CD19 Amplification of B Lymphocyte Ca2+ Responses
2001; Elsevier BV; Volume: 276; Issue: 48 Linguagem: Inglês
10.1074/jbc.m107559200
ISSN1083-351X
AutoresManabu Fujimoto, Jonathan C. Poe, Minoru Hasegawa, Thomas F. Tedder,
Tópico(s)Immunotherapy and Immune Responses
ResumoB lymphocyte antigen receptor (BCR) signals are regulated by CD19, with BCR-induced intracellular calcium ([Ca2+]i ) responses enhanced by CD19 co-ligation. In this study, CD19 engagement using a dimeric anti-CD19 antibody induced [Ca2+]i mobilization and significantly enhanced BCR-induced [Ca2+]i responses without a requirement for CD19/BCR co-ligation. Although simultaneous CD19 and BCR engagement significantly enhanced CD19/Lyn complex formation and [Ca2+]i responses, downstream tyrosine phosphorylation of CD22 and multiple other cellular proteins was inhibited, as was SHP1 recruitment to phosphorylated CD22. CD19 overexpression also enhanced BCR-induced [Ca2+]i responses, but down-regulated tyrosine phosphorylation of CD22 and multiple other cellular proteins following BCR ligation. Because CD19 and Lyn expression are genetically titrated in B cells, CD19 engagement may augment BCR-induced [Ca2+]i responses by sequestering the available pool of functional Lyn away from downstream negative regulatory proteins such as CD22. Consistent with this, simultaneous CD19 engagement did not further enhance the BCR-induced [Ca2+]i responses of Lyn- or CD22-deficient B cells. Thus, CD19 recruitment of Lyn may preferentially activate selective signaling pathways downstream of the CD19/Lyn complex to the exclusion of other downstream regulatory and effector pathways. Other receptors may also utilize a similar strategy to regulate kinase availability and downstream intermolecular signaling. B lymphocyte antigen receptor (BCR) signals are regulated by CD19, with BCR-induced intracellular calcium ([Ca2+]i ) responses enhanced by CD19 co-ligation. In this study, CD19 engagement using a dimeric anti-CD19 antibody induced [Ca2+]i mobilization and significantly enhanced BCR-induced [Ca2+]i responses without a requirement for CD19/BCR co-ligation. Although simultaneous CD19 and BCR engagement significantly enhanced CD19/Lyn complex formation and [Ca2+]i responses, downstream tyrosine phosphorylation of CD22 and multiple other cellular proteins was inhibited, as was SHP1 recruitment to phosphorylated CD22. CD19 overexpression also enhanced BCR-induced [Ca2+]i responses, but down-regulated tyrosine phosphorylation of CD22 and multiple other cellular proteins following BCR ligation. Because CD19 and Lyn expression are genetically titrated in B cells, CD19 engagement may augment BCR-induced [Ca2+]i responses by sequestering the available pool of functional Lyn away from downstream negative regulatory proteins such as CD22. Consistent with this, simultaneous CD19 engagement did not further enhance the BCR-induced [Ca2+]i responses of Lyn- or CD22-deficient B cells. Thus, CD19 recruitment of Lyn may preferentially activate selective signaling pathways downstream of the CD19/Lyn complex to the exclusion of other downstream regulatory and effector pathways. Other receptors may also utilize a similar strategy to regulate kinase availability and downstream intermolecular signaling. B lymphocyte antigen receptor intracellular calcium concentration transgenic mice expressing a human CD19 transgene extracellular signal-regulated kinase horseradish peroxidase mitogen-activated protein kinase polyacrylamide gel electrophoresis phosphatidylinositol 3-kinase phospholipase C SHIP, Src homology-2 domain-containing inositol polyphosphate-5′ phosphatase Src homology-2-containing protein-tyrosine phosphatase-1 monoclonal antibody fluorescein isothiocyanate B lymphocyte development and function are regulated by signals transduced through the B cell antigen receptor (BCR)1 and cell surface regulatory molecules including CD19 (1Tedder T.F. Inaoki M. Sato S. Immunity. 