Enhanced membrane binding of autoantibodies to cultured keratinocytes of systemic lupus erythematosus patients after ultraviolet B/ultraviolet A irradiation.

Although sunlight is known to induce skin lesions in patients with systemic lupus erythematosus (SLE) and to exacerbate systemic manifestations, the underlying mechanisms remain obscure. We report experiments that show enhanced binding of IgG autoantibodies to the cell surface membrane of ultraviolet-B (UVB) irradiated (200-1,600 J/m2) cultured SLE keratinocytes in 10 out of 12 such cell strains. The autoantibody probes showing increased binding were directed against the soluble intracellular antigens, Sm, RNP, SSA/Ro, SSB/La, whereas serum with anti-dsDNA activity did not demonstrate such binding. Control keratinocytes from several sources shared low level binding of autoantibodies after ultraviolet light exposure. In addition, 4/6 UVB-sensitive SLE strains showed increased autoantibody binding to the surface of SLE keratinocytes after UVA exposure (50-150 kJ/m2), but of lower magnitude. When UVB-sensitive nonirradiated SLE strains were exposed to autologous serum, 3/8 sera demonstrated a striking increase in IgG binding, which increased further after UVB exposure. Enhanced expression of saline-soluble intracellular antigens on the cell surface membrane of patient, but not control, keratinocytes may, in part, explain the photosensitivity of patients with SLE.

Induction of antiphospholipid autoantibodies by immunization with beta 2 glycoprotein I (apolipoprotein H).

A subset of patients with systemic lupus erythematosus has autoantibodies to acidic phospholipids. Since lipids are poor immunogens, the mechanism responsible for the induction of these antibodies is unclear. Immunization of a normal rabbit and normal mice with purified human beta 2-glycoprotein I (apolipoprotein H) resulted in the production of high levels of two non-cross-reactive antibody populations, anti-apolipoprotein H, and antiphospholipid. The antiphospholipid antibodies had binding specificities indistinguishable from autoantibodies obtained from human and murine lupus. These findings suggest a novel mechanism for the induction of antiphospholipid autoantibodies.

Activation of cultured vascular endothelial cells by antiphospholipid antibodies.

Circulating antiphospholipid antibodies (aPL) are associated with a syndrome of thrombosis, recurrent fetal loss, and thrombocytopenia. We have demonstrated the activation of cultured human umbilical vein endothelial cells (HUVEC) by IgG from patients with anticardiolipin antibodies (aCL). Incubation of HUVEC for 4 h with purified IgG (100 micrograms/ml) from patients with high-titer aCL induced a 2.3-fold increase in monocyte adhesion over that seen in HUVEC incubated with IgG's from normal subjects. The effect of aCL was not attributable to LPS contamination, Fc receptors, or immune complexes. Monocyte adhesion was not induced when the aCL were added in serum-free media but was restored by the addition of purified beta 2GP1, previously described as a necessary cofactor for aCL reactivity. Purified rabbit polyclonal IgG raised against beta 2GP1 also induced monocyte adhesion when incubated with HUVEC. Preadsorption of patient serum with cardiolipin reduced monocyte adhesion by 60%. Immunofluorescent microscopy demonstrated that endothelial cells incubated with patient IgG expressed cell adhesion molecules, including E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1. These data support the hypothesis that aPL activate vascular endothelial cells, thereby leading to a pro-thrombotic state.

Opposite beta2-glycoprotein I requirement for the binding of infectious and autoimmune antiphospholipid antibodies to cardiolipin liposomes is associated with antibody avidity.

