Specificity of anti-phospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies

Abstract

The antigen specificity of anti-phospholipid antibodies in infectious mononucleosis (IM) was studied using ELISA for the detection of anti-β₂-glycoprotein I (β₂-GPI), anti-annexin V, anti-protein S and anti-prothrombin antibodies and TLC immunostaining for the detection of anti-phospholipid antibodies. This technique enabled us to look at antibodies reacting to ‘pure’ phospholipid antigens in the absence of protein contamination. Sera from 46 patients with IM, 18 with systemic lupus erythematosus (SLE), 21 with primary anti-phospholipid antibody syndrome (PAPS), 50 with Helicobacter pylori infection and 30 healthy blood donors were tested. This study highlights anti-phospholipid antibodies in patients with IM as specific ‘pure’ anti-cardiolipin antibodies, while in PAPS and SLE patients anti-phosphatidylserine and anti-phosphatidylethanolamine antibodies were also found. This investigation also shows that the anti-cardiolipin antibodies found in IM can be present with anti-cofactor protein antibodies. The higher prevalence of anti-cofactor antibodies found in IM sera than in Helicobacter pylori sera may be due to the immunostimulatory effect and/or the polyclonal activation often observed in course of Epstein–Barr virus infection. However, anti-β₂-GPI and, to a lesser extent, anti-prothrombin antibodies occur with a significantly lower prevalence in IM than in PAPS patients. This finding suggests that these antibodies should be regarded as the expression of the broad autoimmune syndrome involving the phospholipid-binding plasma proteins.

INTRODUCTION

Anti-cardiolipin antibodies (aCl) are a subset of anti-phospholipid antibodies (aPl) frequently detected in sera of patients with systemic lupus erythematosus (SLE) or related autoimmune disorders. These autoantibodies are considered responsible for the so-called anti-phospholipid antibody syndrome (APS), which is characterized by arterial and/or venous thromboses and multiple abortions [1,2]. However, they were also reported in many infectious diseases and in apparently healthy individuals [3]. aPl were firstly detected by Wasserman [4] in patients with syphilis and subsequently Pangborn showed that cardiolipin (Cl) was the antigen reacting to these antibodies [5]. However, aCl were also found in many non-syphilitic infections. Sera from subjects with viral infections [6], such as Epstein–Barr virus (EBV), hepatitis A virus, rubella virus and parvovirus infections showed a high frequency of IgM and IgG aCl [7]. More recently, aCl have been detected in a large variety of infectious diseases, including AIDS [8–10], cytomegalovirus infection [11] and hepatitis C [12]. The occurrence of these antibodies in leptospirosis [13], Q fever [14] and bacterial infections [15] has also been reported. However, the clinical features of the APS, such as thrombosis and recurrent pregnancy loss, do not occur in patients with infectious diseases. It is now well recognized that the binding specificity of the antibodies found in APS is different from that reported in infectious conditions. The main difference seems to be the absolute requirement of β₂-glycoprotein I (β₂-GPI) to detect aPl in the former but not in the latter [16–18]. Furthermore, it has been reported that aCl from APS recognize β₂-GPI structure altered by the interaction with an oxygen-modified solid-phase surface [19]. Thus, aPl are a heterogeneous group of antibodies [20,21] detected by different tests, i.e. LAC and aCl ELISA. Moreover, it has been suggested that the presence of plasma proteins such as β₂-GPI, and possibly annexin V, protein S or prothrombin can be essential for the binding of aPl to phospholipids and that these proteins are probably the main target of the antibodies [22].

This study was undertaken in order: (i) to assess the specificity of aPl in a well-defined infectious disease in which these antibodies are virtually present, and (ii) to compare the autoantibody profile with that found in ‘autoimmune patients’ as well as in healthy individuals. Infectious mononucleosis (IM) is an acute infection caused by EBV. In these patients the generation of aCl [23] could be triggered by epitopes on EBV-transformed lymphocytes [24]. In order to clarify the target of these antibodies, we have recently developed a highly selective method for the detection of anti-phospholipid reactivity [25]. This technique is suitable for lipid identification and separation. It relies upon the different lipid partition and permits the analysis of antibodies against ‘pure’ phospholipid antigens, in the absence of protein contamination [25]. An ELISA with delipidated protein antigens was used for the detection of anti-β₂-GPI, anti-annexin V, anti-protein S and anti-prothrombin and TLC immunostaining for the detection of aCl. This study highlights aPl in patients with IM as specific ‘pure’ aCl and shows that they are present with anti-cofactor protein antibodies. We observe that anti-β₂GPI and anti-prothrombin antibodies have a significantly lower prevalence in IM than in primary APS (PAPS) patients.

