RESUMEN
Recognition of antigen by the B cell antigen receptor (BCR) determines the subsequent fate of a B cell and is regulated in part by the involvement of other surface molecules, termed coreceptors. CD22 is a B cell-restricted coreceptor that gets rapidly tyrosyl-phosphorylated and recruits various signaling molecules to the membrane following BCR ligation. Although CD22 contains three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), only the two carboxyl-terminal ITIM tyrosines are required for efficient recruitment of the SHP-1 phosphatase after BCR ligation. Furthermore, Grb2 is inducibly recruited to CD22 in human and murine B cells. Unlike SHP-1, Grb2 recruitment to CD22 is not inhibited by specific doses of the Src family kinase-specific inhibitor PP1. The tyrosine residue in CD22 required for Grb2 recruitment (Tyr-828) is distinct and independent from the two ITIM tyrosines required for efficient SHP-1 recruitment (Tyr-843 and Tyr-863). Individually both Lyn and Syk are required for maximal phosphorylation of CD22 following ligation of the BCR, and together Lyn and Syk are required for all of the constitutive and induced tyrosine phosphorylation of CD22. We propose that the cytoplasmic tail of CD22 contains two domains that regulate signal transduction pathways initiated by the BCR and B cell fate.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos B/fisiología , Moléculas de Adhesión Celular , Lectinas , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas/metabolismo , Receptores de Antígenos de Linfocitos B/fisiología , Transducción de Señal/fisiología , Animales , Secuencia de Bases , Cartilla de ADN , Activación Enzimática , Proteína Adaptadora GRB2 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fenotipo , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Lectina 2 Similar a Ig de Unión al Ácido SiálicoRESUMEN
The balance between cell survival and cell death is critical for normal lymphoid development. This balance is maintained by signals through lymphocyte antigen receptors and death receptors such as CD95/Fas. In some cells, ligating the B cell antigen receptor can protect the cell from apoptosis induced by CD95. Here we report that ligation of CD95 inhibits antigen receptor-mediated signaling. Pretreating CD40-stimulated tonsillar B cells with anti-CD95 abolished B cell antigen receptor-mediated calcium mobilization. Furthermore, CD95 ligation led to the caspase-dependent inhibition of antigen receptor-induced calcium mobilization and to the activation of mitogen-activated protein kinase pathways in B and T cell lines. A target of CD95-mediated caspase 3-like activity early in the apoptotic process is the adaptor protein GrpL/Gads. GrpL constitutively interacts with SLP-76 via its C-terminal SH3 domain to regulate transcription factors such as NF-AT. Cleavage of GrpL removes the C-terminal SH3 domain so that it is no longer capable of recruiting SLP-76 to the membrane. Transfection of a truncated form of GrpL into Jurkat T cells blocked T cell antigen receptor-induced activation of NF-AT. These results suggest that CD95 signaling can desensitize antigen receptors, in part via cleavage of the GrpL adaptor.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas Nucleares , Receptores de Antígenos de Linfocitos T/fisiología , Receptor fas/fisiología , Animales , Caspasa 3 , Caspasas/metabolismo , Línea Celular , Proteínas de Unión al ADN/fisiología , Activación Enzimática , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/fisiología , Factores de Transcripción NFATC , Factores de Transcripción/fisiologíaRESUMEN
The serine/threonine kinase Mst1, a mammalian homolog of the budding yeast Ste20 kinase, is cleaved by caspase-mediated proteolysis in response to apoptotic stimuli such as ligation of CD95/Fas or treatment with staurosporine. Furthermore, overexpression of Mst1 induces morphological changes characteristic of apoptosis in human B lymphoma cells. Mst1 may therefore represent an important target for caspases during cell death which serves to amplify the apoptotic response. Here we report that Mst1 has two caspase cleavage sites, and we present evidence indicating that cleavage may occur in an ordered fashion and be mediated by distinct caspases. We also show that caspase-mediated cleavage alone is insufficient to activate Mst1, suggesting that full activation of Mst1 during apoptosis requires both phosphorylation and proteolysis. Another role of phosphorylation may be to influence the susceptibility of Mst1 to proteolysis. Autophosphorylation of Mst1 on a serine residue close to one of the caspase sites inhibited caspase-mediated cleavage in vitro. Finally, Mst1 appears to function upstream of the protein kinase MEKK1 in the SAPK pathway. In conclusion, Mst1 activity is regulated by both phosphorylation and proteolysis, suggesting that protein kinase and caspase pathways work in concert to regulate cell death.
