 Besides theseroles in immunoreceptor function, Syk is also activated uponligation with cell-surface integrins, 16 such as integrin  IIb  3 inplatelets 17,18 or  2 integrin in neutrophils 19-21 or monocytic celllines. 22,23 Among complement receptors, CR3 (integrin  M  2) and CR4(integrin  X  2) are the main phagocytic receptors and both typesbelong to the integrin receptor family. Considering the particularrole of Syk in integrin signaling, Syk should play a critical role ininnate immunity through complement-mediated phagocytosis.The present study examines the role of Syk in complement-mediated phagocytosis using macrophage-like differentiated HL60cells and C3bi-opsonized zymosan. Our results indicated that Syk plays a crucial role in the process from phagosome formation toengulfment by controlling the accumulation and disassembly of polymerized actin and Rho-GTPase activation. From the Department of Genome Sciences, Kobe University Graduate Schoolof Medicine, Kobe; and Department of Laboratory Medicine and ClinicalPathology, Kawasaki Medical School, Kurashiki, Japan.Submitted September 8, 2005; accepted January 17, 2006. Prepublishedonline as  Blood   First Edition Paper, January 31, 2006; DOI 10.1182/blood-2005-09-3616.Supported in part by a Grant-in-Aid for Scientific Research from the JapanSociety for the Promotion of Science, the 21st Century Centers of Excellence(COE) Program of the Ministry of Education, and the Osaka Medical ResearchFoundation for Incurable Diseases.Y.S., T.K., J.H., S.M.S.M., R.H., and C.T. performed research; Y.T. designedresearch, analyzed data, and wrote the paper; K.T. analyzed data; and H.Y.organized this research project and analyzed data. Reprints:  Yumi Tohyama, Department of Genome Sciences, Kobe UniversityGraduate School of Medicine, Kobe 650-0017, Japan; e-mail: ytohyama@med.kobe-u.ac.jp.The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’in accordance with 18 U.S.C. section 1734. © 2006 by TheAmerican Society of Hematology4554 BLOOD, 1 JUNE 2006    VOLUME 107, NUMBER 11  Materials and methods Reagents and antibodies ZymosanAand RPMI 1640 medium were purchased from Sigma (St Louis,MO). Anti-Syk polyclonal antibody (polyAb; N-19), anti-Syk monoclonalantibody (monoAb; 4D10), anti-PLC  2 polyAb, and anti-Vav polyAb(C-14) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).Anti-C3bi monoAb was obtained from Quidel (Santa Clara, CA).Antiphos-photyrosine (4G10) monoAb was from Upstate Biotechnology (LakePlacid, NY). Hygromycin B was from Wako Pure Chemical Industries(Osaka, Japan). Penicillin-Streptomycin Mixed Solution was from NacalaiTesque (Kyoto, Japan). Antihuman integrin  M (CD11b) for immunoblot-ting was from R&D Systems (Minneapolis, MN). Antihuman CD11b forflow cytometry was from DAKO (Glostrup, Denmark).Antihuman integrin  M (CD11b) activating monoAb VIM12 F(ab) 2  was from Orbigen (SanDiego, CA). 24 A C3-convertase inhibitor, compstatin, is a 13–amino acidcyclic peptide that binds to and inhibits cleavage of C3. 25,26 The acetylatedform of compstatin (Ac-ICVVQDWGHHRCT-NH2) and the control pep-tide (IAVVQDWGHHRAT-NH2) were purchased from Peptide Institute(Osaka, Japan) and Sigma-Aldrich (Tokyo, Japan), respectively. Cells and cell culture The human promyelocytic cell line HL60 was maintained in RPMI 1640medium supplemented with 8% heat-inactivated fetal calf serum (FCS),100 U/mL penicillin, and 100  g/mL streptomycin in 5% carbon dioxide(CO 2 )humidifiedairat37°C.Thecellswereinducedtoundergodifferentia-tion to macrophages by seeding them on dishes at a concentration of 2  10 6  /100-mm dish in the presence of 10  7 M vitamin D 3  and 10 ng/mL12- O -tetradecanoylphorbol-13-acetate (TPA) and incubating for 3 days.Cell differentiation was confirmed morphologically by evaluating CR3 withflow