1997; 6: 107-118Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 2Fearon D.T. Locksley R.M. Science. 1996; 272: 50-54Crossref PubMed Scopus (1427) Google Scholar, 3Buhl A.M. Cambier J.C. Immunol. Rev. 1997; 160: 127-138Crossref PubMed Scopus (38) Google Scholar). CD19 is a member of the immunoglobulin superfamily expressed exclusively on B cells and follicular dendritic cells (4Tedder T.F. Isaacs C.M. J. Immunol. 1989; 143: 712-717PubMed Google Scholar). CD19 has a ∼240-amino acid cytoplasmic domain that is critical for CD19 signaling (4Tedder T.F. Isaacs C.M. J. Immunol. 1989; 143: 712-717PubMed Google Scholar, 5Bradbury L.E. Goldmacher V.S. Tedder T.F. J. Immunol. 1993; 151: 2915-2927PubMed Google Scholar, 6Sato S. Miller A.S. Howard M.C. Tedder T.F. J. Immunol. 1997; 159: 3278-3287PubMed Google Scholar), and CD19-deficient (CD19−/−) mice are hyporesponsive to a variety of transmembrane signals (7Engel P. Zhou L.-J. Ord D.C. Sato S. Koller B. Tedder T.F. Immunity. 1995; 3: 39-50Abstract Full Text PDF PubMed Scopus (484) Google Scholar, 8Rickert R.C. Rajewsky K. Roes J. Nature. 1995; 376: 352-355Crossref PubMed Scopus (578) Google Scholar, 9Sato S. Steeber D.A. Tedder T.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11558-11562Crossref PubMed Scopus (151) Google Scholar). Specifically, CD19 functions as a specialized adapter protein for the amplification of Src family protein-tyrosine kinase (PTK) activity and as an interaction molecule for multiple signaling pathways crucial for modulating intrinsic and antigen receptor-induced signals (10Fujimoto M. Poe J.C. Inaoki M. Tedder T.F. Semin. Immunol. 1998; 10: 267-277Crossref PubMed Scopus (81) Google Scholar, 11Fujimoto M. Fujimoto Y. Poe J.C. Jansen P.J. Lowell C.A. DeFranco A.L. Tedder T.F. Immunity. 2000; 13: 47-57Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 12Fujimoto M. Poe J.C. Jansen P.J. Sato S. Tedder T.F. J. Immunol. 1999; 162: 7088-7094PubMed Google Scholar). The cytoplasmic domains of human CD19 and mouse CD19 are highly homologous (13Zhou L.-J. Ord D.C. Hughes A.L. Tedder T.F. J. Immunol. 1991; 147: 1424-1432PubMed Google Scholar). In fact, human CD19 can replace mouse CD19 function when expressed at the appropriate site density in CD19−/− mice (14Sato S. Steeber D.A. Jansen P.J. Tedder T.F. J. Immunol. 1997; 158: 4662-4669PubMed Google Scholar). Overexpression of CD19 in transgenic mice expressing a human CD19 transgene (CD19TG mice) renders B cells hyperresponsive to transmembrane signals (7, 15, and 16). Thus, CD19 is generally considered a positive regulator of B cell function because it amplifies Src family PTK activation, mitogen-activated protein kinase (MAPK) activation, and proliferation (11, 17, and 18). Moreover, CD19 and BCR co-ligation greatly augments BCR-induced ([Ca2+]i ) responses (19Carter R.H. Tuveson D.A. Park D.J. Rhee S.G. Fearon D.T. J. Immunol. 1991; 147: 3663-3671PubMed Google Scholar) and dramatically lowers the threshold for B cell activationin vitro (17Carter R.H. Fearon D.T. Science. 1992; 256: 105-107Crossref PubMed Scopus (552) Google Scholar, 20Dempsey P.W. Allison M.E.D. Akkaraju S. Goodnow C.C. Fearon D.T. Science. 1996; 271: 348-350Crossref PubMed Scopus (1008) Google Scholar).Although CD19 and BCR co-ligation can significantly enhance [Ca2+]i responses (17Carter R.H. Fearon D.T. Science. 1992; 256: 105-107Crossref PubMed Scopus (552) Google Scholar, 19Carter R.H. Tuveson D.A. Park D.J. Rhee S.G. Fearon D.T. J. Immunol. 