Caracterização morfológica e funcional de células do infiltrado inflamatório na lesão da hanseníase

Abstract

Leprosy presents an ample spectrum of clinical forms with also histopathological differences. In this context, skin macrophages from paucibacillary and multibacillary patients have a different response to Mycobacterium leprae infection. In addition, the relationship of macrophage with others skin cells may have a key role in the development of local immunological response in these groups of patients. This way, the functional and morphological characterization of these macrophages is crucial for a better understanding of tissue lesion in leprosy. By enzymatic dissociation, cells from paucibacillary (n=3), multibacillary patients with (ENL; n=5) and without reaction (n=13) were isolated. For the morphologic and functional analysis of this sample, Giemsa and Ziehl-Neelsen staining was used, as well as, flow cytometry, immunocytochemistry, ELISA and real time PCR. In parallel, skin fragments used for culture, were stored and processed for histopathological and real time RT-PCR analysis. Approximately 40-50% of the cell population of the groups was CD14+, characteristic of macrophages. The CD3+ cells (lymphocyte) analysis showed a major percentage of this cell population in paucibacillary patients. The inverse was observed for CD56+ cells (NK cells), where a greater percentage was verified in multibacillary patients. Moreover, the analysis of TLR-2 and TLR-4 expression demonstrated that the percentage of TLR-2+ was higher than TLR-4+ in paucibacillary and multibacillary patients. In patients with ENL a higher percentage of TLR4+ cells was observed which could be related with the hiper-reactivity of the immune inflammatory response. Giemsa staining showed morphological characteristics of macrophages during the whole period of culture and Ziehl-Neelsen s the bacilli detection respectively in these paucibacillary patients cells. Moreover, the cells presented intense marking for CD68 in immunocytochemistry. During the period of culture the cells secreted TNFα, IFNγ and IL-10, as well as inflammatory mediators. The gene expression for these same citokines was evaluated by real time PCR, and showed an increase in the TNFα and IFNγ expression in cells of paucibacillary compared with the multibacillary patients and a higher expression of IL-10 in multibacillary. A similar profile to the one of the multibacillary patients was observed in the patients with ENL and in the skin fragments. The analysis of the TNFα secretion, IFNγ and IL-10 was carried out and the same profile of the analysis for RT-PCR was observed. These data confirm isolated different profiles of Mфfrom paucibacillary and multibacillary patient s skin in the same way that it is observed in the tecidual injury in leprosy.