cytometry. Separation of human primary monocytes Human monocytes were collected using the magnetic cell sorting (MACS)system and microbeads conjugated with monoclonal mouse anti–humanCD14 antibodies purchased from Miltenyi Biotec (Bergisch Gladbach,Germany). Briefly, peripheral blood mononuclear cells of healthy blooddonors were isolated by density gradient centrifugation using Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden). CD14-expressingcells were positively separated by MACS according to the protocolprovided by the manufacturer. The purity of the CD14  cell fraction wasconsistently over 90%. Plasmid and transfection Human  syk   cDNA, provided by Muller et al, 27 was modified as previouslydescribed. 28 The polymerase chain reaction (PCR) product of the Flag-tagged dominant-negative (DN) form of human  syk   gene expressing onlythe first 261 amino acids of Syk protein was subcloned into pcDNA3.1/ Hygro (Invitrogen, Carlsbad, CA).To inhibit  syk   gene expression, a vector for short interfering RNA (siRNA)expression, the pSilencer 2.1-U6 hygro (Ambion, Austin, TX), was used.Synthetic oligonucleotides for the sense and antisense target sequences of the human Syk-coding sequences including bp 1192 to 1210 and controlsequenceswithstem-loopsequencetoformthesmallhairpinRNA(apairof oligonucleotides for annealing is 5  -GATCCACCGTGGCTGTGAAAAT-ACTTCAAGAGAGTATTTTCACAGCCACGGTTTTTTTGGAAA-3  and5  -AGCTTTTCCAAAAAAACCGTGGCTGTGAAAATACTCTCTTGAA-GTATTTTCACAGCCACGGTG-3  )wereannealedandligatedinto  Bam HI-and HindIII-cleaved backbone of pSilencer 2.1-U6 according to themanufacturer’s instructions. Control sequences with stem-loop sequence toform the small hairpin RNA (a pair of oligonucleotides for annealing is5  -GATCCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTG-ACACGTTCGGAGAATTTTTTGGAAA-3   and 5  -AGCTTTTCCAAA-AAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACG-TTCGGAGAACG-3  ) were annealed in the same way. Plasmids weretransfected into HL60 cells by electroporation, and positive clones wereselected with 1 mg/mL hygromycin B.To support the knockdown effects of Syk-siRNA, we constructed aFlag-rescue–Syk expression vector containing 4 silent mismatches in theknockdown oligonucleotide sequence, 5  -ACGGTCGCAGTCAAAATAC-3  ,corresponding to nucleotides 1192 to 1210 (mutation points are indicatedby the underscores) by two-step overlap extension PCR. To amplify 2fragments between 839 and 867, sense primer (5  -ATCCTGCGACTTGGT-CAGCGGGTGGAATA-3  ) or 1512-1540 antisense primer (5  -CGAAAT-CACTGATCTTGGCGTAATGTTGG-3  ) and the specific mutation primerof the appropriate sense or antisense strand were used. The sense strandsequences of the mutation primers were 1186-1223 sense primer (5  -GTGAAAACGGTCGCTGTGAAAATACTGAAAAACGAGGC-3  ) for1194C  G and 1197G  C and additionally 1192-1229 (5  -ACGGTCG-CAGTCAAAATACTGAAAAACGAGGCCAATGA-3  )for1200T  Aand1203G  C. Two fragments were combined by the PCR with the 839-867sense primer and 1512-1540 antisense primer and digested with  Sca I. The Sca I fragment was inserted between the  Sca I sites of Syk. Mutations wereverified by sequencing both strands (ABI PRISM Cycle Sequencing FSReadyReactionKit;AppliedBiosystems,FosterCity,CA).TheFlag-rescue–Syk was subcloned in the expression vector pcDNA4/TO (Invitrogen).Flag-rescue–SykwasintroducedintoSyk-siRNA/HLcellsbyelectropo-ration and positive clones were selected with 0.2 mg/mL zeocin (Invitro-gen) for pcDNA4/TO. The expression of Flag-tagged DN-Syk or Flag-rescue–Syk was confirmed by immunoblotting analysis with the anti-Flagepitope antibody M2 (Sigma) and anti-Syk polyAb. The effects of siRNAon expression of Syk were also confirmed by immunoblotting analysis withanti-Syk polyAb. Phagocytosis assay To opsonize zymosan with C3bi, complement activation cascade