Background/Purpose:

The spectrum of the vascular pathology affecting SLE patients with secondary antiphospholipid syndrome includes vasculopathy with endothelial cell hyperplasia as in APS nephropathy. Previous studies established a role for endothelial cell activation via the mammalian target of rapamycin (mTORC) but were limited to IgG fractions of primary APS patients. We extend this finding by studying SLE patients with secondary APS and demonstrate that this activity resides in both IgG and IgA fractions.

Methods:

Endothelial cell phenotypes were assessed using an immunofluorescent assay to report the mTORC biomarker, phospho-S6 ribosomal protein (S6RP) using the human microvascular cell line (serum starved HMEC1 with β2GP1 pretreatment) in the presence and absence of purified human IgG, IgGFab2, and IgA with or without LY294002, a mTORC inhibitor (5 min). We studied 8 SLE patients with secondary APS (mean age 46.8 ± .3, 89% female and 67% white), 4 disease controls that were either SLE without APS or asymptomatic Ro+, and 3 controls. Purified IgG and IgA fractions were used as follows: SLE patients 4 IgG and IgA, 3 IgG and I IgA; 4 diseases controls 4 IgG and 2 IgA; and controls 2 IgG and 1 IgA.   Immunofluorescence (IgG TRITC and Hoechst 33342) was reported using both intensity (1-3+) and a staining scale reflecting % positive cells (3 fields) with 1, <10%; 2, 10-30%; 3, 40-50%; and 4 >50%.

Results:

The phenotype of the endothelial cells which were co-incubated with the IgG fractions of 3 (of 8) patients were diffusely positive for phospho-S6RP (i.e. 3+ intensity, >3 on the scale of % positive cells), which was attenuated by co-incubation with LY294002 (1+, 1 % positive cells). Moreover, endothelial cells co-incubated with Fab2 subfraction retained the  diffuse stain for phospho-6SRP. In addition, treatment of endothelial cells with IgA fractions from the same  individuals resulted in an increase of phosphorylation of S6RP, suggesting the importance of both IgG and IgA APS isotypes. The patient with the greatest intensity and highest staining with both IgG and IgA fraction is triple and full house positive (eg positive for LAC as well as very high titer IgG, IgA and IgM ACA and β2GPI).  For the total group of 8 SLE patients with APS antibodies however, there were no associations with APS titers and the ability to elicit the biomarker in HMEC1. The expression of phospho- S6RP by HMEC1 were within the ranges reported for no treatment (1+, <3 to report % positive cells) for IgG fractions isolated from disease controls, healthy controls, and IgG Fab2 from a disease control and a control IgA.

Conclusion:

These data support the novel finding that both IgG and IgA fractions from SLE patients with secondary APS activate endothelial cells via the mTORC pathway as demonstrated by S6RP phosphorylation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/signaling-by-mammalian-target-of-rapamycin-mtorc-highlight-pathological-igg-and-iga-in-sle-patients-with-secondary-aps/