Specificity of antinuclear and antiphospholipid antibodies in sera of patients with autoimmune lymphoproliferative disease (ALD).

OBJECTIVE: A human lymphoproliferative syndrome characterized by a defect of the Fas-mediated apoptosis pathway in the absence of a fas gene mutation (Autoimmune Lymphoproliferative Disease) has recently been described and characterized by autoimmune phenomena. The aim of this study was to investigate the presence of antinuclear and antiphospholipid antibodies and to define their specificity in 5 pediatric patients with this syndrome. METHODS: Antinuclear antibodies were investigated by Western Blot and IIF performed under standard as well as apoptotic conditions. The fine specificity of antiphospholipid antibodies was dissected by an ELISA for anti-beta 2-glycoprotein I, anti-prothrombin, anti-annexin V and anti-protein S antibodies, and by immunostaining on thin layer chromatography plates for antiphospholipid molecule antibodies. RESULTS: This study showed that the autoantibodies found in these patients targeted a broad spectrum of nuclear antigens which undergo redistribution from the nucleus to the cytoplasm and plasma membrane during the course of the apoptotic process. This reactivity does not comprise known specificities such as anti-extractable nuclear antigens or anti-dsDNA. Antiphospholipid antibodies were also found in these sera. A further characterization of the antiphospholipid antibodies showed the presence of a heterogeneous response with antibodies directed to negatively-charged phospholipids and antibodies targeting coagulation-related proteins (beta 2-GPI, prothrombin, annexin V) which are considered relevant antigens in the antiphospholipid syndrome. CONCLUSIONS: These results suggest that lack of tolerance due to a defect of Fas-mediated apoptosis allows the survival of B and T clones involved in the antinuclear and antiphospholipid immune responses.

Beta-2-glycoprotein I expression on monocytes is increased in anti-phospholipid antibody syndrome and correlates with tissue factor expression.

It is well known that monocytes may play an active role in thrombogenesis, since they may express on their surface tissue factor, the major initiator of the clotting cascade. The results of this investigation demonstrate beta-2-glycoprotein I (beta2-GPI) mRNA expression by human peripheral blood monocytes, indicating that these cells synthesize beta2-GPI. In addition, we show beta2-GPI expression on cell surface of these cells by flow cytometric analysis, and the presence of this protein in cell lysate by Western blot. Interestingly, beta2-GPI expression on monocytes is significantly increased in patients with anti-phospholipid syndrome (APS) or systemic lupus erythematosus (SLE) as against healthy blood donors and correlates with tissue factor expression on monocytes. These findings support the view that monocytes are able to synthesize beta2-GPI and suggest that patients with APS may have increased beta2-GPI exposure on cell surface, which leads to persistently high monocyte tissue factor expression and consequently to a prothrombotic diathesis.

Polymorphism of the Duffy erythrocyte chemokine receptor in Italian patients with Behçet's disease.

This study examined the hypothesis that the polymorphism of Duffy antigen receptor for chemokines (DARC) predisposes to and/or influences the clinical manifestations of Behçet's disease. The serum levels of IL-8 and monocyte chemotactic peptide (MCP)-1, two DARC-binding chemokines, were investigated and related to this polymorphism. Twenty-eight patients with Behçet's disease and 30 healthy blood donors were included in the study. No null phenotypes were found among the patients studied, and the frequencies of the other phenotypes (Fy((a+b-)), Fy((a+b+)), and Fy((a-b+))) did not significantly differ from those found in the blood donor group or reported in the general Caucasian population. No difference was found between the single phenotypes in terms of IL-8 and MCP-1 serum levels, and no relevant association between the clinical characteristics, Behçet's disease-associated human leukocyte antigen (HLA)-B51, and single phenotypes was observed. This investigation indicates that DARC is not a genetic trait significantly associated with or predisposing to Behçet's disease, at least in Caucasian Italians. However, the role of this polymorphism in the development and in the clinical course of the disease awaits further investigation.

Anti-tumour necrosis factor (TNF) alpha treatment of rheumatoid arthritis (infliximab) selectively down regulates the production of interleukin (IL) 18 but not of IL12 and IL13.

