Session Information
Title: Antiphospholipid Syndrome: Clinical Manifestations and New Biomarkers in Antiphospholipid Syndrome
Session Type: Abstract Submissions (ACR)
Background/Purpose: Antiphospholipid syndrome (APS) is an autoimmune disorder characterised by recurrent vascular thrombosis (VT) and/or pregnancy morbidity (PM) caused by antiphospholipid antibodies (aPL). Whilst aPL are known to activate monocytes by inducing production of tissue factor and pro-inflammatory cytokines, upstream mechanisms leading to this pattern of cellular activation are not fully understood. There is evidence that aPL from VT and PM patients stimulate different monocyte intracellular pathways but these have not been characterised in detail. We therefore carried out proteomic analysis of human monocytes treated with IgG from VT, PM and healthy controls (HC) using two different techniques.
Methods: Human monocytic cell line U937 and healthy monocytes were treated with 200 µg/ml of IgG purified from patients with VT, PM or HC. Proteomic analysis was performed using two dimensional difference gel electrophoresis (2D DiGE) and Label Free Quantitative LC-MS/MS. Differentially upregulated proteins were identified by mass spectrometry and validated using quantitative PCR (qPCR) and western blotting.
Results:
The table shows the numbers of proteins whose expression in 2D DiGE analysis was regulated by at least two-fold (up or down) in cells exposed to VT IgG or PM IgG compared to HC IgG.
| U 937 | Healthy monocytes | |
| VT IgG | 41 upregulated, 11 downregulated | 119 upregulated, 21 downregulated |
| PM IgG | 22 upregulated, 1 downregulated | 105 upregulated, 22 downregulated |
Monocytes showed more regulated proteins than U937 cells. Far more proteins were upregulated than downregulated in both cell types. 11 proteins showing the most significant regulation were identified by mass spectrometry analysis. Of these, vimentin and zinc finger CCCH domain-containing protein 18 (ZCH18) were the most significantly upregulated in both U937 cells and monocytes. Additionally, Myeloperoxidase (MPO) and CAP Gly domain-containing linker protein 2 (CLIP2) were significantly induced in monocytes. Upregulation of vimentin, ZCH18, MPO and CLIP2 was confirmed by qPCR. Induction of vimentin and MPO was also validated by western blotting. Anti-cardiolipin/vimentin antibodies have been reported in APS. We found anti-vimentin in serum of 35% of patients with APS but no correlation between anti-vimentin level and ability of IgG to up-regulate monocyte vimentin.
LC-MS/MS of healthy monocytes exposed to VT IgG revealed over 100 proteins that were differentially regulated compared to monocytes exposed to HC IgG. Pathway analysis showed that these proteins were involved in cytoskeletal, coagulation and integrin-signaling functions; all relevant to APS. S100 A11 and POTE ankyrin domain family member E were significantly upregulated whereas Plasminogen activator inhibitor 2 and Coronin 1 were downregulated. Targets were validated using qPCR.
Conclusion: Two different proteomic techniques were used to identify novel proteins involved in the actions of aPL on monocytes. Several of these proteins are strongly associated with autoimmune disease and coagulation and could become potential new therapeutic targets for APS.
Disclosure:
V. M. Ripoll,
None;
A. Lambrianides,
None;
W. Heywood,
None;
A. Rahman,
None;
Y. Ioannou,
None;
I. Giles,
None.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/proteomic-analyses-of-monocyte-responses-to-igg-from-patients-with-antiphospholipid-syndrome/