PATIENTS AND METHODS

Patients and sera

Sera were collected from:

• 46 patients with IM (24 female, 22 male; age range 14–42 years), 20 of whom showed acute IM (anti-VCA IgM⁺ IgG⁺ and anti-EA IgG⁺), eight were anti-VCA IgM⁻ IgG⁺, anti-EA IgG⁺, 18 with recent (6–9 months) IM (anti-VCA IgM⁻ IgG⁺ and anti-EA IgG⁻);

• 18 SLE patients (16 female, two male; age range 12–66 years) diagnosed according to ARA revised criteria [26]. None of these patients showed any sign of secondary APS (SLE_(APS−));

• 21 patients with PAPS (17 female, four male; age range 18–48 years). Twenty of these patients showed arterial and/or venous thrombosis, two recurrent miscarriages. The diagnosis of PAPS was made following the new preliminary criteria for the classification of APS [2];

• 50 patients with Helicobacter pylori infection. Exclusion criteria were: age under 18 years, pregnancy or lactation, alcoholism, severe concomitant diseases, penicillin allergy, disorders of clotting, oesophageal reflux disease, gastric ulcer or resection of stomach for gastric neoplasm, antibiotics or bismuth salts therapy during the month prior to inclusion, as well as proton pump inhibitor therapy during the last week. In all cases the identification of H. pylori infection was based on the histological examination of endoscopic biopsy specimens and confirmed by CLO test (Delta West Pty Ltd, W. Australia);

• 30 healthy blood donors.

Detection of aCl by immunostaining on TLC plates

Immunostaining was performed as previously described [25]. Briefly, this assay was performed using 2 μg of five different phospholipid antigens: CL, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine and phosphatidylinositol (PI), purchased by Sigma Chemical Co. (St Louis, MO). Phospholipids were separated by thin-layer chromatography, using aluminium-backed silica gel 60 (20 × 20) high performance thin layer chromatography (HPTLC) plates (Merck, Darmstadt, Germany). Chromatography was performed in chloroform:methanol:CH₃COOH:water (100:75:7:4) (v/v/v/v). The dried chromatograms were soaked for 90 s in a 0·5% (w/v) solution of poly(isobutyl methacrylate) beads (Polysciences, Warrington, PA) dissolved in hexane. After air drying, the chromatograms were incubated for 1 h at 25°C in 0·5% (w/v) gelatin/PBS. The blocking solution was removed and replaced by a washing buffer (PBS). The chromatograms were then incubated for 1 h at 25°C with sera, diluted 1:100 in 0·5% (w/v) gelatin/PBS. Sera were removed and chromatograms were washed three times for 10 min with PBS. Horseradish peroxidase-conjugated goat anti-human IgG (Sigma), diluted 1:500 in 0·5% (w/v) gelatin/PBS, was added and incubated at 25°C for 1 h. The colour reaction was obtained by adding 200 mg of sodium nitroprusside (Sigma) and 80 mg of o-dianisidine (Sigma) dissolved in 100 ml of H₂O, containing 35 μl of 30% H₂O₂.

