title: High dilutions of antimony modulate cytokines production and macrophage - Leishmania (L.) amazonensis interaction in vitro.
abstract: In previous results mice treated with high dilutions of antimony presented reduction of monocyte migration to the site of infection with increase in B lymphocytes population in the local lymph node.
  
  
  
  To know the mechanisms involved, a series of in vitro studies was done, using co-cultures of macrophages (RAW 264.7) and Leishmania (L.) amazonensis treated with different dilutions of antimony (Antimonium crudum or AC), in different times.
  
  
  
  Spreading, phagocytosis, the oxidative activity of macrophages, the viability of free promastigotes and the cytokines/chemokines concentration in the supernatant were evaluated. The assays were performed in quadruplicate.
  
  
  
  Cells treated with AC 30cH (10-58M) and AC 200cH (10-398M) presented a temporary reduction of the spreading after 02h of incubation, followed by increase after 48h, being the most significant increase observed after the AC 200cH treatment. However, the percentage of internalized parasites at 48, 96 and 120h of incubation was also higher in cells treated with AC 200cH. It is suggested that the AC 200cH improves the ability of phagocytes to internalize the parasites, but not to digest them. The cytokines-chemokines panel corroborated these results. Both dilutions potentiated the parasite-induced reduction of cytokines production, especially IL-6, IL 12 p40 and γ-IFN, after 48h of incubation. In addition, the production of MIP-1 beta (CCL4), a chemokine involved in chronic inflammation, was also reduced after 120h. A specific effect of AC 30cH was seen by the inhibition of two peaks of CCL2 (MCP-1) observed in infected macrophages, at 24 and 120h. Since this cytokine is an important chemokine for monocytes, it explains the results obtained formerly in vivo. The morphology of macrophages after acridine orange staining revealed that the treatment with AC 30cH reduced substantially the acid vacuoles in the cytoplasm, indicating a certain inability of these cells to digest the parasites. On the other hand, a large peak of VEGF-A, associated with increase of internalized parasites was observed after 120h of treatment with AC 200cH, which could be associated to the regulation of the chronic inflammation events by M1-M2 polarization. There was no statistical difference among groups regarding the production of TNF, NO and H2O2, showing that the drugs do not alter macrophage cytotoxic activity. A clear quantitative and qualitative variation of the modulatory effects of AC 30cH and 200cH was seen, in function of time.
  
  
  
  Both dilutions were able to potentiate the decrease of most of cytokines and chemokines induced by the parasite infection in vitro, which explains the clinical improvement seen previously in vivo, however, the mechanisms involved and the epidemiological significance of these findings are still under discussion.
authors.affiliation: Graduating Program of Environmental and Experimental Pathology, Universidade Paulista, São Paulo, Brazil.
authors.forename: Fabiana Rodrigues
authors.initials: FR
authors.lastname: de Santana
authors.affiliation: Graduating Program of Environmental and Experimental Pathology, Universidade Paulista, São Paulo, Brazil.
authors.forename: Luciane C
authors.initials: LC
authors.lastname: Dalboni
authors.affiliation: Laboratory of Cell Biology, Universidade Federal do Paraná, Curitiba, Brazil.
authors.forename: Kátia F
authors.initials: KF
authors.lastname: Nascimento
authors.affiliation: Graduating Program of Environmental and Experimental Pathology, Universidade Paulista, São Paulo, Brazil.
authors.forename: Fabiana Toshie
authors.initials: FT
authors.lastname: Konno
authors.affiliation: Graduating Program of Environmental and Experimental Pathology, Universidade Paulista, São Paulo, Brazil.
authors.forename: Anuska M
authors.initials: AM
authors.lastname: Alvares-Saraiva
authors.affiliation: Graduating Program of Environmental and Experimental Pathology, Universidade Paulista, São Paulo, Brazil.
authors.forename: Michelle S F
authors.initials: MSF
authors.lastname: Correia
authors.affiliation: Graduating Program of Environmental and Experimental Pathology, Universidade Paulista, São Paulo, Brazil.
authors.forename: Maristela Dutra Correa
authors.initials: MDC
authors.lastname: Bomfim
authors.affiliation: Faculty of Odontology - Piracicaba, Universidade Estadual de Campinas, Piracicaba, Brazil.
authors.forename: Renato C V
authors.initials: RCV
authors.lastname: Casarin
authors.affiliation: Graduating Program of Environmental and Experimental Pathology, Universidade Paulista, São Paulo, Brazil.
authors.forename: Elizabeth C
authors.initials: EC
authors.lastname: Perez
authors.affiliation: Graduating Program of Environmental and Experimental Pathology, Universidade Paulista, São Paulo, Brazil.
authors.forename: Maria Anete
authors.initials: MA
authors.lastname: Lallo
authors.affiliation: Graduating Program of Environmental and Experimental Pathology, Universidade Paulista, São Paulo, Brazil.
authors.forename: Giovani B
authors.initials: GB
authors.lastname: Peres
authors.affiliation: Faculty of Medicine, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Márcia Dalastra
authors.initials: MD
authors.lastname: Laurenti
authors.affiliation: Faculty of Veterinary Medicine and Zootechny, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Nilson R
authors.initials: NR
authors.lastname: Benites
authors.affiliation: Laboratory of Cell Biology, Universidade Federal do Paraná, Curitiba, Brazil.
authors.forename: Dorly F
authors.initials: DF
authors.lastname: Buchi
authors.affiliation: Graduating Program of Environmental and Experimental Pathology, Universidade Paulista, São Paulo, Brazil. Electronic address: leonibonamin@unip.br.
authors.forename: Leoni Villano
authors.initials: LV
authors.lastname: Bonamin
mesh_terms: D000818:Animals; D000965:Antimony / Q000494:pharmacology*; D007891:Leishmania / Q000276:immunology*; D007896:Leishmaniasis / Q000276:immunology* / Q000473:pathology; D008264:Macrophages / Q000276:immunology* / Q000469:parasitology; D051379:Mice; D015846:Monokines / Q000276:immunology*; D000067996:RAW 264.7 Cells
chemical_list: D015846:Monokines; D000965:Antimony
keywords: Antimonium crudum; Cytokine; Leishmania amazonensis; Macrophage