Characterization of a CD43/Leukosialin-mediated Pathway for Inducing Apoptosis in Human T-Lymphoblastoid Cells
1996; Elsevier BV; Volume: 271; Issue: 44 Linguagem: Inglês
10.1074/jbc.271.44.27686
ISSN1083-351X
AutoresT J Brown, Walt W. Shuford, Wei‐Chun Wang, Steven G. Nadler, Tina S. Bailey, Hans Marquardt, Robert S. Mittler,
Tópico(s)Invertebrate Immune Response Mechanisms
ResumoThe monoclonal antibody (mAb) J393 induces apoptosis in Jurkat T-cells. NH2-terminal amino acid sequence analysis identified the 140-kDa surface antigen for mAb J393 as CD43/leukosialin, the major sialoglycoprotein of leukocytes. While Jurkat cells co-expressed two discrete cell-surface isoforms of CD43, recognized by mAb J393 and mAb G10-2, respectively, only J393/CD43 signaled apoptosis. J393/CD43 was found to be hyposialylated, bearing predominantly O-linked monosaccharide glycans, whereas G10-2/CD43 bore complex sialylated tetra- and hexasaccharide chains. Treatment with soluble, bivalent mAb J393 killed 25-50% of the cell population, while concomitant engagement of either the CD3·TcR complex or the integrins CD18 and CD29 significantly potentiated this effect. Treatment of Jurkat cells with mAb J393 induced tyrosine phosphorylation of specific protein substrates that underwent hyperphosphorylation upon antigen receptor costimulation. Tyrosine kinase inhibition by herbimycin A diminished J393/CD43-mediated apoptosis, whereas inhibition of phosphotyrosine phosphatase activity by bis(maltolato)oxovanadium-IV enhanced cell death. Signal transduction through tyrosine kinase activation may lead to altered gene expression, as J393/CD43 ligation prompted decreases in the nuclear localization of the transcriptional regulatory protein NF-κB and proteins binding the interferon-inducible regulatory element. Since peripheral blood T-lymphocytes express cryptic epitopes for mAb J393, these findings demonstrate the existence of a tightly regulated CD43-mediated pathway for inducing apoptosis in human T-cell lineages. The monoclonal antibody (mAb) J393 induces apoptosis in Jurkat T-cells. NH2-terminal amino acid sequence analysis identified the 140-kDa surface antigen for mAb J393 as CD43/leukosialin, the major sialoglycoprotein of leukocytes. While Jurkat cells co-expressed two discrete cell-surface isoforms of CD43, recognized by mAb J393 and mAb G10-2, respectively, only J393/CD43 signaled apoptosis. J393/CD43 was found to be hyposialylated, bearing predominantly O-linked monosaccharide glycans, whereas G10-2/CD43 bore complex sialylated tetra- and hexasaccharide chains. Treatment with soluble, bivalent mAb J393 killed 25-50% of the cell population, while concomitant engagement of either the CD3·TcR complex or the integrins CD18 and CD29 significantly potentiated this effect. Treatment of Jurkat cells with mAb J393 induced tyrosine phosphorylation of specific protein substrates that underwent hyperphosphorylation upon antigen receptor costimulation. Tyrosine kinase inhibition by herbimycin A diminished J393/CD43-mediated apoptosis, whereas inhibition of phosphotyrosine phosphatase activity by bis(maltolato)oxovanadium-IV enhanced cell death. Signal transduction through tyrosine kinase activation may lead to altered gene expression, as J393/CD43 ligation prompted decreases in the nuclear localization of the transcriptional regulatory protein NF-κB and proteins binding the interferon-inducible regulatory element. Since peripheral blood T-lymphocytes express cryptic epitopes for mAb J393, these findings demonstrate the existence of a tightly regulated CD43-mediated pathway for inducing apoptosis in human T-cell lineages. INTRODUCTIONIn T-lymphocytes CD43/leukosialin, the major sialoglycoprotein of leukocytes (1Remold-O'Donnell E. Kenny D.M. Parkman R. Cairns L. Savage B. Rosen F.S. J. Exp. Med. 1984; 159: 1705-1723Crossref PubMed Scopus (156) Google Scholar, 2Kenny D. Cairns L. Remold-O'Donnell E. Peterson J. Rosen F.S. Parkman R. Blood. 1986; 68: 1329-1332Crossref PubMed Google Scholar), is thought to serve a dual role in regulating cellular immune responses. Due to the repulsive effect of its high numbers of O-linked negatively charged sialic acid sugar residues, CD43 acts as a "barrier molecule" by limiting cell-cell/cell-ligand interactions (3Brown W.R.A. Barclay A.N. Sunderland C.A. Williams A.F. Nature. 1981; 289: 456-460Crossref PubMed Scopus (157) Google Scholar, 4Rosenstein Y. Park J.K. Hahn W.C. Rosen F.S. Bierer B.E. Burakoff S.J. Nature. 1991; 354: 233-235Crossref PubMed Scopus (224) Google Scholar, 5Ardman B. Sikorski M.A. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5001-5005Crossref PubMed Scopus (149) Google Scholar, 6Manjunath N. Johnson R.S. Staunton D.E. Pasqualini R. Ardman B. J. Immunol. 