Asunto(s)
Apoptosis/fisiología , Caspasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor fas/fisiología , Humanos , Hidrólisis , Péptidos y Proteínas de Señalización Intracelular , Fosforilación , Células Tumorales CultivadasRESUMEN
To define how the signaling pathways that mediate the B cell receptor (BCR) death pathway differ from those responsible for CD95/Fas-mediated death, we compared the BCR and Fas death pathways in two human B cell lines, B104 and BJAB. Both BCR- and Fas-induced apoptosis are blocked by the peptide cysteine protease inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD (mlz)), demonstrating a common requirement caspase activity. Despite this common characteristic, the ability of actinomycin D and cycloheximide to block BCR-induced apoptosis, but not apoptosis induced by Fas cross-linking, suggests that a major difference between these two pathways is their differential requirements for new gene and protein synthesis. BCR- and Fas-mediated apoptosis are both accompanied by activation of stress-activated protein kinase and p38 mitogen-activated protein kinase (MAPK). Activation of both stress-activated protein kinase and p38 MAPK was inhibited by ZVAD (mlz), suggesting the involvement of caspases. To determine the role of p38 MAPK activation in BCR- and Fas-induced apoptosis, we employed SB203580, a specific inhibitor of p38 MAPK. SB203580 inhibited BCR-induced apoptosis, but not apoptosis induced by cross-linking Fas. Furthermore, both actinomycin D and SB203580 inhibited BCR-induced, but not Fas-induced, activation of caspase. Collectively, these findings establish a role for p38 MAPK in BCR-induced apoptosis both upstream and downstream of caspase activity. The p38 MAPK pathway may function to regulate transcriptional or translational events that are critical for BCR-induced apoptosis.
Asunto(s)
Apoptosis/inmunología , Subgrupos de Linfocitos B/inmunología , Proteínas Quinasas Activadas por Mitógenos , Receptores de Antígenos de Linfocitos B/fisiología , Transducción de Señal/inmunología , Receptor fas/fisiología , Anexina A5/metabolismo , Anticuerpos Antiidiotipos/farmacología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Subgrupos de Linfocitos B/enzimología , Subgrupos de Linfocitos B/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Cisteína Endopeptidasas/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Humanos , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Cinética , Linfoma de Células B , Unión Proteica/inmunología , Células Tumorales Cultivadas , Receptor fas/inmunología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Mst1 is a ubiquitously expressed serine-threonine kinase, homologous to the budding yeast Ste20, whose physiological regulation and cellular function are unknown. In this paper we show that Mst1 is specifically cleaved by a caspase 3-like activity during apoptosis induced by either cross-linking CD95/Fas or by staurosporine treatment. CD95/Fas-induced cleavage of Mst1 was blocked by the cysteine protease inhibitor ZVAD-fmk, the more selective caspase inhibitor DEVD-CHO and by the viral serpin CrmA. Caspase-mediated cleavage of Mst1 removes the C-terminal regulatory domain and correlates with an increase in Mst1 activity in vivo, consistent with caspase-mediated cleavage activating Mst1. Overexpression of either wild-type Mst1 or a truncated mutant induces morphological changes characteristic of apoptosis. Furthermore, exogenously expressed Mst1 is cleaved, indicating that Mst1 can activate caspases that result in its cleavage. Kinase-dead Mst1 did not induce morphological alterations and was not cleaved upon overexpression, indicating that Mst1 must be catalytically active in order to mediate these effects. Mst1 activates MKK6, p38 MAPK, MKK7 and SAPK in co-transfection assays, suggesting that Mst1 may activate these pathways. Our findings suggest the existence of a positive feedback loop involving Mst1, and possibly the SAPK and p38 MAPK pathways, which serves to amplify the apoptotic response.