OBJECTIVE: To measure interleukin (IL)18 serum concentrations in patients with rheumatoid arthritis (RA) undergoing infliximab treatment (tumour necrosis factor (TNF) alpha blockade) and to evaluate the concomitant modification of IL12 and IL13 serum concentrations, two cytokines belonging to the Th1 and Th2 profile respectively and biologically related to IL18. METHODS: Ten patients with RA not responding to disease modifying antirheumatic drugs (DMARDs) received intravenous infliximab at a dose of 3 mg/kg at baseline and after two and six weeks. Serum samples were collected from all patients before each infusion and assayed for IL18, IL12, and IL13 by enzyme linked immunosorbent assay (ELISA); IL18 was also measured eight weeks after the last infusion. RESULTS: Serum concentrations of IL18 in all patients were already markedly reduced from baseline after two weeks (p<0.005). Serum IL18 was also decreased in a stable manner after six (p<0.01) and 14 weeks (p<0.01) compared with baseline concentrations. No significant modifications were found in serum concentrations of IL12 and IL13 at any time point. CONCLUSION: There was a rapid and persistent decrease in serum concentrations of IL18 in all the patients studied. This result provides evidence of an in vivo regulation of IL18 by TNFalpha and suggests that anti-TNFalpha therapy is likely to interrupt the synergistic effect between these two cytokines.

Evidence for anticoagulant activity and beta2-GPI accumulation in late endosomes of endothelial cells induced by anti-LBPA antibodies.

This investigation was undertaken to test whether anti-LBPA antibodies and IgG from patients with APS interfere with intracellular beta2GPI distribution in EAhy926 endothelial cells and with the coagulation system. Cell incubation with anti-LBPA MoAb or with patients' IgG resulted in antibody binding to late endosomes and caused beta2GPI redistribution and accumulation within perinuclear vesicular structures reminiscent of late endosomes. This finding suggests that aPI may contribute to the pathogenic mechanisms of APS by modifying the intracellular traffic of proteins, by interactions between aPl and LBPA, beta2GPI and/or LBPA-beta2GPI complexes. The anticoagulant activity of anti-LBPA MoAb was analyzed in a sensitized activated partial thromboplastin time (aPTT) system and in a dilute Russell's viper venom time (dRVVT). A significant, concentration-dependent effect of the antibody on both aPTT and dRVVT prolongation was found. These observations suggest that LBPA is an important lipid target for aPl with potential functional implications for the immunopathogenesis of APS.

Cardiolipin on the surface of apoptotic cells as a possible trigger for antiphospholipids antibodies.

This study provides evidence that cardiolipin (CL) molecules are expressed on the surface of apoptotic cells and are recognized by antiphospholipid antibodies, purified from patients with the antiphospholipid antibody syndrome (APS). CL expression on cell surface was demonstrated by high performance thin layer chromatography analysis of phospholipids from plasma membrane purified fractions and by the positive staining with the CL-specific dye nonyl-acridine orange. This finding was complemented with the observation that aCL IgG purified from patients with APS bind to the surface of apoptotic cells. This staining shows a clustered distribution mostly localized on surface blebs. Interestingly, CL exposure on the cell surface preceded the DNA fragmentation, as shown by cytofluorimetric analysis. These findings demonstrate that exposure of CL molecules on the cell plasma membrane is an early event of the apoptotic cellular program that may represent an in vivo trigger for the generation of aCL.

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

The antigen specificity of anti-phospholipid antibodies in infectious mononucleosis (IM) was studied using ELISA for the detection of anti-beta2-glycoprotein I (beta2-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-beta2-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.

Human monoclonal anti-phospholipid antibodies selectively bind to membrane phospholipid and beta2-glycoprotein I (beta2-GPI) on apoptotic cells.

