In vitro study of phenotypical characteristics of BCG granuloma macrophages over the course of granuloma development.

Structural and immunophenotypical characteristics of macrophages, associated with their polarization in the M1 and M2 directions of differentiation and activation, were studied in different morphological types of BCG granulomas by the expression of GM-CSF, IFN-γ, and FGFb cytokines and CD36 and CD16/32 differentiation clusters. The proportion of IFN-γ and FGFb macrophage subpopulations changed over the course of granuloma formation, which led to accumulation of FGFb macrophages in the granulomas. These data indicated that the formation of macrophage granulomas and their subsequent transformation into epithelioid-cell granulomas were associated with dynamic quantitative changes in the subpopulations of macrophages with the morphofunctional characteristics of M1 and M2 phenotypes, determining the antibacterial and destructive potential of granulomas. These data are useful for understanding the contribution of functional polarization of macrophages to the pathogenesis of tuberculous infection and, presumably, its complications.

In vitro expression of IL-1α, GM-CSF, and TNF-α by multinucleated macrophages from BCG-infected mice.

Peritoneal cells from intact and BCG-infected mice were explanted in vitro. In these cultures, multinucleated macrophages in different number of nuclei were formed. The intensity of multinucleated cell formation was higher in cultures from BCG-infected mice. Increasing role of amitosis in the formation of multinucleated macrophages with relatively high number of nuclei was noted with presumable domination of cell fusion mechanism. Relatively high level of IL-1α expression was noted only in the population of binucleated macrophages of BCG-infected mice in comparison with mononuclear cells. It was found macrophages from BCG-infected mice demonstrate a kind of "lineage commitment" towards multinucleated cells, which manifested in culture in initially high and increasing (with increasing the number of nuclei in cells) expression of granulocyte-macrophage CSF and TNF-α as well as initially high amitotic activity of macrophages.

Study of macrophages in BCG granulomas in different compartments of the mononuclear phagocyte system.

We studied in vitro morphological and functional properties of macrophages associated with their M1 and M2 polarization in different mononuclear phagocyte compartments during BCG-induced granuloma formation, namely expression patterns of cytokines IL-1α, GM-CSF, TNF-α, and clusters of differentiation CD36 and CD16/32. We showed the mosaic pattern of macrophage polarization in BCG granulomatosis manifested by simultaneous formation of different macrophage subpopulations with M1 and M2 phenotypes in the population of mononuclear phagocytes of BCG granulomas and various compartments of the mononuclear phagocyte system. These data clarify the role of the functional polarization of macrophages in the pathogenesis of tuberculosis infection.

Dynamics of structural transformations of BCG granulomas and expression of TNF-α and granulocyte-macrophage CSF by macrophages in vitro.

The spleens were isolated from mice at different times after BCG infection and BCG granulomas were explanted and cultured in vitro. Cell migration, chemoattractant potential, and expression of TNF-α and granulocyte-macrophage CSF (GM-CSF) by macrophages migrated from granulomas were evaluated in granulomas. The number of macrophages able to migrate; migrating out of granulomas, expressing TNF-α and GM-CSF decreased with increasing the time after infection. The number of cells in "dissociating" granulomas correlates with the number of macrophages containing live BCG mycobacteria in the cytoplasm.

Phagocytic activity of macrophages against liposomes with conjugates of oxidized dextrans and isonicotinic acid hydrazide during modeling of phagocytosis disturbances in vitro.

We studied phagocytic activity of macrophages against molecular-liposome hybrid compositions consisting of liposomes (diameter 200-450 nm) containing oxidized dextrans with a molecular weight of 35 or 60 kDa conjugated with the basic antituberculosis preparation isonicotinic acid hydrazide (dextrazides) during modeling of various disturbances of endocytosis function of phagocytic cells in vitro. Preincubation of macrophages with trypsin, colchicine, or sodium azide did not change the parameters of adhesion of molecular-liposome hybrid compositions to macrophages. It was found that preincubation of cells with colchicine or sodium azide reduced parameters of phagocytosis of the molecular-liposome hybrid compositions; this reduction did not depend on the molecular weight of dextrans entering the composition of the molecular-liposome hybrid compositions.

Effects of molecular liposomal hybrid compositions with oxidized dextrans and isonicotinic acid hydrazide on production of granulocytic macrophage colony-stimulating factor by macrophages.

The effects of molecular liposomal hybrid compositions consisting of liposomes (200-450 nm) containing oxidized dextrans (dextranals; 35-60 kDa) conjugated with isonicotinic acid hydrazide (dextrazides), their components, and native dextrans on the production of granulocytic macrophage CSF by peritoneal macrophages were studied in vitro. Dextranals proved to be more potent inductors of granulocytic macrophage CSF than native dextrans. Conjugation of nicotinic acid hydrazide with dextranals did not modify their capacity to stimulate the production of granulocytic macrophage CSF. Liposomes in the molecular liposomal hybrid compositions did not attenuate the dextrazide capacity to stimulate the production of granulocytic macrophage CSF. Molecular liposomal compositions containing 60 kDa dextrazide exhibited the most potent stimulatory effect on macrophage production of granulocytic macrophage CSF.

Phagocytosis of hybrid molecular nanosomal compositions containing oxidized dextrans conjugated with isonicotinic acid hydrazide by macrophages.

We studied phagocytic activity of macrophages towards hybrid molecular nanosomal compositions consisting of 150-800-nm nanoliposomes containing oxidized dextrans with a molecular weight of 35 and 60 kDa obtained by chemical ("permanganate") and radiochemical oxidation of dextran conjugated with isonicotinic acid hydrazide (dextrazides, intracellular prolonged antituberculous drugs). Phagocytic activity of macrophages towards hybrid molecular nanosomal compositions containing dextrazides obtained by chemical oxidation of dextrans is higher than activity towards hybrid molecular nanosomal compositions containing dextrazides prepared by radiochemical oxidation and depends on the size of hybrid molecular nanosomal compositions and molecular weight of oxidized dextrans.