1993; 151: 1528-1534PubMed Google Scholar, 7Kojima N. Saito M. Shuichi T. Exp. Cell Res. 1994; 214: 537-542Crossref PubMed Scopus (13) Google Scholar), a property that may negatively regulate T-cell activation (8Manjunath N. Correa M. Ardman M. Ardman B. Nature. 1995; 377: 535-538Crossref PubMed Scopus (187) Google Scholar). In addition, engagement of CD43 by monoclonal antibodies has been shown to induce costimulatory activity in T-cells by a mechanism analogous to a classic ligand-receptor interaction (9Sportsman J.R. Park M.M. Cheresh D.A. Fukuda M. Elder J.H. Fox R.I. J. Immunol. 1985; 135: 158-164PubMed Google Scholar, 10Axelsson B. Youseffi-Etemad R. Hammarstrom S. Perlmann P. J. Immunol. 1988; 141: 2912-2917PubMed Google Scholar, 11Alvarado M. Klassen C. Cerny J. Horejsi V. Schmidt R.E. Eur. J. Immunol. 1995; 25: 1051-1055Crossref PubMed Scopus (45) Google Scholar). Alterations in O-glycan structure and function of CD43 reportedly occur in the immunodeficiency disorders Wiskott-Aldrich syndrome (12Parkman R. Remold-O'Donnell E. Kenny D.M. Perrine S. Rosen F.S. Lancet. 1981; ii: 1387-1389Abstract Scopus (89) Google Scholar, 13Siminovitch K.A. Greer W.L. Axelsson B. Rubin L.A. Novogrodsky A. Peacocke M. Immunodefic. Rev. 1993; 4: 99-108Google Scholar) and AIDS (14Ardman B. Sikorski M.A. Settles M. Staunton D.E. J. Exp. Med. 1990; 172: 1151-1158Crossref PubMed Scopus (66) Google Scholar, 15Lefebvre J.-C. Giordanengo V. Limouse M. Doglio A. Cucchiarini M. Monpoux F. Mariani R. Peyron J.-F. J. Exp. Med. 1994; 180: 1609-1617Crossref PubMed Scopus (59) Google Scholar) as well as in graft versus host disease (16Ellies L.G. Jones A.T. Williams M.J. Ziltener H.J. Glycobiology. 1994; 4: 885-893Crossref PubMed Scopus (35) Google Scholar), acute lymphocytic leukemia (17Saitoh O. Piller F. Fox R.I. Fukuda M. Blood. 1991; 77: 1491-1499Crossref PubMed Google Scholar), and permanent mixed-field polyagglutinability or Tn antigen syndrome (18Thunher M. Rusconi S. Berger E.G. J. Clin. Invest. 1993; 91: 2103-2110Crossref PubMed Scopus (64) Google Scholar).The molecular structure of human CD43 is mucin-like, consisting of an extended rod-shaped extracellular portion bearing approximately 80 sialylated O-glycan sites and a single N-glycan site, a highly conserved transmembrane region, and a long cytoplasmic domain bearing potential serine/threonine phosphorylation sites (19Shelley C.S. Remold-O'Donnell E. Davis A.E. Bruns G.A.P. Rosen F.S. Carroll M.C. Whitehead A.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2819-2823Crossref PubMed Scopus (117) Google Scholar). Based on the exon/intron arrangement within its gene, the observed molecular heterogeneity of CD43 in both mice and man is thought to reflect differential post-translational modifications of a single gene product (20Pallant A. Eskenazi A. Mattei M.-G. Fournier R.E.K. Carlsson S.R. Fukuda M. Frelinger J.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1328-1332Crossref PubMed Scopus (116) Google Scholar, 21Shelly C.S. Remold-O'Donnell E. Rosen F.S. Whitehead A.S. Biochem. J. 1990; 270: 569-576Crossref PubMed Scopus (26) Google Scholar, 22Cyster J. Somoza C. Killeen N. Williams A.F. Eur. J. Immunol. 1990; 20: 875-881Crossref PubMed Scopus (49) Google Scholar). Linear protein epitopes in the native structure of CD43 have been shown to be modified by glycosylation (23Cyster J.G. Shotton D.M. Williams A.F. EMBO J. 1991; 10: 893-902Crossref PubMed Scopus (236) Google Scholar), allowing for the development of isoform-specific antibodies (9Sportsman J.R. Park M.M. Cheresh D.A. Fukuda M. Elder J.H. Fox R.I. J. Immunol. 1985; 135: 158-164PubMed Google Scholar, 11Alvarado M. Klassen C. Cerny J. Horejsi V. Schmidt R.E. Eur. J. Immunol. 1995; 25: 1051-1055Crossref PubMed Scopus (45) Google Scholar, 24Jones A.T. Federsppiel B. Ellies L.G. Williams M.J. Burgener R. Duronio V. Smith C.A. Takei F. Ziltener H.J. J. Immunol. 1994; 153: 3426-3439PubMed Google Scholar). Following T-cell activation, the O-linked oligosaccharides of CD43 change from tetrasaccharides (mAb 1The abbreviations used are: mAbmonoclonal antibodyTcRT-cell receptorFACSfluorescence-activated cell sorterFITCfluorescein isothiocyanatePEphycoerythrinPIpropidium iodidePHAphytohemagglutininBMLOVbis(maltolato)oxovanadium-IVNFκ Bnuclear factor kappa BTNFtumor necrosis factorPBSphosphate-buffered salineMOPS3-[N-morpholino]propanesulfonic acidTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinePTPasephosphotyrosine phosphataseIREinterferon-inducible regulatory element. G10-2 reactive) to more complex hexasaccharides (mAb T-305 reactive) due to the activation-induced expression of core 2 β1→6 N-acetylglucosaminyltransferase (17Saitoh O. Piller F. Fox R.I. Fukuda M. Blood. 