A monoclonal antibody to human macrophage gangliosides inhibits macrophage migration

Abstract

Gangliosides have diverse immunoregulatory properties. The gangliosides endogenous to macrophages may have immunoregulatory properties that distinguish them from other gangliosides. Gangliosides have been indirectly implicated in macrophage migration as putative cell surface receptors for migration inhibitory factor (MIF). In this study, a monoclonal antibody to human macrophage gangliosides (antibody 25F4) was developed and characterized. This is the first report of the development of monoclonal antibodies to gangliosides of macrophages of any species. Thin-layer chromatographic immunostaining indicated that antibody 25F4 recognized major gangliosides of human macrophages but did not recognized those previously identified as containing fucose. Immunofluorescent surface labeling of viable human macrophages indicated that antibody 25F4 recognized a surface-accessible epitope, present on all cells, and that this was abolished with lipid depletion of macrophage membranes. This epitope was not present on several human nonmacrophage cells. Finally, human macrophages pretreated with antibody 25F4 demonstrated striking inhibition of migration of an agarose droplet assay, whereas an irrelevant monoclonal antibody or monoclonal antibodies to nonganglioside surface epitopes of human macrophages had no effect on migration. Migration inhibition occurred even though antibody 25F4 was removed from the extracellular milieu and was not due to formation of cellular aggregates. These studies support a role for human macrophage gangliosides in macrophage migration.

Gangliosides of monocyte-derived macrophages of adults with advanced HIV infection show reduced surface accessibility

“…Volume was expanded with an equal volume of Ca 2ϩ and Mg 2ϩ -free Hanks' balanced salt solution. Peripheral blood mononuclear cells were purified by density centrifugation, as previously detailed [6], and seeded onto 100-mm glass Petri dishes (2 to 3 ϫ 10 6 cells/mL) in RPMI 1640 supplemented with 10% heat-inactivated human AB-positive serum. After incubation at 5% CO 2 , 95% humidity at 37°C for 4-10 days, nonadherent cells were removed with serial rinses of warm phosphate-buffered saline.…”

“…Monoclonal antibody 25F4 (IgG2a) was produced by immunization of BALB/c mice with purified human macrophage gangliosides and purification by affinity chromatography, as previously detailed [6]. Antibody 25F4 has been previously shown to recognize a cell surface epitope on human macrophages that is abolished with lipid-depletion of the cells.…”

“…In all experiments, duplicate wells of the same donor's cells, cultured on the same chamber slide, were concomitantly treated with either protein A-purified SP-2 cell supernatant or an irrelevant IgG2a (11E2) that recognizes outer membrane protein C/D of Moraxella catarrhalis but does not recognize macrophage gangliosides, either in intact macrophages or in ELISA. Both negative controls have had essentially identical background immunofluorescence on human macrophages [6].…”

“…Comparative reactivity of macrophage gangliosides from HIV-seronegative and -seropositive donors with monoclonal antibody 25F4 was determined by ELISA, as previously detailed [6]. Simultaneous determination of immunofluorescent surface labeling of macrophages of all donors, as described earlier, was performed.…”

“…Gangliosides may also regulate antigen presentation by monocytes [5]. In human macrophages, monoclonal antibodies to endogenous gangliosides inhibit macrophage migration without impairing cell viability nor by inducing macrophage aggregation [6].…”