Purified IgG from patients with obstetric but not IgG from non-obstetric antiphospholipid syndrome inhibit trophoblast invasion

Abstract

Problem: Some patients with antiphospholipid syndrome (APS) suffer pregnancy morbidity (PM) but not vascular thrombosis (VT), whilst others suffer VT only. Therefore, we compared the effects of IgG from VT+/PM- and VT-/PM+ subjects on human first-trimester trophoblast (HTR8) cells.

Method of study: HTR-8 cells were incubated with APS VT+/PM-, APS VT-/PM+ or healthy control (HC) IgG. We measured trophoblast invasion by cell invasion assay; mRNA expression of TLR4 and adaptor proteins; phosphorylation of p38 MAPK, NFκB and ERK; and expression of interleukin (IL)-8 and IL-6.

Results: VT-/PM+ IgG, but not VT+/PM- IgG significantly reduced HTR-8 invasion. The effects on invasion were blocked by TLR-4 inhibition. Neither VT+/PM- nor VT-/PM+ IgG altered MyD88 mRNA expression, phosphorylation of signalling molecules or cytokine expression.

Conclusions: VT-/PM+ IgG exert functionally relevant effects on human trophoblast cells but VT+/PM- IgG do not.

Keywords: Antiphospholipid; TLR4; obstetric; trophoblast.

Figure 1

IgG purified from patients with VT−/PM+ APS inhibit HTR-8 cell invasion, which is abrogated when cells are treated with a TLR4 inhibitor. The ability of HTR-8 cells to invade after treatment with 100 μg/mL pooled IgG from VT+/PM− with 83.7 GPLU and 66.0SU binding activity, VT−/PM+ with 47.2GPLU and 63.8SU binding activity and HCs with 0GPLU and SU binding (a) and following pre-treatment with the TLR4 inhibitor CLI-095 or TLR4 antagonist, Ultra Pure Rhodobacter sphaeroides LPS (b) was measured using a transwell invasion assay after 48 hr. HC cell invasion was set at 100%, and the relative invasion of HTR-8 cells exposed to APS-IgG was analysed from this. Graph shows mean ± SEM of quantitative analysis from six (a) and three (b) independent experiments. Statistical analysis was performed as follows: (a) one-way anova (P = 0.01) with Dunn's multiple comparisons test (*P < 0.05); (b) one-way anova (P = 0.03) with Dunn's multiple comparisons test (*P < 0.05).

Figure 2

HTR-8 cells treated with VT−/PM+ IgG but not HTR-8 cells treated with VT+/PM− IgG increase TLR4 and TRIF transcript levels. HTR-8 cells were treated with 100 μg/mL pooled IgG from VT+/PM− with 78.2GPLU and 44.4SU binding activity, VT−/PM+ with 83.4GPLU and 71.5SU binding activity and HCs with 0GPLU and SU binding. TLR4 (a), TRIF (b) and MyD88 (c) mRNA expression at 6 hr was measured by qRT-PCR. TRIF mRNA expression in HTR-8 cells pre-treatment with the TLR4 inhibitor CLI-095 before treatment with 100 μg/mL pooled IgG from VT+/PM−, VT−/PM+ and HCs was also measured (d). The mean ± SEM of quantitative analysis from four independent experiments is shown. Results are expressed as fold change of either TLR4, TRIF or MyD88/GAPDH, relative to untreated cells. No statistically significant differences were found by one-way anova.

Figure 3

APS-IgG do not increase the phosphorylation of p38 MAPK, NFκB p65 or ERK. HTR-8 cells were treated with 100 μg/mL pooled IgG from VT+/PM− with 78.2GPLU and 44.4SU binding activity, VT−/PM+ with 83.4GPLU and 71.5SU binding activity and HCs with 0GPLU and SU binding as well as the positive controls TNF-α (10 ng/mL). Graphs show relative expression at 15 min of p38 MAPK (a), NFκB p65 (b) and ERK (c). The mean ± SEM of quantitative analysis from three independent experiments is shown. No statistically significant differences were found by one-way anova.

Figure 4

APS-IgG do not increase the transcript or protein secretion of the cytokines IL-8 and IL-6 in HTR-8 cells. HTR-8 cells were treated with 100 μg/mL pooled IgG from VT+/PM− with 83.7 GPLU and 66.0SU binding activity, VT−/PM+ with 47.2GPLU and 63.8SU binding activity and HCs with 0GPLU and SU binding as well as the positive controls TNF-α (10 ng/mL). Graphs (a–d) show qRT-PCR analysis of IL-8 (a), and IL-6 (c) mRNA expression at 6 hr and ELISA results of IL-8 (b) and IL-6 (d) protein expression at 72 hr. The mean ± SEM of quantitative analysis from four independent experiments is shown. No statistically significant differences were found by one-way anova.