Purpose Antiphospholipid antibodies (aPL) activate monocytes in antiphospholipid syndrome (APS), although the precise mechanisms by which this activation occurs are not fully understood. We have identified several novel protein targets using proteomic analysis of human monocytes treated with APS-IgG. Amongst these novel targets lysosomal proteases cathepsin B and D were identified. The balance between different cathepsins is important in protein degradation, apoptosis and autophagy. Reduced cathepsin B and D with increased cathepsin L is a phenotype suggesting reduced autophagy. Dysregulation of autophagy may be important in the pathogenesis of APS. Therefore, we aimed to determine the effect of aPL on monocyte cathepsin balance and autophagy.
Methods Healthy control (HC) monocytes were treated with 200 µg/ml of IgG purified from (n=9) patients with APS-IgG or (n=9) HC-IgG for 6 hour. Cathepsin B and D expression were measured by western blot. Cathepsin D, B and L activity were measured using fluorescence-based assays. Intracellular proteolytic activity was determined using DQ-BSA.
Results Consistent with our previous proteomic analysis, western blots confirmed that cathepsin B and cathepsin D were down-regulated in monocytes treated with APS-IgG compared to HC-IgG. Similarly, cathepsin B and D activities were significantly reduced in monocytes treated with APS-IgG (p=0.0188 for B, 0.0323 for D). In contrast, cathepsin L activity was increased in monocytes treated with APS-IgG (p=0.0106). We then examined cathepsin activity in monocytes from patients with APS. Cathepsin L activity was increased significantly when these monocytes were treated with APS-IgG compared to HC-IgG (p=0.0257).
Lysosomal proteolysis is a key process in the late phase of autophagy. We therefore exposed HC monocytes to IgG, treated them with GM-CSF overnight and tested their intracellular proteolytic activity. APS-IgG reduced the lysosomal activity of GM-CSF-treated monocytes whereas HC-IgG had no effect.
Conclusions We found that APS-IgG regulate the expression and activity of lysosomal proteases cathepsins B/D and cathepsin L in opposite directions. Consistent with this finding, APS-IgG reduced lysosomal proteolysis in monocytes. Additional experiments are now underway to increase our understanding of how modulation of cathepsin activity and autophagy may be important in the pathogenesis of APS and to provide new therapeutic targets.
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