1991; 77: 1491-1499Crossref PubMed Google Scholar, 25Bierhuizen M.F.A. Maemura K. Fukuda M. J. Biol. Chem. 1994; 269: 4473-4479Abstract Full Text PDF PubMed Google Scholar). This change in oligosaccharide structure results in a shift from 115 to 140 kDa (9Sportsman J.R. Park M.M. Cheresh D.A. Fukuda M. Elder J.H. Fox R.I. J. Immunol. 1985; 135: 158-164PubMed Google Scholar, 17Saitoh O. Piller F. Fox R.I. Fukuda M. Blood. 1991; 77: 1491-1499Crossref PubMed Google Scholar, 25Bierhuizen M.F.A. Maemura K. Fukuda M. J. Biol. Chem. 1994; 269: 4473-4479Abstract Full Text PDF PubMed Google Scholar, 26Fox R.I. Hueniken M. Fong S. Behar S. Royston I. Singhal S.K. Thompson L. J. Immunol. 1983; 131: 762-767PubMed Google Scholar).There is little information correlating heterogeneity in oligosaccharide structure with ligand specificity or the signal transducing properties of CD43. In the thymus, thymocyte-thymic epithelial cell interactions correlate with the preferential binding of galectin-1 to the mAb T-305/CD43 isoform expressed in immature, cortical thymocytes (27Baum L.G. Pang M. Perillo N.L. Wu T. Delegeane A. Uittenbogarrt C.H. Fukuda M. Seilhamer J.J. J. Exp. Med. 1995; 181: 877-887Crossref PubMed Scopus (255) Google Scholar). In mature T-cells the binding of anti-CD43 antibodies, thought to mimic natural ligands, results in CD28-independent costimulatory activity (11Alvarado M. Klassen C. Cerny J. Horejsi V. Schmidt R.E. Eur. J. Immunol. 1995; 25: 1051-1055Crossref PubMed Scopus (45) Google Scholar, 28Silvermann L.B. Wong R.C.K. Remold-O'Donnell E. Vercelli D. Sancho J. Terhorst C. Rosen F. Geha R. Chatila T. J. Immunol. 1989; 142: 4194-4200PubMed Google Scholar, 29Wong R. Remold-O'Donnell E. Vercelli D. Sancho J. Terhorst C. Rosen F. Geha R. Chatila T. J. Immunol. 1990; 144: 1455-1460PubMed Google Scholar, 30Mentzer S.J. Remold-O'Donnell E. Crimmis M.A.V. Bierer R.E. Rosen F.S. Burakoff S.J. J. Exp. Med. 1987; 165: 1383-1392Crossref PubMed Scopus (123) Google Scholar, 31Sperling A.I. Green J.M. Mosley R.L. Smith P.L. DiPaolo R.J. Klein J.R. Bluestone J.A. Thompson C.B. J. Exp. Med. 1995; 182: 139-146Crossref PubMed Scopus (124) Google Scholar). Interestingly, the L10 antibody directed against a neuraminidase-resistant epitope of CD43 is a strong inducer of T-cell proliferation, whereas the B1B6 antibody directed against a neuraminidase-sensitive epitope is only weakly mitogenic (10Axelsson B. Youseffi-Etemad R. Hammarstrom S. Perlmann P. J. Immunol. 1988; 141: 2912-2917PubMed Google Scholar). Immunoprecipitations from T-lymphoblastoid cell lysates have found CD43 to be associated with CD3/TcR and p56LCK protein tyrosine kinase (11Alvarado M. Klassen C. Cerny J. Horejsi V. Schmidt R.E. Eur. J. Immunol. 1995; 25: 1051-1055Crossref PubMed Scopus (45) Google Scholar), providing both physical and functional evidence for a role for CD43 in signal transduction. The anti-CD43 antibody, mAb MEM-59, which is costimulatory in T-lymphocytes has recently been reported to induce programmed cell death or apoptosis in hematopoietic progenitor cells (32Bazil V. Brandt J. Tsukamoto A. Hoffman R. Blood. 1995; 86: 502-511Crossref PubMed Google Scholar). Clearly, CD43-mediated responses can differ significantly depending on the cell type and the isoform being expressed. While aberrant isoforms of CD43 have been associated with immunodeficiencies resulting in lymphopenia (33Cooper M.D. Chase H.P. Lowman J.T. Krivit W. Good R.A. Clin. Stud. 1968; 44: 499-513Google Scholar, 34Bowen D.L. Lane H.C. Fauci A.S. Ann. Intern. Med. 1985; 103: 704-709Crossref PubMed Scopus (248) Google Scholar), there has been no direct evidence linking CD43-mediated apoptosis with T-cell depletion.Here we report the biochemical and functional characterization of an anti-CD43 mAb designated J393 that recognizes a unique, alternatively glycosylated isoform of CD43 expressed on the surface of the human T-lymphoblastoid cell line, Jurkat. Treatment of Jurkat cells with mAb J393 induces apoptosis in a CD43 isoform-specific manner. The level of apoptosis may be enhanced by concomitant engagement of the TcR or integrin molecules. Moreover, an isoform of CD43 is detected in peripheral blood T-lymphocytes bearing a cryptic epitope for mAb J393. These results describe a potentially novel mechanism for T-cell lineage depletion involving the regulated expression of specific isoforms of CD43.DISCUSSIONThe existence of a CD43-mediated pathway for signaling apoptosis in T-cell lineages has not been previously described. Our observation that the BMS-2 Jurkat cell line co-expressed two antigenically