in serumwas used. Zymosan A was incubated in 50% human serum at 37°C for 30minutes in the presence or absence of compstatin or control peptide andthen washed with PBS twice at 4°C. Binding of C3bi to zymosan wasconfirmed by flow cytometry with anti-C3bi antibody. For comparison,zymosan was also treated with PBS at 37°C or 50% human serum at 4°C. Insome cases proteinAcoupled to agarose (Pierce, Rockford, IL) was used toremove IgG from human serum. Serum IgG concentration was measured bynephelometry using the IMMAGE Immunochemistry System (BeckmanCoulter, Fullerton, CA). As a result of repeated treatment with protein A,IgG concentration in the treated serum was decreased from 12.5 mg/mL tothe undetectable level.C3bi-opsonized or nonopsonized zymosan was added to macrophage-like differentiated HL60 cells (cell–zymosan particle ratio, 1:10) andincubated for indicated times at 37°C in a 5% CO 2  humidified atmosphere.In some cases, phagocytosis was recorded by a time-lapse microscope(Olympus IX71 microscope, Olympus, Tokyo, Japan; and CoolSNAPcf camera, Roper Scientific, Tucson,AZ) every 15 seconds for indicated timesand analyzed by an image analyzing software, Mac SCOPE (Mitani, Fukui,Japan). In the case of quantitative analysis of phagocytosis assay, TexasRed zymosan (Sigma) was used similarly as described for zymosan Aand analyzed by flow cytometry (FACSCalibur; Becton Dickinson, SanJose, CA). In some experiments, cells were treated with serum-opsonized or nonopsonized Texas Red zymosan for 10 minutes at 0°C or37°C, washed with PBS, further incubated for 10 minutes at 37°C, andanalyzed by flow cytometry.To make a distinction whether zymosan particles exist inside or outsidethe cells, AlexaFluor 488 zymosan (Invitrogen) was opsonized and phago-cytosis assay was performed, then the cells were analyzed by fluorescencemicroscopy before and after treatment of 0.2% trypan blue in PBS. Analysis for actin accumulation around phagosomes Phagocytosisassaywasstarted,andthenat10minutesafterincubationwithC3bi-zymosan, cells were washed with medium 2 times to remove unboundC3bi-zymosan and then reincubated for indicated times at 37°C in a 5%CO 2  humidified atmosphere. Cells were fixed and then incubated withAlexaFluor 594–labeled phalloidin (Invitrogen) and observed with a ROLES OF Syk IN COMPLEMENT-MEDIATED PHAGOCYTOSIS 4555BLOOD, 1 JUNE 2006    VOLUME 107, NUMBER 11  confocal laser-scanning microscope (LSM 510 META; Carl Zeiss,Oberkochen, Germany). Immunoprecipitation and immunoblotting Cells were lysed with lysis buffer (0.05% SDS; 0.5% sodium deoxy-cholate; 50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 5 mM EDTA; 2 mMphenylmethylsulfonyl fluoride [PMSF]; 1 mM Na 3 VO 4 ; 20   g/mLaprotinin). Cellular debris was sonicated and removed by centrifugation.In some cases, cell lysate was immunoprecipitated with anti-Syk antibody or anti-Vav antibody at 4°C for 60 minutes and then incubatedwith protein A or G–Sepharose beads for 60 minutes, washed 3 times,and subjected to immunoblotting. For detection of tyrosine phosphoryla-tion proteins, agarose-conjugated antiphosphotyrosine monoAb (clone4G10) was directly used. Cell lysates or immunoprecipitated sampleswere separated in sodium dodecyl sulfate–polyacrylamide gel electro-phoresis (SDS-PAGE) and transferred to a polyvinylidene difluoridemembrane (Millipore, Bedford, MA). The membrane was blockedwith 5% skim milk in T-TBS (25 mM Tris-HCl, pH 8.0; 150 mMNaCl; 0.1% Tween 20) for 60 minutes at room temperature and thenincubated with the appropriate antibodies. The membrane was washed 3times with T-TBS and incubated with horseradish peroxidase–conjugated goat antirabbit or goat antimouse antibodies for 30 minutes,and specific proteins were detected using an enhanced chemilumines-cence immunoblotting system. Preparation of GST-rhotekin RBD proteins For the preparation of GST-rhotekin Rho binding domain (RBD) proteins,RBD cDNA corresponding to the 7-89 amino acid residues of mouserhotekin 29 that were produced by PCR was incorporated into pGEX4T-3and subsequently introduced into  Escherichia coli  DH5  . Expression of theproteins was induced with Isopropyl   - D -thiogalactopyranoside (IPTG;Nacalai Tesque), and the proteins were purified with GST-Sepharose beads. GST pull-down assay for activated RhoA GST pull-down assay was performed as previously described. 