Purified Protein Derivatives Tuberculin (PPD) From Virulent Or Attenuated Strains Induces Cell Death Of Human Macrophages In Different Manner And The Research Of Signaling Pathway In The Development Of Induction

Slection 1Purified protein derivatives tuberculin from virulent or attenuated strains of mycobacterium tuberculosis induces cell death of human macrophages in different manners[Objective] To study the cell death in macrophages (THP-1) stimulated with different agonists (H37Rv-PPD and BCG-PPD) and to investigate the relationship between Toll like receptor (TLR)-2 and THP-1 apoptosis.[Methods] H37Rv-PPD and BCG-PPD were used to stimulate THP-1 cells for 3 h, 8 h,15 h and 24 h, respectively with or without TLR-2 blockade. Cells were analyzed by flow cytometry to detect the TLR-2 expression. Annexin V staining and Hochest staining were performed to evaluate apoptosis.[Results] The apoptosis cells were increased when stimulated with BCG-PPD and the percentage was 30.2%at 24 h, which were confirmed by Hochest staining. However, the expression of TLR-2 did not increase simultaneously with percentage of 8.84%at 24 h. Nevertheless, most cells presented with necrosis form when stimulated with H37Rv-PPD and the expression of TLR-2 remained high level with the percentage of 17.2%at 24 h, while the percent of apoptosis rate was only 7.72%. Under treatment of TLR-2 antibodies, the percentage of apoptosis decreased to 10.5%at 24 h of BCG-PPD stimulation and TLR-2 expressions were down-regulated to less than 3%at all time points; but after H37Rv-PPD stimulation, the percentage of apoptosis and TLR-2 expression did not changed obviously.[Conclusion] The attenuated BCG-PPD induces THP-1 apoptosis predominately, which is partially correlated with TLR-2 expression. While virulent H37Rv-PPD induces THP-1 necrosis predominately. Selection 2Different cell death of THP-1 induced by virulent/attenuated purfied protein derivatives tuberculin (PPD) and the different expression of TNF-αIL-1βand IL-10 in Mycobacterium tuberculosis[Ob j i ct i ve] To study the different response in macrophages treated with different agonists(H37Rv-PPD and BCG-PPD) related with Mycobacterium tuberculosis and the relationship with TNF-α, IL-1βand IL-10.[Methods] Using H37Rv-PPD and BCG-PPD to stimulate THP-1 cell for 3h,8h,15h,24h respectively. Cells were ananlyzed by Hoechst Staining under fluorescence microscopy to assay cell death (apoptosis and necrosis). At each stimulating time, TNF-α, IL-1βand IL-10 were examined by ELISA kits.[Results] Under fluorescence microscopy, it could easily see oval apoptotic bodies of THP-1 stimulated by BCG-PPD. However, the nucleus were often isolated and necrosis-like when cells were stimulated by H37Rv-PPD.In a word, BCG-PPD tend to induce THP-1 cells to apoptosis, but H37Rv-PPD inclined to induce cells to undergo necrosis.In supernatant of cells stimulated by BCG-PPD, the expression of TNF-a and IL-10 were lower than the cells stimulated by H37Rv-PPD,but the expression of IL-1βwas higher than the latter.[Conclusion] It indicated that the necrosis of cells stimulated by H37Rv-PPD was asossiated with the excessive expression of TNF-a and IL-10,and the apoptosis of cells induced by BCG-PPD was IL-1β-related. Perhaps the mechanism of differences in virulence exist in protein of strain, and associated with cytokines IL-1β\TNF-αand IL-10.