transforming-growth-factor-beta and Myasthenia-Gravis

Thymoma is a rare characterized by a unique association with autoimmune diseases, especially myasthenia gravis (MG). However, little is known about the molecular characteristics of MG-associated thymoma individuals. We aim to examine the influences of MG on thymoma by analyzing multiomics data. A total of 105 samples with thymoma was analyzed from TCGA and these samples were divided into subgroups with MG (MGT) or without MG (MGF) according to clinical information. We then characterized the differential gene expression, pathway activity, somatic mutation frequency, and likelihood of responding to chemotherapies and immunotherapies of the two identified subgroups. MGT subgroup was characterized by elevated inflammatory responses and metabolically related pathways, whereas the MGF subgroup was predicted to be more sensitive to chemotherapy and presented with mesenchymal characteristics. More copy number amplifications and deletions were observed in MGT, whereas GTF2I mutations occur at significantly higher frequencies in MGF. Two molecular subtypes were further identified within MGF samples by unsupervised clustering where one subtype was enriched in TGF-β and WNT pathways with higher sensitivity to relevant targeted drugs but hardly respond to immunotherapy. For another subtype, a higher recurrence rate of thymoma and more likelihood of responding to immunotherapy were observed. Our findings presented a comprehensive molecular characterization of thymoma patients given the status of MG, and provided potential strategies to help individualized management and treatment.

Topics: Aged; Disease-Free Survival; DNA Copy Number Variations; Drug Therapy; Female; Gene Expression Regulation, Neoplastic; Humans; Immunotherapy; Male; Middle Aged; Myasthenia Gravis; Neoplasm Proteins; Precision Medicine; Thymoma; Transcription Factors, TFII; Transforming Growth Factor beta; Wnt Signaling Pathway

Myasthenia gravis (MG) and experimental autoimmune MG are T cell-dependent antibody-mediated autoimmune diseases. A dual altered peptide ligand (APL), composed of the tandemly arranged two single amino acid analogs of two myasthenogenic peptides, p195-212 and p259-271, down-regulated in vitro and in vivo MG-associated T cell responses. In the present study, we investigated the role of CD8(+)CD28(-) regulatory cells in the mechanism of action of the dual APL. We demonstrated that treatment of mice with the dual APL concomitant with immunization with a myasthenogenic peptide resulted in an increased population of CD8(+)CD28(-) cells that express forkhead box P3 (Foxp3). The dual APL inhibited the proliferation of lymph node (LN) cells of the Torpedo acetylcholine receptor-immunized WT C57BL/6 mice, whereas the inhibition was abrogated in CD8(-/-) knockout mice. Moreover, the dual APL did not inhibit the secretion of IFN-gamma by LN cells from CD8(-/-) mice immunized with Torpedo acetylcholine receptor. However, the mRNA expression of IL-10 and TGF-beta by LN cells from CD8(-/-) mice was up-regulated similarly to that of the WT mice. Furthermore, the dual APL elevated the proapoptotic markers caspases 3 and caspase 8, whereas it down-regulated the antiapoptotic marker Bcl-xL in both CD8(-/-) and WT mice. Finally, the dual APL-induced CD4(+)CD25(+)Foxp3(+) cells were up-regulated in CD8(-/-) mice to a similar extent to that observed in the WT mice. Thus, we suggest that CD8(+)CD28(-) regulatory cells play a partial role in the mechanism of action by which the dual APL suppresses experimental autoimmune MG-associated T cell responses.

