TNF-alpha secretion and apoptosis of lymphocytes mediated by gene transfer.
Efficient gene transfer of lymphocytes is extremely difficult. Apoptosis may play a role in this gene transfer resistance of lymphocytes. Here we show that transfection of lymphocytes via non-viral vectors leads to induction of apoptosis in a significant proportion of cells. Since apoptosis may be mediated via tumor necrosis factor d (TNF-alpha) and the TNF-alpha receptor pathway, we studied the amount of TNF-alpha secreted by lymphocytes transfected without gene insert. TNF-alpha secretion was dependent on the gene transfer method used. High amounts were detected using receptor-mediated gene transfer and lipofection. In contrast, only low amounts of TNF-alpha were detected after electroporation and retroviral gene transfer. In receptor-mediated gene transfer, TNF-alpha secretion was due to the use of anti-CD3 antibody. Transfection of lymphocytes led to selective decrease in CD120b/TNF-alpha receptor II (TNFR-2)-positive cells. Induction of apoptosis and necrosis mediated by TNF-alpha via TNFR-2 (p80) was partially blocked using a neutralizing anti-TNF-alpha antibody. Blockage of apoptosis and necrosis could be further increased by adding anti-Fas-ligand (FasL) antibody, suggesting that induction of apoptosis via FasL and Fas receptor (Apo-1/CD95) may also play a role. This blockage led to a significant increase in the proliferation rate of lymphocytes transfected with cytokine genes. In conclusion, various gene transfer techniques led to TNF-alpha secretion, apoptosis and necrosis of lymphocytes. Apoptosis and necrosis could be partially blocked using a neutralizing anti-TNF-alpha antibody.
Ebert
O
O
Abteilung für Innere Medizin und Poliklinik m. S. Hämatologie und Onkologie, Humboldt-Universität zu Berlin, Germany.
Röpke
G
G
Märten
A
A
Lefterova
P
P
Micka
B
B
Buttgereit
P
P
Niemitz
S
S
Trojaneck
B
B
Schmidt-Wolf
G
G
Huhn
D
D
Wittig
B
B
Schmidt-Wolf
I G
IG
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis; D016207:Cytokines / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D006801:Humans; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000166:cytology* / Q000378:metabolism; D009336:Necrosis; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism*; D019014:fas Receptor / Q000276:immunology
D000911:Antibodies, Monoclonal; D016207:Cytokines; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor