RESUMO
OBJECTIVE: T cell activation plays a pivotal role in the immunopathogenesis of systemic sclerosis (SSc). Lymphocyte processes are tightly controlled by molecules activating either proliferation or programmed cell death (apoptosis). We investigated whether an imbalance in apoptotic function, increasing the survival rate of autoreactive cells, may lead to persistent autoreactive phenomena. METHODS: We studied peripheral a/b and g/d T lymphocytes of 22 patients with SSc and 22 healthy controls for their spontaneous and stimulated (phytohemagglutinin, dexamethasone) apoptotic rate and surface phenotype including expression of Fas (CD95) and Bcl-2, determined by flow cytometry. sFas and sFas ligand in sera and supernatants were measured by ELISA. Caspase-3 activation in response to agonistic anti-Fas Mab treatment was assessed. RESULTS: Lymphocytes of SSc patients showed a significant decrease in the percentage of apoptotic cells over time, in both unstimulated and stimulated cultures, compared to controls. We observed no difference between patients and controls, in stimulated or unstimulated cells, in the phenotypic expression of apoptotic cells, including surface Fas. SSc T cells were less susceptible to undergoing apoptosis after anti-Fas stimulation. We observed a significant decrease of apoptotic cells from stimulated culture of isolated SSc g/d T cells. Serum levels of sFas in SSc patients were significantly higher compared to controls. Similar data were obtained in the supernatants of stimulated and unstimulated cultures. By contrast, sFas ligand was always reduced. Bcl-2 expression in SSc was significantly elevated. A significant decrease in caspase-3 activity was detected in SSc patients after treatment by agonistic anti-Fas antibody. CONCLUSION: Resistance to apoptosis is present in a/b and g/d T cell lymphocyte subsets of patients with SSc, and several pathways seem to be connected in this setting.
Assuntos
Apoptose , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Escleroderma Sistêmico/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Adulto , Idoso , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Estudos de Casos e Controles , Caspase 3/metabolismo , Células Cultivadas , Dexametasona/farmacologia , Proteína Ligante Fas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitógenos/farmacologia , Fito-Hemaglutininas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Escleroderma Sistêmico/metabolismo , Receptor fas/metabolismoRESUMO
OBJECTIVE: Systemic sclerosis (SSc) is characterized by early endothelial damage evolving to vascular desertification. Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 regulate specific steps in new vessel formation. We undertook this study to determine whether an alteration of the SDF-1/CXCR4 axis might be involved in the pathogenetic mechanisms following ischemic damage during SSc. METHODS: We enrolled 36 SSc patients and 15 controls. Skin biopsy samples were obtained from each subject, and the expression of SDF-1 and CXCR4 was assessed by immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analyses. Furthermore, isolated microvascular endothelial cells (MVECs) from 4 patients with diffuse cutaneous SSc (dcSSc) and 3 controls were analyzed for SDF-1 and CXCR4 by confocal laser scanning microscopy, RT-PCR, and Western blotting. RESULTS: SDF-1 and CXCR4 were up-regulated in the skin of patients with early (edematous) SSc, both in the diffuse and limited cutaneous forms, and progressively decreased, with the lowest expression in the latest phases of both SSc subsets. MVECs from patients with dcSSc expressed significantly higher amounts of both isoforms of SDF-1 in the early stage of disease, with a progressive reduction of SDF-1 and CXCR4 in later stages. On the surface of cultured MVECs from patients with dcSSc, SDF-1 and CXCR4 colocalized in polarized areas, suggesting that they are activated in vivo and that they are under strict genetic control to retain capping function. CONCLUSION: Due to its transient expression, SDF-1 could be considered a future therapeutic target to induce new vessel formation in SSc.
Assuntos
Quimiocinas CXC/genética , Receptores CXCR4/genética , Escleroderma Sistêmico/genética , Pele/patologia , Adulto , Idoso , Biópsia , Quimiocina CXCL12 , Primers do DNA , Endotélio Vascular/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Escleroderma Sistêmico/patologiaRESUMO
OBJECTIVE: To evaluate, in a pilot study, the efficacy of a short term cyclophosphamide (CYC) pulse regimen on alveolitis in a cohort of patients with systemic sclerosis (SSc). METHODS: Twenty-three patients with SSc (17 diffuse SSc and 6 limited SSc) were selected in 5 centers in Italy, based on the findings of an abnormal bronchoalveolar lavage (BAL) cell analysis in association with altered pulmonary function tests (PFT) or recent deterioration in flow volume curve (FVC). Patients were also evaluated by skin score (Rodnan), esophageal manometry and barium swallow radiography, and electrocardiography and 2-mode echocardiography. The pre-enrolment pulmonary evaluation and after 6 months of therapy included evaluation of the clinical status, PFT (FVC, FEV1, DLCO), BAL. standard chest radiograph, and chest high resolution computed tomography. All patients received i.v. CYC (1000 mg/m2 of body surface monthly for 6 mo) and oral prednisone (25 mg daily for the first month and subsequently 5 mg daily of maintenance dosage for the remaining 5 mo). A complete blood count and urinalysis were obtained at monthly intervals. RESULTS: After 6 months of therapy the values for FVC did not change significantly. Individually, 8 of 23 patients showed an improvement (> 15% increase) in FVC after 6 months, while FVC in 13 cases remained stable. Only 2 patients had an important decline in FVC after 6 months of therapy (17 and 24% decrease). Improvement in DLCO was noted in 15 of 23 patients after 6 months of therapy. Four patients were stable and 4 patients had a worsened DLCO at the end of the study. After therapy the mean value of BAL fluid recovery did not change. There was a reduction in total cell number although this value did not reach statistical significance. The levels of neutrophils, eosinophils, lymphocytes, and macrophages did not change significantly. Scans for patients with grades 1, 2, and 3 did not differ significantly after 6 months of therapy, and 14 patients were stable. Changes in appearance, in relation to changes in extent of disease, were seen in 8 patients and consisted of an extension of reticular pattern and transformation from grade 1 to 2 (6/8 patients). All patients showed a ground-glass appearance indicating an acute alveolitis. Improvement in ground-glass was noted in 10 of 23 patients after 6 mo therapy. At the end of the study, 8 patients were stable and 5 patients had a diffusion of the ground-glass to other segments. No side effects were experienced during the treatment except for mild nausea in 4 patients; no patients discontinued therapy during the study. CONCLUSION: CYC pulse regimen seems to stabilize alveolitis in the majority of cases. The association of CYC pulsed modality with prednisone may be useful in SSc patients to control disease evolution in the lung.