Accuri C6 Flow Cytometer System
Originally published online as doi:10.1189/jlb.0403133 on October 2, 2003

Published online before print October 2, 2003
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(Journal of Leukocyte Biology. 2004;75:99-105.)
© 2004 by Society for Leukocyte Biology

Monoclonal LYM-1 antibody-dependent cytolysis by human neutrophils exposed to GM-CSF: auto-regulation of target cell attack by cathepsin G

Luciano Ottonello*,1, Alan L. Epstein{dagger}, Marina Mancini*, Patrizia Dapino* and Franco Dallegri*

* Department of Internal Medicine, University of Genova Medical School, Italy; and
{dagger} Department of Pathology, University of Southern California, Los Angeles

1Correspondence: Dipartimento di Medicina Interna e Specialità Mediche, Viale Benedetto XV n.6, I-16132 Genova, Italy. E-mail: otto{at}unige.it

Murine monoclonal antibody (mAb) Lym-1 is an immunoglobulin G2a specific for certain human leukocyte antigen-DR variants expressed on the surface of malignant B cells. It has been proposed for serotherapy in patients with B lymphomas. We have previously shown that mAb Lym-1 synergizes with granulocyte macrophage-colony stimulating factor to promote Raji B-lymphoid cell lysis by human neutrophils via the intervention of neutrophil Fc receptors type II and D-mannose-inhibitable interactions between CD11b–CD18 integrins and CD66b glycoproteins. Here, we provide evidence that the process is oxygen-independent by inference related to the release of primary granules and is regulated by cathepsin G activity. The lysis was indeed reproduced by replacing normal neutrophils with cells from three patients suffering from chronic granulomatous disease, i.e., neutrophils genetically incapable of generating oxidants. Moreover, the lysis was inhibited by the serine protease inhibitor 3,4-dichloroisocoumarin and by Z-glycyl-leucyl-phenyl-chloromethyl ketone (Z-Gly-Leu-Phe-CMK), which blocks cathepsin G. Conversely, the lysis was unaffected by N-methoxysuccinyl-alanyl-alanyl-prolyl-alanyl-CMK (MeOSuc-Ala-Ala-Pro-Ala-CMK; elastase inhibitor) and MeOSuc-Ala-Ala-Pro-valine (Val)-CMK, which inhibits elastase and proteinase 3. The ability of neutrophils, engaged in cytolysis, to release cathepsin G was proved by detecting this enzymatic activity spectrophotometrically and immunocytochemically. Moreover, inhibition of cathepsin G activity by concentrations of Z-Gly-Leu-Phe-CMK, incapable of affecting elastase activity, was found to reduce the release of elastase and myeloperoxidase from neutrophils under conditions similar to those used for cytolytic assays. These findings suggest that neutrophils auto-regulate their lytic efficiency by controlling the exocytosis of primary granules via their cathepsin G activity.

Key Words: ADCC • lymphoma • primary granules • exocytosis • Fc receptors