Asunto(s)
Apoptosis , Caspasas , Cisteína Endopeptidasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Clorometilcetonas de Aminoácidos/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Caspasa 3 , Línea Celular Transformada , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM , Mamíferos , Datos de Secuencia Molecular , Oligopéptidos/farmacología , Proteínas Serina-Treonina Quinasas/genética , Serpinas/genética , Serpinas/metabolismo , Células Tumorales Cultivadas , Proteínas Virales/genética , Proteínas Virales/metabolismo , Receptor fas/metabolismoRESUMEN
CD22 is a B cell-restricted surface molecule which may play an important role in interactions between B cells and other cells and in regulating signals through the B cell receptor (BCR) complex. Here we have examined whether the mouse is a suitable in vivo model for studying CD22 functions. In primary and secondary lymphoid organs of adult mice CD22 is on all mature B cells, including resting IgM+IgD+ B cells, IgG+ HSA(lo) memory B cells, syndecan+ plasma cells and CD5+ B cells, but it is not on immature IgM+IgD- B cells. Biochemical analysis revealed that murine CD22 is associated with the IgM receptor in some, but not all, CD22+ B leukemic and lymphoma cell lines; as with human CD22, murine CD22 is rapidly phosphorylated on tyrosine after ligation of the BCR. In the CD22- murine pro-B cell line, FEMCL, CD22 expression was inducible by treatment with phorbol 12-myristate 13-acetate. A genomic fragment of the cd22b allele containing 1.3 kb 5' of exon 1 was sequenced in order to identify potential DNA regulatory elements in the CD22 promoter region. Consensus sequences for transcription factor binding sites including PU.1, AP-1, AP-2, C/EBP and SP-1 were present, but no classical TATA elements or initiator motifs were evident at relevant positions. The 1.3-kb promoter fragment 5' of exon 1 was sufficient for directing basal promoter activity in B and T cells. There was no significant sequence similarity between the murine and human cd22 gene promoters, although both contain repetitive elements and Sp-1 and AP1 binding sites. Thus, murine CD22 shares a number of features with human CD22 and the mouse provides a suitable model system for elucidating the function of CD22 in vivo.
Asunto(s)
Antígenos CD/química , Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos B/química , Antígenos de Diferenciación de Linfocitos B/genética , Linfocitos B/metabolismo , Moléculas de Adhesión Celular , Lectinas , Regiones Promotoras Genéticas/inmunología , Animales , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos B/biosíntesis , Linfocitos B/efectos de los fármacos , Secuencia de Bases , Línea Celular , Exones/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , ARN Mensajero/análisis , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The B-lymphocyte-restricted glycoprotein CD22 is expressed on mature IgM+IgD+ B cells, and is capable of binding to ligands on T and B cells. CD22 can interact with both the B-cell antigen receptor (BCR) complex and signalling molecules, including the protein tyrosine phosphatase SHP1 (PTP1C, SHP), a putative negative regulator of BCR signalling. Thus CD22 may facilitate interactions with lymphocytes and regulate the threshold of BCR signalling. To define the in vivo function of CD22, we generated CD22-deficient mice. Here we show that CD22 is required for normal antibody responses to thymus-independent antigens and regulates the lifespan of mature B cells.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos B/fisiología , Linfocitos B/fisiología , Moléculas de Adhesión Celular , Lectinas , Animales , Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos B/genética , Apoptosis , Linfocitos B/inmunología , Calcio/metabolismo , Senescencia Celular/genética , Senescencia Celular/fisiología , Eliminación de Gen , Activación de Linfocitos , Ratones , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Bazo/citología , Timo/citologíaRESUMEN
Despite intensive efforts, the intracellular signaling pathways that mediate apoptosis remain unclear. The human B lymphoma cell line, B104, possesses characteristics that make it an attractive model for analysis of receptor-mediated apoptosis. Although these cells express both membrane IgM (mIgM) and membrane IgD (mIgD) crosslinking mIgM results in significant apoptosis while crosslinking mIgD does not. Our results show that crosslinking mIgM but not mIgD induced a delayed and sustained activation of the mitogen-activated protein kinase (MAPK) family members stress-activated protein kinase (SAPK) and p38 MAPK. The calcium ionophore ionomycin, which also induces apoptosis in B104 cells, stimulated a similar SAPK and p38 MAPK response. Cyclosporin A, a potent inhibitor of apoptosis induced by either mIgM or ionomycin, inhibited activation of both SAPK and p38 MAPK, suggesting that stimulation of these kinases may be required for induction of apoptosis. Collectively, our results indicate that SAPK and p38 MAPK may be downstream targets during mIgM-induced, calcium-mediated, apoptosis in human B lymphocytes.