1991; 147: 3663-3671PubMed Google Scholar), it is unclear whether CD19 ligation can augment BCR-induced [Ca2+]i responses independent of BCR and CD19 coengagement. Moreover, CD19 ligation can also inhibit BCR-induced [Ca2+]i responses and suppress B cell proliferation (21Pezzutto A. Dorken B. Rabinovitch P.S. Ledbetter J.A. Moldenhauer G. Clark E.A. J. Immunol. 1987; 138: 2793-2799PubMed Google Scholar, 22Barrett T.B. Shu G.L. Draves K.E. Pezzutto A. Clark E.A. Eur. J. Immunol. 1990; 20: 1053-1059Crossref PubMed Scopus (38) Google Scholar, 23Callard R.E. Rigley K.P. Smith S.H. Thurstan S. Shields J.G. J. Immunol. 1992; 148: 2983-2987PubMed Google Scholar, 24Rigley K.P. Callard R.E. Eur. J. Immunol. 1991; 21: 535-540Crossref PubMed Scopus (29) Google Scholar, 25De Rie M.A. Schumacher T.N.M. van Schijndel G.M.W. van Lier R.A.W. Miedema F. Cell. Immunol. 1989; 118: 368-381Crossref PubMed Scopus (43) Google Scholar). Although the molecular mechanisms through which CD19 augments or inhibits BCR-induced [Ca2+]i responses are unknown, the above studies suggest that the extent, valency, or timing of CD19 ligation may be crucial to establishing a balance between positive and negative regulatory signals and signals transduced via the BCR. To address these issues, we have generated a dimeric IgA anti-mouse CD19 monoclonal antibody (16Sato S. Ono N. Steeber D.A. Pisetsky D.S. Tedder T.F. J. Immunol. 1996; 157: 4371-4378PubMed Google Scholar) that allows assessment of the functional consequences of CD19 cross-linking independent of BCR ligation. Independent CD19 and BCR engagement was found to augment BCR-induced [Ca2+]i responses and qualitatively regulate BCR signaling through potentially novel mechanisms depending on the extent or valency of CD19 ligation.DISCUSSIONThe current study demonstrates that signal transduction pathways activated by independent CD19 and BCR cross-linking synergize to generate augmented [Ca2+]i responses (Figs. 1 and 2 A) without a requirement for CD19-BCR co-ligation (Fig. 2 A). This supports a model where CD19 ligation can influence signaling thresholds of B cells independent of antigen receptor engagement or regardless of antigen receptor specificity (1Tedder T.F. Inaoki M. Sato S. Immunity. 1997; 6: 107-118Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 46Tedder T.F. Semin. Immunol. 1998; 10: 259-265Crossref PubMed Scopus (44) Google Scholar). This conclusion is supported by the phenotypes of CD19-deficient and -overexpressing mice (1Tedder T.F. Inaoki M. Sato S. Immunity. 1997; 6: 107-118Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Although IgM and heparin sulfate binding to CD19 and C3d binding to the CD21 component of the CD19 complex are the only currently characterized ligands for the CD19 complex (47 and 48), the ability of CD19 ligation to lower signaling thresholds without a requirement for intimate physical association with the BCR complex suggests that CD19 ligands may regulate B cell function regardless of their antigen specificity. Nonetheless, the finding that simultaneous CD19 and BCR ligation resulted in an overall decrease in B cell protein-tyrosine phosphorylation relative to phosphorylation induced by IgM cross-linking alone was unexpected (Fig. 3 A). Overall, BCR-induced protein-tyrosine phosphorylation was also reduced in B cells from mice that overexpress CD19 (Fig. 7 A). Consistent with this, simultaneous BCR and CD19 ligation reduces Vav tyrosine phosphorylation (44Sato S. Jansen P.J. Tedder T.F. Proc. Natl. Acad. Sci., U. S. A. 1997; 94: 13158-13162Crossref PubMed Scopus (100) Google Scholar), whereas independent BCR or CD19 ligation augments Vav phosphorylation and CD19-Vav complex formation (30O'Rourke L.M. Tooze R. Turner M. Sandoval D.M. Carter R.H. Tybulewicz V.L.J. Fearon D.T. Immunity. 