RESUMO
OBJECTIVE: A decline in the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) may enhance cytokine release in Alzheimer's disease (AD) resulting in neuroinflammation. We investigated the GABA-mediated suppression of the synergistic release of interleukin (IL)-6 due to interleukin 1-beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). METHODS: Rat C6 astrocytoma cells were treated with IL-1 beta and TNF-alpha in the absence and presence of GABA. Activation of p38, degradation of I kappaB-alpha and total cellular IL-6 were determined by Western blot analysis. IL-6 release and gene expression were measured by ELISA and RT-PCR, respectively. RESULTS: Although p38 and nuclear factor (NF)-kappaB are essential for the synergistic release of IL-6, GABA did not affect either p38 phosphorylation or I kappaB-alpha degradation. Additionally, GABA suppressed IL-6 release but did not alter cytokine-driven synergistic increases in IL-6 gene expression. Western blot analysis revealed that co-treatments with IL-1 beta and TNF-alpha resulted in an increase in intracellular IL-6 that was prevented by GABA. CONCLUSION: GABA-induced inhibition of IL-6 release appears to coincide with a reduction in cellular IL-6. The GABA-induced suppression of IL-6 release may include inhibition of IL-6 gene translation.
Assuntos
Astrocitoma/genética , Astrocitoma/imunologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Interleucina-6/genética , Ácido gama-Aminobutírico/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/imunologia , Astrócitos/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Proteínas I-kappa B/metabolismo , Interleucina-1beta/farmacologia , Interleucina-6/metabolismo , Inibidor de NF-kappaB alfa , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/imunologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Thymosin fraction-5 (TF5) is a protein preparation of the bovine thymus. TF5 stimulates many assays of T cell-mediated immunity. We found that TF5 substantially suppressed proliferation of the rat C6 glioma and MMQ pituitary adenoma cell lines. Our current research using the promyelocytic cell line HL-60 suggests that TF5 also prevents proliferation of human myeloid leukemia cells. Our objective is the purification and chemical characterization of TF5 peptide components responsible for inhibition of HL-60 proliferative capacity. Using the inhibition of HL-60 cell proliferation, we have chemically characterized TF5 using fast protein liquid chromatography (FPLC), reversed-phase high-performance liquid chromatography (RP-HPLC), and high-performance capillary electrophoresis (HPCE). Vital dye-exclusion, oxidative metabolism of chromogenic dyes, and clonogenic growth profiles were used to determine rates of HL-60 proliferation. Our results identified an approximately 6000 Da component of TF5 capable of inducing HL-60 growth arrest. Synchronized HL-60 cells exposed to TF5 and its various constituents were subjected to cytometric analysis by flow cytometry. TF5-treated HL-60 cells had an increased subdiploid faction (i.e., sub-G1) compared to control cells. TF5 also increased Annexin V staining in randomly cycling HL-60 cells. Thus, a TF5 subfraction possesses growth-suppressive activity for human myeloid neoplasms. Our results indicate that this effect is characterized by at least one hallmark of apoptosis. Future clinical management strategies for certain leukemias may involve the use of thymic peptides.
