ABSTRACT
BACKGROUND: Endogenous complement inhibitors in the brain may protect against the neuroinflammation in Alzheimer's disease. Human neuroblastoma cells were stimulated by Abeta1 - 4 2 to investigate whether the expression of various complement regulator genes is upregulated. METHODS: SK-N-SH cells were incubated overnight with a single dose of 20 microM of Abeta1-42 or 0.5 ng/ml - 5 ng/ml of TNFalpha or both. Actinomycin D (2.5 microM) or cycloheximide (2.5 microM) was also added to the cell suspension. Messenger RNA expression of decay accelerating factor (DAF), membrane cofactor protein (MCP), CD59, complement-receptor 1(CR1), C1 inhibitor (C1-INH), C4-binding protein, factor H, factor I, clusterin and S-protein was measured by RT-PCR. RESULTS: Abeta1-42 and TNFalpha upregulated the expression of C1- INH significantly but increased expression of mRNA for factor H was not statistically significant. The expression of mRNAs for DAF and MCP was at low a level after stimulation. Factor I, CD59 and clusterin were not changed in their mRNA level. The mRNAs for S-protein, C4-binding protein and CR1 were not detected. Actinomycin D suppressed mRNA levels of C1-INH and CD59 significantly. Cycloheximide also inhibited the expression of both C1-INH and CD59. CONCLUSIONS: Early upregulated expression of C1-INH in Abeta1-42 stimulated neuroblastoma cell may contribute to a host defense mechanism against complement-mediated neuronal cell damage.
Subject(s)
Humans , Alzheimer Disease , Amyloid beta-Peptides , Membrane Cofactor Protein , CD55 Antigens , CD59 Antigens , Brain , Clusterin , Complement Factor H , Complement System Proteins , Cycloheximide , Dactinomycin , Fibrinogen , Genes, Regulator , Neuroblastoma , Neurons , RNA, Messenger , Tumor Necrosis Factor-alphaABSTRACT
BACKGROUND: Endogenous complement inhibitors in the brain may protect against the neuroinflammation in Alzheimer's disease. Human neuroblastoma cells were stimulated by Abeta1 - 4 2 to investigate whether the expression of various complement regulator genes is upregulated. METHODS: SK-N-SH cells were incubated overnight with a single dose of 20 microM of Abeta1-42 or 0.5 ng/ml - 5 ng/ml of TNFalpha or both. Actinomycin D (2.5 microM) or cycloheximide (2.5 microM) was also added to the cell suspension. Messenger RNA expression of decay accelerating factor (DAF), membrane cofactor protein (MCP), CD59, complement-receptor 1(CR1), C1 inhibitor (C1-INH), C4-binding protein, factor H, factor I, clusterin and S-protein was measured by RT-PCR. RESULTS: Abeta1-42 and TNFalpha upregulated the expression of C1- INH significantly but increased expression of mRNA for factor H was not statistically significant. The expression of mRNAs for DAF and MCP was at low a level after stimulation. Factor I, CD59 and clusterin were not changed in their mRNA level. The mRNAs for S-protein, C4-binding protein and CR1 were not detected. Actinomycin D suppressed mRNA levels of C1-INH and CD59 significantly. Cycloheximide also inhibited the expression of both C1-INH and CD59. CONCLUSIONS: Early upregulated expression of C1-INH in Abeta1-42 stimulated neuroblastoma cell may contribute to a host defense mechanism against complement-mediated neuronal cell damage.
Subject(s)
Humans , Alzheimer Disease , Amyloid beta-Peptides , Membrane Cofactor Protein , CD55 Antigens , CD59 Antigens , Brain , Clusterin , Complement Factor H , Complement System Proteins , Cycloheximide , Dactinomycin , Fibrinogen , Genes, Regulator , Neuroblastoma , Neurons , RNA, Messenger , Tumor Necrosis Factor-alphaABSTRACT
CD59 antigen is a widely expressed cell surface glycosylphosphatidyl-inositol (GPI) anchored glycoprotein.It acts as an inhibitor to the assembly of the membrane attack complex of homologous complement,binds to CD2,and also transduces activation signals with T cells.In this report,a 396bp DNA fragment was amplified by RT-PCR method from the total RNA of Jurkat cells.The fragment was cloned into pUC18 and pUC19 plas-mids,and further sequenced by Sanger′s-dideory-mediated chain termination.The results showed that this cDNA fragment included 384bp open reading fragment and its sequence was identical to the published sequence encoding human CD59 antigen.Furthermore,the cDNA of CD59 was subcloned into retroviral vector pLXSN and transfec-ted into packaging cell line PA317 to generate stable virus-producing cell lines.Then,mouse thymotase cell line EL-4 and fibroblasts cell line NIH3T3 were infected with the virus resulting in stable expression of CD59 on the cell surface.The transfected cells were tested for their susceptibility to human complement-mediated cytolysis.It was found that the transfected cells expressing CD59 antigen were far less susceptible than the controls,indicating that the gene for CD59 can be expressed in xenotypic cells stably to confer protection against human serum complement.