distinct isoforms of CD43 on its surface provided us with a good model for examining structure-function relationships between these two molecules. While both mAb J393 and mAb G10-2 induced homotypic adhesion, only mAb J393 induced apoptosis. This implied that the apoptotic response was independent of the homotypic adhesion phenomenon and was associated with a specific CD43 isoform. The reactivity of mAb G10-2 required terminal sialic acid moieties, whereas reactivity of mAb J393 was sialic acid-independent. Other investigators have observed sialic acid-independent CD43 antibodies to elicit stronger cellular responses than their sialic acid-dependent counterparts (10Axelsson B. Youseffi-Etemad R. Hammarstrom S. Perlmann P. J. Immunol. 1988; 141: 2912-2917PubMed Google Scholar). We found the carbohydrate structure of J393/CD43 in Jurkat cells to be deficient in oligosaccharide complexity and sialic acid content when compared with that of the G10-2/CD43 isoform expressed in HPB-ALL cells. The majority of serine/threonine residues in the extracellular portion of the J393 antigen contained only terminal GalNAc monosaccharides, similar to the Tn antigen-bearing CD43 molecule expressed in Jurkat cells (50Nakada H. Inoue M. Tanaka N. Numada Y. Kitagawa H. Fukui S. Yama-Shina I. Biochem. Biophys. Res. Commun. 1991; 179: 762-767Crossref PubMed Scopus (26) Google Scholar). Glycopeptides generated from intact mAb J393 antigen lost functional epitope as determined by immunoaffinity chromatography run under conditions that favor interaction of low valency glycopeptides with the antibody. Low valency for antibodies has been reported for glycopeptides containing the cluster antigens of Tn when compared with intact glycoprotein (56Inoue M. Nakada H. Tanaka N. Yamashina I. Cancer Res. 1994; 54: 85-88PubMed Google Scholar). Although mAb J393 likely recognizes the Tn-containing isoform of CD43, it does not react with the surface of certain cell types expressing Tn epitopes. Therefore, we propose that mAb J393 recognizes a unique epitope on CD43 that has not been previously characterized.Little is known about how CD43 functions as a signaling molecule. We find that soluble, bivalent antibody is sufficient to initiate a CD43 signal, unlike the requirement for trimeric ligation by members of the TNF receptor superfamily including CD95/Fas (57Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1831) Google Scholar). In T-lymphocytes, CD43 has been shown to be constitutively phosphorylated in resting cells and hyperphosphorylated following cellular activation (58Piller V. Piller F. Fukuda M. J. Biol. Chem. 1989; 264: 18824-18831Abstract Full Text PDF PubMed Google Scholar). Because CD43 contains no catalytic region or tyrosine residues within the cytoplasmic domain, its phosphorylation is thought to reflect its association with serine/threonine-specific protein kinases (59Chatila T.A. Geha R.S. J. Immunol. 1988; 140: 4308-4314PubMed Google Scholar, 60Axelsson B. Perlmann P. Scand. J. Immunol. 1989; 30: 539-547Crossref PubMed Scopus (21) Google Scholar, 61Fukuda M. Carlsson S.R. Med. Biol. 1986; 64: 335-343PubMed Google Scholar). However, investigators characterizing CD43 as a CD28-independent costimulatory molecule have described the physical association of CD43 with CD3 in a complex containing the SRC family protein tyrosine kinases lck and fyn (11Alvarado M. Klassen C. Cerny J. Horejsi V. Schmidt R.E. Eur. J. Immunol. 1995; 25: 1051-1055Crossref PubMed Scopus (45) Google Scholar), suggesting an involvement of CD43 with tyrosine phosphorylation events. Indeed, pretreatment of Jurkat cells with the tyrosine kinase inhibitor herbimycin A significantly interfered with mAb J393-induced cell death. We found that treatment of Jurkat cells with mAb J393 induced a rapid increase in overall protein tyrosine phosphorylation and, in particular, for proteins in the 50-55-kDa range. Interestingly, this group of proteins was not phosphorylated following engagement of CD3/TcR, suggesting a degree of specificity in the pattern of CD43-induced tyrosine phosphorylation. Moreover, concomitant ligation of J393/CD43 and CD3/TcR resulted in the hyperphosphorylation of these CD43-dependent substrates, providing a biochemical correlate for the synergy observed for these two receptor molecules in mediating apoptosis. Collectively, these findings suggest that protein tyrosine phosphorylation of specific substrates is important in signaling CD43-mediated apoptosis.We found that preventing dephosphorylation of phosphotyrosyl residues by blocking phosphatase activity resulted in a significant enhancement in mAb J393-induced cell death. Thus, the PTPase inhibitor BMLOV mimicked the action of CD3/TcR in potentiating CD43-mediated cell death, seemingly consistent with a hyperphosphorylated state. These results indicate that the catalytic activity of BMLOV-sensitive phosphatases negatively regulates CD43-mediated apoptosis. It is likely that the potentiation of CD43-mediated apoptosis by CD3/TcR engagement involves CD45 phosphatase activity since CD45 is expressed in high abundance in Jurkat cells and is required to activate TcR-associated SRC family kinases allowing for competent antigen-induced signal transduction (62Hovis R.R. Donovan J.A. Musci M.A. Motto D.G. Goldman F.D. Ross S.E. Koretzky G.A. Science. 