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Gc PROTEIN-DERIVED MACROPHAGE ACTIVATING FACTOR (GcMAF) STIMULATES ACTIVATION AND PROLIFERATION OF HUMAN CIRCULATING MONOCYTES
M. Ruggiero*, S. Pacini**, N. Yamamoto
*Department of Experimental Pathology and Oncology, University of Firenze, Italy **Department of Anatomy, Histology and Forensic Medicine, University of Firenze, Italy
Division of Molecular Immunology and Immunotherapy, Socrates Institute for Therapeutic Immunology, Philadelphia, PA, USA
Background. Vitamin D binding protein-macrophage activating factor (GcMAF) is a powerful stimulator of the immune system. Its effects were studied in conditions where macrophage/monocyte function is deficient, from HIV infection to cancer (Transl Oncol 1:65-72, 2008; J Med Virol 81:16-26, 2009), whereas the effects on monocytes from healthy subjects have not been studied. Thus, here we report the results obtained challenging human monocytes from healthy subjects with GcMAF: we demonstrate that the individual degree of responsiveness is dependent on vitamin D receptor (VDR) gene polymorphisms. In addition, since the signal transduction pathway of GcMAF is not fully understood, we studied the effects of GcMAF on the formation of intracellular cAMP. Finally, we studied the effects of GcMAF on normal and cancer cell-induced angiogenesis in the chick embryo chorioallantoic membrane (CAM) assay.
Materials and Methods. GcMAF was prepared by Prof. N. Yamamoto. Peripheral blood monocytes were isolated from healthy subjects using Ficoll-Paque gradient centrifugation. 100L of cells from donors harbouring different VDR polymorphisms (identified by BsmI and FokI restriction enzymes; see Adv Chronic Kidney Dis 15:186-90, 2008) 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. Cell proliferation and viability were assessed by the cellular reduction of tetrazolium salts to colored formazans and measured as optical density (Leuk Lymph 44:1957-62, 2003). cAMP was measured using the Cayman Chemical cAMP assay. CAM assay was performed as described in J Environ Pathol Toxicol Oncol 28:85-8, 2009.
Results. Normal murine and human monocytes, incubated with 10 pg GcMAF/ml for 30 min and cultured for 3-6 h, were highly activated and produced 30-fold increased superoxide generation (Table 1).
Monocytes activated with 10-100 pg GcMAF/ml developed a large amount of Fc-receptors as well as significant variation of receptors that recognize IgG-bound and unbound HIV virions.
Thus, monocyes/macrophages activated by GcMAF preferentially phagocytize IgG-bound HIV virions. Thus, monocyes/macrophages activated by GcMAF preferentially phagocytize IgG-bound HIV virions.
In preliminary experiments with healthy donors, however, we had noticed that individual responses to GcMAF significantly varied. Therefore, donors were selected for VDR genotypes and we observed that the b and F alleles of the VDR gene were associated with the highest responses in terms of cAMP formation and proliferation (Tables 2 and 3).
In fact, subjects harbouring homozygous bb/FF genotypes showed the highest response and 100 pg GcMAF/ml stimulated monocyte proliferation to an extent comparable to that achieved by the highest concentration of lipopolysaccharide (1 g/ml), taken as positive control. Heterozygous subjects (Bb/Ff) showed a smaller, but still significant, response, whereas BB/ff homozygous did not respond. In subjects harbouring bb/FF genotypes, GcMAF sustained cell viability for about 98 h whereas un-stimulated cells were no longer viable after 48 h, as if, in those subjects, GcMAF had rescued monocytes from apoptosis (Cell Death Dis 1, e30; doi:10.1038/cddis.2010.8, 2010).
Chronic HIV infection is associated with deregulated angiogenesis possibly resulting in some of the pathological processes that occur in AIDS patients (Angiogenesis 5:141-51, 2002). In the past, it was demonstrated that GcMAF inhibited growth factor-induced cell proliferation, chemotaxis, and tube formation in vitro by using cultured endothelial cells and in vivo by using a mouse cornea micropocket assay (J Natl Cancer Inst 94:1311-19, 2002).
Its effects on CAM assay (i.e. on an entire developing embryo), however, had not been studied. Table 4, shows that 1 ng GcMAF/ml (i.e. a concentration 10-fold higher than that required to stimulate monocytes) completely inhibited the angiogenesis induced by prostaglandin E2 or by a human breast cancer cell line, MCF-7. GcMAF alone did not modify basal angiogenesis or chick embryo viability and development.
Discussion. These results elucidate the cellular and molecular mechanisms through which stimulation of the immune system leads to eradication of HIV (J Med Virol 81:16-26, 2009; Figs 1, 2 and 3). In fact, we demonstrate that GcMAF has a potent mitogenic activity in vitro and these data are consistent with the observation that intravenous administration of GcMAF increased the systemic cell counts of the activated macrophages to >200-fold. In addition, we demonstrate for the first time that the response of human monocytes to GcMAF is dependent on VDR gene polymorphisms. It is worth noting that the alleles b and F are also associated with the highest sensitivity to vitamin D; a convergence of the vitamin D and GcMAF signalling pathways can thus be hypothesized. Thus, domain I of GcMAF complexes with vitamin D and then binds to monocytes. However, gene-cloned domain III alone fully activates monocytes in a manner identical to entire GcMAF. Whatever the case, these results can prove instrumental in identifying those HIV-positive subjects that could benefit the most from GcMAF treatment.
Figure 3 Figure 2
Acknowledgements: this study was subsidized by grants from the University of Firenze to M.R. and S.P. Author for correspondence: Prof. Marco Ruggiero, MD, PhD: email@example.com