Asunto(s)
Apoptosis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Inmunoglobulina M/fisiología , Proteínas Quinasas Activadas por Mitógenos , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Línea Celular , Membrana Celular/inmunología , Membrana Celular/fisiología , Reactivos de Enlaces Cruzados , Ciclosporina/farmacología , Humanos , Inmunoglobulina D/fisiología , Inmunoglobulina M/inmunología , Ionomicina/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Cinética , Linfoma de Células B , Factores de Tiempo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
B cell antigen receptors are multicomponent complexes consisting of the surface immunoglobulin and accessory molecules with associating protein-tyrosine kinases. A spleen tyrosine kinase, Syk, in porcine B cells and a 72-kDa protein-tyrosine kinase, PTK72, in murine B cells associate with the B cell antigen receptor. Herein, we report the isolation of a full-length cDNA encoding the human homologue of Syk. This cDNA predicted a polypeptide consisting of two NH2-terminal SH2 domains and a COOH-terminal tyrosine kinase domain. Syk is highly conserved between human and swine and is homologous to the T cell-associated protein-tyrosine kinase ZAP-70. Both Syk mRNA and protein were detected in cells derived from multiple hematopoietic lineages. Within the B cell compartment, Syk was expressed from pro-B cells to plasma cells. In vitro kinase assays conducted on the human Syk protein isolated from B cells revealed the presence of autophosphorylation activity on Syk tyrosine residues. Tyrosine phosphorylation of Syk associating with the B cell receptor complex in human was augmented rapidly after surface immunoglobulin cross-linking. The human SYK locus was mapped to chromosome 9 at band q22.
Asunto(s)
Linfocitos B/enzimología , Cromosomas Humanos Par 9 , Precursores Enzimáticos/biosíntesis , Precursores Enzimáticos/aislamiento & purificación , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/aislamiento & purificación , Receptores de Antígenos de Linfocitos B/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Clonación Molecular/métodos , Secuencia Conservada , ADN Complementario/análisis , Precursores Enzimáticos/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Fosforilación , Proteínas Tirosina Quinasas/genética , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Receptores de Antígenos de Linfocitos B/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Bazo/enzimología , Porcinos , Quinasa Syk , Linfocitos T/inmunología , TransfecciónRESUMEN
The B-cell surface molecule CD22, when cross-linked, modulates signaling through the surface IgM (sIgM)-B-cell receptor (BCR) complex. Here we analyzed the basis of this interaction between CD22 and the human sIgM complex. After lysis of B cells or B-cell lines in digitonin, CD22 coimmunoprecipitated a kinase activity that in vitro-phosphorylated two polypeptides of 150 and 130 kDa on tyrosine residues. By immunoblot analysis with a rabbit anti-serum specific for a synthetic peptide of CD22, we found these proteins to be CD22 itself. Furthermore, the phosphorylated 150-kDa CD22 was found in the sIgM-BCR complex maintained by digitonin, along with Ig alpha/mb-1, Ig beta/B29, and a 75-kDa polypeptide precipitated by an antiserum specific to protein-tyrosine kinase PTK72. CD22 is likely to be an important signaling partner in the sIgM-BCR complex since it is very rapidly and strikingly phosphorylated after sIgM is cross-linked and since it contains the antigen recognition homology I (ARHI) motif, present in other antigen receptor molecules.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Linfocitos B/inmunología , Moléculas de Adhesión Celular/metabolismo , Inmunoglobulina M/metabolismo , Lectinas , Proteínas Quinasas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Antígenos CD/aislamiento & purificación , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/aislamiento & purificación , Linfocitos B/metabolismo , Linfoma de Burkitt , Moléculas de Adhesión Celular/aislamiento & purificación , Membrana Celular/inmunología , Membrana Celular/metabolismo , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoglobulina M/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Peso Molecular , Tonsila Palatina/inmunología , Proteínas Quinasas/aislamiento & purificación , Receptores de Antígenos de Linfocitos B/aislamiento & purificación , Homología de Secuencia de Aminoácido , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Células Tumorales CultivadasRESUMEN