1993; 260: 544-546Crossref PubMed Scopus (100) Google Scholar, 63Volarevic S. Niklinska B.B. Burns C.M. June C.H. Weissman A.M. Ashwell J.D. Science. 1993; 260: 541-543Crossref PubMed Scopus (102) Google Scholar). Therefore, in this context CD45 may be positively regulating CD43-mediated apoptosis by directly enhancing kinase activity. Notably, BMLOV treatment of Jurkat cells does not interfere with TcR-induced tyrosine phosphorylation (55Schieven G.L. Wahl A.F. Myrdal S. Grosmaire L. Ledbetter J.A. J. Biol. Chem. 1995; 270: 20824-20831Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) which may explain why BMLOV treatment did not block the synergism we observed for TcR/CD43-induced apoptosis. Conversely, the catalytic activity of SH2 PTPases may be expected to exert a negative regulatory effect on CD43-mediated apoptosis, since PTP1C has been shown to negatively regulate antigen receptor signaling in B-lymphocytes as confirmed in PTP1C-deficient mice expressing the motheaten phenotype (64Thomas M.L. Immunology. 1995; 7: 279-288Google Scholar). Further investigation is necessary to identify which enzymes and substrate sites are involved in this CD43-mediated pathway.It was recently reported that CD3/TcR-induced apoptosis in T-cells is mediated by the autocrine production of the ligand for CD95/Fas (FasL), thereby activating the CD95/Fas receptor signaling pathway (65Ju S.-T. Panka D.J. Cui H. Ettinger R. El-Khatib M. Sherr D.H. Stanger B.Z. Marshak-Rothstein A. Nature. 1995; 373: 444-448Crossref PubMed Scopus (1451) Google Scholar). Interestingly, a requirement for tyrosine kinase activation in CD95/Fas-mediated programmed cell death has been described in conjunction with a requirement for ceramide-initiated RAS activation (66Eischen C.M. Dick C.J. Leibson P.J. J. Immunol. 1994; 153: 1947-1954PubMed Google Scholar, 67Gulbins E. Bissonnette R. Mahboubi A. Martin S. Nishioka W. Brunner T. Baier G. Baier-Bitterlich G. Byrd C. Lang F. Kolesnick R. Altman A. Green D. Immunity. 1995; 2: 341-351Abstract Full Text PDF PubMed Scopus (432) Google Scholar); however, these signals alone were not sufficient for subsequent apoptosis. We found that neither a blocking antibody to CD95/Fas nor the competing fusion protein CD95/Fas-Ig prevented J393/CD43-induced apoptosis, making it unlikely that FasL was mediating this response. Moreover, we observed that combined engagement of J393/CD43-CD3/TcR leads to a rapid reduction in the nuclear localization of NF-κB and IRE regulatory proteins normally associated with transcriptional activation of the FasL gene (68Takahashi T. Int. Immunol. 1994; 6: 1567-1574Crossref PubMed Scopus (412) Google Scholar). Likewise, since sphingomyelinase-dependent NF-κB activation has been reported to lead to apoptosis signaled by the TNF receptor-1 "death domain" (69Wiegmann K. Schutze S. Machleidt T. Witte D. Kronke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (673) Google Scholar), it is unlikely that TNF receptor-1 is involved in J393/CD43-mediated killing. Therefore, engagement of CD43 may define a distinct signaling pathway for programmed cell death that differs from that of the TNF-nerve growth factor receptor family. Perhaps in the BMS-2 Jurkat cells, constitutive activation and nuclear localization of NF-κB and IRE DNA-binding proteins are critical for continued proliferation; hence, a decrease in nuclear localization of these transcriptional factors may lead to molecular events that promote growth arrest and eventual suicide. Recent findings in CD43-deficient T-cells generated by homologous recombination indicate that CD43 acts as a negative regulator of T-cell activation and proliferation (8Manjunath N. Correa M. Ardman M. Ardman B. Nature. 