Expression of ciliary neurotrophic factor receptor in myasthenia gravis.

In myasthenia gravis, high anti-nicotinic receptor (AChR) antibody titers are not always associated with severity of the disease, suggesting the involvement of other pathological effectors. We showed that ciliary neurotrophic factor receptor (CNTFR) gene expression was higher in muscle biopsy tissue from severely affected MG patients regardless of anti-nAChR antibody titer. This increase was also triggered in vitro by a seric factor from MG patients. CNTFR protein expression was decreased in muscles from seropositive MG patients only.Altogether, our data indicate that the alteration of CNTFR expression in some MG patients could contribute to the severity of the disease in a subgroup of patients.

A specific interferon (IFN)-stimulated response element of the distal HLA-G promoter binds IFN-regulatory factor 1 and mediates enhancement of this nonclassical class I gene by IFN-beta.

Type I interferons display a broad range of immunomodulatory functions. Interferon beta increases gene expression at the transcriptional level through binding of factors to the interferon-stimulated response element (ISRE) within the promoters of interferon-inducible genes, such as HLA class I. Despite mutation of the class I ISRE sequence within the nonclassical HLA-G class I gene promoter, we show that interferon beta enhances both transcription and cell surface expression of HLA-G in trophoblasts and amniotic and thymic epithelial cells that selectively express it in vivo. Deletion and mutagenesis analysis of a putative interferon-regulatory factor (IRF)-1 binding site within the HLA-G promoter show that HLA-G transactivation is mediated through an ISRE sequence 746 base pairs upstream from ATG, which is distinct from the interferon-responsive element described within proximal classical class I gene promoters. Electrophoretic mobility shift analysis and supershift analysis further demonstrate that interferon-responsive transcription factors, including IRF-1, specifically bind to the HLA-G ISRE. Our results provide evidence that IRF-1 binding to a functional ISRE within the HLA-G promoter mediates interferon beta-induced expression of the HLA-G gene. These observations are of general interest considering the implication of HLA-G in mechanisms of immune escape involved in fetal-maternal tolerance and other immune privilege situations.

Modulation of acetylcholine receptor expression in seronegative myasthenia gravis.

In a previous study, we demonstrated a compensatory mechanism for regulating acetylcholine receptor (AChR) gene expression in muscle biopsies from seropositive and seronegative (SN) myasthenia gravis (MG) patients. To further characterize the AChR regulation mechanisms involved in SNMG disease, we investigated the effects of MG sera on nicotinic AChR expression (at the protein and messenger RNA [mRNA] levels) in cultured human muscle cells. Sera from SNMG patients induced an in vitro increase in the level of nicotinic AChR beta-subunit mRNA but did not cause a decrease in AChR protein level. This apparent discrepancy was not due to a higher level of AChR synthesis as demonstrated by analysis of AChR turnover. In SN patients, the increase in beta-subunit mRNA level was followed after 48 hours by a slight increase in the amount of AChR surface protein. This regulation of nicotinic receptor expression was due to the purified IgG-containing fraction. Thus, sera from SNMG patients contain an immunoglobulin that induces an increase in AChR mRNA without causing a decrease in AChR protein level, suggesting an indirect regulatory mechanism involving another surface molecule. This model is therefore useful for defining the targets involved in the pathogenesis of SNMG disease.

Establishment of a human thymic myoid cell line. Phenotypic and functional characteristics.

The subset of myoid cells is a normal component of the thymic stroma. To characterize these cells, we immortalized stromal cells from human thymus by using a plasmid vector encoding the SV40 T oncogene. Among the eight cell lines obtained, one had myoid characteristics including desmin and troponin antigens. This new line was designated MITC (myoid immortalized thymic cells). These cells expressed both the fetal and adult forms of muscle acetylcholine receptor (AChR) at the mRNA level, as well as the myogenic transcription factor MyoD1. alpha-Subunit AChR protein expression was detected by flow cytometry and the AChR was functional in patch-clamp studies. In addition, AChR expression was down-modulated by myasthenia gravis sera or by monoclonal antibody anti-AChR on MITC line similarly to TE671 rhabdomyosarcoma cells, making the MITC line an interesting tool for AChR antigenic modulation experiments. Finally, the MITC line expressed LFA-3, produced several cytokines able to act on T cells, and protected total thymocytes from spontaneous apoptosis in vitro. These results are compatible with a role of thymic myoid cells in some steps of thymocyte development. Therefore MITC line appears to be a useful tool to investigate the physiological role of thymic myoid cells.

Regulation of acetylcholine receptor gene expression in human myasthenia gravis muscles. Evidences for a compensatory mechanism triggered by receptor loss.

Myasthenia gravis (MG) is a neuromuscular disorder mediated by antibodies directed against the acetylcholine receptor (nAChR) resulting in a functional nAChR loss. To analyze the molecular mechanisms involved at the muscular target site, we studied the expression of nAChR subunits in muscle biopsy specimens from MG patients. By using quantitative PCR with an internal standard for each subunit, we found that the levels of beta-, delta-, and epsilon-subunit mRNA coding for the adult nAChR were increased in severely affected MG patients, matching our previous data on the alpha-subunit. Messenger levels were highly variable in MG patients but not in controls, pointing to individual factors involved in the regulation of nAChR genes. The fetal subunit (gamma-chain) transcripts were almost undetectable in the extrajunctional region of MG muscle, suggesting that gene regulation in MG differs from that in the denervation model, in which nAChR gamma-subunit mRNA is reexpressed. Nicotinic AChR loss mediated by monoclonal anti-nAChR antibodies in both the TE671 muscle cell line and cultured normal human myotubes induces a similar increase in beta- alphand delta-subunit mRNA levels, suggesting the existence of a new muscular signaling pathway system coupled to nAChR internalization and independent of muscle electrical activity. These data demonstrate the existence of a compensatory mechanism regulating the expression of the genes coding for the adult nAChR in patients with MG.