RESUMO
OBJECTIVE: CD22 is believed to be restricted to normal and neoplastic B cells. Human basophils were found to express CD22 molecules. Among the antibodies against CD22, Leu14, which recognized the ligand binding domain, reacted to basophils, and B3 and 4KB128, which recognized the amino terminus side and carboxy terminus side of the ligand binding epitope, respectively, did not. To clarify the difference of CD22 antigenicity in human B cells and basophils, we investigated RNA sequence and structures of CD22 molecules. MATERIALS AND METHODS: Purified B cells and basophils were obtained from normal human volunteers by using a MACS magnetic cell sorting system and anti-CD19 and anti-Fc epsilon R1 antibodies, respectively. RT-PCR and sequencing of CD22 mRNA were performed in the exons 3 to 8. Western blotting analysis of CD22 was also performed. RESULTS: The sequence of CD22 mRNA extracted from the basophils was the same as that of B cells in exons 3 to 8 (epitopes recognized by Leu14, B3, and 4KB128 were translated from exons 4 and 5). Reduced CD22 peptide extracted from the basophils reacted to Leu14 as well as B3 and 4KB128, and the molecular size of the reduced and nonreduced products was 130 kDa as expected. CONCLUSION: Disulfide bonds and the resulting 3D conformation of the CD22 molecules may have important roles in the difference of antigenicity of CD22 beta in B cells (CD22 beta 1) and basophils (CD22 beta 2). The difference in molecular structure surrounding the ligand-binding domain of CD22 may imply a specialization of the conformational forms of CD22 according to the ligand isoforms.
Assuntos
Antígenos CD/química , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/química , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Basófilos/imunologia , Moléculas de Adesão Celular , Lectinas , Adulto , Anticorpos , Antígenos CD/genética , Antígenos CD19/imunologia , Antígenos de Diferenciação de Linfócitos B/genética , Western Blotting , Separação Celular/métodos , Dissulfetos/química , Éxons , Humanos , Magnetismo , Masculino , Conformação Proteica , RNA Mensageiro/sangue , RNA Mensageiro/química , Receptores de IgE/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Lectina 2 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
CD22, one of the important markers for diagnosing B-lineage acute leukemia, was expressed in mature basophil granulocytes. We then investigated the expression of CD22 and other B cell- and basophil-related molecules in 25 human acute leukemia cell lines to find the phenotype of the virtual common progenitor of B and myeloid lineage. Surface and cytoplasmic expressions of antigens were analyzed using a flow cytometer and an essential antibody panel used for diagnosing acute leukemia as well as cytokine receptors and basophil-related enzymes. Messenger RNA expression of Fc epsilon R1 and CD22 was also analyzed. Peroxidase-positive and -negative myeloid leukemias showed eosinophil- and basophil-type expression of enzymes, respectively. Early myeloid and B-lineage cells expressed basically similar combinations of cytokine receptors and various combinations of mRNA listed above, while T-lineage cells did not. The virtual common progenitor of B and myeloid lineage cells may be defined as immature cells simultaneously expressing B and basophil phenotypes.
Assuntos
Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos B/genética , Linfócitos B/citologia , Moléculas de Adesão Celular , Lectinas/genética , Leucemia Mieloide/patologia , RNA Mensageiro/metabolismo , Receptores de IgE/genética , Doença Aguda , Linfócitos B/metabolismo , Basófilos/citologia , Basófilos/metabolismo , Linhagem da Célula , Humanos , Imunofenotipagem , Leucemia/patologia , Células Progenitoras Mieloides/citologia , RNA Mensageiro/análise , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Células Tumorais CultivadasRESUMO
Dendritic cells (DCs) are potent antigen presenting cells and possess a direct anti-tumor cytotoxic ability. Nevertheless, the mechanism of anti-tumor cytotoxicity by DCs and the methods for its evaluation are not fully elucidated. In order to clarify this mechanism of cytotoxicity, we examined the ability of DCs 1) to suppress [3H] thymidine (3H-TdR) uptake by tumor cells; 2) to induce cytolysis on 51Cr-labeled tumor cells; 3) and to induce DNA fragmentation on 3H-TdR labeled tumor cells (JAM test). Cytolysis and DNA fragmentation are markers of necrotic and apoptotic mechanisms of cytotoxicity in vitro, respectively. DCs inhibited approximately 38.6% to 54.8% of the growth of B4D6, NB4, U937, and Daudi cells as evaluated by the uptake of 3H-TdR. However no cytolysis was verified by 51Cr-release assay. On the other hand, cytotoxicity rates found using the JAM test ranged from 3 to 81% depending on the cell line and the effector to target cell ratio. The discrepancy of cytotoxicity between 51Cr-release assay and the JAM test may be due to the phagocytosis of apoptotic tumor cells or the absorption of released 51Cr by DCs surrounding the target cells. In conclusion, the JAM test was more sensitive than the 4-h and the 10-h 51Cr-release assay to investigate cytotoxicity mediated by DCs toward hematopoietic tumor cell lines in vitro.