Daclizumab (Zenapax) inhibits early interleukin-2 receptor signal transduction events.
Daclizumab, a humanized antibody against the interleukin-2 (IL-2) receptor (R) alpha-chain, is a promising new immunosuppressant in transplantation. As its exact mechanism of action has remained unclear, we examined its short-term effects on primary human T lymphocytes expressing the high-affinity IL-2R. Daclizumab exposure for 20 min neither affected T cell viability nor their surface expression of the IL-2R alpha-, beta-, or gamma-chains. However, after IL-2 stimulation (200 U/ml, 20 min), immunoblots of cell lysates demonstrated attenuation of the IL-2-induced tyrosine phosphorylation of 65-75 kDa proteins by Daclizumab, but not by isotype controls. Since this is the molecular weight of the IL-2R beta- and gamma-chains, which are both tyrosine-phosphorylated by IL-2, we next examined the effect of Daclizumab on their IL-2-induced tyrosine phosphorylation. In immunoblots of IL-2R beta- and gamma-chain-immunoprecipitates the tyrosine phosphorylation of both chains by IL-2, but not by IL-15, was attenuated in the presence of Daclizumab. Furthermore, co-immunoprecipitation experiments showed that Daclizumab inhibited the IL-2-induced association of these chains, a prerequisite for their mutual tyrosine phosphorylation. Lastly, we demonstrated that Daclizumab inhibits the receptor-downstream induction of the IL-2-activated DNA-binding protein STAT5 in gel shift assays. We conclude that Daclizumab directly and specifically interferes with IL-2 signaling at the receptor level by inhibiting the association and subsequent phosphorylation of the IL-2R beta- and gamma-chains induced by ligand binding. Under our experimental conditions, Daclizumab had no effects on cell viability, and it did not modulate the surface expression of the IL-2R alpha-, beta-, or gamma-chains.
Goebel
J
J
Department of Pediatrics, University of Kentucky, Lexington 40536, USA. jwgoeb0@pop.uky.edu
Stevens
E
E
Forrest
K
K
Roszman
T L
TL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D000077561:Daclizumab; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology*; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000494:pharmacology; D051379:Mice; D008894:Milk Proteins; D010766:Phosphorylation; D015375:Receptors, Interleukin-2 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D015534:Trans-Activators / Q000096:biosynthesis; D014181:Transplantation Immunology; D014443:Tyrosine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D004268:DNA-Binding Proteins; D007074:Immunoglobulin G; D007166:Immunosuppressive Agents; D007376:Interleukin-2; D008894:Milk Proteins; D015375:Receptors, Interleukin-2; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D014443:Tyrosine; D000077561:Daclizumab