Pharmacology Department, Faculty of Medical Sciences, New University of Lisbon, Lisbon, Portugal, 1 Centre for Rheumatology, Department of Medicine, University College London, London, 2 Academic Department of Rheumatology, Leeds University Teaching Hospitals, Leeds, UK, 3 Department of Medicine, Division of Rheumatology, University of California at Los Angeles, CA, 4 Section of Nephrology, Department of Medicine, The University of Chicago, Chicago, IL, USA and 5 Division of Rheumatology, Department of Medicine, The Research Institute of the McGill University Health Centre, Montreal, Canada.
Correspondence to: J. Delgado Alves, Faculdade de Ciências Medicas, Departamento Farmacologia, Campo Mártires da Pátria, 1301169-056 Lisboa, Portugal. E-mail: jdalves.farm{at}fcm.unl.pt
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Abstract |
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Methods. Hybridomas producing human and murine aCL and anti-ß2-glycoprotein I (aß2-GPI) monoclonal antibodies were injected into three groups of five female BALB/c severe combined immunodeficiency (SCID) mice. Corresponding hybridomas secreting non-antiphospholipid antibodies of the same isotype were employed as controls. Sera and organs were collected after 30 days. Paraoxonase (PON) activity, peroxynitrite, superoxide, nitric oxide (NO) and nitrotyrosine were measured in plasma. Expression of endothelial nitric oxide synthase and inducible nitric oxide synthase (iNOS) was assessed by western blot and immunohistochemistry.
Results. PON activity and NO (sum of nitrate and nitrite) levels were reduced in the human aCL IgG group (P<0.002 and P<0.04, respectively), whilst peroxynitrite and superoxide and expression of total antioxidant capacity of plasma were increased (P<0.01). PON and NO were decreased in the murine aß2-GPI IgG and IgM aCL groups (P<0.03 and P<0.05, respectively). Nitrotyrosine was elevated in the human aCL IgG group (P<0.03). Western blotting showed reduced iNOS expression in the hearts of the IgG aCL group, confirmed by immunostaining. PON inversely correlated with IgG aCL titres (P<0.001), superoxide (P<0.008) and peroxynitrite levels (P<0.0009). Peroxynitrite and total IgG aCL were independent predictors of PON (P<0.0009 and P<0.02, respectively). Superoxide was the only independent predictor of NO (P<0.008) and of nitrotyrosine (P<0.002).
Conclusion. aCL antibodies are associated with the decreased PON activity and reduced NO that may occur in the preclinical phase of APS.
KEY WORDS: Antiphospholipid antibodies, Oxidative stress, Nitric oxide, Total antioxidant capacity, Paraoxonase
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Introduction |
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Nitric oxide (NO) is the main endothelium vasodilator [7], and interference with NO synthesis induces vascular dysfunction [8], particularly in the early phases of atherosclerosis [9]. Furthermore, oxidation of lipids causes oxidative stress that is associated with atherosclerosis [10], both features of APS [11]. Anticardiolipin (aCL) antibody titres positively correlated to plasma levels of F2-isoprostanes, sensitive markers of in vivo lipid peroxidation [12], indicating enhanced oxidative stress in APS [13], and to decreased urinary excretion of NO metabolites [14].
An emerging concept in atherogenesis relates to paraoxonase (PON). This enzyme is carried in plasma by high-density lipoprotein and its major function is to prevent oxidation of low-density lipoprotein [15]. In primary APS, PON activity is reduced and correlated inversely with aCL titres and directly with the total antioxidant capacity of plasma [16]. Decreased PON activity, with increased oxidative stress and reduction of NO, may be involved in the early phases of APS.
To further evaluate the association between aCL antibodies and oxidative stress in vivo, we have tested whether aCL antibodies may affect the oxidant/antioxidant balance in an experimental non-lupus murine model.
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Materials and methods |
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Cells were cultured in RPMI 1640 medium containing 1% L-glutamine, 1% sodium pyruvate, 2% Minimum Essential Medium (MEM) non-essential amino acids, 1% penicillin/streptomycin, 0.2% gentamicin (all from Gibco, UK) and 10% fetal calf serum (Sigma, UK).
Experimental protocol
Female BALB/c severe combined immunodeficiency (SCID) mice were obtained from Harlan (Bicester, UK) at 8 weeks of age. Mice were all housed in sterile conditions on vented racks. All procedures were carried out in accordance with the Animals (Scientific Procedures) Act 1986.