1995; 377: 535-538Crossref PubMed Scopus (187) Google Scholar), which may reflect our observation that CD43 signaling can suppress the activity of transcriptional regulators of early T-cell activation genes.While mAb J393 reacted strongly with Jurkat cell surfaces, it failed to immunostain the surface of either resting or activated peripheral blood T-lymphocytes. However, immunoblot analysis revealed the presence of several mAb J393-reactive proteins in the lysates of activated T-cells, possibly representing intracellular intermediates of CD43 in the process of O-glycan elongation. Interestingly, in activated T-cells both mAb J393 and mAb G10-2 cross-reacted with a 97-kDa isoform of CD43. Investigators have previously shown that isoform-specific antibodies may cross-react with linear protein epitopes on the deglycosylated molecule and that these epitopes may be modified by glycosylation in conferring antigenic specificity (23Cyster J.G. Shotton D.M. Williams A.F. EMBO J. 1991; 10: 893-902Crossref PubMed Scopus (236) Google Scholar). This suggested that the antigenic specificity for these two antibodies developed as a consequence of activation-induced changes in the glycosylation of the core protein. Furthermore, the mAb J393-specific 140-kDa isoform present in Jurkat cells was replaced with a 160-kDa isoform in activated T-cells. Presumably, the J393/CD43 intermediate in T-lymphocytes contains less sialic acid than its counterpart in Jurkat cells, thus contributing to its reduced electrophoretic mobility (14Ardman B. Sikorski M.A. Settles M. Staunton D.E. J. Exp. Med. 1990; 172: 1151-1158Crossref PubMed Scopus (66) Google Scholar). Unlike T-cells from normal individuals, Jurkat cells have been shown to be deficient in β1,3-galactosyltransferase activity, a key enzyme in the formation of complex O-glycan structures (49Piller V. Piller F. Fukuda M. J. Biol. Chem. 1990; 265: 9264-9271Abstract Full Text PDF PubMed Google Scholar). Thus, we postulate that the epitope for mAb J393 is tightly regulated in T-cell lineages, normally being masked at the cell surface by the presence of complex carbohydrate chains attached to the CD43 protein.Our finding that apoptosis can be induced through a CD43-mediated pathway may have physiologic significance in peripheral lymphoid tissues such as the thymus and lymph nodes, since the inhibition of O-glycan elongation in T-cells potentiates the apoptotic effect of galectin-1, an endogenous ligand for CD43 expressed at these sites (70Perillo N.L. Pace K.E. Seilhamer J.L. Baum L.G. Nature. 1995; 378: 736-739Crossref PubMed Scopus (935) Google Scholar). Concomitant engagement of specific integrin molecules may serve to modulate CD43induced responses as our data suggest, in addition to the specific regulation of integrin expression mediated by CD43 as observed by others (71Hernandez-Caselles T. Martinez-Esparza M. Lazarovits A.I. Aparicio P. J. Immunol. 1996; 156: 3668-3677PubMed Google Scholar). We propose that the truncated O-glycan structure of the J393/CD43 molecule represents a molecular phenotype with an altered affinity or specificity for natural ligands involved in cell-cell interactions and that following its ligation an apoptotic response may be triggered in appropriate cell types.As mentioned previously, abnormalities in O-glycan biosynthesis are found in a variety of pathologic conditions involving hematopoietic and immunologic disorders, often correlating with the appearance of Tn antigen and autoantibodies to cell-surface molecules including CD43 (14Ardman B. Sikorski M.A. Settles M. Staunton D.E. J. Exp. Med. 1990; 172: 1151-1158Crossref PubMed Scopus (66) Google Scholar, 18Thunher M. Rusconi S. Berger E.G. J. Clin. Invest. 1993; 91: 2103-2110Crossref PubMed Scopus (64) Google Scholar, 72Berger E.G. Thurnher M. Dinter A. Transfus. Clin. Biol. 1994; 1: 103-108Crossref PubMed Scopus (4) Google Scholar). These findings suggest that the expression of alternatively glycosylated isoforms of CD43 may contribute to the progression of such diseases by promoting T-cell deficiency and lymphopenia as a consequence of programmed cell death. INTRODUCTIONIn T-lymphocytes CD43/leukosialin, the major sialoglycoprotein of leukocytes (1Remold-O'Donnell E. Kenny D.M. Parkman R. Cairns L. Savage B. Rosen F.S. J. Exp. Med. 1984; 159: 1705-1723Crossref PubMed Scopus (156) Google Scholar, 2Kenny D. Cairns L. Remold-O'Donnell E. Peterson J. Rosen F.S. Parkman R. Blood. 1986; 68: 1329-1332Crossref PubMed Google Scholar), is thought to serve a dual role in regulating cellular immune responses. Due to the repulsive effect of its high numbers of O-linked negatively charged sialic acid sugar residues, CD43 acts as a "barrier molecule" by limiting cell-cell/cell-ligand interactions (3Brown W.R.A. Barclay A.N. Sunderland C.A. Williams A.F. Nature. 1981; 289: 456-460Crossref PubMed Scopus (157) Google Scholar, 4Rosenstein Y. Park J.K. Hahn W.C. Rosen F.S. Bierer B.E. Burakoff S.J. Nature. 1991; 354: 233-235Crossref PubMed Scopus (224) Google Scholar, 5Ardman B. Sikorski M.A. