Gc protein-derived macrophage activating factor (GcMAF) stimulates activation and proliferation of human circulating monocytes
M. Ruggiero1, S. Pacini2, N. Yamamoto3
1University of Firenze, Experimental Pathology and Oncology, Firenze, Italy, 2University of Firenze, Anatomy, Histology and Forensic Medicine, Firenze, Italy, 3Socrates Institute for Therapeutic Immunology, Division of Molecular Immunology and Immunotherapy, Philadelphia, United States
Background: Vitamin D binding protein-macrophage activating factor (GcMAF) has been proposed as a tool to fight HIV infection. Its effects on macrophage activation have been studied in conditions where macrophage function is deficient, from HIV infection to cancer. However, the effects on in vitro activation and proliferation of monocytes from healthy subjects have not been studied. Here we report the results obtained challenging human monocytes with GcMAF.
Methods: Peripheral blood mononuclear cells were isolated from healthy subjects using Ficoll-Paque gradient centrifugation. 100µL of cells were cultured with GcMAF for different time intervals (30 min - 96 h). Each condition was replicated in quadruplicate and each subject served as internal control.
Results: Monocytes, incubated with 10 pg GcMAF/ml for 30 min and cultured for 3 h, were highly activated and produced 30-fold increased superoxide generation. Monocytes activated with 10-100 pg GcMAF/ml developed a large amount of Fc-receptors as well significant variation of receptors that recognize IgG-bound and unbound HIV virions. Thus, monocyes/macrophages activated by GcMAF preferentially phagocytize IgG-bound HIV virions. GcMAF (100 pg/ml) stimulated monocyte proliferation in vitro to an extent comparable to that achieved by the highest concentration of lipopolysaccharide (1 µg/ml) taken as positive control. The effect was dose-dependent and maximal stimulation was obtained with 100 pg GcMAF/ml. The effect was evident after 24 h and lasted for 96 h. At that time (i.e. about 98 h after drawing) un-stimulated cells were no longer viable as if GcMAF had rescued monocytes from apoptosis. Monocyte proliferation induced by GcMAF was not inhibited by 1alpha, 25-dihydroxyvitamin D3.
Conclusions: These results demonstrate that GcMAF has a potent mitogenic activity in vitro and are consistent with the observation that intravenous administration of GcMAF increased the systemic cell counts of the activated macrophages to >200-fold, presumably because of interaction of GcMAF with myeloid progenitors in bone marrow.
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