title: Long-term culture of multibacillary leprosy macrophages isolated from skin lesions: a new model to study Mycobacterium leprae-human cell interaction.
abstract: BACKGROUND
  Leprosy is characterized by a disease spectrum having two polar clinical forms dependent on the presence or not of cell-mediated immunity. In the tuberculoid forms, granuloma-activated macrophages kill Mycobacterium leprae in conjunction with a Th1 response while, in multibacillary (MB) lesions, M. leprae nonactivated macrophages infiltrate the nerves and internal organs together with a Th2 response. The functional properties and activation pathways of macrophages isolated from patients with MB leprosy remain only partially understood.
  
  
  OBJECTIVE
  To establish an ex vivo methodology capable of evaluating the activation pathways, grade and fate of cultured macrophages isolated from MB lesions.
  
  
  METHODS
  Skin biopsies from patients with borderline tuberculoid, bordeline lepromatous and lepromatous leprosy (LL) were characterized by immunohistochemistry and transcriptional analysis. To isolate inflammatory cells, a portion of the samples was submitted to enzymatic digestion. These same cells, maintained in culture for a minimum 7-day period, were characterized morphologically and via flow cytometry at different culture time points. Cytokine [interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10] mRNA levels were quantified by real-time polymerase chain reaction and protein secretion in the culture supernatants was measured by enzyme-linked immunosorbent assay and the nitric oxide levels by Griess reagent.
  
  
  RESULTS
  RNA expression in tuberculoid and MB lesions showed the profile expected of characteristic Th1 and Th2 responses, respectively. The inflammatory cells in all biopsies were successfully isolated. Although the number of cells varied between biopsies, it was highest in LL biopsies. The frequency of isolated CD14+ and CD3+ cells measured by flow cytometry correlated with the percentages of macrophages and lymphocytes in the lesions. Throughout the culture period, CD68+ macrophages showed morphological changes. A progressive increase in cell number and reduction of infected cells were perceptible in the cultures. In contrast to the biopsies, TNF-alpha, IFN-gamma and IL-10 expression in the tuberculoid and MB leprosy cells in 24-h culture and the cytokine levels in the supernatants did not differ significantly. During the culture period, cytokine expression in the MB cells progressively declined, whereas, from days 1 to 7, nitrite levels progressively increased. After day 40, the remaining macrophages were able to ingest fluorescein isothiocyanate-labelled M. leprae. These data need to be confirmed.
  
  
  CONCLUSIONS
  This study confirmed the feasibility of obtaining ex vivo macrophages from leprosy lesions and keeping them in long-term culture. This procedure may open new pathways to studying the interaction between M. leprae and human macrophages, which might, in turn, lead to the development of therapeutic tools capable of overcoming the specific anergy found in patients with MB leprosy.
authors.affiliation: Leprosy Laboratory, Department of Mycobacteriosis, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Av. Brasil 4365, Manguinhos 21040-360, Rio de Janeiro, Brazil.
authors.forename: D F
authors.initials: DF
authors.lastname: Moura
authors.forename: R M B
authors.initials: RM
authors.lastname: Teles
authors.forename: M M
authors.initials: MM
authors.lastname: Ribeiro-Carvalho
authors.forename: R B
authors.initials: RB
authors.lastname: Teles
authors.forename: I M C F
authors.initials: IM
authors.lastname: Santos
authors.forename: H
authors.initials: H
authors.lastname: Ferreira
authors.forename: T O
authors.initials: TO
authors.lastname: Fulco
authors.forename: J A C
authors.initials: JA
authors.lastname: Nery
authors.forename: E P
authors.initials: EP
authors.lastname: Sampaio
authors.forename: E N
authors.initials: EN
authors.lastname: Sarno
mesh_terms: D000328:Adult; D000368:Aged; D002452:Cell Count; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D005240:Feasibility Studies; D005260:Female; D015870:Gene Expression; D006801:Humans; D007918:Leprosy / Q000276:immunology*; D015439:Leprosy, Borderline / Q000276:immunology; D015440:Leprosy, Lepromatous / Q000276:immunology; D015441:Leprosy, Tuberculoid / Q000276:immunology; D008264:Macrophages / Q000276:immunology* / Q000469:parasitology; D008297:Male; D008875:Middle Aged; D008954:Models, Biological; D009166:Mycobacterium leprae / Q000502:physiology*; D009573:Nitrites / Q000378:metabolism; D010587:Phagocytosis / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics; D012867:Skin / Q000276:immunology* / Q000469:parasitology
chemical_list: D016207:Cytokines; D009573:Nitrites; D012333:RNA, Messenger