The ability of an anti-phospholipid (LJ1) and an anti-beta2-GPI (RSP-57) human MoAb to bind to apoptotic but not viable cells was demonstrated in this study. Both MoAbs were derived from patients with systemic lupus erythematosus and anti-phospholipid antibody syndrome. The parallel analysis of the specificity and affinity of four anti-phospholipid human MoAbs suggests that the binding of LJ1 MoAb to apoptotic cells is a specific property of this MoAb. RSP-57 MoAb recognizes apoptotic cells through beta2-GPI which becomes available for binding after the interaction with negatively charged phospholipids. This observation provides evidence that the binding of human anti-phospholipid antibodies to apoptotic cells occurs in both a beta2-GPI-dependent and independent way and involves a restricted group of epitopes. The finding that LJ1 and RSP-57 MoAbs bind apoptotic cells underlines the property of these MoAbs to act as cell membrane markers of apoptosis. Major pathological implications derive from the observation that LJ1 and RSP-57 MoAbs recognize epitopes expressed on 'early' apoptotic cells. The interference with the in vivo clearance and processing of apoptotic cells is a potential pathogenic mechanism of these antibodies.

Treatment of thrombosis associated with immunological risk factors.

The clinical management of the antiphospholipid syndrome is aimed at assessing the thrombotic risk of an individual in order to undertake primary prevention for the asymptomatic subject positive for antiphospholipid antibodies or prophylaxis of major ischaemic events for the patient who has already experienced thrombosis. Lack of immunological parameters with a clear predictive value and the current concept that thrombosis is a multifactorial disease suggest that all the known acquired and genetic thrombotic risk factors should be taken into account in antiphospholipid syndrome. Low-dose aspirin and hydroxychloroquine have been proven useful in primary prevention. While convincing evidence has been provided that aspirin and hydroxychloroquine do not prevent secondary thrombosis, much debate has recently developed on the level of oral anticoagulation needed to guarantee this prevention. Main concerns are also related to duration of anticoagulation therapy, risk of bleeding, and the increased risk of thrombosis as a result of withdrawal of the anticoagulant. Low-molecular-weight heparin has recently emerged as a valid and safe alternative for those conditions that require transient interruption or withdrawal of anticoagulation. Although treatment of the catastrophic antiphospholipid syndrome is largely empirical, the therapeutic approach based on plasmapheresis associated with immunosupression or intravenous immunoglobulin seems to be the most promising. Most attention has recently been paid to the role of oxidative stress in the pathogenesis of antiphospholipid syndrome, as a correlation between lipid peroxidation and antiphospholipid antibodies has been demonstrated. Our studies showed that lipid peroxidation may contribute to the activation of the clotting system observed in antiphospholipid syndrome and that markers of both procoagulant state and increased lipid peroxidation can be modified by experimental antioxidant treatment.

Apoptosis in human autoimmune diseases.

The description of apoptosis or programmed cell death nearly thirty years ago did not initially stimulate a great deal of interest. However, the ways cells die is clearly an essential part of biological homeostasis and well worth of study in its own right as the enormous literature on the subject in the past 15 years confirms. In the past decade new avenues of apoptosis research have opened up as the relationship between this form of cell death and autoimmune disease has come under increasing scrutiny. Although most research to date has been in animal study models, there are a variety of studies which have begun to explore links between apoptosis and a wider range of human autoimmune conditions. In this review we analyse briefly the background to what is known about apoptosis and focus on the increasing likelihood that abnormalities in apoptosis are contributory factors in the development of human autoimmunity.

Decreased immunoreactive beta-endorphin in mononuclear leucocytes from patients with chronic fatigue syndrome.

OBJECTIVE: To investigate beta-endorphin concentrations in the peripheral blood mononuclear cells (PBMC) of patients with chronic fatigue syndrome (CFS). METHODS: Sixteen patients with CFS were enrolled in this study. Ten healthy subjects were studied as controls. Beta-endorphin concentrations were measured in PBMC by radioimmunoassay performed with antibodies specific for the C-terminal portion of human beta-endorphin. RESULTS: Beta-endorphin concentrations in the PBMC of chronic fatigue patients were significantly lower (p < 0.001) than in healthy subjects (mean +/- SD: 8.5 +/- 7.0 vs. 42.6 +/- 22.6). CONCLUSION: Patients with CFS were found to have low levels of PBMC beta-endorphin. This finding may reflect the condition of chronic immune activation in CFS that has been reported in previous investigations. Beta-endorphin concentrations in PBMC seem to mirror the central nervous system homeostasis of the opioid. Therefore, we would postulate that the fatigue and weakness typical of CFS could be related to low beta-endorphin concentrations at the central nervous system level.

