RESUMO
Localized adherence (LA) of enteropathogenic Escherichia coli (EPEC) to epithelial cells results in attaching and effacing of the surface of these cells. LA depends on the gene bfpA, which codes for the BfpA protein. We found that EPEC-E. coli adherence factor (EAF)((+)), expressing BfpA, significantly reduced HeLa cell viability in comparison with EPEC-EAF((-)), as evaluated by the mitochondrial-dependent succinate dehydrogenase conversion of 3'-[4,5,-dimethylthiazol-2yl]2,5-diphenyltetrazolium bromide (MTT) to its formazan. Apoptosis accounts for a substantial loss of the cell viability, because the cells incubated with EPEC-EAF((+)) or with cloned BfpA (data not shown), but not with EPEC-EAF((-)), were positive for annexin-V binding, demonstrated chromatin condensation and nuclei fragmentation and exhibited a high level of caspase-3 activity. Because the blockade of bacterial cell-surface-associated BfpA by anti-BfpA immunoglobulin (Ig)Y antibody suppressed apoptotic death induced by EPEC-EAF((+)), BfpA may be the trigger for apoptosis. Both EPEC-EAF((+)) and EPEC-EAF((-)), as well as recombinant BfpA (data not shown), activated nuclear factor (NF)-kappaB in a similar manner as analysed by the electrophoretic mobility shift assay (EMSA). EMSA supershift analysis demonstrated the presence of p65/RelA in a DNA-binding complex. In contrast to DNA binding, NF-kappaB-dependent reporter gene transactivation was stimulated more strongly by EPEC B171/EAF((+)), suggesting a role for this virulence factor in the regulation of transcriptional activity of NF-kappaB. Because suppression of NF-kappaB activation by BAY11-7085, a NF-kappaB inhibitor, neither induced apoptosis by itself nor blocked apoptosis induction by EPEC-EAF((+)), it may be suggested that apoptosis is not regulated by the NF-kappaB pathway in HeLa cells.
Assuntos
Apoptose , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , NF-kappa B/metabolismo , Escherichia coli/patogenicidade , Células HeLa , Humanos , Fatores de Virulência/metabolismoRESUMO
Two BCG vaccine formulations of the Moreau strain, commercially manufactured for anti-tuberculosis vaccination, ID-BCG, or anti-cancer adjuvant therapy, Onco-BCG, were compared for immunogenic activity in vitro. The growth rates of both vaccines in murine macrophages were the same, however, Onco-BCG induced stronger and longer-lasting secretion of TNF-alpha, IL-6 and nitric oxide. Onco-vaccine was also more potent in inducing NF-kappaB p65/p50 DNA-binding activity whilst in ID-BCG-infected cells the activity was transient and then gradually replaced by the transcriptionally inactive homodimer p50/p50. Comparative analysis of mycobacterial antigens of the two vaccines demonstrated a difference in expression of the 19 kDa and 38 kDa lipoproteins detected only in Onco-BCG extracts. These results suggest that these molecules may be responsible for the vigorous activation of macrophages induced by the Onco-vaccine. The data obtained show that vaccines from the same BCG strain, when manufactured differently, can vary significantly in their antigen expression and, consequently, in their capacity for macrophage activation which could contribute to the difference in their immunopotentiating effects.
Assuntos
Antígenos de Bactérias/análise , Vacina BCG/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , NF-kappa B/metabolismo , Animais , Linhagem Celular , Interleucina-6/biossíntese , Ativação de Macrófagos , Camundongos , Mycobacterium bovis/imunologia , Óxido Nítrico/biossíntese , Fagocitose , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The presence of antibodies against the major stress protein, Hsp70, in patients with autoimmune diseases led us to hypothesize that Hsp70 may occur extracellularly, and could exert chaperoning and regulatory effects on various cells. We examined the action of pure Hsp/Hsc70 on the main physiological functions of human promonocytic U-937 cells. The protein was isolated from calf muscle and was shown to be a mixture of inducible Hsp70 (60%) and constitutive Hsc70 (40%) isoforms. It was observed that the addition of the protein up-regulated two major monocyte/macrophage differentiation markers, CD11c and CD23, by 20-35%, while it had no effect on CD14. The experiments performed to investigate the influence of Hsp/Hsc70 on the reaction of U-937 cells to differentiation stimuli demonstrated that the addition of the protein prior to PMA was able to inhibit binding of proper transcription factors to double-symmetry and cAMP-response elements of the c-fos early response gene promoter. Administration of exogenous Hsp/Hsc70 prior to treatment with the tumor necrosis factor-alpha significantly lowered the number of apoptotic and necrotic cells. In no case did the control protein, ovalbumin, taken in the same concentration give a comparable effect on U-937 cells. Since the Hsp/Hsc70 effects occurred within the first hour of co-incubation, and therefore they might be explained by its interaction with the cell surface, we assayed binding of the biotinylated protein to U-937 cells by immunoenzyme assay, flow cytometry and indirect immunofluorescence. Using these three techniques we were able to detect Hsp/Hsc70 bound to cells after a 20 min incubation. According to flow cytometry data, at this time 32% of cells were positively stained with streptavidin-FITC. Immunofluorescence studies demonstrated Hsp/Hsc70 bound to the cell surface after a 20 min incubation followed by induction of patch and cap-like structures. One hour later, the majority of the protein had been internalized by U-937 cells.