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Sasa Vasilijić

Publications and source records attributed to Sasa Vasilijić.

6 recordsLinked to original sources
PubMed2005

Dendritic cells acquire tolerogenic properties at the site of sterile granulomatous inflammation.

Subcutaneous implantation of polyvinyl sponges represents a suitable model for studying the mechanisms of acute and chronic inflammation, granulomatous foreign-body reaction, as well as wound healing. Using such a model in rats, we studied the phenotypic and functional characteristics of dendritic cells (DC). DC were purified from the sponge exudate using a combination of separation gradients, adherence to plastics, and immunomagnetic sorting. We have shown that the number of DC progressively increased in the sponges, reaching maximal values at day 10 after implantation, followed by their decrease thereafter. Inflammatory DC expressed MHC class II molecules and myeloid markers CD11b, CD11c, and CD68. A subset of DC expressed CD4, R-MC46, DEC-205, R-MC17, and CCR1. Compared to DC isolated in the early phase of inflammation (day 6 DC), DC in the late stage of inflammation (day 14 DC) had a lower capability to stimulate the proliferation of allogeneic lymphocytes and CD4(+) T cells. This finding correlated with the downregulation of CD80, CD86, and CD54 expression and the increased proportion of plasmacytoid MHC class II(+) His 24(+) His 48(+) DC. The suppression of allogeneic lymphocyte proliferation was abrogated by the treatment of DC with lipopolysaccharide. In addition, day 14 DC exerted tolerogenic capability in co-culture with allogenic CD4(+) T cells. These results correlated with the increased levels of IL-10 and TGF-beta in culture supernatants and the sponge exudate.

Animals↗
PubMed2003

Granulocyte-macrophage colony stimulating factor is an anti-apoptotic cytokine for thymic dendritic cells and a significant modulator of their accessory function.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a growth-promoting factor for myeloid-derived dendritic cells (DC) but not for lymphoid DC. The data about its effect on thymic DC (TDC), which are both of lymphoid and myeloid origin, are very scarce. Using an in vitro model, we demonstrated in this work that GM-CSF significantly increased the survival of rat TDC in culture by inhibiting their apoptosis and the effect correlated with up-regulation of Bcl-2 expression. GM-CSF also stimulated differentiation and maturation of TDC as judged by higher expression of MHC class I and II molecules, CD54, CD80 and CD86. These changes correlated with stronger stimulatory activity of GM-CSF-pulsed TDC in syngeneic thymocyte proliferation assay and MLR. The stimulatory potential of TDC was further increased when thymocytes were cultivated with an anti-alphabeta TCR (R73) monoclonal antibody (mAb). The influence of unstimulated TDC on proliferation of thymocytes was inhibited by anti-CD86 but not anti-CD80 mAb, whereas in cultures with GM-CSF-treated TDC both mAbs exerted an additive blocking effect. After separation of TDC on CD11b(+) and CD11b(-) we demonstrated that GM-CSF inhibited apoptosis and potentiated accessory activity of both TDC subsets independently of the myeloid marker expression. Cummulatively, our results suggest that GM-CSF is one of the regulatory cytokine involved in survival, maturation, differentiation and accessory function of TDC.

Animals↗
PubMedDate not supplied

Differentiation of human dendritic cells from monocytes in vitro using granulocyte-macrophage colony stimulating factor and low concentration of interleukin-4.