Detection of anti-cofactor protein antibodies by ELISA

Anti-cofactor protein antibodies were detected by a slight modification of an ELISA method previously reported [27–30]. Irradiated polystyrene plates (Nunc Intermed, Roskilde, Denmark) were coated (100 μl/well) with human β₂-GPI 10 µ g/ml [27] (Calbiochem, La Jolla, CA), delipidated by butanol-diethyl ether extraction [31], annexin V 5 μg/ml [28] (Sigma), protein S 5 μg/ml [29] (American Diagnostica Inc., Greenwich, PT) or prothrombin 5 μg/ml [30] (Calbiochem) in PBS pH 7·4. The plates were blocked by incubation for 2 h at 25°C with a solution of PBS−0·1% Tween 20 (PBS–T) containing 1% albumin (3% albumin for anti-annexin V detection). Serum (100 μl; 1:100 diluted in PBS–T−1% albumin) was added and incubated for 1 h at 25°C. After washing three times with PBS–T, alkaline phosphatase-conjugated affinity-purified goat anti-human IgG (Fc fragment-specific) was added (1:1000 diluted in PBS–T−1% albumin) and incubated for 1 h at 25°C. After washing three times, 100 μl of p-nytrophenylphosphate solution were added and the optical density (OD) was read at 405 nm wavelength when a positive control reached 1·00 OD value. The absorbance of control wells was subtracted to account for non-specific binding. Thirty normal human sera were also tested and a cut-off value was established at a mean of OD ± 3 s.d. of normal human sera for anti-β₂-GPI, anti-protein S, anti-prothrombin and at a mean of OD ± 5 s.d. for anti-annexin V.

RESULTS

Detection of aCl by immunostaining on TLC plates

The TLC-immunostaining technique demonstrated aCl reactivity in 14 out of 46 (30·4%) patients with IM. In particular, aCl antibodies were detected in nine out of 28 patients with acute IM and in five out of 18 patients with recent IM infection (6–9 months). All positive sera reacted specifically with Cl, since no reactivity with phosphatidylserine, PE, PC or PI was found (Fig. 1, Table 1). In order to rule out the possibility that β₂-GPI present in serum samples could influence antibody reactivity, sera reactivities were confirmed at a dilution of 1:1000. To such a dilution, β₂-GPI was provided at a concentration of about 0·2 μg/ml, which is below the minimum requirement (> 0·5 μg/ml) for aCl binding [32].

Fig. 1.

Anti-phospholipid antibody (aPl) pattern in ‘infectious’ and ‘autoimmune’ sera. TLC immunostaining was performed with 2 μg of each phospholipid separated using high performance thin layer chromatography (HPTLC) aluminium-backed silica gel 60 (20 × 20) plates. Sera were diluted 1:100 in 0·5% gelatin/PBS. Horseradish peroxidase-conjugated anti-human IgG detector antibodies were diluted 1:500 in 0·5% gelatin/PBS. The colour reaction was developed by adding sodium nitroprusside. Lane A, reactivity of the serum from a patient with primary anti-phospholipid antibody syndrome (PAPS); lane B, reactivity of the serum from a patient with systemic lupus erythematosus (SLE); lane C, reactivity of the serum from a patient with acute mononucleosis infection; lane D, reactivity of the serum from a patient with Helicobacter pylori infection; lane E, reactivity of the serum from a healthy donor.

Table 1.

Occurrence of anti-phospholipid antibodies (aPl) in ‘infectious’ and ‘autoimmune’ sera

Patients	n	aCl	aPC	aPE	aPSer	aPI
IM	46	14 (30·4%)	0	0	0	0
SLE	18	12 (66·6%)	0	2 (11·1%)	2 (11·1%)	0
PAPS	21	11 (52·3%)	0	5 (23·8%)	6 (28·5%)	0
Helicobacter pylori infection	50	4 (8%)	0	0	0	0
Healthy blood donors	30	0	0	0	0	0

aCl, Anti-cardiolipin antibodies; aPC, anti-phosphatidylcholine antibodies; aPE, anti-phosphatidylethanolamine antibodies; aPI, anti-phosphatidylinositol antibodies; IM, infectious mononucleosis; SLE, systemic lupus erythematosus; PAPS, primary anti-phospholipid antibody syndrome.

aCl were detected in 12 out of 18 (66·6%) SLE_(APS−) sera; two of these sera reacted with phosphatidylserine and PE and none with PC or PI. In the PAPS group the prevalence of aCl antibodies was 52·3% (11 out of 21 sera); six of these sera reacted with phosphatidylserine, five with PE and none with PC or PI (Fig. 1, Table 1). Among the patients with H. pylori infection only four sera reacted with Cl and none with the other phospholipids under test. None of the sera from 30 healthy blood donors showed any presence of aPl.