30 Briefly,2  10 6 cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.2; 1%Triton X-100; 100 mM NaCl; 10 mM MgCl 2 ; and 2 mM PMSF). Aftercentrifugation (15 000 g , 15 minutes, 4°C), lysates were incubated withGST-rhotekin RBD immobilized on glutathione-Sepharose 4B beads (Am-ersham Biosciences, Uppsala, Sweden) for 60 minutes at 4°C. Afterwashing, the proteins on the beads were run on SDS-PAGE. GTP-boundRhoAwas detected by immunoblotting with anti-RhoApolyclonal antibody(Santa Cruz Biotechnology). Statistical analysis In some experiments, statistical significance was determined by theStudent  t   test. Results Syk is tyrosine phosphorylated during complement-mediatedphagocytosis in macrophage-like differentiated HL60 cells We investigated the role of Syk in innate immunity, especially incomplement-mediated phagocytosis, using macrophage-like differ-entiated HL60 cells incubated with vitamin D 3  and TPA for 3 daysas described. 31 Three days thereafter, the cells became morphologi-cally macrophage-like and the cell surface expression of CR3(  M  2 integrin/CD11bCD18) gradually increased (Figure 1A, toppanel), whereas the amount of Syk remained unchanged (Figure1A, bottom panel).We studied the mechanism of phagocytosis mediated bycomplement activation using C3bi-opsonized zymosan, the mem-brane particles of   Saccharomyces cerevisiae,  in human serum at37°C or 4°C. Activation of the complement pathway was thenexamined by flow cytometry using anti-C3bi antibody. C3bi boundto zymosan at 37°C but not at 4°C. In contrast, in the presence of aC3-convertase inhibitor, compstatin, 25,26 binding of C3bi to zymo-san was significantly suppressed but not in the presence of a controlpeptide (Figure 1B). Using complement-opsonized zymosan, weassayed phagocytosis on macrophage-like HL60 cells after incuba-tion with vitamin D 3  andTPAfor 3 days (Day3-HL60). Microscopy Figure 1. Complement-mediated phagocytosis usingmacrophage-likedifferentiatedHL60cellsandserum-treatedzymosan. (A)ExpressionoftheCR3/integrin  Min macrophage-like differentiated HL60 cells. HL60 cellswere treated with 10  7 M vitamin D 3  (VD3) and 10 ng/mLTPA for indicated days and the expression of the CR3/ integrin   M and Syk was examined by immunoblotting(IB) analysis with the corresponding antibodies. The blotis a representative of 3 independent experiments. (B)Binding of C3bi to zymosan. To opsonize zymosan withC3bi, zymosan was incubated in 50% human serum at37°C for 30 minutes in the presence or absence ofcompstatin or control peptide and then washed with PBStwice at 4°C. For comparison, zymosan was also treatedwith PBS at 37°C for 30 minutes or in 50% human serumat 4°C. Binding of C3bi to zymosan was confirmed byflow cytometry with anti-C3bi antibody (thick line) or withcontrol mouse IgG (thin line). The representative flowcytometric patterns (left) and the ratio of mean fluores-cence intensity (anti-C3bi antibody/control IgG) with SDoftriplicateexperimentsattheindicatedconditions(right)are presented. The statistically significant difference wasassessed by the Student  t   test; * P   .05. (C) Phagocyto-sisofzymosanparticlesbyHL60cells.Serum-opsonized(top panel) or nonopsonized zymosan (bottom panel)was added to