RESUMEN
BACKGROUND: Previous immunohistochemical studies of muscle from patients with inclusion body myositis and polymyositis found many more T cells than B cells, suggesting a role for intramuscular cell-mediated immune mechanisms rather than humoral mechanisms. METHODS: Microarray studies were performed on muscle biopsy specimens from 40 patients with inclusion body myositis (IBM; n = 23), polymyositis (PM; n = 6), and without neuromuscular disease (n = 11). Reverse transcription PCR of selected immunoglobulin gene transcripts was performed on two patient samples. Qualitative immunohistochemical studies for B-cell lineage cell surface markers were performed on 28 muscle specimens and quantitative studies performed on a subset of 19 untreated patients with IBM or PM. CD138+ cells were isolated from muscle using laser capture microdissection, and immunoglobulin transcripts were PCR amplified to determine the presence or absence of immunoglobulin gene rearrangements unique to the B-cell lineage. RESULTS: Immunoglobulin gene transcripts accounted for 59% in IBM and 33% in PM of the most stringently defined highest differentially expressed muscle transcripts compared with normal. Plasma cells, terminally differentiated B cells expressing CD138 but not CD19 or CD20, are present in IBM and PM muscle in numbers several times higher than B cells. CONCLUSIONS: There are differentiated B cells in the form of CD138+ plasma cells within the muscle of patients with inclusion body myositis and polymyositis. The principle of linked recognition of B-cell activation predicts several strategies for autoantigen discovery that could not otherwise be pursued through the study of the infiltrating T-cell population alone.
Asunto(s)
Músculo Esquelético/inmunología , Músculo Esquelético/patología , Miositis por Cuerpos de Inclusión/diagnóstico , Miositis por Cuerpos de Inclusión/inmunología , Células Plasmáticas/inmunología , Polimiositis/diagnóstico , Polimiositis/inmunología , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Autoantígenos/genética , Autoantígenos/inmunología , Linfocitos B/inmunología , Biomarcadores/metabolismo , Biopsia , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Inmunohistoquímica , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Músculo Esquelético/fisiopatología , Miositis por Cuerpos de Inclusión/fisiopatología , Células Plasmáticas/patología , Polimiositis/fisiopatología , Proteoglicanos/genética , Proteoglicanos/inmunología , ARN Mensajero/análisis , ARN Mensajero/genética , Sindecano-1 , Sindecanos , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Currently, there is substantial evidence that nuclear lipid metabolism plays a critical role in a number of signal transduction cascades. Previous work from our laboratory showed that stimulation of quiescent fibroblasts with alpha-thrombin leads to the production of two lipid second messengers in the nucleus: an increase in nuclear diacylglycerol mass and an activation of phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid. Diacylglycerol kinase (DGK) catalyzes the conversion of diacylglycerol to phosphatidic acid, making it an attractive candidate for a signal transduction component. There is substantial evidence that this activity is indeed regulated in a number of signaling cascades (reviewed by van Blitterswijk, W. J., and Houssa, B. (1999) Chem. Phys. Lipids 98, 95-108). In this report, we show that the addition of alpha-thrombin to quiescent IIC9 fibroblasts results in an increase in nuclear DGK activity. The examination of nuclei isolated from quiescent IIC9 cells indicates that DGK-theta and DGK-delta are both present. We took advantage of the previous observations that phosphatidylserine inhibits DGK-delta (reviewed by Sakane, F., Imai, S., Kai, M., Wada, I., and Kanoh, H. (1996) J. Biol. Chem. 271, 8394-8401), and constitutively active RhoA inhibits DGK-theta (reviewed by Houssa, B., de Widt, J., Kranenburg, O., Moolenaar, W. H., and van Blitterswijk, W. J. (1999) J. Biol. Chem. 274, 6820-6822) to identify the activity induced by alpha-thrombin. Constitutively active RhoA inhibited the nuclear stimulated activity, whereas phosphatidylserine did not have an inhibitory effect. In addition, a monoclonal anti-DGK-theta antibody inhibited the alpha-thrombin-stimulated nuclear activity in vitro. These results demonstrate that DGK-theta is the isoform responsive to alpha-thrombin stimulation. Western blot and immunofluorescence microscopy analyses showed that alpha-thrombin induced the translocation of DGK-theta to the nucleus, implicating that this translocation is at least partly responsible for the increased nuclear activity. Taken together, these data are the first to demonstrate an agonist-induced activity of nuclear DGK-theta activity and a nuclear localization of DGK-delta.