The aim of this study was to investigate the interaction of antiphospholipid antibodies (aPL) from two different populations (patients with autoimmune or infectious disorders) with cardiolipin (CL) arranged in a defined bilayer. beta2-Glycoprotein I (beta2GPI), an apolipoprotein that plays a critical role in the aPL binding to phospholipids, was quantified by dot blot in purified IgG-aPL samples, further classified according to apparent avidity to CL. In solid-phase assays, beta2GPI increased, preferentially, the binding of low-avidity autoimmune aPL to CL but inhibited the binding of low-avidity syphilitic aPL. In the absence of beta2GPI, both autoimmune and infectious aPL induced the leakage of the entrapped fluorescent probe, carboxyfluorescein (CF), from small unilamellar vesicles containing CL. aPL-induced probe leakage was protein concentration-dependent and characterized by a lag-phase onset of 100-120 min. beta2GPI increased the leakage rate induced by low-avidity autoimmune aPL only and inhibited the leakage induced by all syphilitic aPL. The following conclusions were provided: (1) in the absence of beta2GPI, autoimmune and infectious aPL bind to CL in a bilayer, inducing liposome leakage; (2) the leakage mechanism induced by aPL is suggested to be intravesicular; (3) beta2GPI requirement for phospholipid binding in both solid and fluid phase is associated to aPL avidity; (4) CL alone or the CL-beta2GPI complex are the most likely epitopes for autoimmune aPL; (5) aPL from syphilis patients can only form the CL-aPL complex, supporting that beta2GPI is not (part of) the target epitope.

Thrombosis, recurrent fetal loss, and thrombocytopenia. Predictive value of the anticardiolipin antibody test.

To determine the predictive value of the IgG anticardiolipin antibody (ACA) test for thrombosis, recurrent fetal loss, and thrombocytopenia, the clinical features of 121 patients with varying antibody levels were studied. When patients were grouped into high-positive, low-positive, and normal groups according to their ACA levels, there were strong statistical correlations with arterial thrombosis, venous thrombosis, fetal loss, thrombocytopenia, and a positive Coombs' test. At levels of 7 SD and above, the test was highly specific (greater than 80%) and predictive (greater than 70%) for thrombosis, thrombocytopenia, and recurrent fetal loss. This study suggests that the IgG ACA test may be a useful predictor for thrombosis, recurrent fetal loss, and thrombocytopenia in patients with autoimmune disorders.

Penetration of autoantibodies into living epithelial cells.

The ability of autoantibodies to penetrate living cells is controversial. We have identified immunoglobulin G (IgG) antibodies capable of penetrating an epithelial cell line, COLO-16, in five of 36 (14%) antinuclear antibody positive sera from patients with SLE. Thirty minutes following incubation of cells with dilutions of either whole sera, globulin fractions, or F(ab')2 fragments of IgG, approximately 80-90% of cells demonstrated intranuclear IgG by indirect immunofluorescence. Viability of cells prior to assay was > 98% as determined by trypan blue staining and penetration of IgG into the nuclei did not affect viability or DNA synthesis of the cells in short-term culture. Intracellular IgG could not be detected following exposure of the cells to high-titer reference autoantibodies of known specificities (against DNA, Ro, La, Sm, RNP, or ribosomes). Furthermore, absorption of the sera with either DNA or chromatin failed to abolish intranuclear penetration, indicating that the autoantibodies were not directed against DNA receptors or nucleosomes on the cell surface. Antibody uptake was relatively selective for epithelial cell lines, because intranuclear IgG was not detected in cell lines of lymphoid origin exposed to the sera. Two of the five sera immunoprecipitated proteins of molecular weight 88 kD with or without a 68-kD protein from COLO-16 cells labeled with 125I at the cell surface. These findings indicate that a subset of SLE patients have IgG capable of penetrating a cell line of epithelial origin. These antibodies, most likely, bind to cell surface proteins and are translocated into the cell nucleus. Although direct immunofluorescence of a skin biopsy obtained from one of the five patients with "penetrating IgG" also showed intranuclear staining for IgG, the biologic relevance of these findings remains to be determined.

Antiphospholipid antibody syndrome: immunologic and clinical aspects.

Antiphospholipid antibody is associated with a clinical syndrome of vascular thrombosis, thrombocytopenia, recurrent fetal loss, and livedo reticularis, whether or not a clinical diagnosis of systemic lupus erythematosus (SLE) coexists. A positive antiphospholipid antibody test is defined by enzyme-linked immunosorbent assay (ELISA) (antiphospholipid antibody itself) or by coagulation assay (lupus anticoagulant). These are similar but not identical antibodies. The test for syphilis is less closely related to the preceding two and is less regularly associated with clinical complications. The mechanism of action of either antiphospholipid antibody or lupus anticoagulant is as yet unknown. SLE-induced but not infection-induced antiphospholipid antibody has immunoglobulin G2 (IgG2) and IgG4 predominance. It recognizes all negatively charged phospholipids, but various physical characteristics of the phospholipids alter the recognition patterns. Treatment for the antiphospholipid antibody syndrome has not been clearly defined. Anticoagulation with aspirin, heparin, or warfarin is currently favored. A role for corticosteroid remains to be demonstrated.

Chorea in systemic lupus erythematosus and "lupus-like" disease: association with antiphospholipid antibodies.

Twelve patients with chorea from a population of 500 patients with SLE and "lupus-like" disease were reviewed. Clinical histories, including time relationships of chorea to the systemic illness and other neurologic manifestations, are reported. Chorea appeared early in the course of disease in most patients, but the development of cerebral infarctions or TIAs occurred subsequently in seven of nine patients demonstrating antiphospholipid antibodies. The relationship of chorea to the presence of these antibodies in nine of 12 patients and the therapeutic outcome are briefly discussed.

Pulmonary involvement in systemic sclerosis. Association with anti-Scl 70 antibody and digital pitting.

The association of clinical and serologic features of 34 patients with systemic sclerosis was examined. Anti-Scl 70 antibody was found to identify patients with abnormal pulmonary function, particularly impaired diffusion (p less than 0.005), as well as patients with digital pitting scars (p less than 0.025). In addition, the presence of digital pitting scars correlated with impaired diffusion (p less than 0.005), suggesting that interstitial pulmonary disease in systemic sclerosis may, like digital pitting, be secondary to vascular pathology. Anticentromere antibody-positive patients were less likely to have abnormalities of pulmonary function (p less than 0.001).

Neurologic complications of antiphospholipid antibodies.

Antibodies directed against phospholipids are highly associated with venous and arterial thrombotic episodes, which are often recurrent. There seems to be a skewed frequency of cerebral and ocular events when the arterial circulation is affected. Other neurologic syndromes, including dementia, migraine, chorea, transverse myelopathy, Guillain Barré syndrome, transient global amnesia, seizures, motor neuron disease, myasthenia gravis, and depression, have also been described in association with aPL. Although some of them (for example, dementia, chorea, seizures, and transient global amnesia) could well be the result of aPL-related cerebrovascular disease, the relationship of aPL to the underlying pathophysiology of these syndromes is less clear. Clues that should lead one to consider evaluating for these antibodies include recurrent thrombosis (especially in young people), recurrent fetal loss, and thrombocytopenia. Associated laboratory abnormalities may include a biologically false-positive VDRL test, abnormal ANA or anti-DNA titers, and a high ESR. If the APTT is positive on routine screening and does not correct with mixing studies, a LA should be highly suspected. More sensitive and specific tests are usually necessary to detect aPL, however. Many in vitro and, more recently, in vivo systems strongly suggest that aPL may be directly implicated in the pathogenesis of thrombosis, as opposed to being markers of a procoagulant state. The management of patients with aPL-associated thrombosis can be difficult. Prospective studies are needed to determine the optimal treatment strategies for this group of patients.

Origin of antiphospholipid antibodies.

Our observations and those from others give further support to our hypothesis that "autoimmune aPL" may be generated by immunization with products from bacteria or viruses after incidental exposure or infection. We also were able to generate an APS-like syndrome in a strain of mice susceptible to autoimmunity, indicating that other factors such as genetic factors are likely to be involved in development of APS. Furthermore, not all aPL generated by immunization with bacterial or viral products were pathogenic. Based on the clinical experience and on the numerous reports indicating the presence of aPL in large number of infectious diseases, it may be expected that not all aPL produced during infection are pathogenic. We hypothesize that a limited number aPL induced by certain viral or bacterial products would be pathogenic in certain groups of predisposed individuals. Identification of those bacterial or viral agents may help to find strategies for the prevention of production of "pathogenic" aPL. Alternatively, free peptides may be used to induce tolerance against aPL production.

Lupus psychosis: differentiation from the steroid-induced state.

