RESUMO
In the present study, we investigated the expression of human CD1d antigen on activated mature T cells. Expression of this glycoprotein was found to be highly regulated and dependent on PHA stimulation. Flow cytometry studies using the NOR3.2 antibody, which recognized CD1d under denaturing conditions, showed a clear increase in its expression after PHA stimulation. Expression of this molecule after PHA activation was confirmed by analysis of its corresponding transcript by RT-PCR. A single band representing mRNA for CD1d membrane isoform was observed in activated PBMC as well as in ER3 CD1D-transfected and MOLT-4, pre-T cell lines, which were used as controls. Western blot analysis revealed an activation-dependent increase in CD1d protein expression when PBMC and enriched T cells were activated for different time periods. Activation-dependent expression of CD1d antigen was also confirmed in allogenic-activated T cells, suggesting that this event could have biological significance. Finally, immunocytochemical studies showed the presence of this protein at the plasma membrane accompanied by a cytoplasmic and perinuclear distribution. Results presented herein provide the first experimental evidence showing that CD1d antigen is present on circulating, activated T lymphocytes, suggesting that its expression is dependent on the activation state of the cells. Elucidation of the molecular mechanisms implicated in the activation-dependent expression of this nonclassical antigen will provide new insights into the understanding of antigen presentation and immune regulation.
Assuntos
Antígenos CD1/biossíntese , Ativação Linfocitária , Linfócitos T/imunologia , Linfócitos T/metabolismo , Anticorpos Monoclonais/análise , Antígenos CD1/sangue , Antígenos CD1/genética , Antígenos CD1/imunologia , Antígenos CD1d , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular Transformada , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Ativação Linfocitária/genética , Peso Molecular , Fito-Hemaglutininas/farmacologia , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/química , Frações Subcelulares/imunologia , Linfócitos T/química , Linfócitos T/citologia , Transfecção , Células Tumorais Cultivadas , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
We investigated the regulation of and the intracellular trafficking involved in the membrane expression of CD1c antigen on activated mature T cells. Membrane expression of this glycoprotein was highly regulated and dependent on the activation state of the cells. The presence of the CD1c antigen on activated peripheral blood mononuclear cells (PBMCs) was confirmed by flow cytometry, reverse transcriptase-PCR (RT-PCR), and immunoperoxidase staining. The RT-PCR analysis of the alpha3- and 3'-untranslated regions of CD1C showed that phytohemagglutinin (PHA) activation induced expression of transcripts that encode the three isoforms (soluble, membrane, and cytoplasmic/soluble). Immunocytochemical studies showed a specific association of CD1c with the cell membrane and a cytoplasmic, perinuclear distribution. Although flow-cytometric staining confirmed the intracellular presence of CD1c, membrane expression on PHA blast cells was not detected. We found that membrane detection of CD1c antigen was temperature dependent. Cell surface binding of the anti-CD1c monoclonal antibody (mAb) was consistently negative at 4 and 37 degrees C but was detected at room temperature (18-22 degrees C). At physiologic temperatures, activated PBMCs showed intracellular accumulation of the anti-CD1c mAbs, indicating that CD1c cycled between cell surface and intracellular compartments. The CD1c exocytosis pathway was sensitive to Brefeldin A, cytochalasin B, and chloroquine.
Assuntos
Antígenos CD1/imunologia , Antígenos CD1/metabolismo , Glicoproteínas/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Processamento Alternativo , Antígenos CD1/genética , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Citometria de Fluxo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Técnicas Imunoenzimáticas , Transporte Proteico , RNA Mensageiro/metabolismo , TemperaturaRESUMO
BACKGROUND AND PURPOSE: Excitatory amino acid transporters (EAATs) in the CNS contribute to the clearance of glutamate released during neurotransmission. The aim of this study was to explore the role of EAATs in the regulation of locus coeruleus (LC) neurons by glutamate. EXPERIMENTAL APPROACH: We measured the effect of different EAAT subtype inhibitors/enhancers on glutamate- and KCl-induced activation of LC neurons in rat slices. EAAT2-3 expression in the LC was also characterized by immunohistochemistry. KEY RESULTS: The EAAT2-5 inhibitor DL-threo-ß-benzyloxaspartic acid (100 µM), but not the EAAT2, 4, 5 inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (100 µM) or the EAAT2 inhibitor dihydrokainic acid (DHK; 100 µM), enhanced the glutamate- and KCl-induced activation of the firing rate of LC neurons. These effects were blocked by ionotropic, but not metabotrobic, glutamate receptor antagonists. DHK (100 µM) was the only EAAT inhibitor that increased the spontaneous firing rate of LC cells, an effect that was due to inhibition of EAAT2 and subsequent AMPA receptor activation. Chronic treatment with ceftriaxone (200 mg·kg(-1) i.p., once daily, 7 days), an EAAT2 expression enhancer, increased the actions of glutamate and DHK, suggesting a functional impact of EAAT2 up-regulation on the glutamatergic system. Immuhistochemical data revealed the presence of EAAT2 and EAAT3 surrounding noradrenergic neurons and EAAT2 on glial cells in the LC. CONCLUSIONS AND IMPLICATIONS: These results remark the importance of EAAT2 and EAAT3 in the regulation of rat LC by glutamate. Neuronal EAAT3 would be responsible for terminating the action of synaptically released glutamate, whereas glial EAAT2 would regulate tonic glutamate concentrations in this nucleus.