Our study aims at investigating the effects of Echinacea phytocompounds on monocyte derived-dendritic cell (DC) generation, maturation and biological activity. Phenotypical evaluation, endocytic capacity and the dosage of culture surnatant cytokine are performed on immature and mature DCs, treated or not with E. purpurea, E. pallida, E. angustifolia root extracts (100-150-200 μg/mL). Our results demonstrate that Echinacea extracts don’t affect DC generation, while they interfere with their maturation (shown as a reduced expression of CD40, CD80, CD83 and HLA-DR antigens). Moreover, they seem to reduce the endocytic activity and the production of IL-10. No significant differences among the extracts belonging to the three analysed species have been noticed.Introduction. Echinacea represents a perennial plant indigenous to North America, but also cultured in Europe since a lot of years 1. Ten species belong to this genus 2, but only three species show pharmacological properties: E. angustifolia, E. pallida, E. purpurea 3.Up to date, formulations with Echinacea extracts are usually used for their immunomodulatory, antiiflammatory, antiviral and antioxidant activities, even if the mechanism of the action of their component has not yet been investigated.The goal of our study is to investigate the mechanism of action by which Echinacea phytocompounds have an immunoenhancing activity, focusing on the effects of these extracts on dendritic cell (DC) generation, maturation and biological activity. DCs are the most important antigen-presenting cells: they check both the first phase and the maintenance of adaptative immune responses and the induction of tolerance 4-7.Materials and Methods. DCs are in vitro generated from CD14+ circling monocytes, obtained from mononuclear cells of healthy donors, through an immunomagnetic system (Miltenyi Biotec, Germany). Monocytes are cultured in RPMI 1640 medium, added with fetal bovine serum, L-glutamine, streptomycin and penicillin and supplemented with growth factors (GM-CSF and IL-4) 5, in presence or absence of total root extract of the three specie of Echinacea (E. purpurea, E. pallida, E. angustifolia). These extracts are tested at a concentration of 100, 150 and 200 μg/mL. After 6 days of incubation, LPS is added to the culture for further 24 hours, in order to induce maturation. Then, morphological and phenotypical analysis are performed respectively by cytospin, May-Grunwald Giemsa staining and cytometric assays. The markers used are CD14 antigen, present on monocytes surface, but not on DC surface, CD40 and CD80 costimulatory molecules, HLA class II (HLA-DR) antigen and CD83 antigen, expressed by mature DCs 8.Functional studies on DCs generated in presence of Echinacea include cytometric analysis of endocytotic activity mediated by mannose receptor, using the dextran FITC-conjugated as an antigen, and the dosage of culture surnatant cytokines (IL-6, IL-1, IL-10, IL-12, TNF-α, TGF-β) through an enzyme immunoassay.Results and Discussion. Echinacea phytocompounds don’t compromise DC generation from monocytes (loss of CD14 expression on cells surface).Neverthless, total extracts interfere with the maturation, as the reduced expression of CD83 antigen shows if compared to the control. Moreover, in presence of Echinacea, a reduced expression (both as percentage and mean) of the CD80 and HLA-DR is observed, if compared to the control; as far the CD40 antigen is concerned, its percentage remains unchanged, while the mean decreases in presence of the extracts, compared to the control. No significant differences of cell phenotype among the different concentrations used, nor among the extracts belonging to the three analysed species, is noticed (Fig. 1).When on the 6th day we perform endocytosis assay, in presence of total root extract of Echinacea we observe no differences compared with the control: the cells show a marked phagocytic activity. On the contrary, on the 7th day, the cells treated with E. purpurea keep a more intense activity if compared to the control, even if slightly inferior to that one shown at the immature stage (Fig. 2).Cytokine dosage is carried out on the 7th day. In the sample treated with Echinacea we remark an increase in the release of IL-1 and a decrease in the concentration of IL-10, compared with the control. We notice no relevant differences between the three species we used (Fig. 3).Conclusions. Our study deserves further investigation. In fact, as far as the various and reported pharmacological properties is concerned, up to date the immunomodulatory activity is only partially elucidated. Analysing the literature and our data it seems fundamental to understand which are the compounds responsible of the pharmacological activity of the plant and the mechanism of action by which it is realized. For these reasons, we are going to study the activity of lipophilic and hydrophilic fractions of Echinacea extracts on DCs, but also evaluing the effects of separate active principals on these cells. It could be interesting to identify a possible fraction or species mainly responsible of the observed effects.
Root extracts of three species of Echinacea affect the maturation state of monocyte-generated human dendritic cells / I., Mucci; A., Legitimo; M., Compagnino; Pellati, Federica; P., Nieri; Benvenuti, Stefania; M. R., Metelli; B., Longoni; F., Mosca; P., Macchia; R., Consolini. - In: EUROPEAN JOURNAL OF IMMUNOLOGY. - ISSN 0014-2980. - STAMPA. - 39:(2009), pp. 656-656. (Intervento presentato al convegno 2nd European Congress on Immunology tenutosi a Berlino, Germania nel September 13-16, 2009).