Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca(2+) response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)(2) or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcεRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)(2) fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcεRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family.

Department of Signal Transduction Institute of Molecular Genetics Academy of Sciences of the Czech Republic CZ 14220 Prague Czech Republic

Zobrazit více v PubMed

Okayama Y., Kawakami T. (2006) Development, migration, and survival of mast cells. Immunol. Res. 34, 97–115 PubMed PMC

Halova I., Draberova L., Draber P. (2012) Mast cell chemotaxis-chemoattractants and signaling pathways. Front Immunol. 3, 119. PubMed PMC

Wedemeyer J., Galli S. J. (2000) Mast cells and basophils in acquired immunity. Br. Med. Bull. 56, 936–955 PubMed

Saitoh S., Arudchandran R., Manetz T. S., Zhang W., Sommers C. L., Love P. E., Rivera J., Samelson L. E. (2000) LAT is essential for FcϵRI-mediated mast cell activation. Immunity 12, 525–535 PubMed

Volná P., Lebduška P., Dráberová L., Šímová Š., Heneberg P., Boubelík M., Bugajev V., Malissen B., Wilson B. S., Hořejší V., Malissen M., Dráber P. (2004) Negative regulation of mast cell signaling and function by the adaptor LAB/NTAL. J. Exp. Med. 200, 1001–1013 PubMed PMC

Zhu M., Liu Y., Koonpaew S., Granillo O., Zhang W. (2004) Positive and negative regulation of FcϵRI-mediated signaling by the adaptor protein LAB/NTAL. J. Exp. Med. 200, 991–1000 PubMed PMC

Tkaczyk C., Horejsi V., Iwaki S., Draber P., Samelson L. E., Satterthwaite A. B., Nahm D. H., Metcalfe D. D., Gilfillan A. M. (2004) NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and FcϵRI aggregation. Blood 104, 207–214 PubMed

Draber P., Halova I., Levi-Schaffer F., Draberova L. (2011) Transmembrane adaptor proteins in the high-affinity IgE receptor signaling. Front. Immunol. 2, 95. PubMed PMC

Rivera J. (2005) NTAL/LAB and LAT. A balancing act in mast-cell activation and function. Trends Immunol. 26, 119–122 PubMed

Simeoni L., Lindquist J. A., Smida M., Witte V., Arndt B., Schraven B. (2008) Control of lymphocyte development and activation by negative regulatory transmembrane adapter proteins. Immunol. Rev. 224, 215–228 PubMed

Lebduška P., Korb J., Tůmová M., Heneberg P., Dráber P. (2007) Topography of signaling molecules as detected by electron microscopy on plasma membrane sheets isolated from non-adherent mast cells. J. Immunol. Methods 328, 139–151 PubMed

Dráberová L., Shaik G. M., Volná P., Heneberg P., Tůmová M., Lebduska P., Korb J., Dráber P. (2007) Regulation of Ca2+ signaling in mast cells by tyrosine-phosphorylated and unphosphorylated non-T cell activation linker. J. Immunol. 179, 5169–5180 PubMed

Kimura T., Hisano M., Inoue Y., Adachi M. (2001) Tyrosine phosphorylation of the linker for activator of T cells in mast cells by stimulation with the high affinity IgE receptor. Immunol. Lett. 75, 123–129 PubMed

Tůmová M., Koffer A., Šimíček M., Dráberová L., Dráber P. (2010) The transmembrane adaptor protein NTAL signals to mast cell cytoskeleton via the small GTPase Rho. Eur. J. Immunol. 40, 3235–3245 PubMed

Charrin S., Le Naour F., Oualid M., Billard M., Faure G., Hanash S. M., Boucheix C., Rubinstein E. (2001) The major CD9 and CD81 molecular partner. Identification and characterization of the complexes. J. Biol. Chem. 276, 14329–14337 PubMed

Kotha J., Longhurst C., Appling W., Jennings L. K. (2008) Tetraspanin CD9 regulates β1 integrin activation and enhances cell motility to fibronectin via a PI-3 kinase-dependent pathway. Exp. Cell Res. 314, 1811–1822 PubMed

Levy S., Shoham T. (2005) Protein-protein interactions in the tetraspanin web. Physiology 20, 218–224 PubMed

Hemler M. E., Mannion B. A., Berditchevski F. (1996) Association of TM4SF proteins with integrins. Relevance to cancer. Biochim. Biophys. Acta 1287, 67–71 PubMed