1998; 8: 635-645Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 32Weng W.K. Jarvis L. LeBien T.W. J. Biol. Chem. 1994; 269: 32514-32521Abstract Full Text PDF PubMed Google Scholar, 44Sato S. Jansen P.J. Tedder T.F. Proc. Natl. Acad. Sci., U. S. A. 1997; 94: 13158-13162Crossref PubMed Scopus (100) Google Scholar). Thus, rather than augmenting overall B cell kinase activity, simultaneous CD19 ligation inhibited specific kinase pathways downstream of BCR engagement.The recent observations that CD19 efficiently recruits Src family PTKs (11 and 12) and that phosphorylated CD19 preferentially localizes within detergent-insoluble lipid raft microsignaling domains (49 and 50) provides a mechanistic explanation for why simultaneous BCR and CD19 ligation augments [Ca2+]i responses (Fig. 2) yet down-regulates B cell protein-tyrosine phosphorylation (Fig. 3). Lyn, the primary Src family PTK in B cells, preferentially localizes within lipid rafts and plays a critical role in regulating BCR, CD19, and CD22 function (45DeFranco A.L. Chan V.W.F. Lowell C.A. Semin. Immunol. 1998; 10: 299-308Crossref PubMed Scopus (80) Google Scholar). Because CD19, CD22, and Lyn expression are genetically titrated in B cells (51Satterthwaite A.B. Willis F. Kanchanastit P. Fruman D. Cangley L.C. Helgason C.D. Humphries R.K. Lowell C.A. Simon M. Leitges M. Tarakhovsky A. Tedder T.F. Lesche R. Wu H. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6687-6692Crossref PubMed Scopus (34) Google Scholar), BCR signaling is significantly influenced by alterations in the expression and functional pools of these molecules (7, 8, 14, 45, 52, and 53). Lyn and other Src family PTKs become activated following BCR engagement with increased binding to phosphorylated CD19 and the BCR (11Fujimoto M. Fujimoto Y. Poe J.C. Jansen P.J. Lowell C.A. DeFranco A.L. Tedder T.F. Immunity. 2000; 13: 47-57Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 54Roifman C.M. Ke S. Biochem. Biophys. Res. Commun. 1993; 194: 222-225Crossref PubMed Scopus (32) Google Scholar, 55Uckun F.M. Burkhardt A.L. Jarvis L. Jun X. Stealey B. Dibirdik I. Myers D.E. Tuel-Ahlgren L. Bolen J.B. J. Biol. Chem. 1993; 268: 21172-21184Abstract Full Text PDF PubMed Google Scholar, 56van Noesel C.J. Lankester A.C. van Schijndel G.M. van Lier R.A. Int. Immunol. 1993; 5: 699-705Crossref PubMed Scopus (79) Google Scholar, 57Hasegawa M. Fujimoto M. Poe J.C. Steeber D.A. Lowell C.A. Tedder T.F. J. Immunol. 2001; 167: 2469-2478Crossref PubMed Scopus (45) Google Scholar). In turn, phosphorylated CD19 functions as an intrinsic adapter protein for Lyn binding and the amplification of Src family PTK activity (11 and 12). In agreement with this, simultaneous CD19 engagement during BCR signaling significantly augmented CD19/Lyn complex formation (Fig.3 B). CD19 is likely to regulate the spatial distribution of Lyn at the membrane/cytoplasm interface because both Lyn and tyrosine-phosphorylated CD19 preferentially localize within lipid rafts, and ligation of the CD19 complex results in a translocation of CD19 into lipid rafts regardless of concomitant BCR ligation (49 and 50). The ability of phosphorylated CD19 to efficiently recruit and form CD19/Lyn complexes may sequester functionally active Lyn to a sufficient degree that multiple downstream signaling pathways and CD22 phosphorylation are inhibited (11Fujimoto M. Fujimoto Y. Poe J.C. Jansen P.J. Lowell C.A. DeFranco A.L. Tedder T.F. Immunity. 2000; 13: 47-57Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 58Fujimoto M. Bradney A.P. Poe J.C. Steeber D.A. Tedder T.F. Immunity. 1999; 11: 191-200Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Although the amount of Lyn physically associated with CD19 at any instance only represents a small percentage of total cellular Lyn (11Fujimoto M. Fujimoto Y. Poe J.C. Jansen P.J. Lowell C.A. DeFranco A.L. Tedder T.F. Immunity. 