Detection of anti-cofactor protein antibodies

None of the patients with IM had detectable levels of anti-β₂-GPI antibodies. However, anti-β₂-GPI antibodies were detected in seven sera from the PAPS group (33·3%) and in SLE_(APS−) patients (16·6%) (Fig. 2). None of the sera from patients with H. pylori infection or from the healthy blood donors showed the presence of anti-β₂-GPI antibodies.

Fig. 2.

Anti-cofactor protein antibody pattern in ‘infectious’ and ‘autoimmune’ sera. The occurrence of anti-cofactor protein antibodies in infectious mononucleosis (IM), systemic lupus erythematosus (SLE), primary anti-phospholipid antibody syndrome (PAPS), Helicobacter pylori infection patients and healthy blood donors was detected by ELISA.

The prevalence of anti-annexin V antibodies in patients with IM was 26·0%, as against 61·9% found in PAPS patients and 22·2% in SLE_(APS−) patients. Anti-annexin V antibodies were also found in one patient with H. pylori infection (2%) and in one healthy blood donor (3·3%).

Anti-protein S antibodies were detected in 10 out of 46 patients with IM (21·7%), in five out of 21 (23·8%) sera from patients with PAPS and in four out of 18 (22·2%) SLE_(APS−) sera. They were also found in two patients with H. pylori infection (4%) and in no healthy blood donor.

Three out of 46 sera from patients with IM (6·5%) showed reactivity against prothrombin. Anti-prothrombin antibodies were detected by ELISA in seven out of 21 PAPS sera (33·3%), in two out of 50 (4%) sera from patients with H. pylori infection and in no sera from SLE_(APS−) patients or healthy blood donors.

Relationship between anti-cofactor protein antibodies and aCl

Autoantibodies directed against cofactor protein antigens were mostly present in association with detectable levels of aCl. Indeed, seven patients showed aCl, anti-annexin V and anti-protein S antibodies, three aCl and anti-annexin V, one aCl, anti-annexin V and anti-prothrombin and one aCl and anti-protein S. Among the other patients, only two were positive for Cl and three for anti-cofactor protein antibodies (anti-annexin V, anti-prothrombin, anti-prothrombin + anti-protein S).

DISCUSSION

This study, performed with a highly specific technique, demonstrates the presence of ‘pure’ aCl in IM patients. These antibodies are relatively often present with anti-cofactor protein antibodies not including anti-β₂-GPI.

‘Anti-phospholipid antibodies’ are a heterogeneous group of antibodies, and it has been suggested that the presence of plasma proteins such as β₂-GPI, annexin V, protein S or prothrombin may be essential for the binding of ‘autoimmune’ aPl to phospholipids [20–22]. The possibility that cofactor proteins are only involved in the assay but have no pathogenic role ‘in vivo’ cannot be excluded.

TLC immunostaining allows the detection of antibodies to ‘pure’ phospholipid antigens, in the absence of protein contamination [25]. Thanks to this method, we ascertained that ‘true’ aPl can be found in 30% of patients with IM, but they are also highly prevalent in ‘autoimmune sera’. However, in patients with IM aPl are specifically directed to Cl, while in both PAPS and SLE_(APS−) patients anti-phosphatidylserine and anti-PE antibodies were also detected. These findings are in agreement with previous studies which reported the different specificity of aPl in ‘autoimmune’ as against infectious diseases [33,34], and are reinforced by our TLC immunostaining analysis [25]. This demonstrated that aPl in patients with syphilis are specifically directed to Cl.

In our study we detected specific anti-β2-GPI antibodies in patients with PAPS or APS, but not in patients with IM. This finding is in agreement with previous reports [16,18], which provided evidence that syphilis-associated aCl differ from ‘autoimmune’ aCl. In fact, in syphilis these autoantibodies were found to bind Cl in solid phase as well in fluid phase, without requirement of β₂-GPI. In addition, Forastiero and colleagues demonstrated that IgG aCl from APS patients recognized β₂-GPI on irradiated plates in the absence of phospholipids, while IgG purified from syphilis patients bound to Cl alone [17].

