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. 2016 Oct;68(10):2540-9.
doi: 10.1002/art.39743.

Enrichment of Scleroderma Vascular Disease-Associated Autoantigens in Endothelial Lineage Cells

Zsuzsanna H McMahan  1 , Tricia R Cottrell  1 , Fredrick M Wigley  1 , Brendan Antiochos  1 , Elias T Zambidis  1 , Tea Soon Park  1 , Marc K Halushka  1 , Laura Gutierrez-Alamillo  1 , Raffaello Cimbro  1 , Antony Rosen  1 , Livia Casciola-Rosen  2
Affiliations

Enrichment of Scleroderma Vascular Disease-Associated Autoantigens in Endothelial Lineage Cells

Zsuzsanna H McMahan et al. Arthritis Rheumatol. 2016 Oct.

Abstract

Objective: Scleroderma patients with autoantibodies to CENPs and/or interferon-inducible protein 16 (IFI-16) are at increased risk of severe vascular complications. This study was undertaken to determine whether these autoantigens are enriched in cells of the vasculature.

Methods: Successive stages of embryoid bodies (EBs) as well as vascular progenitors were used to evaluate the expression of scleroderma autoantigens IFI-16 and CENP by immunoblotting. CD31 was included to mark early blood vessels. IFI-16 and CD31 expression were defined in paraffin-embedded skin sections from scleroderma patients and from healthy controls. IFI-16 expression was determined by flow cytometric analysis in circulating endothelial cells (CECs) and circulating hematopoietic progenitor cells.

Results: Expression of CENP-A, IFI-16, and CD31 was enriched in EBs on days 10 and 12 of differentiation, and particularly in cultures enriched in vascular progenitors (IFI-16, CD31, and CENPs A and B). This pattern was distinct from that of comparator autoantigens. Immunohistochemical staining of paraffin-embedded skin sections showed enrichment of IFI-16 in CD31-positive vascular endothelial cells in biopsy specimens from scleroderma patients and normal controls. Flow cytometric analysis revealed IFI-16 expression in circulating hematopoietic progenitor cells but minimal expression in CECs.

Conclusion: Our findings indicate that expression of the scleroderma autoantigens IFI-16 and CENPs, which are associated with severe vascular disease, is increased in vascular progenitors and mature endothelial cells. High level, lineage-enriched expression of autoantigens may explain the striking association between clinical phenotypes and the immune targeting of specific autoantigens.

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Figures

Figure 1
Figure 1. Biochemical validation of the human ES/EB progenitor cell differentiation system
Lysates made from human ES cells and EBs harvested at different times of differentiation over a 12-day period were immunoblotted with antibodies against specific differentiation markers (Oct4, Nanog, NCAD and CD31). Equal protein amounts were electrophoresed in each gel lane and β-actin was immunoblotted as a loading control.
Figure 2
Figure 2. Scleroderma autoantigen expression levels in the progenitor cell differentiation system
(A) Lysates made from human ES cells and EBs harvested at different times of differentiation over a 12-day period were immunoblotted with commercial antibodies against IFI16, CENP-B, POLR3A, topoisomerase I, and TIF1γ, or patient sera with high titer antibodies against PARP or CENP-A. (B) Lysates made from day 12 EBs and vascular progenitors (“VP”) were immunoblotted as above; a rabbit polyclonal antibody was used for the CENP-A blot. (A & B): Equal protein amounts were electrophoresed in each gel lane. The same lysates were immunoblotted with an antibody against β–actin as a loading control (lowest panels of each set).
Figure 3
Figure 3. Progenitors expressing CD31, a marker of early blood vessels, have high levels of IFI16 and CENP-A expression
EB day 12 cultures were embedded, cryosectioned and double stained with CD31 and antibodies against either IFI16 or CENP-A as described in the Methods section. Top panel (10×): IFI16 (green), CD31 (red), and a merged image with DAPI (blue nuclear stain) shows enrichment of IFI16 in cells expressing CD31 in day 12 EB's. Middle panel (63×): Higher magnification of the image shown in the top panel. Bottom panel (63×): CENP (red) is enriched in CD31 expressing cells (green). A merged image with DAPI (blue nuclear stain) is also shown. Scale bar represents 100 microns (top panel) and 20 microns (middle and lowest panels).
Figure 4
Figure 4. IFI16 expression is enriched in vascular endothelial cells in skin biopsies from both normal individuals and scleroderma patients
Skin paraffin sections from 20 scleroderma patients (9 with limited disease and 11 with diffuse disease) and 8 normal controls were double stained with a rabbit polyclonal antibody against CD31 (depicted in green) and a mouse monoclonal anti-IFI16 antibody (depicted in red). All sections were counterstained with DAPI (represented in blue). Representative data (merged images) from 4 scleroderma patients with limited disease (tissue numbers 2915, 3213, 3585 and 0943), 4 scleroderma patients with diffuse disease (tissue numbers 2001, 3426, 2804, and 3340) and 4 normal controls (tissue numbers 0120, 0059, 0122 and 9044) is shown. Robust levels of IFI16 staining are found in vascular endothelial cells in both normal controls and scleroderma patients. Scale bar represents 20 microns. Tissues 2915, 3213, 2001 and 3426 are from patients with digital ulcers or digital gangrene; all other scleroderma tissue was from patients with Raynaud's phenomoneon without digital ulcers.
Figure 5
Figure 5. IFI16 expression is enriched in circulating hematopoietic progenitor cells
(A) Gating strategy of a representative PBMC flow cytometry staining. Live cells were defined by FSC-A and SSC-Area. FSC-Height vs FSC-Width and SSC-Height vs SSC-Width were used to exclude cell aggregates from the analysis. Within the CD3 negative gate circulating endothelial cells (CEC) and circulating progenitor cells (CPC) were identified by their differential expression of CD31 and CD45. Histogram representations of the percentage of IFI16 positive cells for CEC and CPC are shown. (B) Percentage of IFI16 positive cells (± standard deviation) in CEC and CPC in PBMCs prepared from five healthy donors, 6 patients with diffuse scleroderma and 5 patients with limited scleroderma.
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References

    1. Varga JA, Trojanowska M. Fibrosis in systemic sclerosis. Rheum Dis Clin North Am. 2008 Feb;34(1):115–43. vii. - PMC - PubMed
    1. Greidinger EL, Flaherty KT, White B, Rosen A, Wigley FM, Wise RA. African-American race and antibodies to topoisomerase I are associated with increased severity of scleroderma lung disease. Chest. 1998 Sep;114(3):801–7. - PubMed
    1. Khimdas S, Harding S, Bonner A, Zummer B, Baron M, Pope J, et al. Associations with digital ulcers in a large cohort of systemic sclerosis: results from the Canadian Scleroderma Research Group registry. Arthritis Care Res (Hoboken) 2011 Jan;63(1):142–9. - PubMed
    1. Tiev KP, Diot E, Clerson P, Dupuis-Simeon F, Hachulla E, Hatron PY, et al. Clinical features of scleroderma patients with or without prior or current ischemic digital ulcers: post-hoc analysis of a nationwide multicenter cohort (ItinerAIR-Sclerodermie) J Rheumatol. 2009 Jul;36(7):1470–6. - PubMed
    1. Domsic RT. Scleroderma: the role of serum autoantibodies in defining specific clinical phenotypes and organ system involvement. Curr Opin Rheumatol. 2014 Nov;26(6):646–52. - PMC - PubMed

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