Topics: Animals; bcl-X Protein; Caspase 3; Caspase 8; CD28 Antigens; CD4-Positive T-Lymphocytes; CD8 Antigens; CD8-Positive T-Lymphocytes; Cell Proliferation; Cells, Cultured; Down-Regulation; Female; Forkhead Transcription Factors; Immunization; Interleukin-10; Interleukin-2 Receptor alpha Subunit; Ligands; Mice; Mice, Knockout; Myasthenia Gravis; Peptides; Receptors, Nicotinic; Torpedo; Transforming Growth Factor beta; Up-Regulation

We report a case of myasthenia gravis associated with marked expansion of an unusual CD16(+)CD56(-)2B4(+) NK subset. These atypical cells were characterized by poor cytotoxicity against CD48(+) target cells and high proliferation due to 2B4/CD48 interaction. IL18, IFN-gamma and TGF-beta levels were profoundly different in this patient than in healthy donors. Immunosuppressive treatment induced disease remission and decreased the CD16(+)CD56(-)2B4(+)NK cells count. Our data suggest that expansion of this NK subset in myasthenia gravis patients may account for the deleterious NK cell functioning that occurs in this autoimmune disease.

Topics: Aged; Anti-Inflammatory Agents; Antigens, CD; Azathioprine; CD56 Antigen; Cholinesterase Inhibitors; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Immunosuppressive Agents; Interferon-gamma; Interleukin-18; Killer Cells, Natural; Lymphocyte Subsets; Male; Membrane Glycoproteins; Myasthenia Gravis; Phenotype; Pyridostigmine Bromide; Receptors, IgG; Receptors, Immunologic; Signaling Lymphocytic Activation Molecule Family; Transforming Growth Factor beta

Topics: Antigens, CD; Antigens, Differentiation, B-Lymphocyte; Cell Adhesion Molecules; Child; DNA-Binding Proteins; Humans; Hyperplasia; Immunohistochemistry; Keratins; Lectins; Myasthenia Gravis; Nerve Tissue Proteins; Nuclear Proteins; Receptors, Steroid; Receptors, Thyroid Hormone; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialic Acid Binding Ig-like Lectin 2; Thymoma; Thymus Gland; Transforming Growth Factor beta; Transforming Growth Factor beta1

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T-cell regulated, antibody-mediated diseases. Peptides p195-212 and p259-271 of the human acetylcholine receptor (AChR) alpha-subunit, were previously shown to be immunodominant T cell epitopes in MG patients as well as in SJL and BALB/c mice, respectively. A dual altered peptide ligand (APL) composed of the two single amino acid analogs of the myasthenogenic peptides was shown to inhibit, in vitro and in vivo, MG-associated autoimmune responses. Furthermore, the dual APL was shown to down-regulate the clinical manifestations of an established EAMG in C57BL/6 mice injected with Torpedo AChR (TAChR). In the present study we attempted the elucidation of the mechanism(s) by which the dual APL down-regulates EAMG-associated responses. It is shown here that the dual APL acts by actively suppressing, in a specific manner, myasthenogenic T cell responses. The active suppression is mediated, at least partially, by the up-regulation of the secretion of TGF-beta following administration of the dual APL. The up-regulated secretion of TGF-beta is accompanied by down-regulation of IFN-gamma and IL-2 [T helper (Th) 1-type cytokine] secretion and by an up-regulation of IL-10 secretion (Th2-type cytokine). Furthermore, the inhibitory effect of the dual APL could be adoptively transferred to p195-212 or TAChR-immunized mice. The down-regulation of IL-2 secretion and the ability of recombinant IL-2 to rescue lymph node cells of mice treated with the dual APL from a state of unresponsiveness suggests that the dual APL acts also, at least partially, by causing the cells to undergo anergy.

Topics: Amino Acid Sequence; Animals; Cytokines; Female; Immunization; Interleukin-2; Interleukin-4; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Myasthenia Gravis; Peptide Fragments; Receptors, Cholinergic; T-Lymphocytes; Torpedo; Transforming Growth Factor beta