macrophage-like differentiated Day3-HL60cells (cell-zymosan ratio, 1:10) and incubated at 37°C.Phagocytosis was recorded by a time-lapse microscopeevery 15 seconds and analyzed by MacSCOPE imageanalyzing software. An LCPlan 20   /0.40 numeric aper-ture(NA)objectivewasusedtovisualizetheimages.Thebar indicates 10  m. 4556 SHI et al BLOOD, 1 JUNE 2006    VOLUME 107, NUMBER 11  showed that C3bi-opsonized, but not nonopsonized, zymosanparticles promptly attached to and were absorbed by the cells(Figure 1C). Zymosan incubated with serum in the presence of compstatin or incubated at 4°C did not become attached (data notshown). In addition, to confirm that our methods are also adequatefor primary phagocytes, we isolated human monocytes from theperipheral blood and performed phagocytosis assay. Primarymonocytes also adsorbed C3bi-opsonized but not nonopsonizedzymosan particles (data not shown).To determine whether Syk is involved in complement-mediatedphagocytosis, Day3-HL60 cells were incubated with zymosanpretreated with PBS, human serum, IgG-removed serum, serum inthe presence of compstatin, or a control peptide. Figure 2A (toppanel) shows that Syk was tyrosine phosphorylated after incubationwith zymosan pretreated with both serum and IgG-removed serum(C3bi-zymosan) but not with zymosan pretreated with either PBSor with serum in the presence of compstatin. Further, cross-linkingwith CR3-activating F(ab) 2  antibody VIM12 caused tyrosinephosphorylation of Syk (Figure 2A, top panel). At 5 minutes afterthe onset of phagocytosis, Syk obviously accumulated in the regionof forming phagosomes (Figure 2B). Dominant-negative Syk and Syk-siRNAdo not affect thecomplement receptor 3 expression To further investigate the effect of Syk on the mechanism of complement-mediated phagocytosis, DN-Syk (a truncated formof human Syk that contains only tandem SH2 domains with nocatalytic domain that interrupts endogenous active Syk kinase)or Syk-siRNA was introduced into HL60 cells, and cell clonesstably expressing DN-Syk (DN-Syk/HL) and cell clones stablyexpressing Syk-siRNA (Syk-siRNA/HL) were isolated. Further,to support the effects of Syk-siRNA, Flag-rescue–Syk wastransferred into Syk-siRNA/HL clone 6 (cl6) cells and stablemutant was isolated. The expression was confirmed by immuno-blotting analyses (Figure 3A-B). In DN-Syk/HL, tyrosinephosphorylation of Syk induced by C3bi-zymosan was sup-pressed (Figure 3A, bottom panel). Introduction of Syk-siRNAsignificantly reduced the expression of endogenous Syk (clone2, 85%; clone 6, 80% suppression) but transfer of Flag-rescue–Syk showed the restoration of Syk expression (Figure 3B). Flowcytometry showed that the level of CR3 surface expression inthese cell clones was identical to that of parental Day3-HL60cells (Figure 3C). Transfer of either DN-Syk or Syk-siRNAresulted in theinhibition of phagocytosis To determine whether Syk is required for complement-mediatedphagocytosis, we analyzed phagocytic activity using fluorescence-labeled zymosan. Day3-HL60 cells were incubated with zymosanparticles that were pretreated with PBS, serum, IgG-removedserum, serum containing compstatin (10   M), or the controlpeptide to compstatin (10   M) for 30 minutes at 37°C and thenanalyzed by flow cytometry to detect phagocytosis of fluorescentzymosan. Figure 4A shows the representative patterns of flowcytometry and Figure 4B shows the result of quantitative analysis.The fluorescence intensity was increased and shifted to the M2region in the cells incubated with C3bi-zymosan but not in thosewith PBS-treated zymosan. Removal of IgG from the serumrevealed the similar phagocytosis of C3bi-zymosan, whereas thisphagocytosis was clearly inhibited in the presence of compstatinbut not in the presence of a control peptide. These results indicatedthat C3bi-CR3 