Steroid induced psychosis in SLE is rare but clinically important, and often difficult to distinguish from lupus cerebritis. We report a patient with SLE who became depressed following an increase in her steroid dosage. Based on her clinical presentation and high levels of antibodies to P ribosomal proteins (both in CSF and serum) a diagnosis of lupus cerebritis was made. Steroid dosage, time intervals, and the duration of mental changes may help in differentiating steroid psychosis from lupus cerebritis. No single laboratory test sufficient to establish a definitive diagnosis of lupus cerebritis is available at the present time. However, elevated levels of antibodies to P ribosomal proteins may assist in confirming the diagnosis of this condition.

Cerebral infarction in systemic lupus: association with anticardiolipin antibodies.

We report fifteen patients, thirteen with systemic lupus and two patients with a "lupus-like" illness who developed cerebral infarction. All fifteen patients were shown to have elevated anticardiolipin antibody levels using a newly devised solid phase radioimmunoassay. The lupus anticoagulant was detected in all eleven patients tested. It is proposed that anticardiolipin antibodies and the lupus anticoagulant make up a population of antiphospholipid antibodies capable of causing cerebral vascular injury and thrombosis resulting in cerebral infarction. These antibodies may also play a pathogenic role in autoimmune disorders other than lupus where cerebral thrombotic disease is a prominent feature.

Enzyme immunoassays for the detection of IgG and IgM anti-dsDNA antibodies: clinical significance and specificity.

A solid phase Enzyme-Linked Immunosorbent Assay (ELISA) was developed for the measurement of IgG and IgM antibodies to double-stranded DNA (anti-dsDNA). This method is sensitive, specific, relatively simple and suitable for routine use. Thus, we evaluated sera from 224 Greek patients with the following autoimmune rheumatic diseases: 54 patients with classical rheumatoid arthritis (RA), 50 patients with primary Sjögren's syndrome (SS), 41 patients with systemic lupus erythematosus (SLE), 30 patients with scleroderma, 20 patients with idiopathic Raynaud's phenomenon (IRP) and 29 patients with juvenile rheumatoid arthritis (JRA). Sera from 119 age- and sex-matched healthy blood donors were tested as normal controls. The presence of both IgG and IgM anti-dsDNA highly correlated with SLE. However, IgM anti-dsDNA levels were significantly lower. Serum complement C3 and C4 levels correlated negatively with anti-dsDNA levels in the SLE group. Finally, in sequential sera from five SLE patients, the anti-dsDNA activity proved to be a relatively sensitive marker of SLE activity.

Enhancement of cryoprecipitation from hypotonic serum: a comparative study in normals and in patients with connective tissue diseases.

In a study of 17 normals and 26 patients we have established a normal range for cryoglobulins in a standard and a simple hypotonic system. In a comparison of standard and hypotonic cryoprecipitates in patients with connective tissue diseases we showed a significant increase in protein content (p less than 0.001), IgM (p less than 0.01) and IgM rheumatoid factor (p less than 0.01) in the hypotonic samples. The ratios of IgM RF/IgM in hypotonic cryoglobulins compared to standard cryoglobulins were significantly increased as shown by a chi 2 analysis in both normals (p less than 0.05) and patients (p less than 0.01). Estimation of cryoglobulins in a hypotonic system is a useful simple test which may detect distinct groups of proteins and rheumatoid factors. Precipitates were demonstrated in patients who had previously been considered serologically normal.

Renal vein thrombosis in systemic lupus erythematosus: association with the "lupus anticoagulant".

Two patients with lupus erythematosus and renal vein thrombosis are described. Both patients had the "lupus anti-coagulant" in their serum. It is postulated that in these patients the clotting tendency could be favoured by a cross reaction of the "lupus anti-coagulant" with phospholipids in the endothelial cell membrane, resulting in inhibition of prostacyclin release.

Antiphospholipid antibody syndrome.