2000; 13: 47-57Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), the proportion of cellular Lyn that is functionally active or available for the CD19 or BCR signaling pathways is unknown. Therefore, small changes in the available pool of Lyn may be of profound importance. Consistent with the hypothesis that CD19 cross-linking augments [Ca2+]i responses by sequestering Lyn away from CD22 and other negative regulatory molecules, BCR-induced [Ca2+]i responses are significantly enhanced in Lyn−/− B cells (Fig.5 B, Ref. 36Chan V.W.F. Lowell C.A. DeFranco A.L. Curr. Biol. 1998; 8: 545-553Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Thus, BCR, CD19, and Lyn cross-talk are likely to be precisely balanced to regulate [Ca2+]i responses and B cell function.Effector molecules participating in BCR-induced [Ca2+]i responses include PI 3-kinase and CD22, which are recruited to or regulated by tyrosine-phosphorylated CD19 (31Tuveson D.A. Carter R.H. Soltoff S.P. Fearon D.T. Science. 1993; 260: 986-989Crossref PubMed Scopus (281) Google Scholar, 58Fujimoto M. Bradney A.P. Poe J.C. Steeber D.A. Tedder T.F. Immunity. 1999; 11: 191-200Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). PI 3-kinase binding does not appear to be the sole mechanism by which CD19 augments BCR signaling because inhibiting PI 3-kinase activity did not ablate the ability of CD19 to augment BCR-induced [Ca2+]i responses (Fig. 4). Current studies suggest that CD19 ligation may enhance BCR-induced [Ca2+]i responses by sequestering Lyn and thereby inactivating selective downstream inhibitory signaling pathways, including CD22 phosphorylation (Fig. 3 F). In support of this, Lyn is primarily responsible for CD22 phosphorylation (28Poe J.C. Fujimoto M. Jansen P.J. Miller A.S. Tedder T.F. J. Biol. Chem. 2000; 275: 17420-17427Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 45DeFranco A.L. Chan V.W.F. Lowell C.A. Semin. Immunol. 1998; 10: 299-308Crossref PubMed Scopus (80) Google Scholar) and CD19 ligation during BCR engagement down-regulated CD22 phosphorylation and SHP1 recruitment by CD22 (Fig. 3, H andI). In addition, BCR-induced [Ca2+]i responses were enhanced in B cells that overexpressed CD19 (Fig. 6), whereas CD22 phosphorylation and SHP1 recruitment by CD22 following BCR ligation were inhibited (Fig. 7, C and D). Augmented BCR-induced [Ca2+]i responses are a hallmark of CD22-deficient B cells (Fig. 5 A, Refs. 26Sato S. Miller A.S. Inaoki M. Bock C.B. Jansen P.J. Tang M.L.K. Tedder T.F. Immunity. 1996; 5: 551-562Abstract Full Text PDF PubMed Scopus (388) Google Scholar, 59Nitschke L. Carsetti R. Ocker B. Kohler G. Lamers M.C. Curr. Biol. 1997; 7: 133-143Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 60O'Keefe T.L. Williams G.T. Davies S.L. Neuberger M.S. Science. 1996; 274: 798-801Crossref PubMed Scopus (468) Google Scholar, 61Otipoby K.L. Andersson K.B. Draves K.E. Klaus S.J. Farr A.G. Kerner J.D. Perlmutter R.M. Law C.-L. Clark E.A. Nature. 1996; 384: 634-637Crossref PubMed Scopus (359) Google Scholar), but BCR-induced [Ca2+]i responses in CD22- or Lyn-deficient B cells were not augmented by simultaneous CD19 ligation (Fig. 5 A). Although it remains unknown exactly how CD22 exerts inhibitory effects on B cell [Ca2+]i responses, CD22 regulation is likely to involve recruitment of the SHP1 and SHIP phosphatases (28, 36, 52, 62, and 63). Lyn is reported to be a target for SHP1 dephosphorylation (64Somani A. Yuen K. Xu F. Zhang J. Branch D.R. Siminovitch K.A. J. Biol. Chem. 2001; 276: 1938-1944Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), consistent with the