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Blood, 1 June 2002, Vol. 99, No. 11, pp. 4166-4173
PHAGOCYTES
Antidisialoganglioside/granulocyte macrophage-colony-stimulating
factor fusion protein facilitates neutrophil antibody-dependent
cellular cytotoxicity and depends on Fc RII (CD32) and
Mac-1 (CD11b/CD18) for enhanced effector cell adhesion and azurophil
granule exocytosis
Leonid S. Metelitsa,
Stephen D. Gillies,
Michael Super,
Hiroyuki Shimada,
C. Patrick Reynolds, and
Robert
C. Seeger
From the Department of Pediatrics, Division of
Hematology-Oncology, Children's Hospital Los Angeles and Keck School
of Medicine, University of Southern California, Los Angeles; and
Lexigen Pharmaceuticals, Lexington, MA.
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 Fc RII
and FcRIII were required for ADCC with hu14.18 alone or mixed with
GM-CSF, but that only FcRII 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  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 FcRII and Mac-1-dependent enhanced adhesion and degranulation.
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