Antidisialoganglioside/granulocyte macrophage–colony-stimulating factor fusion protein facilitates neutrophil antibody-dependent cellular cytotoxicity and depends on FcγRII (CD32) and Mac-1 (CD11b/CD18) for enhanced effector cell adhesion and azurophil granule exocytosis

Abstract

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 multiparameter 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. IntroductionAntibody-cytokine fusion proteins combine the targeting ability of antibodies with the immune stimulation of cytokines for cancer immunotherapy. 1 Studies in mice of antidisialoganglioside (anti-GD2)/interleukin-2 and anti-GD2/lymphotoxin-␣ fusion proteins (immunocytokines) demonstrated the eradication of hepatic metastases of neuroblastoma 2,3 and the induction of protective immunity against melanoma. 4 These and other immunocytokine strategies have been based on the stimulation of NK-cell-or T-cell-mediated responses. Polymorphonuclear leukocytes (PMNs) constitute the largest population of white blood cells, and they can mediate antibody-dependent cellular cytotoxicity (ADCC) against tumor cells. [5][6][7][8][9] In vitro studies demonstrated the strong facilitation of PMN ADCC against neuroblastoma cells by mixing granulocytemacrophage colony-stimulating factor (GM-CSF) with an anti-GD2 antibody and by a human-mouse chimeric anti-GD2/GM-CSF immunocytokine. 5,10 Fc receptors (FcR) involved in PMN ADCC may include Fc␥RII (CD32) and Fc␥RIII (CD16), which are constitutively expressed, and Fc␥RI (CD64), which is induced by interferon-␥ and G-CSF. 11,12 In addition, Fc␣RI of PMNs may be activated by constructing bispecific monoclonal antibodies (mAbs) that recognize a target cell molecule and Fc␣RI. 13 Binding of an antibody-cytokine fusion protein to FcR may be affected by the physical presence of the cytokine or by the cytokinemodulating FcR expression. 14,15 FcRs used by PMNs in ADCC with anti-GD2/GM-CSF immunocytokines have not yet been determined.␤ 2 -Integrin receptors, particularly Mac-1 (␣ m ␤ 2 , CD11b/CD18, and complement receptor type 3), have been reported to have an essential role in PMN ADCC against neuroblastoma, lymphoma, and breast cancer cell lines. 5,13,16 Mac-1 is a major PMN ␤ 2 -integrin receptor that, on activation, acquires the ability to bind multiple ligands and to mediate several adhesion-dependent processes, including phagocytosis, superoxide production, and degranulation. 17 It recently has been demonstrated that Mac-1 function is necessary for PMN spreading on antibody-coated targets and for tumor cytolysis. 13 FcR and GM-CSF receptors activate Mac-1 For personal use only. on May 12, 2018. by ...

Targeting FcαRI on Polymorphonuclear Cells Induces Tumor Cell Killing through Autophagy

“…An explanation for the different observations might be that SK-BR-3 cells were used in suspension during the 20-h assay and detachment of cells induces anoikis (apoptosis triggered by cell detachment) (24). A nonapoptotic early-onset tumor cell death, induced by PMNs, is supported by data of Metelitsa et al (42). Although in this study tumor cell death mechanism was not thoroughly investigated, EM photomicrographs of ADCC samples clearly demonstrated absence of apoptotic features in nonviable neuroblastoma cells.…”

The New Look at Neutrophilic Granulocytes: Rethinking Old Dogmas. Part 2

“…[29]. Экспрессия на мембране НГ CD32, CD16 важна в осуществлении фагоцитарной функции и ан-тителозависимой клеточной цитотоксичности (АЗКЦ), которая ассоциируется с CD11b/CD18-зависимым повышением адгезии, дегрануляци-ей и киллингом [39]. CD64, CD32, CD16 -триг-герные молекулы, запускающие иммунный фа-гоцитоз и процессы киллинга [14] (рис.…”

The New Look at Neutrophilic Granulocytes: Rethinking Old Dogmas. Part 1

“…[4,8,9,10,45,48]. Мы полагаем, что выяснение связи между структурой и функ-цией хроматина НГ, его способностью к ре-структуризации при различных заболеваниях является весьма актуальной задачей.…”