Xu D., Sharma C., Hemler M. E. (2009) Tetraspanin12 regulates ADAM10-dependent cleavage of amyloid precursor protein. FASEB J. 23, 3674–3681 PubMed PMC

Berditchevski F., Odintsova E. (1999) Characterization of integrin-tetraspanin adhesion complexes. Role of tetraspanins in integrin signaling. J. Cell Biol. 146, 477–492 PubMed PMC

Berditchevski F. (2001) Complexes of tetraspanins with integrins. More than meets the eye. J. Cell Sci. 114, 4143–4151 PubMed

Little K. D., Hemler M. E., Stipp C. S. (2004) Dynamic regulation of a GPCR-tetraspanin-G protein complex on intact cells: central role of CD81 in facilitating GPR56-Gαq/11 association. Mol. Biol. Cell 15, 2375–2387 PubMed PMC

Murayama Y., Shinomura Y., Oritani K., Miyagawa J., Yoshida H., Nishida M., Katsube F., Shiraga M., Miyazaki T., Nakamoto T., Tsutsui S., Tamura S., Higashiyama S., Shimomura I., Hayashi N. (2008) The tetraspanin CD9 modulates epidermal growth factor receptor signaling in cancer cells. J. Cell Physiol. 216, 135–143 PubMed

Sala-Valdés M., Ursa A., Charrin S., Rubinstein E., Hemler M. E., Sánchez-Madrid F., Yáñez-Mó M. (2006) EWI-2 and EWI-F link the tetraspanin web to the actin cytoskeleton through their direct association with ezrin-radixin-moesin proteins. J. Biol. Chem. 281, 19665–19675 PubMed

Stipp C. S., Kolesnikova T. V., Hemler M. E. (2001) EWI-2 is a major CD9 and CD81 partner and member of a novel Ig protein subfamily. J. Biol. Chem. 276, 40545–40554 PubMed

Zhang X. A., Bontrager A. L., Hemler M. E. (2001) Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific β1-integrins. J. Biol. Chem. 276, 25005–25013 PubMed

Hemler M. E. (2005) Tetraspanin functions and associated microdomains. Nat. Rev. Mol. Cell Biol. 6, 801–811 PubMed

Rubinstein E. (2011) The complexity of tetraspanins. Biochem. Soc. Trans. 39, 501–505 PubMed

Dráber P., Zikán J., Vojtíšková M. (1980) Establishment and characterization of permanent murine hybridomas secreting monoclonal anti-thy-1 antibodies. J. Immunogenet. 7, 455–474 PubMed

Smrž D., Lebduška P., Dráberová L., Korb J., Dráber P. (2008) Engagement of phospholipid scramblase 1 in activated cells. Implication for phosphatidylserine externalization and exocytosis. J. Biol. Chem. 283, 10904–10918 PubMed

Rudolph A. K., Burrows P. D., Wabl M. R. (1981) Thirteen hybridomas secreting hapten-specific immunoglobulin E from mice with Iga or Igb heavy chain haplotype. Eur. J. Immunol. 11, 527–529 PubMed

Rivera J., Kinet J. P., Kim J., Pucillo C., Metzger H. (1988) Studies with a monoclonal antibody to the β subunit of the receptor with high affinity for immunoglobulin E. Mol. Immunol. 25, 647–661 PubMed

Brdička T., Imrich M., Angelisová P., Brdičková N., Horváth O., Špička J., Hilgert I., Lusková P., Dráber P., Novák P., Engels N., Wienands J., Simeoni L., Osterreicher J., Aguado E., Malissen M., Schraven B., Hořejší V. (2002) Non-T cell activation linker (NTAL). A transmembrane adaptor protein involved in immunoreceptor signaling. J. Exp. Med. 196, 1617–1626 PubMed PMC

Tolar P., Tůmová M., Dráber P. (2001) Folia Biol. 47, 215–217 PubMed

Dráberová L., Amoui M., Dráber P. (1996) Thy-1-mediated activation of rat mast cells. The role of Thy-1 membrane microdomains. Immunology 87, 141–148 PubMed PMC

Kovářová M., Tolar P., Arudchandran R., Dráberová L., Rivera J., Dráber P. (2001) Structure-function analysis of Lyn kinase association with lipid rafts and initiation of early signaling events after Fcϵ receptor I aggregation. Mol. Cell Biol. 21, 8318–8328 PubMed PMC