Anti-beta 2-glycoprotein I antibodies bind to central nervous system.

Anti-beta 2-GPI antibodies (a beta 2-GPI) were found in serum from patients with anti-phospholipid syndrome (APS) and/or systemic lupus erythematosus (SLE). Since a beta 2-GPI are often found in patients with anti-cardiolipin antibodies (aCL), their role in thrombosis as well as other central nervous system (CNS) manifestations in APS is unclear. We, therefore, investigated whether affinity-purified a beta 2-GPI bind the CNS. Astrocyte and neuron cell lines and histological sections were used as CNS substrates. Indirect immunofluorescence and/or streptavidin-biotin-peroxidase techniques revealed that astrocytes, neurons and vascular endothelium were bound by purified a beta 2-GPI (mouse monoclonal, rabbit polyclonal, human serum Ig a beta 2-GPI). This suggests a potential role for a beta 2-GPI in the CNS damage, as a beta 2-GPI might contribute to CNS pathology by either a direct interaction with astrocytes and neurons or an interaction with cerebral vascular endothelial cells. CNS immunoreaction was also demonstrated using six a beta 2-GPI-positive sera from patients (four with neurological manifestations). No binding to CNS was seen using a beta 2-GPI-negative sera, i.e. five from SLE patients (two with CNS involvement) and six healthy donors, or a monoclonal aCL without a beta 2-GPI immunoreactivity. Thus, the CNS reactivity by the a beta 2-GPI-positive sera appears specifically due to a beta 2-GPI and independent from aCL. Because of the presence of aCL in all patient sera, and the CNS involvement in three control patients, it is not possible to attribute a direct role for a beta 2-GPI in neurological diseases in this study.

Apoptosis and antiphospholipid antibodies.

OBJECTIVE: To analyze the potential links between antiphospholipid antibodies (aPL) and apoptosis in the pathogenesis of the antiphospholipid antibody syndrome (APS). METHODS: A review was undertaken of the most relevant scientific literature on apoptosis and autoimmune phenomena. Experimental and human pathology were reviewed to substantiate the hypothesis that apoptosis is involved in the generation of aPL. RESULTS: Several considerations suggest that exposure of phospholipids (PL) during apoptosis may be a driving antigenic stimulus to the production of aPL. Furthermore, the molecular PL-protein complexes formed during apoptosis are targeted by "pathogenic" aPL. The binding and the clearance of apoptotic cells by these autoantibodies likely further enhances the aPL immune response. Experimental models and human pathology suggest that a restricted genetic background is key to the development of this immune response. CONCLUSIONS: Abnormalities of apoptosis observed in the course of autoimmune conditions likely provide an antigenic stimulus to the production of aPL.

Enhanced lipid peroxidation in patients positive for antiphospholipid antibodies.

The mechanism leading to the formation of antiphospholipid antibodies (aPL) is still unknown. Because an in vitro study suggested that aPL may derive from pro-oxidant conditions, we sought a relationship between aPL and isoprostanes, indices of lipid peroxidation in vivo. Thirty patients with systemic lupus erythematosus have been studied. Seventeen (56.6%) were positive for aPL because they had lupus anticoagulant and/or high titer of anticardiolipin antibodies (aCL). Plasma levels of tumor necrosis factor (TNF ) and urinary excretion of two isoprostanes, 8-epi-PGF2alpha and IPF2alpha -I, free radical catalyzed oxidation products of arachidonic acid, were measured. Patients with systemic lupus erythematosus had higher urinary excretion of 8-epi-PGF2alpha and IPF2alpha -I than controls; urinary excretion of the two isoprostanes was highly correlated (Rho = 0.74, P < .0001). Urinary 8-epi-PGF2alpha was highly correlated with both aCL titer (Rho = 0. 70, P < .0001) and TNF (Rho = 0.84, P < .0001), a measure of disease severity. Excretion of this isoprostane was also higher in those patients who exhibited aPL (P < .0001). Comparable correlations were observed with the isoprostane IPF2alpha -I. No difference of 8-epi-PGF2alpha was observed between patients with and without previous history of thrombosis. This study, showing the existence of a close association between aPL and increased in vivo lipid peroxidation, supports the hypothesis that these antibodies may result from pro-oxidative conditions and suggests that inflammation may play an important role.

