IgE-immunotoxins. II. IgE-ricin A-chain.
In order to develop a reagent capable of killing cells with high-affinity IgE Fc receptors, such as mast cells and basophils, ricin A-chain (the toxic portion of ricin) was conjugated to rat IgE myeloma protein, IR 162, via derivatization of the IgE by n-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) thus creating an IgE-immunotoxin. Monensin (10(-7)-10(-8)M), a carboxylic ionophore, facilitated IgE-ricin A-chain (3 X 10(-7)M) toxicity in a dose-related fashion ith significant reductions in [3H]leucine incorporation compared to cells exposed only to monensin. This enhanced toxicity could be inhibited by the addition of both anti-ricin A-chain or anti-IgE, suggesting that different routes of intracellular processing may play a role in determining the toxicity of the IgE-ricin A-chain conjugate. Ricin B-chain (5 X 10(-7) and 5 X 10(-8)M) added to free ricin A-chain (10(-6)-10(-8)M) reproducibly facilitated toxicity, and this toxicity could be inhibited (30-90%) by lactose (50 mM). Ricin B-chain also facilitated IgE-ricin A-chain (2.75 X 10(-8)M) toxicity; however, this toxicity was not affected by lactose. The data suggest that ricin B-chain potentiates the cytosolic access of internalized IgE-immunotoxin and that the binding and internalization of the toxin was mediated via the IgE Fc receptor. A second type of IgE-ricin A-chain conjugate was synthesized whereby both IgE and ricin A-chain were derivatized with SPDP. RBL cells were killed in a dose-dependent manner by this IgE-ricin A-chain conjugate (2.5 X 10(-6)-2.5 X 10(-9)M) without requiring the addition of monensin or ricin B-chain. These data indicate that the intracellular route and processing of internalized immunotoxin is critical to eliciting toxicity.
D000818:Animals; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D007073:Immunoglobulin E / Q000276:immunology*; D000922:Immunotoxins / Q000032:analysis / Q000494:pharmacology*; D007785:Lactose / Q000494:pharmacology; D008985:Monensin / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011817:Rabbits; D011961:Receptors, Fc / Q000378:metabolism; D012276:Ricin / Q000494:pharmacology*
D000922:Immunotoxins; D011961:Receptors, Fc; D007073:Immunoglobulin E; D012276:Ricin; D008985:Monensin; D007785:Lactose