binding in this system was specific. Next, phago-cytic activity was analyzed in primary monocytes and macrophage-like differentiated HL60 cells under the 2 binding conditions of C3bi-zymosan to CR3: at 0°C or 37°C before incubation at 37°C.In both cells the percentage of C3bi-zymosan–positive cellsincreased depending upon C3bi binding at 37°C (Figure 4C). Theeffects of DN-Syk and Syk-siRNA on phagocytic activity wereanalyzed using C3bi-opsonized zymosan. About 30% of parentalDay3-HL60 cells were zymosan positive but both the transfer of DN-Syk and the transfer of Syk-siRNAreduced this ratio to 10% to20%. Transfer of control siRNA did not affect the phagocytosis,and transfer of Flag-rescue–Syk restored the phagocytosis (Figure 4D).ThesedatashowedthatSykplaysasignificantroleintheprocessof C3bi-CR3–mediated phagocytosis. Syk is essential for pathogen engulfment rather than forattachment via complement receptor To determine whether fluorescent zymosan particles are intracellu-larly or extracellularly located, the cells were stained with trypan Figure 2. Syk is tyrosine phosphorylated in the process of complement-mediated phagocytosis.  (A) Tyrosine phosphorylation of Syk by the treatment withserum-opsonized zymosan. Macrophage-like differentiated Day3-HL60 cells wereincubated with zymosan particles that were treated with PBS, serum, IgG-removedserum, serum containing compstatin (10   M), or the control peptide to compstatin(10  M) for 15 or 30 minutes (top). Macrophage-like differentiated Day3-HL60 cellswere stimulated with CR3-activating F(ab) 2  antibody VIM12 (2  g/mL; bottom). Celllysates were immunoprecipitated (IP) with antiphosphotyrosine (anti-PY) monoAband immunoblotting analysis was performed with anti-Syk polyAb. As to the wholecell lysates, immunoblotting analysis was also done with anti-Syk polyAb. (B)AccumulationofSykaroundthephagocytosedzymosan.At5minutesaftertheonsetof phagocytosis, Day3-HL60 cells were fixed and stained with anti-Syk polyAband AlexaFluor 488–conjugated secondary antibody. The arrows indicate thephagocytosed zymosan (the bar indicates 10  m). A 63   /1.4 NA oil objective wasused to visualize the images. DIC indicates differential interference contrast. ROLES OF Syk IN COMPLEMENT-MEDIATED PHAGOCYTOSIS 4557BLOOD, 1 JUNE 2006    VOLUME 107, NUMBER 11  blue, which quenches the fluorescence of extracellular zymosan. 32 After the phagocytosis assay with C3bi-opsonized fluorescentzymosan, the cells were analyzed before and after trypan bluestaining by fluorescence microscopy and fluorescent zymosanparticles were counted.Figure 5 shows that before quenching, Day3-HL60, DN-Syk/ HL,Syk-siRNA/HL,controlsiRNA/HL,andFlag-rescue–Syk/Syk-siRNA/HL cells captured 1.24  0.24, 0.28  0.08, 0.46  0.20,1.31  0.35, and 1.18  0.21 C3bi-opsonized zymosan particlesper cell, respectively. To determine whether capture was impaireddue to reduced binding to CR3 or reduced engulfment afterbinding, cells were stained with trypan blue and only intracellularzymosan particles were counted. The numbers of C3bi-opsonizedzymosan particles inside the parental Day3-HL60, DN-Syk/HL,Syk-siRNA/HL, control siRNA/HL, and Flag-rescue–Syk/Syk-siRNA/HL cells were 0.9  0.2, 0.1  0, 0.1  0, 0.9  0.2, and0.8  0.1 per cell, respectively.These results indicated that most of the C3bi-zymosan attached to the parental HL60 cells was rapidlyengulfed and that the number of C3bi-zymosan outside the cellswas approximately the same among the parental HL60 cells,DN-Syk/HL cells, and Syk-siRNA/HL cells (Figure 5). These dataindicated that the defective complement-mediated phagocytosis of DN-Syk/HL cells and Syk-siRNA/HL cells was not due to de-creased C3bi binding to CR3 but rather to loss of internalization of the CR3-bound zymosan. Syk affects actin dynamics around the C3bi-mediatedphagosomes Because the engulfment of particles by phagocytosis is induced byactin polymerization at the forming phagosome, we analyzed theeffect of Syk on actin dynamics during complement-mediatedphagocytosis. To synchronize phagocytosis, cells were incubatedwith C3bi-zymosan for 10 minutes, washed once, and then thephagocytic reaction was restarted. At 5 minutes after synchroni-zation, actin fibers obviously accumulated in the region of theforming phagosome that surrounded the C3bi-zymosan particlesin HL60 cells (Figure 6A) as reported. 