The roles that thymus cytokines might play in regulating thymic atrophy are not known. Reversing thymic atrophy is important for immune reconstitution in adults. We have studied cytokine mRNA steady-state levels in 45 normal human (aged 3 days to 78 years) and 34 myasthenia gravis thymuses (aged 4 to 75 years) during aging, and correlated cytokine mRNA levels with thymic signal joint (sj) TCR delta excision circle (TREC) levels, a molecular marker for active thymopoiesis. LIF, oncostatin M (OSM), IL-6, M-CSF, and stem cell factor (SCF) mRNA were elevated in normal and myasthenia gravis-aged thymuses, and correlated with decreased levels of thymopoiesis, as determined by either decreased keratin-positive thymic epithelial space or decreased thymic sjTRECs. IL-7 is a key cytokine required during the early stages of thymocyte development. Interestingly, IL-7 mRNA expression did not fall with aging in either normal or myasthenia gravis thymuses. In vivo administration of LIF, OSM, IL-6, or SCF, but not M-CSF, i.p. to mice over 3 days induced thymic atrophy with loss of CD4+, CD8+ cortical thymocytes. Taken together, these data suggest a role for thymic cytokines in the process of thymic atrophy.

Topics: Adolescent; Adult; Aged; Aging; Animals; Atrophy; Child; Child, Preschool; Epithelial Cells; Extracellular Space; Female; Gene Expression Regulation; Gene Rearrangement, delta-Chain T-Cell Antigen Receptor; Growth Inhibitors; Humans; Infant; Infant, Newborn; Injections, Intraperitoneal; Interleukin-6; Leukemia Inhibitory Factor; Lymphokines; Macrophage Colony-Stimulating Factor; Mice; Mice, Inbred BALB C; Middle Aged; Myasthenia Gravis; Oncostatin M; Peptides; RNA, Messenger; Stem Cell Factor; Thymus Gland; Transforming Growth Factor beta

The mucosal administration of the native antigen or peptide fragments corresponding to immunodominant regions is effective in preventing or treating several T cell-dependent models of autoimmune disease. No data are yet available on oral tolerance with immunodominant T-cell peptides in experimental autoimmune myasthenia gravis (EAMG), an animal model of B cell-dependent disease. We report that oral administration of the T-cell epitope alpha146-162 of the Torpedo californica acetylcholine receptor (TAChR) alpha-subunit suppressed T-cell responses to AChR and ameliorated the disease in C57Bl/6 (B6) mice. Protection from EAMG was associated with reduced serum Ab's to mouse AChR and reduced AChR loss in muscle. The effect of Talpha146-162 feeding was specific; treatment with a control peptide did not affect EAMG manifestations. The protective effect induced by peptide Talpha146-162 was mediated by reduced production of IFN-gamma, IL-2, and IL-10 by TAChR-reactive cells, suggesting T-cell anergy. TGF-beta-secreting Th3 cells did not seem to be involved in tolerance induction. We therefore demonstrate that feeding a single immunodominant epitope can prevent an Ab-mediated experimental model of autoimmune disease.

Topics: Administration, Oral; Animals; Cytokines; Dose-Response Relationship, Drug; Down-Regulation; Epitopes, T-Lymphocyte; Mice; Myasthenia Gravis; Peptides; Receptors, Cholinergic; Th1 Cells; Th2 Cells; Transcription, Genetic; Transforming Growth Factor beta

Oral tolerization with acetylcholine receptor (AChR) and myelin basic protein (MBP) prior to immunization with AChR+MBP+ complete Freund's adjuvant (CFA) alleviated clinical signs of experimental autoimmune myasthenia gravis (EAMG)+experimental allergic encephalomyelitis (EAE) and AChR- or MBP-specific T and B cell responses. Tolerance induced via the nasal route needs much less tolerogen and may still be as effective as oral tolerance induction. We now immunized Lewis rats with AChR+MBP+bovine peripheral nerve myelin (BPM)+CFA, which resulted in a multiphasic clinical picture with a combination of clinical signs of the EAMG+EAE+experimental allergic neuritis (EAN), accompanied by massive macrophage infiltrations in sections of muscle, spinal cord and sciatic nerve, and strong T and B cell responses to AChR, MBP and BPM in lymphoid organs. Nasal administration of microg doses of AChR+MBP+BPM prior to immunization with a mixture of these antigens+CFA effectively suppressed the incidence and severity of clinical disease, reduced macrophage infiltrations in sections of muscle, spinal cord and sciatic nerve, and down-regulated autoreactive T cell responses to the three antigens in lymphoid organs. Numbers of AChR-, MBP-, BPM-reactive Th1 type of cytokine interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha mRNA expression in lymph node cells were markedly suppressed, while transforming growth factor-beta (TGF-beta) mRNA expression was upregulated from nasally tolerized rats, suggesting an active suppression mechanism may act partly in the induction of tolerance. The results implicate the possibility to establish multiple autoantigen-based vaccination for the prevention of autoimmune diseases in humans.

