Molecular organization of the non-bilayer phospholipid arrangements that induce an autoimmune disease resembling human lupus in mice

Abstract

Non-bilayer phospholipid arrangements are three-dimensional structures that can form when anionic phospholipids with an intermediate form of the tubular hexagonal phase II (H(II)), such as phosphatidic acid, phosphatidylserine or cardiolipin, are present in a bilayer of lipids. The drugs chlorpromazine and procainamide, which trigger a lupus-like disease in humans, can induce the formation of non-bilayer phospholipid arrangements, and we have previously shown that liposomes with non-bilayer arrangements induced by these drugs cause an autoimmune disease resembling human lupus in mice. Here we show that liposomes with non-bilayer phospholipid arrangements induced by Mn²⁺ cause a similar disease in mice. We extensively characterize the physical properties and immunological reactivity of liposomes made of the zwitterionic lipid phosphatidylcholine and a H(II)-preferring lipid, in the absence or presence of Mn²⁺, chlorpromazine or procainamide. We use an hapten inhibition assay to define the epitope recognized by sera of mice with the disease, and by a monoclonal antibody that binds specifically to non-bilayer phospholipid arrangements, and we report that phosphorylcholine and glycerolphosphorylcholine, which form part of the polar region of phosphatidylcholine, are the only haptens that block the binding of the tested antibodies to non-bilayer arrangements. We propose a model in which the negatively charged H(II)-preferring lipids form an inverted micelle by electrostatic interactions with the positive charge of Mn²⁺, chlorpromazine or procainamide; the inverted micelle is inserted into the bilayer of phosphatidylcholine, whose polar regions are exposed and become targets for antibody production. This model may be relevant in the pathogenesis of human lupus.

Lepromatous leprosy patients produce antibodies that recognise non-bilayer lipid arrangements containing mycolic acids

“…When liposomes made of these phospholipids are suspended in a buffer devoid of inductors of non-bilayer phospholipid arrangements, such as divalent cations, their lipid bilayer is smooth, which is consistent with low granularity. In the presence of Mn 2+ , PA molecules form intermediate structures of the H II tridimensional arrangements within the bilayers formed by PC molecules; these structures are known as non-bilayer phospholipid arrangements or lipidic particles and they can be detected as liposome granularity increases (Aguilar et al 1999, Wong-Baeza et al 2012. Consistent with these findings, the liposomes containing a conic phospholipid, such as those made of PC/PA, have increased granularity in the presence of Mn 2+ (Fig.…”

“…The dot plots of size [forward scatter vs. granularity side scatter (SSC)] and histograms of granularity (SSC) are shown. The Kolmogorov-Smirnov test was used to compare the granularity histogram of each sample with the negative control (liposome suspension without MnCl 2 or CPZ); D ≥ 0.5 with p ≤ 0.001 was considered significant and revealed the presence of non-bilayer phospholipid arrangements in the liposome membranes (Aguilar et al 1999, Wong-Baeza et al 2012). …”

“…2B) because, in this case, no conic phospholipids are available to form the lipidic particles. Cationic drugs with a conical molecular shape, such as CPZ, can also form lipidic particles with PA , Wong-Baeza et al 2012) and accordingly, liposomes made of PC/PA have increased granularity in the presence of CPZ (Fig. 2C).…”

“…Detection of antibodies that bind non-bilayer phospholipid arrangements by liposomal ELISA -The ability of the antiphospholipid antibodies in the sera to bind liposomes with non-bilayer phospholipid arrangements was also assessed using the liposomal ELISA method (Aguilar et al 1999, Wong-Baeza et al 2012). An aliquot of 100 µL of PC/PA liposome suspension, with or without 5 mM MnCl 2 treatment, was added to each well of a 96-well flatbottom plate (Costar Co, Cambridge, MA, USA) and left at room temperature (RT) for 12 h. The plate was decanted and 200 µL of blocking solution (8% foetal bovine serum in TS buffer) was added to each well.…”

“…Cationic molecules with a conical molecular shape, such as the drugs chlorpromazine (CPZ) and procainamide, can induce the formation of intermediate structures of the hexagonal phase II (H II ) arrangements by some anionic conic phospholipids, including phosphatidic acid (PA), phosphatidylserine and cardiolipin; these intermediate forms of H II arrangements are inserted into the bilayer arrangement of the other phospholipids and distort it to form non-bilayer phospholipid arrangements or lipidic particles (Aguilar et al 1999, Wong-Baeza et al 2012. Furthermore, when injected intrasplenically in mice, non-bilayer phospholipid arrangements induce the production of specific antibodies and antiphospholipid antibodies that recognise non-bilayer phospholipid arrangements have been reported in patients with antiphospholipid syndrome ) and patients with preeclampsia (Campos et al 2006).…”