Evaluation of the immunomodulatory activities of royal jelly components in vitro.

In this work the effect of different components isolated from royal jelly (RJ) was studied using an in vitro rat T-cell proliferation assay. We found that lower concentrations of MEL 174 (final water extract of RJ) and MEL 147 (3-10-dihydroxydecanoic acid) stimulated T-cell proliferation, triggered by concanavalin A (Con-A) and the process was followed by an increase in the production of interleukin-2 (IL-2). Higher concentrations of MEL 174, MEL 247 (dry powder of RJ) and MEL 138 (trans-10-hydroxydec-2-enoic acid) inhibited T-cell proliferation. The inhibition of T-cell proliferation in the presence of MEL 174 was followed by a decrease in IL-2 production, which was partly abrogated by exogenous IL-2, a decrease in nitric oxide (NO) production and increased apoptosis. In conclusion, our results showed the complexity of biological activity of RJ and suggest that its water extract possesses the most potent immunomodulatory activity in vitro.

Fatty acids isolated from royal jelly modulate dendritic cell-mediated immune response in vitro.

Royal jelly (RJ), especially its protein components, has been shown to possess immunomodulatory activity. However, almost nothing is known about the influence of RJ fatty acids on the immune system. In this work we studied the effect of 10-hydroxy-2-decanoic acid (10-HDA) and 3,10-dihydroxy-decanoic acid (3,10-DDA), isolated from RJ, on the immune response using a model of rat dendritic cell (DC)-T-cell cocultures. Both fatty acids, at higher concentrations, inhibited the proliferation of allogeneic T cells. The effect of 10-HDA was stronger and was followed by a decrease in interleukin-2 (IL-2) production and down-regulation of IL-2 receptor expression. Spleen DC, cultivated with 10 microg/ml of fatty acids down-regulated the expression of CD86 and the production of IL-12, but up-regulated the production of IL-10. In contrast, DC, pretreated with 100 microg/ml of 3,10-DDA, up-regulated the expression of CD86 and augmented the proliferation of allogeneic T cells. The highest dose (200 microg/ml) of both fatty acids which was non-apoptotic for both T cells and DC, down-regulated the expression of MHC class II and CD86, decreased the production of IL-12 and made these DC less allostimulatory. The immunosuppressive activity of 3,10-DDA was also confirmed in vivo, using a model of Keyhole lymphet hemocyanine immunization of rats. In conclusion, our results showed the immunomodulatory activity of RJ fatty acids and suggest that DC are a significant target of their action.

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.

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.

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.

Different roles of a rat cortical thymic epithelial cell line in vitro on thymocytes and thymocyte hybridoma cells: phagocytosis, induction of apoptosis, nursing and growth promoting activities.

In this work, the interaction between a rat cortical thymic epithelial cell (TEC) line (R-TNC.1) with nursing activity and thymocytes as well as BWRT 8 thymocyte hybridoma (TH) cells has been studied. The R-TNC.1 cell line significantly bound thymocytes and TH. Binding was stronger during the first 30 min of cell incubation and was followed by a progressive deadhesion. Among adherent thymocytes the proportion of apoptotic cells increased with culture time which was a consequence of higher capacity of the line for binding of apoptotic than viable cells and induction of apoptosis in a subset of adherent thymocytes. Emperiopolesis activity of this thymic nurse cell (TNC) line was manifested by engulfment of thymocytes as well as TH cells. A subset of viable intra-TNC thymocytes has been triggered to die by apoptosis, whereas other internalized thymocytes have been stimulated to proliferate, as measured by an increase in the percentage of cells in mitosis and higher incorporation of bromodeoxyuridine (BrdU), in comparison to thymocytes cultivated alone. A significant stimulation of proliferation of engulfed TH cells was also observed. The R-TNC.1 cell line efficiently phagocytosed both apoptotic thymocytes and TH, and the process is followed by intra-TNC destruction of ingested cells. Cumulatively, these results suggest different role of the R-TNC.1 clone: phagocytosis of apoptotic cells; induction of apoptotic cell death in a subset of both bound and internalized thymocytes and stimulation of proliferation of a subset of intra-TNC thymocytes or TH cells.