Hibbs M. L., Tarlinton D. M., Armes J., Grail D., Hodgson G., Maglitto R., Stacker S. A., Dunn A. R. (1995) Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease. Cell 83, 301–311 PubMed

Hájková Z., Bugajev V., Dráberová E., Vinopal S., Dráberová L., Janáček J., Dráber P., Dráber P. (2011) STIM1-directed reorganization of microtubules in activated mast cells. J. Immunol. 186, 913–923 PubMed

Surviladze Z., Dráberová L., Kovářová M., Boubelík M., Dráber P. (2001) Differential sensitivity to acute cholesterol lowering of activation mediated via the high-affinity IgE receptor and Thy-1 glycoprotein. Eur. J. Immunol. 31, 1–10 PubMed

Hálová I., Dráberová L., Dráber P. (2002) A novel lipid raft-associated glycoprotein, TEC-21, activates rat basophilic leukemia cells independently of the type 1 Fcϵ receptor. Int. Immunol. 14, 213–223 PubMed

Philimonenko A. A., Janáček J., Hozák P. (2000) Statistical evaluation of colocalization patterns in immunogold labeling experiments. J. Struct. Biol. 132, 201–210 PubMed

Carpenter A. E., Jones T. R., Lamprecht M. R., Clarke C., Kang I. H., Friman O., Guertin D. A., Chang J. H., Lindquist R. A., Moffat J., Golland P., Sabatini D. M. (2006) CellProfiler. Image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100. PubMed PMC

Fiskum G., Craig S. W., Decker G. L., Lehninger A. L. (1980) The cytoskeleton of digitonin-treated rat hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 77, 3430–3434 PubMed PMC

Bangham A. D., Horne R. W., Glauert A. M., Dingle J. T., Lucy J. A. (1962) Action of saponin on biological cell membranes. Nature 196, 952–955 PubMed

Iwaki S., Spicka J., Tkaczyk C., Jensen B. M., Furumoto Y., Charles N., Kovarova M., Rivera J., Horejsi V., Metcalfe D. D., Gilfillan A. M. (2008) Kit- and FcϵRI-induced differential phosphorylation of the transmembrane adaptor molecule NTAL/LAB/LAT2 allows flexibility in its scaffolding function in mast cells. Cell Signal 20, 195–205 PubMed PMC

Zhang J., Billingsley M. L., Kincaid R. L., Siraganian R. P. (2000) Phosphorylation of Syk activation loop tyrosines is essential for Syk function. An in vivo study using a specific anti-Syk activation loop phosphotyrosine antibody. J. Biol. Chem. 275, 35442–35447 PubMed

Linnekin D. (1999) Early signaling pathways activated by c-Kit in hematopoietic cells. Int. J. Biochem. Cell Biol. 31, 1053–1074 PubMed

Qi J. C., Wang J., Mandadi S., Tanaka K., Roufogalis B. D., Madigan M. C., Lai K., Yan F., Chong B. H., Stevens R. L., Krilis S. A. (2006) Human and mouse mast cells use the tetraspanin CD9 as an alternate interleukin-16 receptor. Blood 107, 135–142 PubMed PMC

Krämer B., Schulte D., Körner C., Zwank C., Hartmann A., Michalk M., Söhne J., Langhans B., Nischalke H. D., Coenen M., Möhl C., Vogt A., Hennenberg M., Sauerbruch T., Spengler U., Nattermann J. (2009) Regulation of NK cell trafficking by CD81. Eur. J. Immunol. 39, 3447–3458 PubMed

Kaji K., Takeshita S., Miyake K., Takai T., Kudo A. (2001) Functional association of CD9 with the Fcγ receptors in macrophages. J. Immunol. 166, 3256–3265 PubMed

Kuroda K., Ozaki Y., Qi R., Asazuma N., Yatomi Y., Satoh K., Nomura S., Suzuki M., Kume S. (1995) FcγII receptor-mediated platelet activation induced by anti-CD9 monoclonal antibody opens Ca2+ channels which are distinct from those associated with Ca2+ store depletion. J. Immunol. 155, 4427–4436 PubMed

Qi R., Ozaki Y., Kuroda K., Asazuma N., Yatomi Y., Satoh K., Nomura S., Kume S. (1996) Differential activation of human platelets induced by Fcγ receptor II cross-linking and by anti-CD9 monoclonal antibody. J. Immunol. 157, 5638–5645 PubMed

