AB0161 Antiphospholipid antibodies differentially regulate the expression & activity of the lysosomal proteases with effects upon monocyte autophagy
Abstracts Accepted for Publication
Antiphosholipid antibodies (aPLs) are known to activate monocytes in the pathogenesis of antiphospholipid syndrome (APS), although the precise mechanisms by which this activation occurs are not fully understood. We have recently identified several novel protein targets using a comprehensive proteomic analysis of human monocytes treated with IgG from patients with APS. Amongst these novel targets lysosomal proteases, including cathepsin B and cathepsin D were identified. These proteases are
... proteases are important in protein degradation, clearance of autolysosomes, apoptosis and autophagy. Dysregulation of these homeostatic cellular functions may be important in the exposure of autoantigens and pathogenesis of the APS. Therefore, we have now studied the effects of APS IgG upon the expression/activity of different cathepsins and their effects upon autophagy. Objectives: Determine the effect of pathogenic aPL antibodies on monocyte autophagy and its association with the regulation of lysosomal activity. Methods: Healthy monocytes were treated with 200 μg/ml of IgG purified from (n=9) patients with APS or (n=9) healthy control (HC) IgG for 6 h. The expression of cathepsin B and cathepsin D were measured by western blotting. Activity assays for lysosomal proteases cathepsin D, cathepsin B and cathepsin L were performed using fluorescence based assays (RayBio ® ). Intracellular proteolytic activity of monocytes was determined using DQ-BSA (Molecular probes) and flow cytometry analysis. Autophagy was induced by treating monocytes with 50 μg/ml GM-CSF for 14 h. Results: Consistent with our previous label free quantification mass spectrometry proteomic analysis, western blot analysis confirmed that levels of cathepsin B and cathepsin D were decreased in monocytes treated with APS IgG compared to HC IgG. Similarly, enzymatic assays revealed that cathepsin B and cathepsin D activities were significantly reduced in monocytes treated with IgG from patients with APS compared to HC (p=0.0188, 0.0323). In contrast, levels of enzymatic activity of cathepsin L were increased in monocytes treated with APS IgG compared to HC IgG (p=0.0106). To determine the effect of APS IgG on autophagy, we exposed healthy monocytes to IgG and induced autophagy by treating them with GM-CSF for 14 h. Subsequently we tested the intracellular proteolytic activity with DQ-BSA. Stimulation of monocytes with APS IgG reduced the lysosomal activity of GM-CSF-treated monocytes whereas HC IgG had no effect, indicating that APS IgG disrupts lysosomal degradation during monocyte autophagy. Conclusions: We found that IgG from patients with APS regulate the expression and activity of lysosomal proteases cathepsins B/D and cathepsin L in opposite directions. Activity of cathepsin B and D was down-regulated by exposure to IgG from patients with APS whereas cathepsin L was up-regulated. Furthermore, we found APS IgG disrupts lysosomal degradation during monocyte autophagy. 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 provide new therapeutic targets.