Several laboratories have developed culture systems that allow the generation of large numbers of human dendritic cells (DC) from monocytes using granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-4 (IL-4). In this work we provided evidence that GM-CSF (100 ng/ml) in combination with a low concentration of IL-4 (5 ng/ml) was efficient in the generation of immature, non-adherent, monocyte-derived DC as the same concentration of GM-CSF, and ten times higher concentration of IL-4 (50 ng/ml). This conclusion was based on the similar phenotype profile of DC, such as the expression of CD1a, CD80, CD86, and HLA-DR, down-regulation of CD14, and the absence of CD83, as well as on their similar allostimulatory activity for T cells. A higher number of cells remained adherent in cultures with lower concentrations of IL-4 than in cultures with higher concentrations of the cytokine. However, most of these adherent cells down-regulated CD14 and stimulated the proliferation of alloreactive T cells. In contrast, adherent cells cultivated with GM-CSF alone were predominantly macrophages, as judged by the expression of CD14 and the inefficiency to stimulate alloreactive T cells. DC generated in the presence of lower concentrations of IL-4 had higher proapoptotic potential for the Jurkat cell line than DC differentiated with higher concentrations of IL-4, suggesting their stronger cytotoxic, anti-tumor effect.

Apoptosis↗
PubMedDate not supplied

Comparison of two different protocols for the induction of maturation of human dendritic cells in vitro.

BACKGROUND: Dendritic cells (DC) have been used for immunotherapy of malignant tumors, different kinds of infections, and other clinical conditions. For that purpose, optimal conditions for the generation of functionally mature DC in vitro are required. Two different protocols for the induction of maturation of monocyte-derived DC (MDDC) were compared in this study. METHODS: MDDC were generated in vitro by cultivating adherent monocytes of healthy volunteers with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) during 6-days period. The immature DC thus prepared were induced to mature using two protocols. DC were stimulated for 2 days with lipopolysaccharide (LPS), or with a cocktail of proinflammatory mediators (PM) containing IL-1beta, IL-6, tumor necrosis factor alpha (TNFalpha), and prostaglandin E2 (PGE2), respectively. Phenotypic characteristics of MDDC and their endocytic activity were studied by flow cytometry. Allostimulatory activity of these cells was tested in the mixed leukocyte reaction (MLR), whereas the production of cytokines was determined by ELISA kits. RESULTS: MDDC matured with PM (PM-DC) were predominantly non-adherent cells, while about 30% of LPS-matured DC were adherent cells. In comparison with LPS-DC, PM-DC expressed higher levels of CD86 and CD83, had lower endocytic activity, produced higher levels of IL-10 and lower levels of IL-12, and more strongly stimulated proliferation of allogeneic lymphocytes. CONCLUSION: The protocol based on the combination of proinflammatory cytokines and PGE2 is better for the induction of maturation of human MDDC in vitro than the protocol using LPS alone.

Cell Culture Techniques↗
PubMedDate not supplied

R-MC46 monoclonal antibody stimulates adhesion and phagocytosis by rat macrophages.

BACKGROUND: In our previous experiments it was shown that R-MC46 monoclonal antibody (mAb), produced at our Institute, stimulated homotypic aggregation of rat granulocytes and production ofproinflammatory cytokines. The aim of this study was to examine antigen expression and function, recognized by R-MC46 mAb on macrophages. METHODS: The expression of R-MC46 antigen on thymic and peritoneal macrophages was investigated using immunocytochemistry and flow cytometry methods. Its biochemical characterization was performed by Western blot. The ability of R-MC46 mAb to modulate adhesion and phagocytosis by macrophages was studied by using co-culture experiments with autologous thymocytes. RESULTS: R-MC46 mAb stained thymic macrophages more strongly than peritoneal macrophages. After in vivo treatment of peritoneal macrophages with Pristane, a significant up-regulation of the R-MC46 antigen expression was observed Western blot analysis showed that the mAb recognized a low molecular weight antigen of about 5.5 kDa. R-MC46 mAb significantly enhanced binding and phagocytosis of thymocytes by both thymic and peritoneal macrophages. These processes were completely blocked by WT.3 (anti-CD18) mAb. The stimulation of binding thymocyte to macrophages was higher with the use of thymic macrophages,while the phagocytosis of these cells was higher in the presence of peritoneal macrophages. CONCLUSION: R-MC46 mAb recognized a new molecule expressed by rat macrophages. The antigen is most probably involved in 82 integrin-mediated adhesion and phagocytosis, as well as proinflammatory functions of macrophages.

Animals↗