To characterize the signal transduction through the antigen receptor (AgR) on human B lymphocytes, we analyzed its association with other molecular components. The surface IgM (sIgM) complex isolated in digitonin contains two surface expressed polypeptides--the previously described Ig alpha and Ig beta proteins--covalently linked to each other in a 48/39-kDa heterodimer. We show herein that the human sIgM complex isolated from the Burkitt's lymphoma cell line, Ramos, or from dense tonsillar B cells contains additional molecules--160 kDa and 75 kDa in size--and enzymatic activities able to phosphorylate on tyrosine as well as serine/threonine residues the 39-, 48-, 75- and 160-kDa polypeptides. By specific immunoprecipitation with antibodies to src-family kinases, we consistently detected p56lyn in the sIgM complex. In the Ramos cell line, both p56lck and p59fyn activity were also observed, although to a much lesser extent than p56lyn. These kinases are associated with sIgM before cell stimulation. As shown by two-dimensional electrophoresis, they interact in a tight complex with multimeric forms of the Ig alpha and Ig beta components. The kinases are active in vitro but must be highly regulated in vivo: Western blotting with anti-phosphotyrosine antibodies revealed that stimulation of the AgR on viable B cells increased detectable phosphotyrosine residues on the components present in the sIgM complex. Based on these phosphorylation changes, the 39-, 48-, 75- and 160-kDa molecules are likely to be functionally active elements in an IgM complex crucial for the transduction of the antigenic signal.
Asunto(s)
Linfocitos B/inmunología , Inmunoglobulina M/análisis , Proteínas Quinasas/análisis , Proteínas Tirosina Quinasas/análisis , Receptores de Antígenos de Linfocitos B/análisis , Línea Celular , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Fosforilación , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-fynRESUMEN
We have examined signal transduction via membrane IgM (mIgM) in resting and cycling human B cells. Crosslinking mIgM on all of the cell types studied transduced a signal through the phosphatidylinositol pathway, producing inositol 1,4,5-trisphosphate and release of intracellular free calcium. These second messengers were formed regardless of quantitative or qualitative differences in the surface expression of mIgM: cells that had low levels of surface IgM (T-51) or had no light chain associated with surface heavy chain (DB) signaled phosphatidylinositol pathway activation after mIgM crosslinking. Production of specific lipid products in nonquiescent B cells differed from that in normal resting cells. Ligation of surface immunoglobulin on resting B cells resulted in sustained increases of both diacylglycerol and phosphatidic acid, two lipids that can influence PKC activation. Whereas PKC was strongly activated in normal tonsillar B cells, several cell lines had reduced PKC activation following crosslinking of mIgM. The reduction in protein kinase C activation correlated with the absence or reduced levels of phosphatidic acid or diacylglycerol following stimulation: protein kinase C translocated and was activated only in cells that had elevated levels of both diacylglycerides and phosphatidic acid. Anti-IgM-induced phosphorylation of a protein kinase C substrate protein CD20, also increased in those cells having PKC activation and not in cells in which kinase activity was reduced. CD20 phosphorylation also increased following the direct addition of exogenous phosphatidic acid to resting B cells. Together, these observations show that the generation of lipid products following mIgM crosslinking in resting cells can vary from that in cycling cells and may relate to the different levels of PKC activation. In a companion study we report that ligation of surface IgM activates both an acyltransferase and phospholipase D to form phosphatidic acid.
