Figure 1.
Antiphospholipid antibodies induce trophoblast IL-1β processing and secretion.
Trophoblast cells were either not treated (NT) or treated with (i) the anti-β2GPI mAb, IIC5 (20 µg/ml) or mouse IgG1 control (mIgG; 20 µg/ml); or (ii) human aPL-IgG (500 µg/ml) or normal human IgG control (hIgG; 500 µg/ml) for 72 hrs. (A) Barcharts show levels of secreted IL-1β as determined by ELISA. Data are from 5 independent experiments. *p<0.01 versus the NT control. (B) Trophoblast cells were evaluated for active IL-1β (17 kDa) expression by Western blot (representative blots are shown). Barcharts below show quantification of protein expression as determined by densitometry and normalized to β-actin. Data are from 3 independent experiments. *p<0.05 versus the NT control.
Figure 2.
Antiphospholipid antibody-induced trophoblast IL-1β is dependent upon caspase-1.
Trophoblast cells were either not treated (NT) or treated with the anti-β2GPI mAb, IIC5 (20 µg/ml), all in the presence of media or the caspase-1 inhibitor (5 µM) for 72 hrs. (A) Active IL-1β (17 kDa) expression was evaluated by Western blot. (B) Barchart shows IIC5-induced IL-1β secretion as determined by ELISA and expressed as fold change relative to the untreated control. Data are from 4 independent experiments (*p<0.05).
Figure 3.
Antiphospholipid antibody-induced trophoblast IL-1β is dependent on ASC and Nalp3.
Trophoblast transfected to express either (A & C) shRNA for ASC (sh-ASC) or a control sequence (sh-control) (n = 5); or (B & D) shRNA for Nalp3 (sh-Nalp3) or a Nalp3 mutated targeting sequence (Nalp3-mut) (n = 4), were either not treated (NT) or treated with the anti-β2GPI mAb, IIC5 (20 µg/ml) for 72 hrs. Culture supernatants were measured for (A & B) IL-1β and (C & D) IL-8 by ELISA. *p<0.05; **p<0.001 versus the NT control, unless indicated otherwise.
Figure 4.
Antiphospholipid antibodies induce trophoblast uric acid production by trophoblasts via TLR4, which in turn triggers IL-1βproduction.
(A) Trophoblast cells were either not treated (NT) or treated with (i & ii) the anti-β2GPI mAb, IIC5 (20 µg/ml) or mouse IgG1 control (mIgG; 20 µg/ml) for 24, 48 and 72 hrs; or (iii) aPL-IgG (500 µg/ml) for 72 hrs. Cell lysates (intracellular) and supernatants (secreted) were measured for uric acid. Data are from 4–6 independent experiments. *p<0.05; **p<0.001 versus the NT control. (B) Trophoblast cells were pretreated with either media or the TLR4 antagonist, LPS-RS (10 µg/ml) for 30 mins before cells were then treated with or without the anti-β2GPI mAb, IIC5 (20 µg/ml) for 72 hrs. Barcharts show levels of IIC5-induced: (i) secreted uric acid and (ii) secreted IL-1β, expressed as fold change relative to the untreated control. Data are from 4 independent experiments (*p<0.01). (C) Trophoblast cells were treated with or without the anti-β2GPI mAb, IIC5 (20 µg/ml) in the presence of media or uricase for 72 hrs. Barchart shows levels of IIC5-induced IL-1β secretion, expressed as fold change relative to the untreated control. Data are from 3 independent experiments (*p<0.05).
Table 1.
Uric acid levels in serum from aPL− and aPL+ PROMISSE patients at 20–23 weeks gestation.