Topics: Administration, Intranasal; Animals; Autoantigens; Autoimmune Diseases; Cattle; Encephalomyelitis, Autoimmune, Experimental; Epitopes; Female; Immune Tolerance; Immunoglobulin G; Immunohistochemistry; Immunosuppressive Agents; Interferon-gamma; Interleukin-10; Leukocytes, Mononuclear; Muscle Weakness; Myasthenia Gravis; Myelin Basic Protein; Myelin Sheath; Neuritis, Autoimmune, Experimental; Rats; Rats, Inbred Lew; Receptors, Cholinergic; RNA, Messenger; T-Lymphocytes; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Adolescent; Adult; Aged; Cells, Cultured; Child; Female; Humans; Hyperplasia; Interferon-gamma; Interleukin-4; Leukocytes, Mononuclear; Male; Middle Aged; Myasthenia Gravis; Reference Values; Thymoma; Thymus Gland; Thymus Neoplasms; Transforming Growth Factor beta

Tolerance to experimental autoimmune myasthenia gravis by nasal administration of microgram amounts of acetylcholine receptor (AChR) has been reported. To elucidate the mechanisms behind tolerance induction via the respiratory tract and the involvement of CD4(+) T cells, we established AChR-specific CD4(+)CD8(-) T cell clones from nasally tolerized rats. Nasal tolerance decreased leukocyte function-associated antigen-1 (LFA-1) expression in CD4(+) T cells from tolerized rats. There was no difference between nasally tolerized and control rats in expression of intercellular adhesion molecule-1. The levels of transforming growth factor-beta (TGF-beta) mRNA-expressing cells were upregulated in CD4(+) T cell clones after tolerance induction. These findings suggest that decreased LFA-1 expression in CD4(+) T cells contributes to reduction of the infiltration of inflammatory CD4(+) T cells, while upregulated TGF-beta may inhibit lymphocyte functions.

Topics: Administration, Intranasal; Animals; Autoantigens; CD4-Positive T-Lymphocytes; Clone Cells; Cytokines; Disease Models, Animal; Down-Regulation; Female; Intercellular Adhesion Molecule-1; Lymphocyte Function-Associated Antigen-1; Muscle, Skeletal; Myasthenia Gravis; Rats; Rats, Inbred Lew; Receptors, Cholinergic; RNA, Messenger; Self Tolerance; Transforming Growth Factor beta; Up-Regulation

Clinical remission in myasthenia gravis after thymectomy and immune suppressive treatment may be reflected by changes of immunoregulatory cytokines. Mononuclear cells from paired blood samples from fifteen patients with myasthenia gravis before and after thymectomy and immunosuppressive therapy were examined by in situ hybridization for acetylcholine receptor-induced mRNA expression of the T helper type 1 proinflammatory cytokines interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha) and TNF-beta, the T helper type 2 cytokines interleukin 4 (IL-4) and IL-10 and the immune response downregulating cytokine tumor growth factor beta (TGF-beta). A significant change was observed for TGF-beta, with increased expression after treatment and induction of clinical remission. Augmentation of TGF-beta may play a role in the induction of remission in myasthenia gravis.

