Colocalization of the IL-12 receptor and FcgammaRIIIa to natural killer cell lipid rafts leads to activation of ERK and enhanced production of interferon-gamma.
Natural killer (NK) cells express an activating receptor for the Fc portion of IgG (FcgammaRIIIa) that mediates interferon (IFN)-gamma production in response to antibody (Ab)-coated targets. We have previously demonstrated that NK cells activated with interleukin-12 (IL-12) in the presence of immobilized IgG secrete 10-fold or more higher levels of IFN-gamma as compared with stimulation with either agent alone. We examined the intracellular signaling pathways responsible for this synergistic IFN-gamma production. NK cells costimulated via the FcR and the IL-12 receptor (IL-12R) exhibited enhanced levels of activated STAT4 and Syk as compared with NK cells stimulated through either receptor alone. Extracellular signal-regulated kinase (ERK) was also synergistically activated under these conditions. Studies with specific chemical inhibitors revealed that the activation of ERK was dependent on the activation of PI3-K, whose activation was dependent on Syk, and that sequential activation of these molecules was required for NK cell IFN-gamma production in response to FcR and IL-12 stimulation. Retroviral transfection of ERK1 into primary human NK cells substantially increased IFN-gamma production in response to immobilized IgG and IL-12, while transfection of human NK cells with a dominant-negative ERK1 abrogated IFN-gamma production. Confocal microscopy and cellular fractionation experiments revealed that FcgammaRIIIa and the IL-12R colocalized to areas of lipid raft microdomains in response to costimulation with IgG and IL-12. Chemical disruption of lipid rafts inhibited ERK signaling in response to costimulation and significantly inhibited IFN-gamma production. These data suggest that dual recruitment of FcgammaRIIIa and the IL-12R to lipid raft microdomains allows for enhanced activation of downstream signaling events that lead to IFN-gamma production.
Kondadasula
Sri Vidya
SV
Department of Molecular Virology, Immunology, and Medical Genetics, Human Cancer Genetics program, The Arthur G. James Comprehensive Cancer Center and Solove Research Institute, The Ohio State University, Columbus, OH, USA.
Roda
Julie M
JM
Parihar
Robin
R
Yu
Jianhua
J
Lehman
Amy
A
Caligiuri
Michael A
MA
Tridandapani
Susheela
S
Burry
Richard W
RW
Carson
William E
WE
D002784:Cholesterol / Q000172:deficiency; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology; D007371:Interferon-gamma / Q000096:biosynthesis*; D018664:Interleukin-12 / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D007694:Killer Cells, Natural / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology*; D021962:Membrane Microdomains / Q000201:enzymology*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D017452:Receptors, IgG / Q000276:immunology*; D053707:Receptors, Interleukin-12 / Q000276:immunology*; D050797:STAT4 Transcription Factor / Q000378:metabolism; D000072377:Syk Kinase; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C496124:FCGR3A protein, human; D007074:Immunoglobulin G; D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D017452:Receptors, IgG; D053707:Receptors, Interleukin-12; D050797:STAT4 Transcription Factor; D018664:Interleukin-12; D007371:Interferon-gamma; D002784:Cholesterol; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D051057:Proto-Oncogene Proteins c-akt; D048031:JNK Mitogen-Activated Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases