ABSTRACT
The tolerance of exercise and its effects on quality of life in myasthenia gravis are not currently backed up by strong evidence. The aim of this study was to determine whether exercise as an adjunct therapy is well tolerated and can improve health-related quality of life (HRQoL) in stabilized, generalized autoimmune myasthenia gravis (gMG). We conducted a parallel-group, multi-center prospective RCT using computer-generated block randomization. Adults with stabilized, gMG, and no contra-indication to exercise, were eligible. Participants received usual care alone or usual care and exercise. The exercise intervention consisted of 3-weekly 40 min sessions of an unsupervised, moderate-intensity home rowing program over 3 months. The primary endpoint was the change in HRQoL from randomization to post-intervention. Assessor-blinded secondary endpoints were exercise tolerance and effects on clinical, psychological and immunological status. Of 138 patients screened between October 2014 and July 2017, 45 were randomly assigned to exercise (nâ¯=â¯23) or usual care (nâ¯=â¯20). Although exercise was well tolerated, the intention-to-treat analysis revealed no evidence of improved HRQoL compared to usual care (MGQOL-15-F; mean adjusted between-groups difference of -0.8 points, 95%CI -5.4 to 3.7). Two patients hospitalized for MG exacerbation were from the usual care group.
Subject(s)
Exercise Therapy/methods , Myasthenia Gravis/therapy , Adult , Aged , Exercise , Exercise Tolerance , Female , Humans , Male , Middle Aged , Prospective Studies , Quality of LifeABSTRACT
Myasthenia gravis (MG) is an autoimmune disease caused by antibodies targeting the neuromuscular junction of skeletal muscles. Triple-seronegative MG (tSN-MG, without detectable AChR, MuSK and LRP4 antibodies), which accounts for ~10% of MG patients, presents a serious gap in MG diagnosis and complicates differential diagnosis of similar disorders. Several AChR antibody positive patients (AChR-MG) also have antibodies against titin, usually detected by ELISA. We have developed a very sensitive radioimmunoprecipitation assay (RIPA) for titin antibodies, by which many previously negative samples were found positive, including several from tSN-MG patients. The validity of the RIPA results was confirmed by western blots. Using this RIPA we screened 667 MG sera from 13 countries; as expected, AChR-MG patients had the highest frequency of titin antibodies (40.9%), while MuSK-MG and LRP4-MG patients were positive in 14.6% and 16.4% respectively. Most importantly, 13.4% (50/372) of the tSN-MG patients were also titin antibody positive. None of the 121 healthy controls or the 90 myopathy patients, and only 3.6% (7/193) of other neurological disease patients were positive. We thus propose that the present titin antibody RIPA is a useful tool for serological MG diagnosis of tSN patients.
Subject(s)
Autoantibodies/blood , Connectin/immunology , Myasthenia Gravis/blood , Myasthenia Gravis/diagnosis , Enzyme-Linked Immunosorbent Assay , Female , Humans , International Cooperation , LDL-Receptor Related Proteins/immunology , Male , Myasthenia Gravis/epidemiology , Radioimmunoprecipitation Assay , Receptor Protein-Tyrosine Kinases/immunology , Receptors, Cholinergic/immunologyABSTRACT
Seronegative myasthenia gravis (MG) presents a serious gap in MG diagnosis and understanding. We applied a cell based assay (CBA) for the detection of muscle specific kinase (MuSK) antibodies undetectable by radioimmunoassay. We tested 633 triple-seronegative MG patients' sera from 13 countries, detecting 13% as positive. MuSK antibodies were found, at significantly lower frequencies, in 1.9% of healthy controls and 5.1% of other neuroimmune disease patients, including multiple sclerosis and neuromyelitis optica. The clinical data of the newly diagnosed MuSK-MG patients are presented. 27% of ocular seronegative patients were MuSK antibody positive. Moreover, 23% had thymic hyperplasia suggesting that thymic abnormalities are more common than believed.
Subject(s)
Autoantibodies/blood , Myasthenia Gravis/blood , Myasthenia Gravis/diagnosis , Receptor Protein-Tyrosine Kinases/immunology , Adult , Aged , Female , Flow Cytometry , Humans , International Cooperation , LDL-Receptor Related Proteins/immunology , Male , Middle Aged , Myasthenia Gravis/pathology , Neuromyelitis Optica/diagnosis , Radioimmunoassay , Receptors, Cholinergic/immunology , Thymus Gland/pathology , Thymus Hyperplasia/diagnosisABSTRACT
Natural thymic T regulatory (tTreg) cells maintain tolerance to self-antigen. These cells are generated in the thymus, but how this generation occurs is still controversial. Furthermore, the contribution of thymus epithelial cells to this process is still unclear, especially in humans. Using an exceptional panel of human thymic samples, we demonstrated that medullary thymus epithelial cells (mTECs) promote the generation of tTreg cells and favor their function. These effects were mediated through soluble factors and were mTEC specific since other cell types had no such effect. By evaluating the effects of mTECs on the absolute number of Treg cells and their state of proliferation or cell death, we conclude that mTECs promote the proliferation of newly generated CD25+ cells from CD4+CD25- cells and protect Treg cells from cell death. This observation implicates Bcl-2 and mitochondrial membrane potential changes, indicating that the intrinsic cell death pathway is involved in Treg protection by mTECs. Interestingly, when the mTECs were cultured directly with purified Treg cells, they were able to promote their phenotype but not their expansion, suggesting that CD4+CD25- cells have a role in the expansion process. To explore the mechanisms involved, several neutralizing antibodies were tested. The effects of mTECs on Treg cells were essentially due to interleukin (IL)-2 overproduction by thymus CD4+ T cells. We then searched for a soluble factor produced by mTECs able to increase IL-2 production by CD4+ cells and could identify the inducible T-cell costimulator ligand (ICOSL). Our data strongly suggest a « ménage à trois ¼: mTEC cells (via ICOSL) induce overproduction of IL-2 by CD25- T cells leading to the expansion of tTreg cells. Altogether, these results demonstrate for the first time a role of mTECs in promoting Treg cell expansion in the human thymus and implicate IL-2 and ICOSL in this process.
Subject(s)
Epithelial Cells/immunology , Inducible T-Cell Co-Stimulator Ligand/immunology , Interleukin-2/immunology , T-Lymphocytes, Regulatory/immunology , Thymus Gland/cytology , Adolescent , Adult , CD4-Positive T-Lymphocytes/immunology , Carrier Proteins/immunology , Cell Cycle Proteins , Cells, Cultured , Child, Preschool , Coculture Techniques , Epithelial Cells/cytology , Female , Humans , Infant , Infant, Newborn , Male , T-Lymphocytes, Regulatory/cytology , Thymus Gland/immunology , Transcription Factors , Young AdultABSTRACT
Myasthenia gravis is characterized by muscle weakness and abnormal fatigability. It is an autoimmune disease caused by the presence of antibodies against components of the muscle membrane localized at the neuromuscular junction. In most cases, the autoantibodies are directed against the acetylcholine receptor (AChR). Recently, other targets have been described, such as muscle-specific kinase protein (MuSK) or lipoprotein related protein 4 (LRP4). The origin of the autoimmune response is not known, but thymic abnormalities and defects in immune regulation certainly play a major role in patients with anti-AChR antibodies. Genetic predisposition probably influences the occurrence of the disease. Sex hormones seem to play a role in the early form of the disease. Muscle weakness is fluctuating and worsens with exercise. Myasthenia gravis could be classified according to the location of the affected muscles (ocular versus generalized), the age of onset of symptoms, thymic abnormalities and profile of autoantibodies. These criteria are used to optimize the management and treatment of patients. In this review, we analyze the latest concepts of the pathophysiology of myasthenia gravis according to the different subgroups of the disease, including a description of the role of immunological, genetic and environmental factors. The potential viral hypothesis of this disease is discussed. Finally, we also discuss the biological assays available to validate the diagnosis.
Subject(s)
Autoantibodies/blood , Myasthenia Gravis/physiopathology , Humans , LDL-Receptor Related Proteins/immunology , Neuromuscular Junction/immunology , Receptor Protein-Tyrosine Kinases/immunology , Receptors, Cholinergic/immunology , Thymus Gland/physiopathologyABSTRACT
Double-seronegative myasthenia gravis (dSN-MG, without detectable AChR and MuSK antibodies) presents a serious gap in MG diagnosis and understanding. Recently, autoantibodies against the low-density lipoprotein receptor-related protein 4 (LRP4) have been identified in several dSN-MG sera, but with dramatic frequency variation (â¼2-50%). We have developed a cell based assay (CBA) based on human LRP4 expressing HEK293 cells, for the reliable and efficient detection of LRP4 antibodies. We have screened about 800 MG patient sera from 10 countries for LRP4 antibodies. The overall frequency of LRP4-MG in the dSN-MG group (635 patients) was 18.7% but with variations among different populations (range 7-32.7%). Interestingly, we also identified double positive sera: 8/107 anti-AChR positive and 10/67 anti-MuSK positive sera also had detectable LRP4 antibodies, predominantly originating from only two of the participating groups. No LRP4 antibodies were identified in sera from 56 healthy controls tested, while 4/110 from patients with other neuroimmune diseases were positive. The clinical data, when available, for the LRP4-MG patients were then studied. At disease onset symptoms were mild (81% had MGFA grade I or II), with some identified thymic changes (32% hyperplasia, none with thymoma). On the other hand, double positive patients (AChR/LRP4-MG and MuSK/LRP4-MG) had more severe symptoms at onset compared with any single positive MG subgroup. Contrary to MuSK-MG, 27% of ocular dSN-MG patients were LRP4 antibody positive. Similarly, contrary to MuSK antibodies, which are predominantly of the IgG4 subtype, LRP4 antibodies were predominantly of the IgG1 and IgG2 subtypes. The prevalence was higher in women than in men (female/male ratio 2.5/1), with an average disease onset at ages 33.4 for females and 41.9 for males. Overall, the response of LRP4-MG patients to treatment was similar to published responses of AChR-MG rather than to MuSK-MG patients.
Subject(s)
LDL-Receptor Related Proteins/immunology , Myasthenia Gravis/epidemiology , Myasthenia Gravis/immunology , Serologic Tests/methods , Thymus Gland/pathology , Adolescent , Adult , Age of Onset , Aged , Autoantibodies/blood , Child , Child, Preschool , Disease Progression , Female , HEK293 Cells , Humans , Hyperplasia , Immunoglobulin G/blood , Infant , Infant, Newborn , International Cooperation , Male , Middle Aged , Myasthenia Gravis/diagnosis , Receptor Protein-Tyrosine Kinases/immunology , Receptors, Cholinergic/immunology , Sex Factors , Young AdultABSTRACT
In autoimmune Myasthenia Gravis (MG), a neuromuscular disease generally mediated by autoantibodies against the acetylcholine receptor (AChR), the muscle is the target organ of the autoimmune attack, while the thymus seems to be the primary production site of the autoantibodies. In the majority of patients with anti-AChR antibodies, it is characterized by the presence of germinal centers, which contain B cells that produce anti-AChR antibodies. In this review, we summarize recent results regarding neoangiogenic processes, cell infiltration and modified chemokine expression in the MG thymus, which are typical features of secondary lymphoid organs. The structural and functional changes in the MG thymus therefore allow us to declare it to be an archetype for tertiary lymphoid neogenesis providing optimal settings for the interaction between lymphocytes and antigen presenting cells in order to elicit an immune response. We further discuss factors that may have a key role in the transformation of the MG thymus into a tertiary lymphoid organ, such as IFN type I and dsRNA signaling. These factors could also be of importance in other autoimmune diseases, especially those characterized by tertiary lymphoid neogenesis.
Subject(s)
Lymphoid Tissue/immunology , Myasthenia Gravis/physiopathology , Thymus Gland/physiology , Animals , Autoantibodies/metabolism , Humans , Lymphoid Tissue/growth & development , Myasthenia Gravis/pathology , Thymus Gland/pathology , Thymus Hyperplasia/immunology , Thymus Hyperplasia/metabolism , Virus Diseases/complications , Virus Diseases/immunologyABSTRACT
BACKGROUND: Genetic and environmental factors are thought to contribute to the etiology of the autoimmune disease myasthenia gravis (MG). Viral involvement has long been suspected, but direct evidence of involvement has not been found. We recently reported that Toll-like receptor 4 (TLR4)-a key activator of innate immunity-was overexpressed in the thymus of some patients with MG, suggesting that thymic infection by pathogens might be involved in MG pathogenesis. We searched for evidence of intrathymic infection in patients with MG. METHODS: Twenty-seven MG thymuses (6 involuted, 7 hyperplastic, 5 thymitis, and 9 thymoma) previously tested for TLR4 expression, 18 nonpathologic control thymuses, and 10 pathologic control thymuses from patients without MG (8 thymoma and 2 hyperplastic) were analyzed for cytomegalovirus, varicella-zoster virus, herpes simplex virus types 1 and 2, eubacteria, respiratory syncytial virus, and enteroviruses using PCR techniques. Immunohistochemistry and double immunofluorescence were used to detect enterovirus capsid protein VP1 in thymic specimens and analyze TLR4 expression in VP1-positive cells. RESULTS: Poliovirus was detected in 4 MG thymuses (14.8%; 2 thymitis and 2 thymoma). No virus was detected in any control thymus. A linear correlation between plus and minus strand poliovirus RNA levels was observed in all 4 thymuses, suggesting persistent thymic infection. VP1 protein was detected in the cytoplasm of CD68-positive macrophages scattered through thymic medulla in all PV-positive thymuses. VP1 and TLR4 colocalized in infected cells. CONCLUSIONS: Poliovirus-infected macrophages are present in thymus of some patients with myasthenia gravis, suggesting a viral contribution to the intrathymic alterations leading to the disease.
Subject(s)
Macrophages/virology , Myasthenia Gravis/immunology , Myasthenia Gravis/virology , Poliomyelitis/complications , Poliovirus/immunology , Thymus Gland/virology , Antigens, CD/analysis , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/analysis , Antigens, Differentiation, Myelomonocytic/metabolism , Capsid Proteins/analysis , Capsid Proteins/metabolism , Fluorescent Antibody Technique , Immunohistochemistry , Macrophages/pathology , Myasthenia Gravis/physiopathology , Poliovirus/genetics , Predictive Value of Tests , RNA, Viral/genetics , Thymus Gland/cytology , Toll-Like Receptor 4/analysis , Toll-Like Receptor 4/metabolismABSTRACT
Desmoglein (Dsg) 1 is a transmembrane glycoprotein of the desmosome allowing cell-cell adhesion between keratinocytes, whose expression is restricted to stratified squamous epithelia-like epidermis. Dsg1 is the target autoantigen of pathogenic autoantibodies produced by pemphigus foliaceus and 50% of pemphigus vulgaris patients in a Dsg1-specific T-cell-dependent pathway. Herewith, we show that mRNA of the DSG1 gene is present in normal human thymus and show by quantitative real-time polymerase chain reaction analysis that the expression of DSG1 transcript increases with age. Although immunoblot analysis on human thymus extracts using different anti-Dsg1 antibodies did not allow to detect the protein, we show by double-immunofluorescence assay that Dsg1 is expressed at protein level by CD19+ CD63+ cells located in the medulla. These data provide another illustration of the thymic expression of a tissue-specific autoantigen involved in an organ-specific autoimmune disease, which may participate in the tolerance acquisition and/or regulation of Dsg1-specific T cells.
Subject(s)
Autoantigens/metabolism , Desmoglein 1/metabolism , Pemphigus/immunology , Thymus Gland/immunology , Adolescent , Adult , Aging , Autoantigens/immunology , Autoimmunity , Child , Child, Preschool , Desmoglein 1/genetics , Desmoglein 1/immunology , Epidermal Cells , Epidermis/immunology , Epidermis/metabolism , Female , Humans , Infant , Infant, Newborn , Keratinocytes/cytology , Keratinocytes/immunology , Keratinocytes/metabolism , Male , Middle Aged , Pemphigus/genetics , Pemphigus/metabolism , Thymus Gland/cytology , Thymus Gland/metabolismABSTRACT
Thymic myoid cells correspond to a muscle-like cell population present in the thymic medulla. They are well conserved throughout species evolution, but their biological role is not known. We demonstrated that myoid cells protected thymocytes from apoptosis as evidenced by a strong decrease of annexin-V-FITC positive thymocytes. This effect was (1) specific of myoid cells compared to thymic epithelial cells; (2) dependent on direct cell-to-cell contacts and (3) triggered rapidly after 2 h in cocultures. This protective phenomenon was due to the activation of prosurvival mechanisms. Indeed, myoid cells activated extracellular-regulated kinases (ERK1/2) and Akt in thymocytes. Myoid cells also influenced thymocyte maturation. We observed an increase in CD4(+) and a decrease in CD8(+) single positive (SP) thymocytes when cocultured with myoid cells, independently of a CD8(+)SP increased death or a CD4(+)SP overproliferation. Consequently, thymic myoid cells protect thymocytes from apoptosis and could also modulate their differentiation process.
Subject(s)
Apoptosis , Cell Differentiation , Signal Transduction , Thymus Gland/cytology , Thymus Gland/metabolism , Annexin A5/metabolism , Blotting, Western , Cell Line, Transformed , Coculture Techniques , Enzyme Activation , Flow Cytometry , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Time FactorsABSTRACT
The present study is aimed at exploring the regulatory CD4(+)CD25(+) T cells in the thymus from myasthenia gravis (MG) patients. In early-onset MG, the thymus is hyperplastic and contains autoreactive activated T cells. Preliminary studies indicate that these CD4(+)CD25(+) cells include activated autoreactive T cells. Studies to characterize the phenotype and suppressive capacity of these cells will be discussed.
Subject(s)
CD4 Antigens/metabolism , CD4-Positive T-Lymphocytes/immunology , Myasthenia Gravis/pathology , Receptors, Interleukin-2/metabolism , Thymus Gland/pathology , CD8 Antigens/immunology , Flow Cytometry/instrumentation , Flow Cytometry/methods , Humans , Myasthenia Gravis/metabolism , ThymectomySubject(s)
Myasthenia Gravis/drug therapy , Receptors, Antigen, T-Cell/therapeutic use , Adolescent , Adult , Child , Child, Preschool , Enzyme-Linked Immunosorbent Assay , HLA-DR3 Antigen/immunology , Humans , Immunoglobulin G/metabolism , Infant , Myasthenia Gravis/blood , Myasthenia Gravis/immunology , Peptides/therapeutic use , Receptors, Antigen, T-Cell/classification , Receptors, Antigen, T-Cell/immunology , Thymus Hyperplasia/blood , Thymus Hyperplasia/complications , Thymus Hyperplasia/therapyABSTRACT
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.
Subject(s)
Muscle, Skeletal/metabolism , Myasthenia Gravis/metabolism , Receptor, Ciliary Neurotrophic Factor/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Blood Proteins/pharmacology , Cells, Cultured/drug effects , Cells, Cultured/immunology , Cells, Cultured/metabolism , Female , Gene Expression/drug effects , Gene Expression/immunology , Humans , Male , Middle Aged , Muscle, Skeletal/pathology , Muscle, Skeletal/physiopathology , Myasthenia Gravis/immunology , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Receptors, Cholinergic/genetics , Receptors, Cholinergic/immunology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
We have previously shown that intrathymic (i.t.) injection of staphylococcal enterotoxin B (SEB) to mice induces both T cell clonal deletion and IL-2-dependent anergy. In the present study, we have used a quantitative RT-PCR to demonstrate that i.t. administration of SEB induced a significant decrease in the levels of the IL-2 and IFN-gamma mRNAs in total splenocytes, from day 7 to day 28 post-injection. I.t. SEB injection also induced a significant increase in the levels both of IL-10 and TGF-beta mRNAs on day 7, leading to a significant enhance in the IL-10 + TGF-beta/IL-2 + IFN-gamma mRNA ratio on days 7 and 28. By contrast, IL-10 and TGF-beta mRNAs were unchanged after intraperitoneal (i.p.) or subcutaneous (s.c.) SEB injections, although both IL-2 and IFN-gamma mRNA levels were decreased. The cytokine mRNA ratio was enhanced on days 7 and 28 after i.p. injection. Interestingly, a cytokine mRNA ratio of a least 10 in favour of IL-10 plus TGF-beta mRNAs was correlated with the hyporesponsive state observed in vitro after i.t. and i.p. injections. Our results clearly demonstrate that i.t. SEB administration induces a switch from Th1-type to Th2-type cytokine expression in the spleen. The deviation from IL-2 plus IFN-gamma towards IL-10 plus TGF-beta expression could be responsible for the immunoregulatory effect exerted upon SEB-reactive T cells, which is characterized by an IL-2-dependent, specific anergy in vitro. Moreover, it highlights the crucial role of the route of SEB injection in the pattern of cytokine expression.
Subject(s)
Enterotoxins/administration & dosage , Interferon-gamma/antagonists & inhibitors , Interleukin-10/agonists , Interleukin-2/antagonists & inhibitors , RNA, Messenger/metabolism , Spleen/immunology , Transforming Growth Factor beta/agonists , Animals , Clonal Anergy/drug effects , Cytokines/drug effects , Cytokines/genetics , Cytokines/metabolism , Female , Injections, Intralymphatic , Injections, Intraperitoneal , Injections, Subcutaneous , Interferon-gamma/genetics , Interferon-gamma/metabolism , Interleukin-10/genetics , Interleukin-10/metabolism , Interleukin-2/genetics , Interleukin-2/metabolism , Mice , Mice, Inbred BALB C , RNA, Messenger/drug effects , Spleen/cytology , Th1 Cells/metabolism , Th2 Cells/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolismABSTRACT
Most thymocytes are deleted by thymic selection. The mechanisms of cell death are far from being clear. Peroxynitrite is a powerful oxidant produced in vivo by the reaction of superoxide (O2*-) with nitric oxide (NO*) and is able to mediate apoptosis. The aim of this study was to analyze whether NO and peroxynitrite could play a role in human thymocyte apoptosis. The results indicate that 3-(4-morpholinyl)-sydnonimine (SIN-1, an O2*- and NO* donor) and chemically synthesized peroxynitrite, but not S-nitroso-N-acetyl-D,L-penicillamine (SNAP, an NO* donor), have a strong apoptotic effect on human thymocytes (annexin V staining and TUNEL reaction). This effect was inhibited by exogenous superoxide dismutase (SOD), which interacts with O2*- and inhibits the formation of peroxynitrite. Because peroxynitrite formation requires NO*, thymic stromal cells were investigated to determine if they produced NO*. Inducible NOS was synthesized in cultured thymic epithelial cells in certain conditions of cytokine stimulation, as shown by messenger RNA levels, protein analysis, and nitrite production in the supernatants. SIN-1-treated thymocytes had high levels of tyrosine nitration, abolished by the addition of exogenous SOD. Tyrosine nitration was also detected in thymus extracts and sections, suggesting the presence of peroxynitrite in situ. In thymus sections, clusters of nitrotyrosine-positive cells were found in the cortex and corticomedullary areas colocalized with cells positive in the TUNEL reaction. These data indicate an association between human thymocyte apoptosis and nitrotyrosine formation. Thus, the results support the notion of a physiologic role for peroxynitrite in human thymocyte apoptosis. (Blood. 2001;97:3521-3530)
Subject(s)
Apoptosis , Thymus Gland/cytology , Thymus Gland/metabolism , Tyrosine/analogs & derivatives , Tyrosine/metabolism , Annexin A5/analysis , Apoptosis/drug effects , Cells, Cultured , Child, Preschool , Enzyme Inhibitors/pharmacology , Epithelial Cells/metabolism , Gene Expression , Humans , In Situ Nick-End Labeling , Molsidomine/analogs & derivatives , Molsidomine/pharmacology , Nitrates/metabolism , Nitrates/pharmacology , Nitric Oxide/metabolism , Nitric Oxide Donors/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , Stromal Cells/metabolism , Superoxide Dismutase/pharmacology , Superoxides/metabolism , T-Lymphocytes/cytology , Thymus Gland/chemistry , Tyrosine/analysisABSTRACT
Three distinct dendritic cell (DC) subsets capable of stimulating allogeneic naive T cells were isolated from human thymus. The most abundant subset was represented by plasmacytoid DCs (pDCs), which secreted high amounts of IFN-alpha upon stimulation with inactivated influenza virus and thus likely correspond to the recently identified peripheral blood natural IFN-alpha/beta-producing cells (IPCs). Like those latter cells, thymic pDCs had distinctive phenotypic features (i.e., Lin(-), HLA-DR(int), IL-3R alpha(hi), CD45RA(hi), CD11c(-), CD13(-), and CD33(lo)) and developed into mature DCs upon culture in IL-3 and CD40L. Of the two other DC subsets, one displayed a phenotype of immature myeloid DCs (imDCs) (HLA-DR(int), CD11c(+), CD13(+), CD33(+)), and the other represented HLA-DR(hi) CD11c(+) mature DCs (mDCs). Since they also expressed DC-LAMP, these mDCs appear to correspond to interdigitating dendritic cells (IDCs). Thymic pDCs, but not myeloid imDCs, strongly expressed lymphoid-specific transcripts such as pre-T alpha, lambda-like, and Spi-B, thereby suggesting a possible lymphoid origin. The detection of Spi-B mRNA, not only upon in vitro maturation of pDCs, but also in freshly purified IDCs, suggests that in vivo pDCs may differentiate into IDCs.
Subject(s)
Dendritic Cells/classification , Integrin alphaXbeta2 , Thymus Gland/cytology , Adolescent , CD40 Ligand/pharmacology , Cell Separation , Child , Child, Preschool , Dendritic Cells/immunology , Dendritic Cells/metabolism , Humans , Infant , Interferon-alpha/pharmacology , Interleukin-3/pharmacology , Orthomyxoviridae/immunology , RNA, Messenger , Receptors, Interleukin-3/geneticsABSTRACT
To establish new tools for studying human thymic stromal cells, we transfected adherent cells from a human postnatal thymus using a plasmid encoding SV40 large T antigen. Among the cell lines obtained, we characterized four epithelial cell lines (LT-TEC1 to LT-TEC4) and one thymic myoid cell line (MITC). Several morphological, functional and phenotypic differences were observed between these 2 cell types. Epithelial cells were heterogeneous and larger than myoid cells. Untreated LT-TEC lines expressed MHC class I, ICAM-1 and LFA-3 antigens and not MHC class II antigens, similarly to primary thymic epithelial cells (PTEC), while MITC line expressed only class I and LFA-3 antigens. After IFN-gamma treatment, MHC class II and ICAM-1 antigens were markedly upregulated in LT-TEC lines but not in MITC, indicating the absence or a dysfunction of regulatory factors in MITC line. Myoid cells expressed mRNA for all the subunits of the acetylcholine receptor (AChR) while epithelial cells expressed only the alpha, beta and epsilon subunits. Strikingly, LT-TEC produced much more C-C chemokines and IL-6 than MITC cells, while these latter produced higher levels of IL-8 and TNF-alpha. Altogether, these results reveal phenotypic and functional differences between these two stromal cell types, suggesting a potential involvement of myoid cells in the thymic function.
Subject(s)
Thymus Gland/cytology , Animals , Antigens, Polyomavirus Transforming/genetics , CD58 Antigens/biosynthesis , Cell Line, Transformed , Cells, Cultured , Cytokines/biosynthesis , Histocompatibility Antigens Class I/biosynthesis , Histocompatibility Antigens Class II/biosynthesis , Humans , Infant , Intercellular Adhesion Molecule-1/biosynthesis , Interferon-gamma/immunology , Interferon-gamma/pharmacology , Mice , Mice, Inbred BALB C , Phenotype , Receptors, Cholinergic/biosynthesis , Stromal Cells/classification , Stromal Cells/cytology , Stromal Cells/drug effects , Stromal Cells/physiologyABSTRACT
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.
Subject(s)
DNA-Binding Proteins/metabolism , Genes, MHC Class I , HLA Antigens/genetics , Histocompatibility Antigens Class I/genetics , Interferon-beta/pharmacology , Phosphoproteins/metabolism , Promoter Regions, Genetic , Response Elements , Amnion/metabolism , Female , HLA-G Antigens , Humans , Interferon Regulatory Factor-1 , RNA, Messenger/analysis , Transcriptional Activation , Trophoblasts/metabolism , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Myasthenia Gravis/blood , Myasthenia Gravis/immunology , Receptors, Cholinergic/immunology , Adolescent , Adult , Female , Humans , Male , Middle Aged , Muscles/immunology , RNA, Messenger/blood , RNA, Messenger/immunology , Receptors, Cholinergic/bloodABSTRACT
Myasthenia Gravis (MG) is an autoimmune disease mediated by antibodies directed against the acetylcholine receptor (AChR). Treatment by IVIg is effective in acute forms of myasthenia gravis. In order to determine the in vivo effects of the various fractions of human immunoglobulins, we used an experimental model of myasthenia gravis in SCID mice. To this end, thymic cells from MG patients are transferred to these mice according to a well defined protocol. When establishing of the model, we noticed the appearance of anti-AChR antibodies and the loss of AChR expression at the muscle level. After treatment with IVIgG or IVIgM, the mice displayed a lower anti-AChR antibody titer compared to control mice (albumin treated) and the loss of the AChR number at the muscle was significantly reduced. These results obtained from one MG patient indicate that the human immunoglobulin preparations induce significant effects on pathogenic parameters in the SCID mouse model. Therefore this model is interesting to approach the mechanisms of action of human immunoglobulins and deserves further investigation.