Antidisialoganglioside/granulocyte macrophage-colony-stimulating factor fusion protein facilitates neutrophil antibody-dependent cellular cytotoxicity and depends on FcgammaRII (CD32) and Mac-1 (CD11b/CD18) for enhanced effector cell adhesion and azurophil granule exocytosis.
Polymorphonuclear leukocytes (PMNs) mediate antibody-dependent cellular cytotoxicity (ADCC), which is increased by the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF). We sought to determine whether PMN ADCC also would be increased by the addition of an antibody/GM-CSF fusion protein and whether this would be associated with the up-regulation and activation of Mac-1 (CD11b/CD18) and with azurophil granule exocytosis. ADCC against LA-N-1 human neuroblastoma cells was evaluated with 4-hour calcein acetoxymethyl ester (calcein-AM) microcytotoxicity assay, electron microscopy, and multi-parameter flow cytometry. With the calcein-AM assay, LA-N-1 cell survival was 10%, 55%, and 75% when PMN ADCC was mediated by the antidisialoganglioside (anti-GD2) immunocytokine hu14.18/GM-CSF, by monoclonal antibody (mAb) hu14.18 mixed with GM-CSF, and by hu14.18 alone. Function-blocking mAbs demonstrated that FcgammaRII and FcgammaRIII were required for ADCC with hu14.18 alone or mixed with GM-CSF, but that only FcgammaRII was required for ADCC with hu14.18/GM-CSF. ADCC mediated by hu14.18 and hu14.18/GM-CSF was Mac-1 dependent. Electron microscopy demonstrated the greatest PMN adhesion, spreading, and lysis of targets with hu14.18/GM-CSF. Monoclonal antibodies blocking Mac-1 function allowed the tethering of PMN to targets with hu14.18/GM-CSF but prevented adhesion, spreading, and cytolysis. Flow cytometry showed that hu14.18 with or without GM-CSF and hu14.18/GM-CSF all mediated Mac-1-dependent PMN-target cell conjugate formation but that GM-CSF was required for the highest expression and activation of Mac-1, as evidenced by the mAb24-defined beta(2)-integrin activation epitope. Hu14.18/GM-CSF induced the highest sustained azurophil granule exocytosis, almost exclusively in PMNs with activated Mac-1. Thus, hu14.18/GM-CSF facilitates PMN ADCC against neuroblastoma cells associated with FcgammaRII and Mac-1-dependent enhanced adhesion and degranulation.
Metelitsa
Leonid S
LS
Department of Pediatrics, Division of Hematology-Oncology, Children's Hospital Los Angeles and Keck School of Medicine, University of Southern California, Los Angeles 90027, USA.
Gillies
Stephen D
SD
Super
Michael
M
Shimada
Hiroyuki
H
Reynolds
C Patrick
CP
Seeger
Robert C
RC
D000906:Antibodies / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000920:Antibody-Dependent Cell Cytotoxicity / Q000187:drug effects / Q000276:immunology*; D003594:Cytoplasmic Granules / Q000502:physiology; D005089:Exocytosis; D005732:Gangliosides / Q000276:immunology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006801:Humans; D016177:Macrophage-1 Antigen / Q000276:immunology*; D009447:Neuroblastoma; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology*; D017452:Receptors, IgG / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000906:Antibodies; D000911:Antibodies, Monoclonal; D005732:Gangliosides; D016177:Macrophage-1 Antigen; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; C025447:sialogangliosides; D016178:Granulocyte-Macrophage Colony-Stimulating Factor