observation that Lyn phosphorylation may be increased by simultaneous CD19 and BCR engagement (Fig. 3 C). Thus, simultaneous CD19 and BCR engagement may augment and/or maintain Lyn activity through multiple mechanisms: CD19 amplification of Lyn kinase activity, decreased CD22 phosphorylation, and diminished SHP1 and SHIP binding to CD22. CD22 may also be excluded from lipid rafts, which may enhance CD19/Lyn phosphorylation if these proteins remain within lipid rafts following CD19 engagement. Consistent with these observations, CD19 phosphorylation was equally high following BCR or simultaneous CD19 and BCR ligation in CD22−/− B cells. 2M. Fujimoto, J. E. Poe, and T. F. Tedder, unpublished observations. Thus, the ability of CD19 ligation to down-regulate phosphorylation of CD22, and perhaps other negative regulatory molecules, may enhance CD19 function.CD19 was originally regarded to be both a positive and negative regulator of BCR signal transduction because CD19 cross-linking could either augment or inhibit BCR signaling and [Ca2+]i responses (21Pezzutto A. Dorken B. Rabinovitch P.S. Ledbetter J.A. Moldenhauer G. Clark E.A. J. Immunol. 1987; 138: 2793-2799PubMed Google Scholar, 22Barrett T.B. Shu G.L. Draves K.E. Pezzutto A. Clark E.A. Eur. J. Immunol. 1990; 20: 1053-1059Crossref PubMed Scopus (38) Google Scholar, 23Callard R.E. Rigley K.P. Smith S.H. Thurstan S. Shields J.G. J. Immunol. 1992; 148: 2983-2987PubMed Google Scholar, 24Rigley K.P. Callard R.E. Eur. J. Immunol. 1991; 21: 535-540Crossref PubMed Scopus (29) Google Scholar). Consistent with this, high valency CD19 ligation inhibited BCR-induced [Ca2+]i responses, whereas lower valency ligation augmented [Ca2+]i responses (Fig. 2, B and C). In addition, antibodies reactive with the CD81 component of the CD19 complex are potent inhibitors of B cell proliferation (65Levy S. Todd S.C. Maecker H.T. Annu. Rev. Immunol. 1998; 16: 89-110Crossref PubMed Scopus (435) Google Scholar). Thus, CD19 may serve both positive and negative roles, depending on its level of engagement. High valency CD19 ligation may efficiently sequester the available pool of functional Lyn because Syk and CD79a phosphorylation were not induced when CD19 was engaged using anti-CD19 antibody at high concentrations. Under these conditions, Lyn, Syk, and CD79a were only phosphorylated at levels equivalent to those induced by CD19 ligation (40 μg/ml) alone (Fig. 3 and data not shown). This proposed mechanism explains why high valency CD19 ligation inhibited BCR-induced [Ca2+]i responses (Fig.2 C), even in the absence of CD22 expression (Fig.5 B). That high valency CD19 ligation inhibited BCR-induced [Ca2+]i responses in Lyn−/− B cells (Fig. 3 B) may be explained by the observation that CD19 binds and amplifies Src family PTKs in addition to Lyn (11, 57, and 58). Alternatively, CD19 cross-linking by antibody may sequester CD19 away from the BCR complex and thereby down-regulate signaling. Arguing against this possibility is the observation that tyrosine phosphorylation of cellular proteins was decreased in CD19 overexpressing B cells following BCR engagement in the absence of CD19 ligation (Fig. 7 A). Moreover, CD19 ligation with high antibody levels reduced BCR-induced [Ca2+]i responses to the levels induced by CD19 ligation alone. Because anti-IgM-induced [Ca2+]i responses are near normal in CD19-deficient B cells (14Sato S. Steeber D.A. Jansen P.J. Tedder T.F. J. Immunol. 