Asunto(s)
Linfocitos B/inmunología , Diglicéridos/biosíntesis , Ácidos Fosfatidicos/biosíntesis , Antígenos CD/química , Antígenos CD20 , Antígenos de Diferenciación de Linfocitos B/química , Linfocitos B/metabolismo , Línea Celular , Activación Enzimática , Humanos , Proteína Quinasa C/análisis , Proteína Quinasa C/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Transducción de SeñalRESUMEN
To understand further the roles that negative regulatory signals may play in B cell immune responses, we compared three inhibitors of B cell proliferation: cross-linking CD19 with monoclonal antibody (mAb), signaling through Fc receptors by intact anti-mu mAb, and transforming growth factor-beta (TGF-beta). Each agent was tested for its ability to block proliferation and specific activation events induced in human tonsilar B cells activated by either cross-linking surface immunoglobulin, signaling through CD20, or direct activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate. We found that each inhibitor was functionally distinct. Both anti-CD19 mAb and anti-mu mAb inhibited anti-immunoglobulin activated cells and anti-CD20-activated cells, but neither inhibited cells activated by phorbol 12-myristate 13-acetate. TGF-beta, on the other hand, inhibited equally profoundly cells activated by each of the three regimens. These results suggest that TGF-beta blocks B cell activation at a step following the activation of PKC, whereas both signaling through CD19 and Fc receptor block early steps in the PKC activation pathway. Signaling through anti-CD19 mAb was unique in that proliferation of anti-immunoglobulin-activated cells was reduced on day 3 and then augmented subsequently. With all other inhibitory combinations the block was permanent. We conclude that each of these three inhibitors has unique important functions and therefore suggest that the effectiveness of negative signaling in B cell immune regulation will depend on the combinations of specific inhibitors modulating a specific activation program.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos B/fisiología , Linfocitos B/inmunología , Activación de Linfocitos/fisiología , Receptores Fc/fisiología , Factores de Crecimiento Transformadores/fisiología , Anticuerpos Antiidiotipos/fisiología , Antígenos CD19 , Antígenos CD20 , Antígenos HLA-D/biosíntesis , Humanos , Interfase/fisiología , Proteína Quinasa C/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-myc , ARN Mensajero/metabolismo , Sistemas de Mensajero Secundario/fisiología , Transducción de Señal/inmunología , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
The effects of the cytokine IL-4 on resting and activated human B cells were compared with the effects of known "competence" signals able to drive resting B cells into the cell cycle, including anti-Ig, PMA, anti-CD20, and a recently described competence signal, anti-Bgp95. In proliferation assays, IL-4 was costimulatory with anti-Ig and anti-Bgp95 but not with anti-CD20 or PMA. IL-4 alone triggered increases in expression of class II DR/DQ and CD40, but it did not trigger increases in intracellular free calcium [Ca2+]i in resting B cells or induce resting B cells to leave G0 and enter the G1 phase of the cell cycle. Although IL-4 has some characteristics of competence signals, it was most effective if added to B cells up to 12 h after anti-Ig or anti-Bgp95 rather than before, and thus, in this respect, works more like a progression signal. Like IL-4, all four competence signals for B cells triggered increases in class II and CD40, but only IL-4 consistently induced increases in CD23 surface levels. IL-4 was costimulatory only with anti-Ig and anti-Bgp95, each of which can trigger increases in [Ca2+]i and new protein synthesis of the proto-oncogene c-myc, and can increase attachment of protein kinase C to the plasma membrane. IL-4 was not costimulatory with signals that 1) did not affect [Ca2+]i yet induced c-myc protein synthesis (anti-CD20), 2) only stimulated the translocation of protein kinase C (PMA), or 3) only stimulated increases in [Ca2+]i (calcium ionophore). These results suggest that resting human B cells require at least two intracytoplasmic signals before IL-4 can effectively promote B cell proliferation.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Linfocitos B/fisiología , Interleucina-4/fisiología , Activación de Linfocitos , Transducción de Señal , Animales , Anticuerpos Antiidiotipos/farmacología , Antígenos CD20 , Antígenos de Diferenciación de Linfocitos B/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Calcio/metabolismo , Humanos , Interfase/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Glicoproteínas de Membrana/farmacología , Ratones , Microesferas , Fenotipo , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas , Proto-Oncogenes MasRESUMEN
Although all CD4+ cells theoretically are at risk for infection by human immunodeficiency viruses or the related simian immunodeficiency viruses found in Old World monkeys, only a small proportion of CD4+ lymphocytes from infected individuals have detectable virus. This suggests that immunodeficiency viruses may replicate predominantly in a minor subset or activated form of CD4+ T cells, a possibility we examined in macaques infected with a simian immunodeficiency virus isolate, SIV/Mne. Macaque CD4+ lymphocytes could be divided into two subtypes that differed in their level [high (hi) or low (lo)] of expression of a class of heterotypic adhesion receptors (HARs). In blood from animals infected with SIV/Mne, HARhi CD4+ T cells were lost selectively compared to HARlo CD4+ cells and, when cultured, exhibited 50-fold more recoverable reverse transcriptase activity. The HARhi CD4+ subset was also markedly more susceptible to productive infection following exposure to SIV/Mne in vitro. Both subsets are composed primarily of small resting lymphocytes. However, HARhi cells respond differentially to mitogenic stimulation and may thus be more likely to provide the cellular factors necessary to initiate or enhance virus replication. Thus, HAR expression may prove useful both as a prognostic indicator in immunodeficiency virus infection and as a tool to analyze pathogenesis of immunodeficiency viruses.
