Cytokine activation sensitizes human monocytic and endothelial cells to the cytotoxic effects of an anti-CD40 immunotoxin.
G28-5 sFv-PE40 is a single-chain immunotoxin that is cytotoxic toward malignant B cells expressing CD40. Human monocytes, which also express cell surface CD40, were found to be insensitive to the immunotoxin. Activation of the monocytic cell line THP-1, or freshly isolated peripheral blood monocytes with IFN-gamma, but not IL-3, GM-CSF, IL-6, or TNF-alpha, greatly sensitized the cells toward G28-5 sFv-PE40, lowering the EC50 value from > 10 micrograms/ml to 80 ng/ml. This sensitization could not be explained simply by the two- to threefold increase in cell surface CD40 expression induced by IFN-gamma since TNF-alpha or the combination of TNF-alpha and IL-6 gave similar increases in CD40 expression but did not sensitize the cells to the immunotoxin. Internalization of G28-5 sFv-PE40 after IFN-gamma activation was also increased threefold, reflective of the increase in CD40 expression. IFN-gamma-treated but not -untreated THP-1 cells produced IL-6 and TNF-alpha following incubation with G28-5 sFv-PE40, indicating an association between CD40 signaling, which induces cytokine production, and sensitivity to the immunotoxin. HUVECs also express CD40 but were found to be insensitive to the anti-CD40 immunotoxin. A combination of IFN-gamma and TNF-alpha, but neither cytokine alone, sensitized the endothelial cells to G28-5 sFv-PE40. These data show that activation with cytokines can sensitize monocytes and endothelial cells to an immunotoxin targeted to CD40, most likely by altering the trafficking and/or processing of the immunotoxin after receptor-mediated internalization.
Francisco
J A
JA
Department of Molecular Immunology, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121, USA.
Kiener
P A
PA
Moran-Davis
P
P
Ledbetter
J A
JA
Siegall
C B
CB
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D019013:CD40 Antigens / Q000096:biosynthesis / Q000276:immunology* / Q000494:pharmacology*; D002478:Cells, Cultured; D004351:Drug Resistance; D004705:Endocytosis; D004730:Endothelium, Vascular / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D005098:Exotoxins / Q000494:pharmacology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology*; D015850:Interleukin-6 / Q000494:pharmacology; D007948:Leukemia, Monocytic, Acute / Q000473:pathology; D009000:Monocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D019013:CD40 Antigens; D005098:Exotoxins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000922:Immunotoxins; D007371:Interferon-gamma; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A