1997; 158: 4662-4669PubMed Google Scholar), it is not clear how antibody sequestration of CD19 away from IgM would have a more profound effect on BCR-induced [Ca2+]i responses than the total absence of CD19. Thus, high valency CD19 ligation may sequester Lyn and other Src family PTKs to a sufficient degree that multiple downstream signaling pathways required for [Ca2+]i responses are inhibited.These results collectively demonstrate that CD19 engagement may alter the quality of BCR-mediated signaling without a requirement for co-ligation of these two receptor complexes. The ability of CD19 to amplify Lyn kinase activity (11 and 12) in combination with its potential to extinguish CD22 negative regulation may explain its potent synergy with BCR signal transduction. Thus, the current results suggest that the BCR, CD19, and other positive and negative regulatory cell surface proteins may turn on and off other receptor functions by recruiting or sequestrating a shared pool of Lyn. Sequestration of a limiting supply of Lyn in mast cells is a proposed basis for antagonistic FcεRI function (66Torigoe C. Inman J.K. Metzger H. Science. 1998; 281: 568-572Crossref PubMed Scopus (135) Google Scholar). Undoubtedly, other receptors are likely to sequester limited supplies of downstream effector molecules after engagement and may thereby utilize a similar strategy to regulate intermolecular signaling and direct the activation of downstream pathways. B lymphocyte development and function are regulated by signals transduced through the B cell antigen receptor (BCR)1 and cell surface regulatory molecules including CD19 (1Tedder T.F. Inaoki M. Sato S. Immunity. 1997; 6: 107-118Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 2Fearon D.T. Locksley R.M. Science. 1996; 272: 50-54Crossref PubMed Scopus (1427) Google Scholar, 3Buhl A.M. Cambier J.C. Immunol. Rev. 1997; 160: 127-138Crossref PubMed Scopus (38) Google Scholar). CD19 is a member of the immunoglobulin superfamily expressed exclusively on B cells and follicular dendritic cells (4Tedder T.F. Isaacs C.M. J. Immunol. 1989; 143: 712-717PubMed Google Scholar). CD19 has a ∼240-amino acid cytoplasmic domain that is critical for CD19 signaling (4Tedder T.F. Isaacs C.M. J. Immunol. 1989; 143: 712-717PubMed Google Scholar, 5Bradbury L.E. Goldmacher V.S. Tedder T.F. J. Immunol. 1993; 151: 2915-2927PubMed Google Scholar, 6Sato S. Miller A.S. Howard M.C. Tedder T.F. J. Immunol. 1997; 159: 3278-3287PubMed Google Scholar), and CD19-deficient (CD19−/−) mice are hyporesponsive to a variety of transmembrane signals (7Engel P. Zhou L.-J. Ord D.C. Sato S. Koller B. Tedder T.F. Immunity. 1995; 3: 39-50Abstract Full Text PDF PubMed Scopus (484) Google Scholar, 8Rickert R.C. Rajewsky K. Roes J. Nature. 1995; 376: 352-355Crossref PubMed Scopus (578) Google Scholar, 9Sato S. Steeber D.A. Tedder T.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11558-11562Crossref PubMed Scopus (151) Google Scholar). Specifically, CD19 functions as a specialized adapter protein for the amplification of Src family protein-tyrosine kinase (PTK) activity and as an interaction molecule for multiple signaling pathways crucial for modulating intrinsic and antigen receptor-induced signals (10Fujimoto M. Poe J.C. Inaoki M. Tedder T.F. Semin. Immunol. 1998; 10: 267-277Crossref PubMed Scopus (81) Google Scholar, 11Fujimoto M. Fujimoto Y. Poe J.C. Jansen P.J. Lowell C.A. DeFranco A.L. Tedder T.F. Immunity. 2000; 13: 47-57Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 12Fujimoto M. Poe J.C. Jansen P.J. Sato S. Tedder T.F. J. Immunol. 1999; 162: 7088-7094PubMed Google Scholar). The cytoplasmic domains of human CD19 and mouse CD19 are highly homologous (13Zhou L.-J. Ord D.C. Hughes A.L. Tedder T.F. J. Immunol. 1991; 147: 1424-1432PubMed Google Scholar). In fact, human CD19 can replace mouse CD19 function when expressed at the appropriate site density in CD19−/− mice (14Sato S. Steeber D.A. Jansen P.J. Tedder T.F. J. Immunol. 