Mice were acclimatized for 1 week and then primed with 500 µl intraperitoneally (i.p.) of pristane (2,6,10,14-tetramethylpentadecane; Sigma), which stimulates macrophages to produce growth factors and create an optimal environment for hybridoma cell growth. Ten days later, all mice were injected with the same amount of hybridoma cells (1x106 cells in 500 µl of RPMI 1640).
Mice were divided into eight groups of five mice each. Three groups were injected with hybridomas producing antiphospholipid antibodies: IS4 producing human IgG aCL; 12A1-A17.3 producing murine IgG1 anti-ß2-GPI; and 16A3-14.11 producing murine IgM aCL [18]. Another three groups were injected with hybridomas secreting antibodies matched for the same isotype and species but that did not have antiphospholipid specificity: TW producing human IgG; 29J3-119 producing murine IgG1; and 16B4-2 producing murine IgM. As negative controls, one group of mice was injected with CBF7, the non-secreting mousehuman heteromyeloma fusion partner cell, and one group was not injected with hybridoma cells.
Throughout the experimental period, proteinuria (Albustix; Bayer Diagnostics, Berkshire, UK) and weight was assessed every 72 h. Mice were killed when the development of ascites resulted in a 20% increase in body weight, which occurred on average 1.2 months after pristane priming. To avoid possible disparities due to the timing of ascites formation, animals in the experimental and control groups were matched by the time they were killed. All investigations, including organ collection for histopathology analysis, were done at the time of killing.
Human IgG enzyme-linked immunosorbent assay (ELISA)
Polystyrene 96-well plates (Maxisorp; Nunc, Roskilde, Denmark) were coated with 2.5 µg/ml of goat anti-human IgG (Sigma) diluted in phosphate-buffered saline (PBS) (pH 7.2). After incubation for 16 h at 4°C, plates were washed three times with PBS and blocked with 1% (w/v) bovine serum albumin (BSA) (Sigma) in PBS for 1 h at 37°C. Serum samples were diluted (1:200) in PBS containing 0.05% Tween 20 (Sigma), and were incubated on the plate for 1 h at 37°C. Following three washes, bound antibodies were detected by incubation for 1 h at 37°C with a 1:1000 dilution of affinity-purified goat anti-human IgG conjugated to alkaline phosphatase conjugate (Sigma) and, following washing, developed with the substrate p-nitrophenol phosphate (Sigma). Optical density was measured at 405 nm with a reference filter at 490 nm. The sample concentrations were calculated by reference to the linear portion of a standard curve of purified human IgG (Sigma) run on every plate.
Anti-cardiolipin ELISA (human and murine)
IgG (human) and IgM (murine) aCL were measured by ELISA, using microtitre plates (Polysorp; Nunc, Life Technologies, Paisley, UK), which were coated overnight at 4°C with 50 µg/ml cardiolipin (bovine heart; Sigma-Aldrich, Poole, UK) in 70% ethanol. Blocking was performed with 1% (w/v) BSA (Sigma) in PBS for 1 h at 37°C. Plates were then washed once using PBS. Samples (50 µl) (diluted 1:100 for human sera and 1:200 for murine sera), and positive IgG and IgM controls were added to duplicate wells for 1 h at 25°C. After washing three times with PBS, 100 µl of alkaline phosphatase-conjugated anti-human IgG and anti-mouse IgM (both 1:1000 in the blocking agent) were added and incubated for 1 h. p-Nitrophenyl phosphate (100 µl) (Sigma) in diethanolamine buffer (pH 9.8) was added; samples were incubated at 37°C for colour development, and the absorbance was read at 405 nm after 1 h. Results were expressed as a percentage of the positive control: OD (sample)/OD (positive control)x100.
aß2GPI ELISA (murine)
IgGß2GPI was measured by ELISA, using a commercial kit (Diastat anti-ß2GPI; Axis-Shield Diagnostics, Dundee, UK) based on a method previously described [19]. Murine aß2GPI was assayed by adaptation of the human kit to detect murine antibodies with goat anti-mouse IgGalkaline phosphatase conjugate (Sigma). Assays were developed as described above.