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5001-5005Crossref PubMed Scopus (149) Google Scholar, 6Manjunath N. Johnson R.S. Staunton D.E. Pasqualini R. Ardman B. J. Immunol. 1993; 151: 1528-1534PubMed Google Scholar, 7Kojima N. Saito M. Shuichi T. Exp. Cell Res. 1994; 214: 537-542Crossref PubMed Scopus (13) Google Scholar), a property that may negatively regulate T-cell activation (8Manjunath N. Correa M. Ardman M. Ardman B. Nature. 1995; 377: 535-538Crossref PubMed Scopus (187) Google Scholar). In addition, engagement of CD43 by monoclonal antibodies has been shown to induce costimulatory activity in T-cells by a mechanism analogous to a classic ligand-receptor interaction (9Sportsman J.R. Park M.M. Cheresh D.A. Fukuda M. Elder J.H. Fox R.I. J. Immunol. 1985; 135: 158-164PubMed Google Scholar, 10Axelsson B. Youseffi-Etemad R. Hammarstrom S. Perlmann P. J. Immunol. 1988; 141: 2912-2917PubMed Google Scholar, 11Alvarado M. Klassen C. Cerny J. Horejsi V. Schmidt R.E. Eur. J. Immunol. 1995; 25: 1051-1055Crossref PubMed Scopus (45) Google Scholar). Alterations in O-glycan structure and function of CD43 reportedly occur in the immunodeficiency disorders Wiskott-Aldrich syndrome (12Parkman R. Remold-O'Donnell E. Kenny D.M. Perrine S. Rosen F.S. Lancet. 1981; ii: 1387-1389Abstract Scopus (89) Google Scholar, 13Siminovitch K.A. Greer W.L. Axelsson B. Rubin L.A. Novogrodsky A. Peacocke M. Immunodefic. Rev. 1993; 4: 99-108Google Scholar) and AIDS (14Ardman B. Sikorski M.A. Settles M. Staunton D.E. J. Exp. Med. 1990; 172: 1151-1158Crossref PubMed Scopus (66) Google Scholar, 15Lefebvre J.-C. Giordanengo V. Limouse M. Doglio A. Cucchiarini M. Monpoux F. Mariani R. Peyron J.-F. J. Exp. Med. 1994; 180: 1609-1617Crossref PubMed Scopus (59) Google Scholar) as well as in graft versus host disease (16Ellies L.G. Jones A.T. Williams M.J. Ziltener H.J. Glycobiology. 1994; 4: 885-893Crossref PubMed Scopus (35) Google Scholar), acute lymphocytic leukemia (17Saitoh O. Piller F. Fox R.I. Fukuda M. Blood. 1991; 77: 1491-1499Crossref PubMed Google Scholar), and permanent mixed-field polyagglutinability or Tn antigen syndrome (18Thunher M. Rusconi S. Berger E.G. J. Clin. Invest. 1993; 91: 2103-2110Crossref PubMed Scopus (64) Google Scholar).The molecular structure of human CD43 is mucin-like, consisting of an extended rod-shaped extracellular portion bearing approximately 80 sialylated O-glycan sites and a single N-glycan site, a highly conserved transmembrane region, and a long cytoplasmic domain bearing potential serine/threonine phosphorylation sites (19Shelley C.S. Remold-O'Donnell E. Davis A.E. Bruns G.A.P. Rosen F.S. Carroll M.C. Whitehead A.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2819-2823Crossref PubMed Scopus (117) Google Scholar). Based on the exon/intron arrangement within its gene, the observed molecular heterogeneity of CD43 in both mice and man is thought to reflect differential post-translational modifications of a single gene product (20Pallant A. Eskenazi A. Mattei M.-G. Fournier R.E.K. Carlsson S.R. Fukuda M. Frelinger J.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1328-1332Crossref PubMed Scopus (116) Google Scholar, 21Shelly C.S. Remold-O'Donnell E. Rosen F.S. Whitehead A.S. Biochem. J. 1990; 270: 569-576Crossref PubMed Scopus (26) Google Scholar, 22Cyster J. Somoza C. Killeen N. Williams A.F. Eur. J. Immunol. 1990; 20: 875-881Crossref PubMed Scopus (49) Google Scholar). Linear protein epitopes in the native structure of CD43 have been shown to be modified by glycosylation (23Cyster J.G. Shotton D.M. Williams A.F. EMBO J. 1991; 10: 893-902Crossref PubMed Scopus (236) Google Scholar), allowing for the development of isoform-specific antibodies (9Sportsman J.R. Park M.M. Cheresh D.A. Fukuda M. Elder J.H. Fox R.I. J. Immunol. 1985; 135: 158-164PubMed Google Scholar, 11Alvarado M. Klassen C. Cerny J. Horejsi V. Schmidt R.E. Eur. J. Immunol. 1995; 25: 1051-1055Crossref PubMed Scopus (45) Google Scholar, 24Jones A.T. Federsppiel B. Ellies L.G. Williams M.J. Burgener R. Duronio V. Smith C.A. Takei F. Ziltener H.J. J. Immunol. 1994; 153: 3426-3439PubMed Google Scholar). Following T-cell activation, the O-linked oligosaccharides of CD43 change from tetrasaccharides (mAb 1The abbreviations used are: mAbmonoclonal antibodyTcRT-cell receptorFACSfluorescence-activated cell sorterFITCfluorescein isothiocyanatePEphycoerythrinPIpropidium iodidePHAphytohemagglutininBMLOVbis(maltolato)oxovanadium-IVNFκ Bnuclear factor kappa BTNFtumor necrosis factorPBSphosphate-buffered salineMOPS3-[N-morpholino]propanesulfonic acidTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinePTPasephosphotyrosine phosphataseIREinterferon-inducible regulatory element. G10-2 reactive) to more complex hexasaccharides (mAb T-305 reactive) due to the activation-induced expression of core 2 β1→6 N-acetylglucosaminyltransferase (17Saitoh O. Piller F. Fox R.I. Fukuda M. Blood. 