Screening of an endothelial cDNA library identifies the C-terminal region of Nedd5 as a novel autoantigen in systemic lupus erythematosus with psychiatric manifestations

Paola Margutti 2 Maurizio Sorice 1 Fabrizio Conti 0 Federica Delunardo 2 Mauro Racaniello 2 Cristiano Alessandri 0 Alessandra Siracusano 2 Rachele Rigan 2 Elisabetta Profumo 2 Guido Valesini 0 Elena Ortona 2 0 Dipartimento di Clinica e Terapia Medica Applicata, Cattedra di Reumatologia, Universita 'La Sapienza' , Rome , Italy 1 Dipartimento di Medicina Sperimentale e Patologia, Cattedra di Reumatologia, Universita 'La Sapienza' , Rome , Italy 2 Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanita , Rome , Italy Anti-endothelial-cell antibodies are associated with psychiatric manifestations in systemic lupus erythematosus (SLE). Our primary aim in this study was to seek and characterize molecules that behave as endothelial autoantigens in SLE patients with psychiatric manifestations. By screening a cDNA library from human umbilical artery endothelial cells with serum from an SLE patient with psychosis, we identified one positive strongly reactive clone encoding the C-terminal region (C-ter) of Nedd5, an intracytoplasmatic protein of the septin family. To evaluate anti-Nedd5 serum immunoreactivity, we analyzed by ELISA specific IgG responses in 17 patients with SLE and psychiatric manifestations (group A), 34 patients with SLE without psychiatric manifestations (group B), 20 patients with systemic sclerosis, 20 patients with infectious mononucleosis, and 35 healthy subjects. IgG specific to Nedd5 C-ter was present in 14 (27%) of the 51 SLE patients. The mean optical density value for IgG immunoreactivity to Nedd5 C-ter was significantly higher in patients of group A than in those of group B, those with infectious mononucleosis, or healthy subjects (0.17 0.14 vs, respectively, 0.11 0.07, P = 0.04; 0.11 0.06, P = 0.034; - and 0.09 0.045, P = 0.003, on Student's t-test). Moreover, IgG immunoreactivity to Nedd5 C-ter was significantly higher in patients with systemic sclerosis than in patients of group B or healthy subjects (0.18 0.18 vs, respectively, 0.11 0.07, P = 0.046; and 0.09 0.045, P = 0.003). The percentage of patients with anti-Nedd5 C-ter serum IgG was higher in group A than in group B (8 (47%) of 17, vs 6 (17%) of 34, P = 0.045, on Fisher's exact test). In order to clarify a possible mechanism by which Nedd5 might be autoantigenic, we observed that Nedd5 relocated from cytoplasm to the plasma membrane of EAhy926 endothelial cells after apoptotic stimuli. In conclusion, Nedd5 is a novel autoantigen of potential clinical importance that could be successfully used for a more thorough investigation of the pathogenesis of psychiatric manifestations in SLE. Although anti-Nedd5 autoantibodies are not specific to SLE, they are significantly associated with neuropsychiatric SLE and may represent immunological markers of psychiatric manifestations in this pathology. Introduction Symptoms originating from the central nervous system occur in 14 to 75% of patients with systemic lupus erythematosus (SLE) and are extremely diverse, including neurological and psychiatric syndromes [1]. In 1999, the American College of Rheumathology defined 19 distinct neuropsychiatric syndromes associated with SLE, including psychosis and depression [2,3]. Neuropsychiatric SLE remains an enigmatic manifestation in lupus. In fact, conflicting results have been reported to clarify associations between neuropsychiatric AECA = anti-endothelial-cell antibody; BSA = bovine serum albumin; C-ter = C-terminal region; ELISA = enzyme-linked immunosorbent assay; FITC = fluorescein isothiocyanate; HUAEC = human umbilical artery endothelial cell; OD = optical density; PBS = phosphate-buffered saline; SD = standard deviation; SLE = systemic lupus erythematosus; SLEDAI = SLE Disease Activity Index; TNF- = tumor necrosis factor . manifestations and serum antibodies against neuronal antigens, ribosomes, and phospholipids [4]. Recently, we demonstrated an association between the presence of antiendothelial-cell antibodies (AECAs) and psychiatric manifestations, such as psychosis and depression in SLE, suggesting a possible mechanism underlying psychiatric symptoms [5]. By activating endothelial cells, AECAs up-regulate the expression of adhesion molecules as well as the secretion of cytokines and chemokines. Until recently, few published data have been available on the identity of endothelial cell autoantigens in immune disorders [6-11]. Identifying endothelial autoantigens involved in the autoimmune processes during neuropsychiatric SLE could help to explain the pathogenetic mechanisms involved in the initiation and progression of psychiatric symptoms. Our primary aim in this study was to seek and characterize molecules that behave as endothelial autoantigens in neuropsychiatric SLE. By screening a cDNA library from human umbilical artery endothelial cells (HUAECs) with serum from an SLE patient with psychosis, we identified one strongly reactive clone encoding the C-terminal region (C-ter) of Nedd5, an intracytoplasmatic protein of the septin family. To evaluate antiNedd5 serum immunoreactivity, we used ELISA to measure specific IgG responses in patients with SLE classified according to the presence of psychiatric manifestations, such as psychosis and depression. Data were compared with those of patients with systemic sclerosis (an autoimmune disease characterized by endothelium damage and the presence of AECAs), of patients with infectious mononucleosis, and of healthy subjects. Finally, we investigated by immunofluorescence the intracellular redistribution of Nedd5 in endothelial cells after apoptotic stimuli. Materials and methods Patients For the present investigation, we studied sera from an SLE cohort of 51 outpatients attending the Rheumatology Division of the University of Rome 'La Sapienza'. All patients were diagnosed according to the American College of Rheumatology revised criteria for the classification of SLE [2]. This population of SLE patients had been previously characterized with regard to their psychiatric and autoantibody profiles [5]. For our present purposes, we studied 50 of the 51 sera, because the serum from one SLE patient with mood disorder had run out. In this study, we also included the serum from a patient seen in the meantime with SLE and acute psychosis, a very rare manifestation of neuropsychiatric SLE. Patients were categorized as being in group A or group B on the basis of the clinical psychiatric examination, which was performed by means of the Structured Clinical Interview for Psychiatric Diagnosis [12]. A psychiatric diagnosis was assigned according to the Diagnostic and Statistical Manual of Mental Disorders IV [13]. Group A consisted of patients with psychosis (n = 3) and mood disorders (n = 14). Group B included patients without psychiatric manifestations (n = 18) and patients whose only psychiatric manifestation was anxiety disorder (n = 16). We did not include patients with anxiety disturbance in group A, becau (...truncated)