Worthington R. E., Carroll R. C., Boucheix C. (1990) Platelet activation by CD9 monoclonal antibodies is mediated by the Fcγ II receptor. Br. J. Haematol. 74, 216–222 PubMed

Gupta N., Wollscheid B., Watts J. D., Scheer B., Aebersold R., DeFranco A. L. (2006) Quantitative proteomic analysis of B cell lipid rafts reveals that ezrin regulates antigen receptor-mediated lipid raft dynamics. Nat. Immunol. 7, 625–633 PubMed

Liu Y., Belkina N. V., Park C., Nambiar R., Loughhead S. M., Patino-Lopez G., Ben-Aissa K., Hao J. J., Kruhlak M. J., Qi H., von Andrian U. H., Kehrl J. H., Tyska M. J., Shaw S. (2012) Constitutively active ezrin increases membrane tension, slows migration, and impedes endothelial transmigration of lymphocytes in vivo in mice. Blood 119, 445–453 PubMed PMC

Parameswaran N., Matsui K., Gupta N. (2011) Conformational switching in ezrin regulates morphological and cytoskeletal changes required for B cell chemotaxis. J. Immunol. 186, 4088–4097 PubMed PMC

Treanor B., Depoil D., Bruckbauer A., Batista F. D. (2011) Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity. J. Exp. Med. 208, 1055–1068 PubMed PMC

Staser K., Shew M. A., Michels E. G., Mwanthi M. M., Yang F. C., Clapp D. W., Park S. J. (2013) A Pak1-PP2A-ERM signaling axis mediates F-actin rearrangement and degranulation in mast cells. Exp. Hematol. 41, 56–66 PubMed PMC

Yonemura S., Hirao M., Doi Y., Takahashi N., Kondo T., Tsukita S., Tsukita S. (1998) Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2. J. Cell Biol. 140, 885–895 PubMed PMC

Fleming T. J., Donnadieu E., Song C. H., Laethem F. V., Galli S. J., Kinet J. P. (1997) Negative regulation of FcϵRI-mediated degranulation by CD81. J. Exp. Med. 186, 1307–1314 PubMed PMC

Ortega E., Schweitzer-Stenner R., Pecht I. (1988) Possible orientational constraints determine secretory signals induced by aggregation of IgE receptors on mast cells. EMBO J. 7, 4101–4109 PubMed PMC

Gary R., Bretscher A. (1995) Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. Mol. Biol. Cell 6, 1061–1075 PubMed PMC

Reczek D., Bretscher A. (1998) The carboxyl-terminal region of EBP50 binds to a site in the amino-terminal domain of ezrin that is masked in the dormant molecule. J. Biol. Chem. 273, 18452–18458 PubMed

Hirao M., Sato N., Kondo T., Yonemura S., Monden M., Sasaki T., Takai Y., Tsukita S., Tsukita S. (1996) Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association. Possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway. J. Cell Biol. 135, 37–51 PubMed PMC

Gonzalez-Espinosa C., Odom S., Olivera A., Hobson J. P., Martinez M. E., Oliveira-Dos-Santos A., Barra L., Spiegel S., Penninger J. M., Rivera J. (2003) Preferential signaling and induction of allergy-promoting lymphokines upon weak stimulation of the high affinity IgE receptor on mast cells. J. Exp. Med. 197, 1453–1465 PubMed PMC

Tetraspanins in the regulation of mast cell function

Mast Cell Migration and Chemotaxis Assayed by Microscopy

Cytoskeletal Protein 4.1R Is a Positive Regulator of the FcεRI Signaling and Chemotaxis in Mast Cells

Positive and Negative Regulatory Roles of C-Terminal Src Kinase (CSK) in FcεRI-Mediated Mast Cell Activation, Independent of the Transmembrane Adaptor PAG/CSK-Binding Protein

Tetraspanins and Transmembrane Adaptor Proteins As Plasma Membrane Organizers-Mast Cell Case

Signal transduction and chemotaxis in mast cells

New Regulatory Roles of Galectin-3 in High-Affinity IgE Receptor Signaling

Negative regulatory roles of ORMDL3 in the FcεRI-triggered expression of proinflammatory mediators and chemotactic response in murine mast cells

Transmembrane adaptor protein PAG/CBP is involved in both positive and negative regulation of mast cell signaling