Autoimmune aPL are associated with a well-defined clinical syndrome of vascular thromboses, recurrent fetal loss, thrombocytopenia, livedo reticularis, and valvular and neurologic abnormalities. A clinical diagnosis of SLE need not be present, and aPL syndrome in the absence of other well-defined autoimmune disease is termed PAPS. A positive test for aPL is defined by enzyme-linked immunoassay (aCL) or by functional coagulation assay (LAC). Anticardiolipin antibody and LAC are similar but probably not identical antibodies. The false-positive test for syphilis is less closely associated with clinical complications than are aCL and LAC. The mechanism of action of aPL is not yet known, although many theories have been advanced. Recent identification of beta 2-glycoprotein I, a serum glycoprotein, as an aPL cofactor suggests that inhibition of this protein's anticoagulant activity may be important. Autoimmune aPL differ from infection-induced aPL in important antibody characteristics, including IgG subclass, light chain preference, antibody avidity, and cofactor requirement. Both recognize negatively charged phospholipids, but various physical characteristics of the phospholipids alter the recognition patterns. Treatment of the aPL syndrome is not well defined. Anticoagulation with heparin, coumadin, or aspirin are currently widely used. Although corticosteroid, immunosuppressive therapy, and plasmapheresis may be used for severe, fulminant thrombosis, the efficacy of this treatment has yet to be proved.

IgA anticardiolipin and anti-beta2-glycoprotein I are the most prevalent isotypes in African American patients with systemic lupus erythematosus.

BACKGROUND: Ethnicity plays a role in the prevalence, isotype distribution, and clinical significance of anticardiolipin (aCL) and anti-beta2-glycoprotein I (anti-beta2-GPI) antibodies in patients with systemic lupus erythematosus (SLE). Few studies have been done in the African American population. METHODS: Serum samples from 100 African American patients with SLE were tested for IgG, IgM, and IgA aCL and anti-beta2-GPI antibodies by enzyme-linked immunosorbent assay (ELISA). Computerized clinical data on these patients were reviewed with a specific focus on clinical manifestations of antiphospholipid syndrome (APS). RESULTS: Positivity for at least one isotype of aCL antibodies was found in 33% of the patients, whereas 28% were positive for at least one isotype of anti-beta2-GPI antibodies. IgA was the most prevalent isotype for both antibodies; 24% of the patients in the aCL ELISA and 19% in the anti-beta2-GPI ELISA were positive for IgA. Positivity for both aCL and anti-beta2-GPI in the same patient was seen more frequently with the IgA isotype. Fewer than half of the patients positive for aCL antibodies had medium-to-high levels of antibodies. A few patients had presented thrombotic manifestations, and these patients were positive for aCL (P = 0.01) and anti-beta2-GPI antibodies (P = 0.02). No other manifestations of APS could be significantly correlated with the presence of these antibodies. CONCLUSIONS: Our results show that IgA is the most prevalent isotype among the African American patients with SLE studied. The predominance of the IgA isotype and the low prevalence of medium-to-high levels of aCL antibodies may account for the low frequency of clinical manifestations of APS in these patients.

Arterial occlusion causing large bowel infarction--a reflection of clotting diathesis in SLE.

The patient, a 44-year old woman with systemic lupus erythematosus, (SLE), developed infarction of the bowel and spleen after occlusion of the inferior mesenteric and splenic arteries, necessitating colectomy and splenectomy. She had had previous cerebral thromboses and a lower limb deep vein thrombosis. Histological examination of the involved vessels showed the presence of thrombus only with the total absence of any vasculitis. The patient demonstrated antibodies to phospholipid - the "lupus anticoagulant" (LA) and antibodies to cardiolipin in serum, both strongly associated with thromboses.

Origin and pathogenesis of antiphospholipid antibodies.

Antiphospholipid antibodies (aPL) are a heterogeneous group of antibodies that are detected in the serum of patients with a variety of conditions, including autoimmune (systemic lupus erythematosus), infectious (syphilis, AIDS) and lymphoproliferative disorders (paraproteinemia, myeloma, lymphocytic leukemias). Thrombosis, thrombocytopenia, recurrent fetal loss and other clinical complications are currently associated with a subgroup of aPL designating the antiphospholipid syndrome. In contrast, aPL from patients with infectious disorders are not associated with any clinical manifestation. These findings led to increased interest in the origin and pathogenesis of aPL. Here we present the clinical features of the antiphospholipid syndrome and review the origin of aPL, the characteristics of experimentally induced aPL and their historical background. Within this context, we discuss the most probable pathogenic mechanisms induced by these antibodies.