1991; 77: 1491-1499Crossref PubMed Google Scholar, 25Bierhuizen M.F.A. Maemura K. Fukuda M. J. Biol. Chem. 1994; 269: 4473-4479Abstract Full Text PDF PubMed Google Scholar). This change in oligosaccharide structure results in a shift from 115 to 140 kDa (9Sportsman J.R. Park M.M. Cheresh D.A. Fukuda M. Elder J.H. Fox R.I. J. Immunol. 1985; 135: 158-164PubMed Google Scholar, 17Saitoh O. Piller F. Fox R.I. Fukuda M. Blood. 1991; 77: 1491-1499Crossref PubMed Google Scholar, 25Bierhuizen M.F.A. Maemura K. Fukuda M. J. Biol. Chem. 1994; 269: 4473-4479Abstract Full Text PDF PubMed Google Scholar, 26Fox R.I. Hueniken M. Fong S. Behar S. Royston I. Singhal S.K. Thompson L. J. Immunol. 1983; 131: 762-767PubMed Google Scholar).There is little information correlating heterogeneity in oligosaccharide structure with ligand specificity or the signal transducing properties of CD43. In the thymus, thymocyte-thymic epithelial cell interactions correlate with the preferential binding of galectin-1 to the mAb T-305/CD43 isoform expressed in immature, cortical thymocytes (27Baum L.G. Pang M. Perillo N.L. Wu T. Delegeane A. Uittenbogarrt C.H. Fukuda M. Seilhamer J.J. J. Exp. Med. 1995; 181: 877-887Crossref PubMed Scopus (255) Google Scholar). In mature T-cells the binding of anti-CD43 antibodies, thought to mimic natural ligands, results in CD28-independent costimulatory activity (11Alvarado M. Klassen C. Cerny J. Horejsi V. Schmidt R.E. Eur. J. Immunol. 1995; 25: 1051-1055Crossref PubMed Scopus (45) Google Scholar, 28Silvermann L.B. Wong R.C.K. Remold-O'Donnell E. Vercelli D. Sancho J. Terhorst C. Rosen F. Geha R. Chatila T. J. Immunol. 1989; 142: 4194-4200PubMed Google Scholar, 29Wong R. Remold-O'Donnell E. Vercelli D. Sancho J. Terhorst C. Rosen F. Geha R. Chatila T. J. Immunol. 1990; 144: 1455-1460PubMed Google Scholar, 30Mentzer S.J. Remold-O'Donnell E. Crimmis M.A.V. Bierer R.E. Rosen F.S. Burakoff S.J. J. Exp. Med. 1987; 165: 1383-1392Crossref PubMed Scopus (123) Google Scholar, 31Sperling A.I. Green J.M. Mosley R.L. Smith P.L. DiPaolo R.J. Klein J.R. Bluestone J.A. Thompson C.B. J. Exp. Med. 1995; 182: 139-146Crossref PubMed Scopus (124) Google Scholar). Interestingly, the L10 antibody directed against a neuraminidase-resistant epitope of CD43 is a strong inducer of T-cell proliferation, whereas the B1B6 antibody directed against a neuraminidase-sensitive epitope is only weakly mitogenic (10Axelsson B. Youseffi-Etemad R. Hammarstrom S. Perlmann P. J. Immunol. 1988; 141: 2912-2917PubMed Google Scholar). Immunoprecipitations from T-lymphoblastoid cell lysates have found CD43 to be associated with CD3/TcR and p56LCK protein tyrosine kinase (11Alvarado M. Klassen C. Cerny J. Horejsi V. Schmidt R.E. Eur. J. Immunol. 1995; 25: 1051-1055Crossref PubMed Scopus (45) Google Scholar), providing both physical and functional evidence for a role for CD43 in signal transduction. The anti-CD43 antibody, mAb MEM-59, which is costimulatory in T-lymphocytes has recently been reported to induce programmed cell death or apoptosis in hematopoietic progenitor cells (32Bazil V. Brandt J. Tsukamoto A. Hoffman R. Blood. 1995; 86: 502-511Crossref PubMed Google Scholar). Clearly, CD43-mediated responses can differ significantly depending on the cell type and the isoform being expressed. While aberrant isoforms of CD43 have been associated with immunodeficiencies resulting in lymphopenia (33Cooper M.D. Chase H.P. Lowman J.T. Krivit W. Good R.A. Clin. Stud. 1968; 44: 499-513Google Scholar, 34Bowen D.L. Lane H.C. Fauci A.S. Ann. Intern. Med. 1985; 103: 704-709Crossref PubMed Scopus (248) Google Scholar), there has been no direct evidence linking CD43-mediated apoptosis with T-cell depletion.Here we report the biochemical and functional characterization of an anti-CD43 mAb designated J393 that recognizes a unique, alternatively glycosylated isoform of CD43 expressed on the surface of the human T-lymphoblastoid cell line, Jurkat. Treatment of Jurkat cells with mAb J393 induces apoptosis in a CD43 isoform-specific manner. The level of apoptosis may be enhanced by concomitant engagement of the TcR or integrin molecules. Moreover, an isoform of CD43 is detected in peripheral blood T-lymphocytes bearing a cryptic epitope for mAb J393. These results describe a potentially novel mechanism for T-cell lineage depletion involving the regulated expression of specific isoforms of CD43.
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