1997; 158: 4662-4669PubMed Google Scholar). Overexpression of CD19 in transgenic mice expressing a human CD19 transgene (CD19TG mice) renders B cells hyperresponsive to transmembrane signals (7, 15, and 16). Thus, CD19 is generally considered a positive regulator of B cell function because it amplifies Src family PTK activation, mitogen-activated protein kinase (MAPK) activation, and proliferation (11, 17, and 18). Moreover, CD19 and BCR co-ligation greatly augments BCR-induced ([Ca2+]i ) responses (19Carter R.H. Tuveson D.A. Park D.J. Rhee S.G. Fearon D.T. J. Immunol. 1991; 147: 3663-3671PubMed Google Scholar) and dramatically lowers the threshold for B cell activationin vitro (17Carter R.H. Fearon D.T. Science. 1992; 256: 105-107Crossref PubMed Scopus (552) Google Scholar, 20Dempsey P.W. Allison M.E.D. Akkaraju S. Goodnow C.C. Fearon D.T. Science. 1996; 271: 348-350Crossref PubMed Scopus (1008) Google Scholar). Although CD19 and BCR co-ligation can significantly enhance [Ca2+]i responses (17Carter R.H. Fearon D.T. Science. 1992; 256: 105-107Crossref PubMed Scopus (552) Google Scholar, 19Carter R.H. Tuveson D.A. Park D.J. Rhee S.G. Fearon D.T. J. Immunol. 1991; 147: 3663-3671PubMed Google Scholar), it is unclear whether CD19 ligation can augment BCR-induced [Ca2+]i responses independent of BCR and CD19 coengagement. Moreover, CD19 ligation can also inhibit BCR-induced [Ca2+]i responses and suppress B cell proliferation (21Pezzutto A. Dorken B. Rabinovitch P.S. Ledbetter J.A. Moldenhauer G. Clark E.A. J. Immunol. 1987; 138: 2793-2799PubMed Google Scholar, 22Barrett T.B. Shu G.L. Draves K.E. Pezzutto A. Clark E.A. Eur. J. Immunol. 1990; 20: 1053-1059Crossref PubMed Scopus (38) Google Scholar, 23Callard R.E. Rigley K.P. Smith S.H. Thurstan S. Shields J.G. J. Immunol. 1992; 148: 2983-2987PubMed Google Scholar, 24Rigley K.P. Callard R.E. Eur. J. Immunol. 1991; 21: 535-540Crossref PubMed Scopus (29) Google Scholar, 25De Rie M.A. Schumacher T.N.M. van Schijndel G.M.W. van Lier R.A.W. Miedema F. Cell. Immunol. 1989; 118: 368-381Crossref PubMed Scopus (43) Google Scholar). Although the molecular mechanisms through which CD19 augments or inhibits BCR-induced [Ca2+]i responses are unknown, the above studies suggest that the extent, valency, or timing of CD19 ligation may be crucial to establishing a balance between positive and negative regulatory signals and signals transduced via the BCR. To address these issues, we have generated a dimeric IgA anti-mouse CD19 monoclonal antibody (16Sato S. Ono N. Steeber D.A. Pisetsky D.S. Tedder T.F. J. Immunol. 1996; 157: 4371-4378PubMed Google Scholar) that allows assessment of the functional consequences of CD19 cross-linking independent of BCR ligation. Independent CD19 and BCR engagement was found to augment BCR-induced [Ca2+]i responses and qualitatively regulate BCR signaling through potentially novel mechanisms depending on the extent or valency of CD19 ligation. DISCUSSIONThe current study demonstrates that signal transduction pathways activated by independent CD19 and BCR cross-linking synergize to generate augmented [Ca2+]i responses (Figs. 1 and 2 A) without a requirement for CD19-BCR co-ligation (Fig. 2 A). This supports a model where CD19 ligation can influence signaling thresholds of B cells independent of antigen receptor engagement or regardless of antigen receptor specificity (1Tedder T.F. Inaoki M. Sato S. Immunity. 1997; 6: 107-118Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 46Tedder T.F. Semin. Immunol. 1998; 10: 259-265Crossref PubMed Scopus (44) Google Scholar). This conclusion is supported by the phenotypes of CD19-deficient and -overexpressing mice (1Tedder T.F. Inaoki M. Sato S. Immunity. 1997; 6: 107-118Abstract Full Text Full Text PDF PubMed
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