Human autoantibodies to diacyl-phosphatidylethanolamine recognize a specific set of discrete cytoplasmic domains

Abstract

The aim of this study was to characterize a novel human autoantibody-autoantigen system represented as cytoplasmic discrete speckles (CDS) in indirect immunofluorescence (IIF). A distinct CDS IIF pattern represented by 3-20 discrete speckles dispersed throughout the cytoplasm was identified among other cytoplasmic speckled IIF patterns. The cytoplasmic domains labelled by human anti-CDS-1 antibodies did not co-localize with endosome/lysosome markers EEA1 and LAMP-2, but showed partial co-localization with glycine-tryptophan bodies (GWB). CDS-1 sera did not react with several cellular extracts in immunoblotting and did not immunoprecipitate recombinant GW182 or EEA1 proteins. The typical CDS-1 IIF labelling pattern was abolished after delipidation of HEp-2 cells. Moreover, CDS-1 sera reacted strongly with a lipid component co-migrating with phosphatidylethanolamine (PE) in high performance thin-layer chromatography (HPTLC)-immunostaining of HEp-2 cell total lipid extracts. The CDS-1 major molecular targets were established by electrospray ionization-mass spectrometry (ESI-MS), HPTLC-immunostaining and chemiluminescent enzyme-linked immunosorbent assay as diacyl-PE species, containing preferentially a cis-C18 : 1 fatty acid chain at C-2 of the glycerol moiety, namely 1,2-cis-C18 : 1-PE and 1-C16 : 0-2-cis-C18 : 1-PE. The clinical association of CDS-1 sera included a variety of systemic and organ-specific autoimmune diseases but they were also observed in patients with no evidence of autoimmune disease.

Fig. 1

Dual immunofluorescence confocal microscopy in HEp-2 cells with human CDS-1 sera and mouse monoclonal antibodies to LAMP-2, EEA1 or GW182. Cells were incubated sequentially with human serum diluted 1 : 100, traced with fluorescein isothiocyanate (FITC)-labelled goat antibody to human IgG, and monoclonal antibodies (mAb) traced with Cy3-labelled goat antibody to mouse IgG. (a) CDS-1 serum no. 35. (b) Anti-LAMP-2 mAb. (c) Merged image. (d) CDS-1 prototype serum no. 11. (e) Anti-EEA1 mAb. (f) Merged image. (g) CDS-1 prototype serum no. 11. (h) Anti-GW182 mAb. (i) Merged image, arrows point to immunoco-localization spots. Bars represent 20 µm.

Fig. 2

Indirect immunofluorescence of CDS-1 and anti-EEA1 sera on delipidated HEp-2 cells. Sera were diluted 1 : 100. (a, b) CDS-1 serum no. 11. (c, d) Human anti-EEA1 serum. (a, c) Non-delipidated HEp-2 cells (control); (b, d) HEp-2 cells extracted with isopropyl alcohol/hexane/water (delipidated). Antibodies were traced with fluorescein isothiocyanate (FITC)-labelled goat antibody to human IgG. Bars represent 20 µm.

Fig. 3

Characterization of CDS-1 sera reactivity against total lipid extract of HEp-2 cells by high performance thin-layer chromatography (HPTLC). (a) HPTLC-immunostaining. 1, human anti-EEA1 serum; 2, human anti-cardiolipin serum; 3, human anti-Scl-70 serum; 4 and 5, normal human sera; 6–15, CDS-1 sera (nn. 38, 51, 52, 53, 35, 11, 43, 36, 54 and 61). Phospholipid standards: phosphatidylethanolamine (PE); phosphatidylcholine (PC); phosphatidylserine (PS). (b) HPTLC of total lipid extract of HEp-2 cells and phospholipid standards. HL, HEp-2 cell total lipids; PE, PS and PC, phospholipid standards. Lipids were visualized after exposure to iodine vapour. C, HPTLC-immunostaining. A replica of the plate used in (b) was assayed with a pool of six CDS-1 sera (nos 11, 35, 38, 51, 52 and 53). O, origin.

Fig. 4

ESI-MS (negative-ion mode) analysis of HEp-2 cell phosphatidylethanolamine (PE) fraction reactive with CDS-1 sera. (a) electrospray ionization–mass spectrometry (ESI-MS) spectrum. HEp-2 cell total lipids were separated by two-dimensional chromatography on silica-gel high performance thin-layer chromatography (HPTLC) plate (inset). The first dimension run was performed using chloroform : methanol : 0·02% CaCl₂ (90 : 40 : 13·5, v/v/v), and the second dimension was carried out in chloroform : methanol : water (65 : 25 : 4, v/v/v). The lipids were visualized after exposure to iodine vapour. The encircled area represents the spot scrapped off for ESI-MS analysis. (b and c) Tandem ESI-MS spectra of ions m/z 478·4 and 742·6, respectively. Fragmentation assignments and putative structures for the two PE species are depicted. O, origin; NL, neutral lipids; LPE, lyso-phosphatidylethanolamine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine; LPC, lyso-phosphatidylcholine; m/z, mass to charge ratio.

Fig. 5

Reactivity of CDS-1 sera on high performance thin-layer chromatography (HPTLC)-immunostaining and chemiluminescent enzyme-linked immonosorbent assay (CL-ELISA) using different phospholipid standards. HPTLC was performed using chloroform : methanol : CaCl₂ 0·02% (90 : 60 : 13·5, v/v/v) as solvent. (a) HPTLC. Phospholipid standards (7 nmol each) were visualized with iodine vapour. (b) HPTLC-immunostaining. A replica of the plate used in (a) was assayed with a pool of six CDS-1sera (nn. 11, 35, 38, 51, 52 and 53). (c) CL-ELISA. All phospholipid standards were assayed by CL-ELISA, at 0·88 nmol/well, using the pool of six CDS-1 sera (black bars) and a pool of 10 normal human sera (white bars). O, origin.

Fig. 6

Abolishment of immunofluorescence reactivity and high performance thin-layer chromatography (HPTLC)-immunostaining of CDS-1 serum by absorption of anti-phosphatidylethanolamine (PE) antibodies. CDS-1 serum no. 11 prediluted 1 : 1000 in 0·5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) was absorbed extensively with PE as described in Material and methods. (a) Indirect immunofluorescence of HEp-2 cells stained with anti-CDS-1 serum before absorption. (b) Indirect immunofluorescence of HEp-2 cells stained with anti-CDS-1 serum after absorption. Bars represent 10 µm. (c) HPTLC-immunostaining of PE standard with anti-CDS-1 serum no. 11 before (lane 1) and after (lane 2) absorption. (d) The same HPTLC plate as in (c) visualized with iodine vapour, depicting the PE band in both lanes. O, origin.