Coexistence of anti-phospholipid antibodies and endothelial perturbation in systemic lupus erythematosus patients with ongoing prothrombotic state.

BACKGROUND: Anti-phospholipid antibodies (aPLs) were associated with an ongoing prothrombotic state in patients with systemic lupus erythematosus (SLE). Because aPLs are able to shift endothelial function toward procoagulant activity in vitro, we investigated the relationship among aPLs, ongoing prothrombotic state, and endothelial perturbation in SLE patients. METHODS AND RESULTS: We measured aPLs, anti-EC antibodies, circulating levels of prothrombin fragment F1 + 2 (F1 + 2), tumor necrosis factor-alpha (TNF-alpha), tissue-type plasminogen activator (TPA), and von Willebrand factor (vWF) in 43 SLE patients and 25 healthy subjects. Patients positive for aPLs (n = 23) had a higher prevalence of anti-EC antibodies (P = .02) and higher levels of F1 + 2 (P = .003) than aPL(-) patients. Endothelial perturbation, defined by elevated plasma levels of both TPA and vWF, was significantly associated with aPL positivity (P = .001). F1 + 2 > 1 nmol/L (mean +/- 2 SD of controls) was detected in all but one patient in whom aPL positivity and endothelial perturbation coexisted and in no aPL(+) patient without endothelial perturbation (P = .0039). F1 + 2 was significantly correlated with vWF (rho = .6, P = .004) and TPA (114 = .70, P = .0006) only in aPL(+) patients. Endothelial perturbation was closely associated with high values of TNF-alpha (P = .0001), anti-phospholipid (P = .001), and anti-EC antibodies (P = .012). In 31 patients without a clinical history of thrombosis followed up for 3 years, aPL(+) patients with endothelial perturbation showed higher F1 + 2 and TNF-alpha values than aPL(+) patients without endothelial dysfunction. CONCLUSIONS: This study shows that in SLE patients, aPL positivity is associated with an ongoing prothrombotic state only in the presence of endothelial perturbation. Our findings also suggest that aPLs and TNF-alpha might cooperate in inducing endothelial perturbation.

Relationship between cutaneous and pulmonary involvement in systemic sclerosis.

OBJECTIVE: Pulmonary disease may shorten survival in patients affected by systemic sclerosis (SSc). However, pulmonary involvement may commonly be silent, whereas skin fibrosis is usually the clinical feature drawing most attention. We investigated the relationship between cutaneous and pulmonary involvement during SSc. METHODS: We studied 52 patients (mean age 50.2 +/- 13.7 years) affected by SSc (mean duration of disease 13.8 +/- 9.5 years). Twenty-eight had the diffuse form of the disease (dSSc) and 24 the limited form (lSSc). All patients underwent pulmonary function studies, high resolution computed tomography (HRCT) of the lungs, and complete echocardiographic examination. Pulmonary artery systolic pressure was measured by Doppler echocardiography. Pulmonary interstitial fibrosis and skin fibrosis were evaluated using a point system. RESULTS: Mean percentages of predicted values of forced vital capacity and total lung capacity were significantly reduced in patients with dSSc compared to lSSc (80.0 +/- 18.9 vs 98.4 +/- 16.8%, p < 0.001; and 81.3 +/- 13.9 vs 92.1 +/- 14.2%, p < 0.01, respectively). The overall HRCT score was 6.1 +/- 4.9, with no significant differences between disease subgroups. However, a HRCT score of 10 or more was present in 10 patients with dSSc vs 2 patients with lSSc (p = 0.02). Pulmonary hypertension was present in 27 patients, 15 with lSSC and 12 with dSSc (p = NS). No significant correlation was observed between skin score and lung volumes, carbon monoxide diffusing capacity, HRCT score, or pulmonary artery systolic pressure for all patients and subgroups. CONCLUSION: Extent and severity of cutaneous and pulmonary involvement in SSc are not directly correlated. Nevertheless, different patterns of pulmonary involvement between SSc subgroups were observed. Restrictive lung disease was more frequent in patients with dSSc, while a trend to higher prevalence of pulmonary hypertension was observed in patients with lSSc.