Nitrotyrosine ELISA
Nitrotyrosine was measured by ELISA using a commercial kit (Hbt nitrotyrosine; HyCult Biotechnology, Uden, Netherlands) in accordance with the instructions of the manufacturer. Standard curves were performed in each plate, using samples with known nitrotyrosine concentrations included in the kit.
Paraoxonase activity
Serum PON activity was measured as described by Eckerson et al. [20] with some modifications. Briefly, paraoxon (1.0 mM), freshly prepared in 300 µl of 50 mM glycine buffer containing 1 mM calcium chloride (pH 10.5), was incubated at 37°C with 5 µl of serum for 15 min in 96-well plates (Polysorp). p-Nitrophenol formation was monitored at 412 nm for 15 min to exclude early paraoxon hydrolysis due to irreversible binding of albumin and non-specific esterases. Activity was expressed as µmol p-nitrophenol per ml serum per min.
Total antioxidant capacity (TAC) of plasma
Two different methods assessed the total antioxidant capacity of plasma. The first employed peroxynitrite (ONOO) quenching: 100 µl of phosphate buffer (50 mM, pH 7.4) containing Pholasin® (1.7 µg/ml) was pipetted into a microcuvette. Plasma or buffer for control (5 µl) was added. The reaction was initiated by adding 3-morpholino-sydnonimine HCl (SIN-1) (2 µl of 2 mg/ml in water), and light emission was measured continuously at 5 min intervals until the maximum reading was obtained. Antioxidant capacity was expressed as the time at which maximum light was emitted. Lower values reflected a decreased TAC of plasma (peroxynitrite-related).
The second method employed superoxide anion () quenching: 100 µl of phosphate buffer (50 mM, pH 7.4) containing Pholasin (0.5 µg/ml) and adjuvant K (50 µl/ml) was pipetted into a microcuvette, and plasma or buffer for control (5 µl) was added. Next, 5 µl of xanthine (50 mM) was added, and the reaction was initiated by the addition of 20 µl of xanthine oxidase (0.5 U/ml phosphate buffer). The signal was recorded over a period of 5 min on a 121 LKB recorder (Bromma, Sweden). Antioxidant capacity was expressed as the counts (mV) at maximum light emission. Higher values reflected a reduced TAC of plasma (superoxide-related).
Serum nitrate and nitrite (NO)
The sum of nitrate and nitrite was determined using the Griess reaction, as previously reported [21]. Briefly, serum was diluted 1:4 with PBS, and 200 µl of this solution was ultrafiltered by centrifugation at 10 000 g for 1 h, using 10 kDa molecular weight filters (Ultrafree-MC; Millipore). Only clear and colourless filtrates were tested. The assay was performed in a standard flat-bottomed 96-well polystyrene microtitre plate, containing 50 µl/well of standard or sample. The assay was blanked against PBS. Fifty microlitres of nitrate reductase and 50 µl ß-NADPH was added to each well, giving final concentrations of 300 U/l and 25 µmol/l, respectively. The plate was incubated at room temperature for 3 h. Excess ß-NADPH was consumed by addition of 50 µl of PBS containing L-glutamic dehydrogenase, -ketoglutaric acid and NH4Cl (final concentrations 500 U/l, 4 mmol/l and 100 mmol/l, respectively), followed by 10 min of incubation at 37°C. NO concentration was then determined by the addition of 50 µl each of Griess reagents 1 and 2. After incubation for 10 min at room temperature, the absorbance was read at 540 nm.
Haematoxylin and eosin histological stain
Formalin-fixed, paraffin-embedded kidney sections from the SCID mice were stained with haematoxylin and eosin. An experienced histopathologist examined these sections for morphological evidence of vascular damage, thrombosis or other abnormalities. The histopathologist was blind to the mouse treatments.
Western blot analysis for inducible and endothelial nitric oxide synthase
Immunoblot assays were performed according to previously described methods [22] with some modifications. In brief, homogenates from mouse heart were obtained after sonication, and proteins were separated under reducing conditions on 7% polyacrylamide gels. Proteins were then transferred to polyvinylidene difluoride membranes (PVDF; Millipore) and quenched with blocking buffer containing 5% non-fat milk in 0.1% PBSTween-20 for 1 h at room temperature. After one wash, membranes were incubated overnight at 4°C with primary antibody [rabbit anti-endothelial western blot analysis (iNOS and eNOS) (eNOS) or rabbit anti-inducible nitric oxide synthase (iNOS); Santa Cruz Biotechnology, USA], 1:1000 in blocking buffer. After several consecutive washes, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (donkey anti-rabbit; Santa Cruz Biotechnology, CA, USA) in blocking buffer for 1 h. Assays were performed using the enhanced chemiluminescence (ECL) detection system (Pharmacia Biotech, Piscataway, NJ, USA).
