Autoantibody profile in systemic lupus erythematosus with psychiatric manifestations: a role for anti-endothelial-cell antibodies

This study included 51 unselected outpatients with SLE (44 women, 7 men; mean age 36.8 years, range 22–54 years; mean disease duration 9.4 years, range 0.5–26 years) attending the Rheumatology Division of the University of Rome 'La Sapienza'. All patients fulfilled the ACR revised criteria for the classification of SLE [19]. Informed consent was obtained from each patient and the local ethics committee approved the study protocol. A blood sample was taken from each patient and was stored at -20°C until assay.

Psychiatric diagnosis was assigned in accordance with the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) [20]. The Structured Clinical Interview for DSM-IV axis I Disorders [21] was administered to all patients by the same psychiatrist. Patients were categorized in group A or B on the basis of clinical psychiatric examination. Those with more severe psychopathology such as psychosis and mood disorders (recurrent major depressive disorder, dysthymic disorder, or depressive disorder not otherwise specified) were included in group A. Group B included patients without psychiatric manifestations other than anxiety. We did not include in group A patients with anxiety disturbance alone because in most SLE patients anxiety is considered a secondary stress reaction and not a direct manifestation of NPSLE [1, 2, 9‐11].

Current SLE disease activity was measured using the SLE Disease Activity Index (SLEDAI) [22]. The rheumatologist responsible for assessment of SLEDAI was blind to the psychiatric evaluation.

Human umbilical-vein endothelial cells were isolated by collagenase perfusion from normal-term umbilical cord veins as previously described [23] and were cultured in M199 medium (Sigma Chemical Co, St. Louis, MO, USA) supplemented with 20% FCS. These cells (third to fourth passage) were used to detect anti-endothelial-cell antibodies (AECA) of IgG isotype using a cell-surface ELISA on living cells allowed to grow to confluence in microtiter plates. After three washes with Hank's balanced salt solution (HBSS), nonspecific binding sites were blocked for 2 hours at room temperature with 3% bovine serum albumin/HBSS. After two washes with HBSS, the wells were incubated in duplicate with 100 μl of the sera diluted 1:50 in HBSS for 2 hours at room temperature. After three washes with HBSS, the bound antibodies were detected with alkaline-phosphatase-conjugated goat antibodies antihuman IgG (Sigma), using 1 mg/ml p-nitrophenylphosphate. Optical density (OD) was measured at 405 nm wavelength and AECA were expressed as a binding index (BI), equal to 100 × (S-A)/(B-A), where S is the OD of the sample tested, A is the OD of a negative control, and B that of a positive reference serum. AECA were considered positive when the BI was higher than the cutoff value (mean + 2 SD of 66 healthy controls) corresponding to 50% of a positive reference serum from an SLE patient [24]. Finally, we evaluated AECA IgG reactivity in a disease control group of 34 consecutive patients with systemic sclerosis (SSc) (32 women, 2 men; mean age 51.1 years, range 21–77; mean disease duration 9.5 years, range 1.2–35; 8 with diffuse SSc and 26 with limited SSc) attending the Rheumatology Division of the University of Rome 'La Sapienza'. The diagnosis of SSc was made in accordance with the criteria of the American Rheumatism Association [25].

Human glial fibrillary acidic protein (GFAP) purified from human brain was purchased from Biogenesis (Poole, UK; BH 17 7 DA). GFAP at 5 μg/ml in carbonate-bicarbonate buffer (pH 9.6) was used to coat 96-well microtiter plates overnight at 4°C. After three washes with PBS, plates were blocked for 2 hours at room temperature with 3% FCS–0.05% Tween 20 in PBS. After four washes in 1% FCS–0.1% Tween 20 in PBS, plates were incubated for 1 hour at room temperature with sera of patients diluted 1/100 in 0.1% Tween 20 in PBS. Mouse monoclonal IgG₁ anti-GFAP (Sigma) diluted 1/1000 in 0.1% Tween 20 in PBS was used as positive control. Subsequently, after four washes, plates were incubated for 1 hour at room temperature with alkaline phosphatase conjugated antihuman and antimouse IgG from goat and rabbit respectively (Sigma) diluted 1/1000 in 0.1% Tween 20 in PBS. After four washes, p-nitrophenylphosphate tablets in ethanolamine were used for the enzyme reaction. Plates were read at 405 nm wavelength and results were expressed as OD. All assays were performed in duplicate. Anti-GFAP assays were considered positive when OD was higher than the cutoff value (mean ± 3 SD of 44 healthy controls).

Anti-Ro, anti-Ro52, anti-La, anti-CL, anti-P (P0, P1, P2), and anti-β₂-GPI ELISA kits were obtained from Diamedix (Miami, FL, USA). Anti-dsDNA and anti-nucleosome antibodies were obtained from Orgentec Diagnostika (Mainz, Germany). ELISA was performed in accordance with the manufacturer's instructions. All assays were performed in duplicate. A positive control and several normal human sera were run in the same assay to confirm the specificity of the results.