The Proteasome Inhibitor PS-341 Induces Early Apoptosis of CD14+ Dendritic Cell (DC) Precursors and of CD1a+ Immature DC.

DC play an essential role in the initiation of (allo)immune responses. Downregulation of NF-kB activity has been shown to prevent the differentiation and maturation of DC. PS-341 a novel antimyeloma terapeutic agent, promotes the degradation of NF-kB. In this study we have investigated whether PS-341 affects DC differentiation and/or maturation. DC were generated by culturing immunomagnetically purified CD14+ monocytes for 1–5 days with GM-CSF (50 ng/ml) and IL-4 (800 U/ml) in the presence or absence of PS-341 at 0.1 to 100 ng/ml. Addition of PS-341 at doses as low as 10 ng/ml significantly reduced the recovery of viable cells, as determined by trypan blue exclusion assay, both after 24h and 48h of culture (3±7% vs 35±25% in control cultures and 3±7% vs 32±25%, respectively, at 24 and 48 hours) (n=5 experiment, p<0.05). Doses higher than 10 ng/ml had a comparable effect. No viable cells were observed after 3 days of culture. Addition of 1 ng/ml PS-341 slightly reduced cell recovery both after 24h (22±26%) and 48h (25±28%) (p=n.s.), whereas cell recovery was not altered in the presence of 0.1 ng/ml PS-341. To test whether the reduced recovery of viable cells was due to apoptosis induction, the expression of annexin V was determined by flow cytometry. Upon addition of 10 ng/ml PS-341, annexin V+ cells increased both at 24h (87±1 vs 55±13% in control cultures) and 48h (93±4% vs 27±13%) (n=5 exps) (p<0.05), whereas no difference was observed with 1 ng/ml and 0.1 ng/ml PS-341. Induction of apoptosis was confirmed by microscopical analysis of cytospins, that showed nuclear fragmentation and massive cytoplasmic vacuolization, features suggestive of apoptosis. Differentiation of monocytes to dendritic cells, as measured by the progressive loss of CD14 and acquisition of CD1a expression, was not altered in cultures containing either 1 or 0.1 ng/ml PS-341. To test whether apoptosis induction by PS-341 required the presence of IL-4, that is necessary to DC differentiation, monocytes were cultured in the presence of GM-CSF only. 10 ng/ml PS-341 signifcantly reduced monocyte recovery after 48 hours of culture (4.5±3.1% vs 69±31% in control cultures, n=3 experiments, p<0.05), due to the induction of early apoptosis (75±4% annexin V+ cells vs 36±1% at 24 hours and 95±1% vs 26±19% at 48 hours in PS-341 and control cultures, respectively). To test whether PS-341 induced apoptosis of immature dendritic cells as well, monocytes were cultured for 6 days with GM-CSF and IL-4 then 1 ng/ml LPS was added for the last 48 hours with or without PS-341 at either 0.1, 1 or 10 ng/ml. Addition of PS-341 at 10 ng/ml strongly reduced day 8 cell recovery as compared to control cultures (3±1% vs 20±21%) (n=2 experiments). Following 48 hours of culture in the presence of 10 ng/ml PS-341, DC were mostly apoptotic (96±2% annexin V+ cells as compared to 28±5% in control cultures). However, PS-341 doses of either 0.1 or 1 ng/ml did not affect cell recovery as well as expression of CD40, CD80 and CD86 costimulatory molecules. Thus clinically relevant doses of PS-341 (in the range of 10⁻⁸ M) may effectively and rapidly induce apoptosis of CD14+ pre-DC as well as CD1a+ immature DC in vitro. Our data suggest that PS-341 might be tested in vivo in the prophylaxis of DC-triggered immunologic diseases, such as graft versus host disease.