Abstract
Dendritic cells (DCs) play a key-role in the immune response against intracellular bacterial pathogens, including mycobacteria. Monocyte-derived dendritic cells (MoDCs) are considered to behave as inflammatory cell populations. Different immunomagnetic methods (positive and negative) can be used to purify monocytes before their in vitro differentiation and their culture behavior can be expected to be different. In this study we evaluated the reactivity of two dendritic cell populations towards the Bacillus Calmette-Gu?rin (BCG) antigen. Monocytes were obtained from the blood of healthy donors, using positive and negative immunomagnetic separation methods. The expression of DC-SIGN, CD86, CD80, HLA-DR and CD40 on MoDCs was estimated by flow cytometry. The level of IL-12p70, IL-10 and TNF-? was measured by ELISA. Neither of the tested methods affected the surface marker expression of DCs. No significant alteration in immunological response, measured by cytokine production, was noted either. After BCG stimulation, the absence of IL-12, but the IL-23 production was observed in both cell preparations. Positive and negative magnetic separation methods are effective techniques to optimize the preparation of monocytes as the source of MoDCs for potential clinical application.
Highlights
Dendritic cells play a significant role in the induction and regulation of a protective response against intracellular bacterial pathogens, including mycobacteria [1,2,3,4]
It is suggested that the mechanism behind Dendritic cells (DCs) uptake of Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis Bacillus Calmette–Guérin (BCG) is mediated by the intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) [7], which is present on the surface of human monocyte-derived dendritic cells (MoDCs), dermal DCs, lung interstitial DCs and in lymph nodes [8,9]
Within the group of MoDCs obtained by the positive separation method we observed a significant increase in CD86 (p=0.027), CD80 (p=0.009) and CD40 (p=0.04) expression after LPS stimulation compared with unstimulated MoDCs (Table 1)
Summary
Dendritic cells play a significant role in the induction and regulation of a protective response against intracellular bacterial pathogens, including mycobacteria [1,2,3,4]. Upon infection with Mtb or BCG in vitro, human DCs mature, produce Th1-promoting cytokines, and activate IFNγ-producing T cells [10,11,12]. Hanekom et al [3] and Larsen et al [13] showed that human DCs infected with Mtb or BCG had decreased levels of MHC class II and costimulatory molecule CD80, produced TNF-α and IL-10 instead of IL-12, and had an impaired capacity to activate T cells. There are some difficulties in dendritic cell isolation due to their low concentration (about 0.01%) in peripheral blood. For these reasons, several methods have been developed to generate DCs in vitro [14]. Human DCs can be generated in vitro from peripheral blood CD14+ monocytes ( they are termed monocyte-derived dendritic cells) or from CD34+ progenitors
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