5 However, Syk alsoaccumulated at the same time point around phagosomes (Figure6A-B). At 30 minutes after synchronization, actin began todisassemble, but the accumulation of Syk around phagosomeswas sustained (Figure 6A-B).To understand the role of Syk in actin dynamics, we analyzedactin accumulation around the phagosomes of the parental Day3-HL60, DN-Syk/HL, Syk-siRNA/HL, control siRNA/HL, and Flag-rescue–Syk/Syk-siRNA/HL cells. In both DN-Syk/HL cells andSyk-siRNA/HL cells, the numbers of phagosomes surrounded byactin were clearly decreased at 5 minutes after synchronization, butthe accumulation of actin was sustained even at 30 minutes.Transfer of control siRNA did not affect the actin accumulationaround the phagosomes and transfer of Flag-rescue–Syk restoredthe accumulation (Figure 6B).Since recent reports suggest a contribution of PLC   to thephagosome enclosure, 5 we analyzed the association of PLC  2 withSyk. Prompt PLC  2 coprecipitation with Syk after the onset of C3bi-CR3–mediated phagocytosis became attenuated at 30 min-utes (Figure 6C). Syk activates the RhoApathway in C3bi-CR3 signaling Rho family GTPases are thought to control actin polymerizationand the extension of membrane protrusions to form a closedphagosome. We therefore further analyzed whether Syk regulatesRhoAsignaling in complement-mediated phagocytosis using GST-rhotekin RBD pull-down assays to detect GTP-bound RhoA.Incubation of Day3-HL60 cells with C3bi-opsonized zymosan led Figure 3. Neither DN-Syk nor Syk-siRNA affects the expression of the complement receptor CR3.  DN-Syk was transferred into HL60 cells and stable mutant clones(DN-Syk/HL, cl3 and cl8) were isolated. Syk-siRNA or control siRNA was also transferred into HL60 cells and stable mutant clones (Syk-siRNA/HL, cl2 and cl6; controlsiRNA/HL, cl1 and cl4) were isolated. Flag-rescue–Syk was transferred into Syk-siRNA/HL cl6 and stable mutant was isolated. Protein expression was examined in thesemutant clones and parental HL60 cells. (A) Expression of transferred Flag-tagged DN-Syk protein in DN-Syk/HL(cl3 and cl8) cells was confirmed by immunoblotting analysisusing anti-Syk polyAb and anti-Flag monoAb (top panel). Macrophage-like differentiated Day3-HL60 and DN-Syk/HL cells were incubated with serum-treated zymosan. Celllysates were immunoprecipitated with antiphosphotyrosine monoAb and immunoblotting analysis was performed with anti-Syk polyAb. As to the whole cell lysates,immunoblotting analysis was also done with anti-Syk polyAb (bottom panel). (B) Expression of endogenous Syk was examined in Syk-siRNA/HL (cl2 and cl6), controlsiRNA/HL (cl1 and cl4), and HL60 cells by immunoblotting analysis using anti-Syk polyAb (top panel). Expression of Flag-rescue–Syk was examined by immunoblottinganalysis using anti-Syk polyAb and anti-Flag monoAb (bottom panel). (C) Cell-surface expression of CR3 (integrin   M  2) on macrophage-like differentiated Day3-HL60,DN-Syk/HL(cl3andcl8),Syk-siRNA/HL(cl2andcl6),controlsiRNA/HL(cl1andcl4),andFlag-rescue–Syk/Syk-siRNA/HLcellswereanalyzedbyflowcytometrywithanti-CR3monoAb (thick line) or with control mouse IgG (thin line). 4558 SHI et al BLOOD, 1 JUNE 2006    VOLUME 107, NUMBER 11
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