Human monoclonal IgG anticardiolipin antibodies induce nitric oxide synthase expression
Introduction
Antiphospholipid antibodies are the hallmark of the antiphospholipid antibody syndrome (APS), an autoimmune disease characterised clinically by venous and arterial thromboses and recurrent foetal losses. These antibodies have varying affinities for anionic (negatively charged) phospholipids (e.g. cardiolipin, phosphatidylglycerol, phosphatidylinositol and phosphatidylserine), plasma phospholipid-binding proteins (e.g. β2-glycoprotein 1[B2-GP1], prothrombin and protein C) or complexes of these proteins and phospholipids [1]. Antiphospholipid (aPL) antibodies may have pro-coagulant effects through the inhibition of the anticoagulant activity of β2-GP1 [2], decreased activation of protein C and antithrombin III, reduction in levels of free protein S and inhibition of factor XII/prekallikrein-mediated fibrinolytic activity [3]. Other reported actions of aPL include complement activation [4], platelet activation and aggregation [5], increased tissue factor expression [6] and decreased function of annexin V [7]. An interaction between aPL antibodies and the vascular endothelium has also been reported, and enhanced expression of adhesion molecules by endothelial cells after incubation with anticardiolipin (aCL) antibodies, suggests a direct activation of the endothelium by aCL [8].
Oxidative stress is now recognised as one of the major factors contributing to vascular disease in general and atherosclerosis in particular, and also seems to play an important role in the pathophysiology of APS [9]. F2-isoprostanes are elevated in patients with systemic lupus erythematosus (SLE) and with aCL antibodies [10], and the lipid peroxidation levels correlate with aCL antibody titres in patients with APS [11]. Furthermore, the activity of paraoxonase (an antioxidant enzyme present in high-density lipoprotein particles), is reduced in patients with APS, as is the total antioxidant capacity (TAC) of plasma [12].
One additional mechanism by which reactive oxygen species might contribute to vascular thrombosis is by rapid chemical inactivation of the vasodilator and antithrombotic mediator NO derived from the endothelium. However, a direct relationship between aCL antibodies and NO activity has never been established, although NO metabolites in urine have been reported to be decreased in patients with APS and their levels to inversely correlate with the aCL antibody titres [9].
We, therefore, examined the effects of aCL antibodies on NO pathways and vascular function in vivo and in vitro. We find that aCL antibodies are proinflammatory and can induce iNOS expression and function and increase oxidant stress in blood vessels, which may contribute to the increased risk of thrombosis in patients with APS.
Section snippets
Hybridoma cells and antibodies
IS4, a human IgG monoclonal antibody which binds to cardiolipin and β2-GP1, was derived from patients with antiphospholipid syndrome (a kind gift from Pojen Chen, UCLA, CA, USA) [13]. Cells were cultured in RPMI 1640 medium containing 1% l-glutamine, 1% sodium pyruvate, 2% MEM non-essential amino-acids, 1% penicillin/streptomycin, 0.2% gentamycin (all from Gibco, UK) and 10% FCS (Sigma, UK). Antibodies were purified in an LPS free environment (Cymbus Biotechnology limited). Human non-specific
Organ chamber experiments
The PE contraction of rat aortic rings was markedly decreased in vessels pre-treated with IS4 for 4 h, when compared to control IgG or vehicle (n = 8, p < 0.001; Fig. 1A). The impairment in PE contraction induced by IS4 was equal in magnitude to that produced by LPS (n = 4).
The impaired response to PE in IS4 and LPS treated vessels was abolished by the addition of l-NAME or 1400 W indicating that the PE hyporeactivity was secondary to an increased activation of iNOS (Fig. 1A and B).
PE hyporeactivity to
Discussion
In this study the ex vivo model demonstrated that short-term incubation with IS4 leads to phenylephrine and acetylcholine hyporeactivity in rat aortic tissue, with no change in response to the endothelium-independent vasodilator sodium nitroprusside. These changes are reversible following incubation with l-NAME or 1400 W and were prevented when the antibody was pre-incubated with its antigen cardiolipin. Addition of l-arginine pre-constricted tissues led to significant relaxation in the IS4
References (23)
Autoantibodies to phospholipid-binding plasma proteins: a new view of lupus anticoagulants and other “antiphospholipid” autoantibodies
Blood
(1994)- et al.
Endothelial activation by aPL: a potential pathogenetic mechanism for the clinical manifestations of the syndrome
J Autoimmun
(2000) - et al.
Enhanced lipid peroxidation in patients positive for antiphospholipid antibodies
Blood
(1997) - et al.
Downregulation of endothelial constitutive nitric oxide synthase expression by lipopolysaccharide
Biochem Biophys Res Commun
(1996) - et al.
Mechanism of action of beta2-glycoprotein I-dependent lupus anticoagulants
Lupus
(1998) Mechanisms of autoantibody-mediated thrombosis
Lupus
(1998)- et al.
The antiphospholipid antibody syndrome
- et al.
Platelet activation markers and the primary antiphospholipid syndrome (PAPS)
Lupus
(1998) - et al.
Increased levels of tissue factor mRNA in mononuclear blood cells of patients with primary antiphospholipid syndrome
Thromb Haemost
(1999) - et al.
Placental pathology in antiphospholipid syndrome
Lupus
(1998)
Antioxidant susceptibility of pathogenic pathways in subjects with antiphospholipid antibodies: a pilot study
Lupus
Cited by (7)
Infliximab improves endothelial dysfunction in a mouse model of antiphospholipid syndrome: Role of reduced oxidative stress
2015, Vascular PharmacologyCitation Excerpt :Injection of anti-β2GP1-IgG induced a significant decrease in eNOS mRNA expression, while iNOS and gp91phox mRNA expression levels were enhanced. Such an increase in iNOS mRNA levels was reported in a rat model of APS after passive transfer of monoclonal anticardiolipin antibodies [24]. This effect was associated with a decrease in eNOS activity suggested by a depressed aortic response to acetylcholine [24].
Mechanistic analysis of nonoxygenated hypothermic machine perfusion's protection on warm ischemic kidney uncovers greater enos phosphorylation and vasodilation
2014, American Journal of TransplantationAdvances in the Research on Anticardiolipin Antibody
2019, Journal of Immunology ResearchAuto-antibodies as emergent prognostic markers and possible mediators of ischemic cardiovascular diseases
2013, Clinical Reviews in Allergy and ImmunologyIdiopathic intracranial hypertension may be caused by reactivation of latent cerebral toxoplasmosis. Effect of various diseases and clinical states
2012, Neuroinflammation: Pathogenesis, Mechanisms and Management