Title: Antiphospholipid Syndrome
Session Type: Abstract Submissions (ACR)
Background/Purpose: Patients with the Antiphospholipid Syndrome (APS) have circulating antiphospholipid antibodies which cause vascular thrombosis (VT) and/or pregnancy morbidity (PM). Previously we have shown that IgG isolated from patients with APS-VT alone caused activation of p38MAPK and NFκB signalling pathways and up-regulation of tissue factor activity in monocytes. These effects were not seen with IgG from patients with APS-PM alone or healthy controls. TF up-regulation caused by the APS-VT samples was reduced by p38MAPK, NFκB, and TLR4 inhibitors, thus implicating a TLR4-MyD88 dependent signalling mechanism (J Immunol, 2010;184:6622). Therefore, in this study we examine whether IgG isolated from patients with different manifestations of the APS have differential effects upon similar pathways leading to activation and migration of trophoblast cells which are more relevant to PM.
Methods: IgG was isolated by protein G purification from serum of 5 patients with APS and VT alone (IgG-VT), 5 patients with APS and PM alone (IgG-PM) and 5 healthy controls. To investigate the intracellular signalling pathways induced by these IgG, a single sample for each of the 3 groups was produced by pooling IgG from the 5 subjects in that group. A human first trimester trophoblast (HTR-8) cell line was incubated with 100 μg/mL IgG from each group. Time course experiments were then performed and mRNA expression of TLR4 and related adaptor protein TRIF was measured using quantitative PCR. The activation of p38MAPK, ERK and NFκB signalling pathways was also examined in cell extracts by immunoblot. Trophoblast migration was determined using a collagen-based cell invasion assay.
Results: Only IgG-PM increased TLR4 mRNA expression (at 6 and 24 hours) and TLR4 (non-MyD88 dependent) adaptor protein TRIF mRNA levels (at 6 hours) compared with IgG-VT and healthy control IgG in HTR-8 cells. None of the APS (VT or PM)-IgG however, increased phosphorylation of TLR4-MyD88 dependent p38MAPK, ERK or NFκB signalling pathways at any (15 minutes, 2 and 6 hour) time points measured in these cells compared with control IgG. IgG-PM caused the greatest inhibition of trophoblast (HTR-8) migration (with 27% inhibition) compared to untreated controls at 48 hours. In contrast, IgG-VT caused minimal (8%) inhibition of trophoblast migration comparable to levels seen with healthy control IgG.
Conclusion: IgG isolated from patients with APS-related PM preferentially inhibit trophoblast cell migration compared with APS-IgG from patients with VT alone. IgG isolated from patients with APS-related PM also activate trophoblast cells via a TLR4 MyD88 independent pathway. This is the reverse of our previous finding in monocytes that were only activated by APS-IgG from patients with VT and not from those with PM. Further experiments are now required to characterise the mechanistic and prognostic implications of these findings.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/purified-igg-from-antiphospholipid-syndrome-patients-with-pregnancy-morbidity-alone-inhibit-trophoblast-migration-and-activate-a-tlr4-myd88-independent-pathway/