rHP986 induces Fas expression and apoptosis of cultured macrophages <i>via</i> a TNFR1 dependant pathway.

<p>Panel A - rHP986 induced Fas expression on the surface of differentiated macrophages as quantified by flow cytometry with anti-Fas antibody. Dose dependent increase in Fas expression in response to treatment with increasing concentration of rHP986 (0.5 µg/ml, 2.5 µg/ml and 5.0 µg/ml) observed across varied time intervals (12 hrs, 24 hrs and 48 hrs) is indicated by graph with pink line while the graphs with green line indicate response of cells without protein stimulation. Panel B - Enhanced Fas expression on cell surface after stimulation with rHP986 as compared to cells without recombinant protein stimulus. Panel C - Quantification of apoptosis in the cells treated with increasing concentration of rHP986 for varied time intervals. Results are represented as (mean±SE) percent apoptotic cells per 300 cells. Panel D - Morphological identification of apoptotic cells by acridine orange and ethidium bromide staining method. Arrow head indicates live cells with normal morphology (green fluorescence) and arrow shows cells that have undergone apoptosis after stimulation with rHP986. Black arrows indicate condensed marginal nucleus, apoptotic body formation and membrane blebbing. Panel E - Bar diagram showing inhibition of apoptosis when the cells were stimulated with rHP986 in the presence of neutralizing antibody against TNFR1 (rHP986+TNFR1) as compared to cells stimulated with only rHP986. Oleandrin was used as positive control; US - unstimulated cells. Results are shown as mean ± SE.