Asunto(s)
Receptores Inmunológicos/análisis , Virus de la Inmunodeficiencia de los Simios/fisiología , Linfocitos T Colaboradores-Inductores/microbiología , Replicación Viral , Animales , Antígenos de Diferenciación de Linfocitos T/análisis , Adhesión Celular , Ciclo Celular , Supervivencia Celular , Células Cultivadas , ADN/análisis , Citometría de Flujo , Activación de Linfocitos , Macaca , Mitógenos/farmacología , Fenotipo , ADN Polimerasa Dirigida por ARN/metabolismo , Receptores Mensajeros de Linfocitos , Infecciones por Retroviridae/inmunología , Linfocitos T Colaboradores-Inductores/análisisRESUMEN
A series of mouse monoclonal antibodies (mAb) to human differentiation antigens known to have agonistic activity for human T or B cells was found to bind specifically to macaque T or B cell subsets. Most of these mAb also stimulated macaque lymphocyte proliferation, implying that they recognize functional homologues in monkeys. Anti-CD3, anti-CD28 (9.3), and anti-Lp220 (CD45R) mAb stimulated proliferation of both human and macaque T cells; similarly, anti-IgM and anti-CDw40 mAb stimulated both human and macaque B cells. In contrast, anti-CD20 and anti-CD39 mAb, which are known to stimulate human B cells, did not stimulate macaque B cells. A human low-molecular weight B cell growth factor (BCGF) and anti-IgM were co-stimulatory for macaque splenic B cells but not for blood B cells, suggesting that B cell subpopulations may differ in their responsiveness to BCGF. The results show that functional epitopes on some lymphocyte surface molecules such as CD28 or CDw40 are conserved in primate evolution. Functional epitopes on other cell surface molecules such as CD3 and CD20 may have more complex evolutionary constraints.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Diferenciación/inmunología , Linfocitos B/inmunología , Activación de Linfocitos , Macaca fascicularis/inmunología , Macaca nemestrina/inmunología , Macaca/inmunología , Linfocitos T/inmunología , Animales , Humanos , Interleucina-4 , Interleucinas/farmacología , Especificidad de la Especie , Bazo/citología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Large granular lymphocyte (LGL) leukemia is a rare disease characterized by clonal expansion of LGL associated with chronic neutropenia, multiple auto-antibodies, and occasionally polyarthritis. We studied cell surface antigen expression and functional activity of leukemic LGL from ten such patients. Using two-color flow cytometric analysis, we found that leukemic LGL from all ten patients expressed the CD3 and HNK-1 markers, while cells from only four patients expressed IgG Fc receptors (FcR). The LGL leukemic cells had little or no NK activity (defined as MHC-nonrestricted cytotoxicity against K562 target cells); however, NK activity could be induced in leukemic LGL by in vitro treatment with as little as 0.05 microgram/mL of anti-CD3 monoclonal antibody. Cell sorting experiments demonstrated that NK activity was induced in CD3+ leukemic LGL (either CD3+, HNK-1+ or CD3+, FcR+) with anti-CD3 monoclonal antibody but not in normal CD3+, FcR- T cells. Treatment with purified interleukin 2 (IL 2) also caused direct activation of some CD3+ leukemic LGL. Despite induction with anti-CD3 MAb or IL 2, activated leukemic LGL did not proliferate or express high density IL 2 receptors detectable by cell sorter analysis. Treatment with alpha interferon had minimal effect on NK activity of LGL leukemic cells. These results suggest that leukemic LGL may provide a useful model for examining the signals required for LGL maturation and activation.