Graciela P. Serebrinsky

Cyclophosphamide augments ADCC by increasing the expression of Fc-receptors

Previous reports demonstrated that cyclophosphamide (Cy) enhances two Fc gamma receptor (Fc gamma R) mediated functions: antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis. In this paper we examine the mechanisms whereby Cy modifies the cytotoxic capacity of mouse splenocytes. The results indicate that the observed augmentation of ADCC could not be attributed to a higher proportion of macrophages and/or polymorphonuclear leukocytes (PMN), but rather to an enhanced activity per effector cell. Binding studies showed that this augmentation was associated with an increased number, but not an increased avidity of Fc gamma R sites. The possibility that the enhanced Fc gamma R expression by Cy may result in the alteration of other Fc gamma R-mediated functions is discussed.

Cyclophosphamide modulates arachidonic acid metabolism by peritoneal macrophages.

The treatment of mice with a single dose of cyclophosphamide (Cy) (200 mg/kg) enhanced the chemiluminescence (CL) response of peritoneal macrophages (PM) triggered with opsonized zymosan (OpZ). The enhanced CL response could not be attributed to the stimulation of the cyanide-insensitive respiratory burst, since neither superoxide anion release nor immune complex-triggered cytotoxicity, an oxygen-dependent lytic mechanism, were increased in Cy-PM. Then, products of the oxidative metabolism of arachidonic acid were measured. It was found that Cy-PM exhibited increased release of prostaglandin E2 and leukotriene C4 in response to OpZ when compared with resident PM. In contrast, similar levels of thromboxane B2 production were observed in both populations. The activation of macrophage arachidonic acid metabolism reported here may contribute to the immunomodulating action of Cy.

Neutrophil Cytotoxicity Induced by Immune Complexes prepared with Cationized Antibodies

Here we analyse the ability of soluble imtnune complexes (IC) prepared with cationized antibodies to induce cytotoxic responses mediated by neutrophils. While eationized IC induced high levels of cytotoxicity, control IC induced very low levels of response. Inhibition of cytotoxicity by catalase but not by three haemenzyme inhibitors suggests that oxygen‐dependent but myeloperoxidase‐independent mechanisms are responsible for cytolysis. While the response induced by control IC was enhanced by cytochalasin B and was not modified by colchicine, that induced by cationized IC was markedly inhibited by cytochalasin B and significantly enhanced by colchicine. Cytotoxicity induced by cationized IC was completely abrogated by monoclonal antibodies to FcγRII. Using control IC, a partial inhibition was observed employing either anti‐FcγRII or anti‐FcγRIII monoclonal antibodies. Treatment of neutrophils with ehemotrypsine or pronase significantly enhanced cytotoxicity induced by cationized IC but not by control IC. We also found that non‐specific absorptive mechanisms appear to play an important role in the binding of cationized IC, but not control IC, to the neutrophil surface. The significance of these results is discussed.

Inhibition of lymphocyte and monocyte antibody-dependent cellular cytotoxicity by immune complexes: effect of normal human serum

Antibody-dependent cellular cytotoxicity (ADCC) mediated by peripheral blood mononuclear cells (PBMC) and by isolated populations of lymphocytes and monocytes was compared for susceptibility to inhibition by soluble immune complexes (IC) and by heat-aggregated IgG (HAIgG). It was found that the decrease of ADCC was significantly higher in lymphocytes than in monocytes at all IC and HAIgG concentrations employed (P less than 0.001). The degree of inhibition of PBMC-mediated ADCC was similar to that observed in monocyte ADCC. In previous reports, we demonstrated that IC inhibition of PBMC-mediated ADCC could be reversed by normal human serum (NHS) used as a source of complement (C). In this paper, we study the effects of NHS on isolated populations of monocytes and lymphocytes. It was found that NHS was unable to modify the capacity of IC-blocked monocytes to mediate ADCC. On the contrary, NHS efficiently reversed the inhibition of both ADCC and Fc gamma R expression in IC-blocked lymphocytes. We propose that the regulation of Fc gamma R-IC interactions by C may constitute a physiological way to preserve Fc gamma R expression in lymphocytes.

Signal transduction during antibody-dependent cellular cytotoxicity mediated by U937 cells.

Stimulation of the human promonocytic cell line U937 with antibody-coated chicken red blood cells (Ab-CRBC), leads to inositol phosphate (IP) release in the effector cells. Neomycin (5 x 10(-4) M) completely inhibits activation of phosphoinositide breakdown, while ADCC is suppressed in a dose-dependent manner. Bordetella pertussis toxin (PT) (0.5 micrograms/ml), entirely inhibits IP release, while ADCC activity is markedly suppressed. The PKC inhibitors H-7 and propranolol also suppress ADCC. HA-1004, which has far lower PKC inhibitory activity than H-7, has a minimal effect on ADCC. The calmodulin antagonists W-7 and TFP are strongly inhibitory. These results indicate that stimulation of U937 cells for ADCC is associated to an increase in IP levels, which may provide positive transduction signals for the activation of this lytic mechanism.

Opposite effects of amines on lymphocyte- and monocyte-mediated ADCC

The effect of different amines on antibody-dependent cellular cytotoxicity (ADCC) activity of human mononuclear cells was tested. Whereas monocyte cytotoxic capacity was significantly stimulated in the presence of methylamine (MA), dansylcadaverine (DC) and glycine ethylester (GEE), lymphocyte ADCC was markedly suppressed by these agents. The pharmacological actions of these compounds in our system are not related to their ability to inhibit transglutaminase (TGase) enzymes, since tertiary amines such as sarcosine ethylester (SEE) and chloroquine (CQ) elicited identical responses to MA, DC and GEE. The calmodulin (CAM) inhibitors trifluoperazine (TFP) and the more specific N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide (W-7) [Hidaka, Sasaki, Tanaka, Endo, Ohno, Fujii & Nagata (1981) Proc. natn. Acad. Sci. U.S.A., 78, 4353-4357] mimicked the effects of amines on ADCC, suggesting the possibility that a CAM-regulated process might be involved in the functional changes provoked by amines on ADCC. Finally, binding of 125I-immune complexes to the effector cells in the presence of amines showed lack of correlation between alterations in ADCC capacity and Fc gamma R expression.