calcimycin and Sjogren-s-Syndrome

To investigate whether a change in the CD95-related apoptosis of T lymphocytes might have a share in the development of the disease in patients with primary Sjögren's syndrome (SS).. Two-color cytometric analysis was used to study the phenotype of freshly separated mononuclear cells, while Western blotting was used to detect CD95 ligand (CD95L) expression in total homogenates of isolated CD4+ T lymphocytes. The ability of various subpopulations of T cells to undergo apoptosis was investigated in 1-day cultures in medium alone or following various (anti-CD3, anti-CD95 monoclonal antibody, calcium ionophore) treatments. Apoptosis was detected using 7-aminoactinomycin D dye.. Compared with the findings in healthy controls, the number of CD4+ T lymphocytes was decreased, while their expression of CD95, HLA-DR, and CD45RO was significantly increased in patients with primary SS. A positive correlation was found between the activity of disease, the decrease in the CD4+ T cell number, and the increase in the expression of CD95, CD95L, HLA-DR, and CD45RO molecules within the CD4+ T cell subset. An increased rate of spontaneous, anti-CD3-, or anti-CD95-induced apoptosis was found in the T cells of SS patients, and this was more pronounced in the CD4+ T cell population, correlated with the decreased CD4+ T cell number, increased CD45RO expression, and activity of disease, and concerned mainly the CD95+ cells.. These observations indicate that the increased susceptibility to apoptosis of peripheral CD4+ T cells from SS patients correlates with disease activity. These findings support the hypothesis that the chronic activation of the immune system that occurs in this autoimmune disease is the primary mechanism responsible for this cell-deletion process.

Topics: Adult; Aged; Antibodies, Monoclonal; Apoptosis; Calcimycin; CD4-Positive T-Lymphocytes; Cell Count; Cells, Cultured; fas Receptor; Female; Humans; Lymphocyte Activation; Middle Aged; Muromonab-CD3; Phenotype; Sjogren's Syndrome; T-Lymphocytes

Human autoantibodies offer unique tools for the study of cellular constituents since they usually recognize highly conserved components, the most difficult to detect due to their low immunogenicity. The serum from a patient with Sjögren's syndrome (RM serum) showing a very high reactivity to the Golgi complex has been shown to immunoprecipitate and to immunodetect by Western blotting experiments a protein mol wt 210,000 (p210) that was shown to be peripheral and cytoplasmically disposed. A close examination of the p210 labeling revealed some differences with Golgi markers: RM serum staining was slightly more extensive than several Golgi markers and showed a discontinuous or granular appearance. Nocodazole induced a specific and early segregation of many p210-associated vesicles or tubules from Golgi apparatus. Upon brefeldin A treatment, p210 did not redistribute in the ER as did other Golgi proteins. In contrast, it exhibited a vesicular pattern reminiscent to that displayed by proteins residing in the intermediate compartment. Double staining immunofluorescence using the RM serum and the marker of the intermediate compartment, p58, revealed segregation of both proteins in control conditions but colocalization in BFA-treated cells. We have further demonstrated by combining different drug treatments that p210-containing elements in brefeldin A-treated cells belong indeed to the intermediate compartment. Experiments on brefeldin A recovery suggested that these p210 elements might play a role in reformation and repositioning of the Golgi apparatus. Ultrastructural localization performed by immunoperoxidase staining allowed us to establish that p210 interacted with the external side of an abundant tubulo-vesicular system on the cis side of the Golgi complex which extended to connecting structures and vesicles between saccules or stacks of cisternae, p210 appears to be a novel protein residing in the cis-Golgi network that may cycle between the Golgi apparatus and the intermediate compartment.

Topics: Autoantigens; Brefeldin A; Calcimycin; Cell Compartmentation; Cell Line; Cyclopentanes; Electrophoresis, Gel, Two-Dimensional; Fluorescent Antibody Technique; Golgi Apparatus; HeLa Cells; Humans; Immunologic Techniques; In Vitro Techniques; Intracellular Membranes; Isoelectric Point; Membrane Proteins; Molecular Weight; Nocodazole; Sjogren's Syndrome

Anti-CD2-induced T cell proliferation was analyzed in the peripheral blood samples of 31 primary and 8 secondary untreated Sjögren's syndrome patients. Anti-CD2-stimulated PBMC proliferation was very low in about one-third of primary Sjögren's syndrome samples, despite the number of CD2+ cells being similar in primary and secondary Sjögren's syndrome and normal PBMC samples. The depressed response to anti-CD2 was mainly found in anti-Ro+/La+ patients. Experiments on purified T cells demonstrated that a defect at the T cell level was responsible for the anti-CD2 unresponsiveness. Cell proliferation failure was associated with poor IL-2 and IL-2 receptor mRNA expression and, consequently, IL-2 and IL-2 receptor synthesis. Since defective anti-CD2-induced mitogenesis could be reversed by phorbol myristate acetate, but not calcium ionophore A23187, it is probably correlated with impaired protein kinase C activation. Comparison of anti-CD2-triggered PBMC proliferation in treated and untreated patients and a long-term study of nine patients showed that the defect is a stable characteristic in primary Sjögren's syndrome patients, but that it can be reversed by pharmacological immunosuppression.

Topics: Adrenal Cortex Hormones; Adult; Antibodies, Monoclonal; Antigen-Presenting Cells; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Calcimycin; CD2 Antigens; Female; Gene Expression; Humans; Interleukin-1; Interleukin-2; Leukocytes, Mononuclear; Lymphocyte Activation; Male; Middle Aged; Receptors, Immunologic; Receptors, Interleukin-2; Sjogren's Syndrome; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription, Genetic