Immunohistochemistry (iNOS and eNOS)
Paraffin-embedded sections of kidney blood vessels were dewaxed and antigen was retrieved by boiling the sections for 180 s in Tris-EDTA (pH 9.0). Following flushing in tap water, slides were rinsed in 0.05% Tween 20 in Tris-buffered saline (TBS/Tween). Endogenous peroxidase activity was blocked for 30 min using Dako Peroxidase block (Dako, Ely, UK). The sections were rinsed in TBS/Tween and primary antibody was applied for 60 min at room temperature (iNOS and eNOS were diluted 1/100 in TBS). The sections were rinsed in TBS/Tween and EnVision reagent (Dako) was applied for 60 min, then rinsed again in TBS/Tween, and EnVision DAB (Dako) was applied for 10 min. The slides were counterstained with haematoxylin before being dehydrated and mounted.
Statistical analysis
Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA). Non-parametric tests were employed to compare differences between groups (KruskalWallis test) and to evaluate associations between variables (Spearman's rank test). A stepwise multiple regression analysis tested the independence of the associations found by univariate analysis.
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Results |
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Characterization of oxidative status
PON activity
Values of all the oxidative status variables are shown in Table 1. PON activity was reduced in the IS4 group (human IgG aCL) when compared with its matched control TW (human IgG), CBF7 and the no cells groups (P always <0.01).
Mice positive for murine IgG aß2-GPI showed decreased PON activity compared with the matched murine control group, but the difference did not reach statistical significance. There were no differences regarding PON activity in the remaining groups.
TAC (peroxynitrite quenching)
TAC assessed by peroxynitrite quenching was low in the IS4 group (human IgG aCL), when compared with the human IgG control, with CBF7 and with the no cells groups (P<0.01, P<0.01 and P<0.008, respectively). TAC was also significantly low in mice injected with hybridomas producing murine IgG aß2-GPI and IgM aCL when compared with the respective control group (irrelevant murine IgG or IgM) (P<0.03 and P<0.05, respectively).
TAC (superoxide quenching)
TAC assessed by superoxide quenching was decreased in the IS4 (human IgG aCL) group, when compared with the human IgG control, CBF7 and no cells controls (P always <0.01) It should be noted that, in the case of superoxide quenching, reduced superoxide quenching is reflected by higher values of plasma TAC. All other groups had similar levels.
NO and nitrotyrosine
NO (sum of nitrate and nitrite) was decreased in the IS4 group (human IgG aCL) when compared with the control groups (P<0.04). In contrast, there was no significant difference between the groups positive for murine IgG aß2-GPI or IgM aCL and their relevant control groups. Nitrotyrosine was found to be elevated only in the IS4 group (human IgG aCL). This was significant when compared with all the control groups (P<0.03).
Relationship between antioxidant variables
PON activity showed an inverse correlation with both total human IgG titres (P<0.001) and TAC (superoxide) (P<0.008) in mice injected with human IgG-producing hybridomas. There was also a direct correlation between PON activity and TAC assessed as peroxynitrite quenching (P<0.0009). NO levels were inversely correlated with TAC assessed as both superoxide quenching (P<0.008) and nitrotyrosine levels (P<0.01).
Multiple regression models
To evaluate factors that could independently predict PON activity, NO levels and nitrotyrosine, stepwise multiple regressions were performed and the following variables were considered: antibody type, total IgG, total IgM, and TAC (peroxynitrite and superoxide).
TAC (peroxynitrite) and total IgG levels were independent predictors of PON activity [P<0.0009 for TAC (peroxynitrite) and P<0.02 for total IgG titre]. TAC (superoxide) was the only independent predictor for NO levels (P<0.008) and nitrotyrosine (P<0.002).