Interestingly, we detected anti-annexin V antibodies in patients with IM. As far as we know, this is the first evidence for the presence of anti-annexin V antibodies in patients with infectious diseases. However, these antibodies seem to occur at a lower frequency in IM than in PAPS. Annexin V has a high calcium-dependent binding affinity for negatively charged phospholipids and blood platelets and shows anticoagulant effects in vitro. Anti-annexin V antibodies, primarily detected in sera from patients with rheumatoid arthritis, have been shown to display LAC activity [35], to be responsible for apoptosis of umbilical vein endothelial cells [36] and to be associated with thrombotic events in the course of SLE [29]. In this study, we provide evidence that these antibodies are also present in IM, although with a lower prevalence than in PAPS.

Moreover, we found the presence of anti-protein S antibodies in IM as well as in SLE and PAPS patients' sera. These antibodies seem to target protein S specifically, since they bind to this protein in the absence of phospholipids [37,38]. As protein S is a vitamin K-dependent plasma glycoprotein which acts as cofactor for activated protein C, its inhibition by autoantibodies may affect the fundamental anticoagulant activity of this protein. Alternatively, these antibodies may modify the anti-prothrombinase activity and thereby compete with prothrombin for binding to factor Va [39]. However, the occurrence of these antibodies in IM patients did not significantly differ from that found in PAPS or SLE patients.

We found that the occurrence of anti-prothrombin antibodies was significantly lower in IM patients than in PAPS patients. Conflicting data have emerged from the literature regarding the association of anti-prothrombin antibodies with the clinical features of APS [28,40,41]. Most studies have shown that the presence of antibodies against prothrombin is associated with thrombosis in patients with SLE and APS [40,41]. Our study indicates that anti-prothrombin antibodies occur in IM, although these antibodies correlate with the clinical features of APS only in ‘autoimmune’ patients.

Both aCl and anti-cofactor protein antibodies seem to persist in the serum for a few months, since they were still present in several patients with previous infection (6–9 months). However, according to our follow-up study, no antibodies were found in most of these patients within 12–15 months (unpublished observation).

Taken as a whole, these findings support the view that aPl play a pathogenic role depending on the accessibility of their protein targets. Indeed, antibodies found in association with infections seem to be mainly directed against the phospholipid molecules themselves. The mechanism by which antibodies directed to phospholipid molecules increase during infections remains to be ascertained. It has been hypothesized that this phenomenon is linked to the adjuvant action displayed by membrane constituents of EBV-transformed cells. The apparent restriction of the specificity of autoantibodies can be explained by two mechanisms: (i) the formation of a complex between viruses and host antigens could induce an immune response once the complex has been exposed after lysis or cell damage; (ii) the autoantibodies are induced by polyclonal activation and their specificities are preferentially directed by the immune repertoire. In this respect, the higher prevalence of autoantibodies against Pl-binding proteins found in IM rather than in H. pylori sera may be due to the immunostimulatory effect and the polyclonal activation often observed in the course of EBV infection.

The majority of autoimmune-type aPl are dependent on the presence of β₂-GPI [42,43] and, possibly, other plasma proteins, such as prothrombin and annexin V [21,22]. Progress in this field will probably depend on the accurate classification of aPl on the basis of their specific targets. The use of techniques to detect antibodies against different phospholipid-binding plasma proteins offers a certain promise in this respect. This study demonstrates that anti-prothrombin antibodies can be found in patients with EBV, although at a significantly lower frequency than in PAPS patients. However, ‘pure’ aCl, anti-annexin V and protein S antibodies were not found to occur at a significantly different frequency in the ‘autoimmune’ patients as against the ‘infectious’ ones. This observation reinforces the concept that anti-β₂-GPI and anti-prothrombin antibodies represent specific independent factors associated with the clinical manifestations of the APS. These antibodies should be regarded as the expression of the broad autoimmune syndrome involving the phospholipid-binding plasma proteins [44].