Human immunology

Abstract

Introduction: We have earlier shown that stimulation with the main P. brasiliensis antigen (gp43) of PBMC from patients with PCM lead to decreased IL-12, IL-2 and IFN-γ secretion but high IL-10 levels. Modulation of in vitro patients´ responses with IL-12 resulted in increased gp43-induced IFN-γ and the addition of neutralizing a-IL-10 Ab also increased the IFN-γ production. Concurrent addition of a-IL-10 and IL-12 not only increased further the IFN-γ but also restored the proliferative responses to gp43. The IL-12R β2 subunit was poorly expressed by patients' lymphocytes with gp43 compared to controls, but normally or near normally expressed with CMA. These findings suggested an imbalance in IL-12/IL-10 regulation. Some studies have suggested that these cytokines can modulate the expression of the same member of the family of the "signal transducers and activators of transcription" (STATs) and to modulate its biological activities. E.g., IFN-γ secretion can induce STAT1 expression in macrophages and increase its function. Methods and Results: Here, we examined intracellular inactive and active STAT 1 expression in PBMC´ s patients and healthy controls by flow cytometry. The suspension of mononuclear cells was isolated by Ficoll-hypaque and cultivated in 96 wells plates in the presence of medium alone, PMA+ ionomycin or gp43. After 24h, the cells was harvested and incubated with monoclonal antibodies for STAT1. STAT1 expression was analyzed by flow cytometry. The expression of inactive or active STAT 1 in PBMC of 6 patients with active PCM was always lower than that in controls cells (9,2 ± 1,4 in control cells vs 5,5 ± 3,9 for patients cells cultivated with PMA+ ionomycin). In cells of controls stimulated with gp43 the STAT 1 expression was 25 ± 3,7, while in the patients was of 2,7 ± 0,9 under the same stimulation. The expression of active STAT1 also was superior in cells from controls when compared with cells of patients (respectively 8,6 ± 2.1 versus 4,6 ± 0,6 for PMA + ionomycin and 28,4 ± 2,8 versus 5.1 ± 1,8 for gp43). Conclusion: Our data corroborates findings in experimental models of infectious diseases that describe reduction in the expression of STAT1 as important factor for reduction of the Th-1 response and reduction of phagocytosis for macrophages. Financial support: FAPESP 02/14026-7, 02/07306-3; LIM56 HC.