Western blot analysis (iNOS and eNOS)
Expression of iNOS in the hearts of mice that were inoculated with the hybridoma producing IS4 (human IgG aCL) was practically abolished compared with heart samples from control groups; that is, mice inoculated with the hybridoma producing the human IgG and mice inoculated with the hybridoma cells alone (CBF7). The expression of iNOS was also low in the non-inoculated group of mice: these mice did not develop the peritonitis necessary to support the growth of the hybridomas. The other control groups that developed hybridoma-induced peritonitis all showed strong iNOS expression (Fig. 1A). The expression of iNOS in the murine Ig groups showed no differences (not shown). There were no significant differences in the expression of eNOS in heart tissues collected from both the human (Fig. 1B) and the murine (not shown) Ig groups.
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Immunostaining for iNOS was markedly decreased in vessels of mice that received the IS4-producing hybridoma when compared with the relevant control groups (Fig. 2A). In contrast, there were no evident changes in staining for eNOS in any of these groups (Fig. 2B).
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Discussion |
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TAC represents the capacity of plasma to resist oxidation. One of the major oxidative pathways involves the generation of superoxide and its subsequent reaction with NO to form peroxynitrite [23]. Peroxynitrite can oxidize different molecules, including lipids and lipoproteins [24], and its persistent generation allows the formation of nitrotyrosine moieties on proteins, which may represent a marker of ongoing nitrosative stress [25].
TAC is a good indicator of oxidative potential at any given time. By the peroxynitrite quenching method, all mice groups injected with hybridomas secreting aCL (human or murine) or aß2-GPI (murine) antibodies showed reduced TAC, suggesting a link between aCL and peroxynitrite formation. Since PON prevents peroxynitrite-mediated lipid peroxidation [26], a reduction of PON activity, as seen in the IgG aCL and aß2-GPI groups, contributes to increased oxidative stress. The superoxide quenching method provides further evidence for a pro-oxidant tendency, in that higher values of TAC were related to increased level of plasma superoxide in the IS4 antibody group.
Mice and humans with SLE generate significant amounts of NO [27, 28], this has never been related to aCL antibodies, whereas in primary APS low NO inversely correlated to IgG aCL [14]. Here we confirm the previous observation, since plasma NO level was lower in the group injected with IS4-producing hybridoma cells.
IgG aCL (IS4, in particular) can activate endothelial cells [29], hence one might have anticipated increased NO production, due to increased expression of iNOS. Instead, western blotting and immunohistochemistry revealed reduced iNOS expression, with no significant differences in the expression of eNOS, consistent with reduced generation of NO.
The high levels of nitrotyrosine found in the IS4 group may explain this discrepancy. Because nitrotyrosine indicates nitrosative stress in vivo [25, 30], its elevation may indicate previous elevated endothelial NO production induced by aCL. In an oxidative environment NO is shifted towards peroxynitrite formation, the latter inducing down-regulation of iNOS. If NO was not generated at all, there would not be any measurable peroxynitrite. On the other hand, down-regulation of iNOS expression may follow a period of increased activity, as reported, but not in autoimmune diseases [31]. The expression of iNOS and eNOS is controlled by different feedback mechanisms, the most important of which relates to NO itself [32]. Increased local levels of NO down-regulate iNOS by inhibiting the transcriptional factor NF-ß in macrophages and endothelial cells [33], causing lowered NO production [34, 35]. A by-product of lipid peroxidation, 4-hydroxynonenal, inhibits NF-
ß activation and consequent iNOS expression [36], whereas IgG aCL induces NF-
ß in endothelial cells [37]. An alternative explanation would be that in the face of iNOS down-regulation eNOS still provides a source of NO to scavenge superoxide.
In conclusion, we replicated in a mouse model our findings from APS patients [16]. Whereas in humans it is difficult to state when these processes begin, in the mouse model they appear as early as 30 days. For this very reason we could not detect any (micro)thrombosis or vascular damage. A diet enriched in cholesterol would have been necessary to favour the appearance of atherosclerotic changes in this short span of time, but increased oxidation induced by hypercholesterolaemia may have confounded our results and a second hit would have been required to induce thrombosis. Nevertheless, IgG aPL may induce in mice a sequence of decreased PON activity, enhanced peroxynitrite formation and inhibition of iNOS expression. This is the first report to show intimate relationships between oxidative/nitrosative pathways and IgG aPL; these could be explored in more suitable models to test their association with vascular manifestations of APS.
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The authors have declared no conflicts of interest.
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References |
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