Topics: Adolescent; Adult; Aged; Cytokines; Female; Humans; Immunosuppressive Agents; Male; Middle Aged; Myasthenia Gravis; Remission Induction; RNA, Messenger; Thymectomy; Transforming Growth Factor beta

Oral and nasal administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR and complete Freund's adjuvant (CFA) resulted in prevention or marked decrease of the severity of experimental autoimmune myasthenia gravis (EAMG) and suppression of AChR-specific B-cell responses and of AChR-reactive T-cell function. To examine the involvement of immunoregulatory cytokines and the underlying mechanisms involved in tolerance induction, in situ hybridization with radiolabeled cDNA oligonucleotide proves was adopted to enumerate mononuclear cells (MNC) expressing mRNA for the proinflammatory cytokine interferon-gamma (IFN-gamma), the B cell-stimulating interleukin-4 (IL-4), and the immunosuppressive transforming growth factor-beta (TGF-beta). Popliteal and inguinal lymph nodes from EAMG rats contained elevated numbers of AChR-reactive IFN-gamma, IL-4, and TGF-beta mRNA-expressing cells, compared to control rats receiving PBS orally or nasally and injected with CFA only. Oral and nasal tolerance was accompanied by decreased numbers of AChR-reactive IFN-gamma and IL-4 mRNA-expressing cells and strong up-regulation of TGF-beta mRNA-positive cells in lymphoid organs when compared to nontolerized EAMG control rats. The results suggest that IFN-gamma and IL-4 are central effector molecules in the development of EAMG and that TGF-beta plays an important role in tolerance induction to EAMG.

Topics: Administration, Intranasal; Administration, Oral; Animals; Antibody Formation; Antigens; Disease Models, Animal; Down-Regulation; Female; Gene Expression; Humans; Immune Tolerance; Interferon-gamma; Interleukin-4; Intestinal Mucosa; Monocytes; Muscle, Skeletal; Myasthenia Gravis; Rats; Rats, Inbred Lew; Receptors, Cholinergic; RNA, Messenger; T-Lymphocyte Subsets; Time Factors; Transforming Growth Factor beta

CD4+ T cells have been shown to be crucial in the development of experimental autoimmune myasthenia gravis (EAMG). The role of CD8+ T cells in EAMG is less well established. We previously showed that antibody depletion of CD8+ T cells in rats effectively suppresses EAMG. To further study the role and relationship of CD4+ versus CD8+ T cells in induction of EAMG, CD4-/-, CD8-/-, and CD4-8- mutant C57BL/6 mice and the parent CD4+8- wild-type mice were immunized with Torpedo acetylcholine receptor (AChR) plus complete Freund's adjuvant. Clinical EAMG was nearly completely prevented in CD4-8-, CD4-/-, and CD8-/- mice. This was associated with strongly reduced AChR-specific T and B cell responses, and with reduced levels of AChR-reactive interferon gamma (IFN-gamma) and interleukin 4 (IL-4) mRNA-expressing cells in lymphoid organs when compared with CD4+8+ wild-type mice. We conclude that (a) both CD4+ and CD8+ T cells are essential for development of EAMG, and a collaboration between these cell types may be necessary; (b) CD4+ as well as CD8+ T cells secrete IFN-gamma and IL-4, and both cytokines are involved in the development of EAMG; and (c), besides T cells, other immune cells might also be responsible for help of anti-AChR antibody production.

Topics: Animals; Autoantibodies; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Female; Gene Expression; Interferon-gamma; Interleukin-4; Lymphocyte Activation; Lymphocyte Cooperation; Mice; Mice, Inbred C57BL; Mice, Knockout; Myasthenia Gravis; Receptors, Nicotinic; RNA, Messenger; T-Lymphocytes, Helper-Inducer; Transforming Growth Factor beta

Various mouse and rat strains show different susceptibilities to experimental autoimmune myasthenia gravis (EAMG) that can be induced by immunization with acetylcholine receptor (AChR) and Freund's complete adjuvant, and represents a model for the antibody-mediated myasthenia gravis in humans. We examined AChR-induced B and T cell responses and cytokine mRNA expression to study the mechanisms behind susceptibility to EAMG in Lewis rats and resistance in Wistar Furth (WF) rats. Both strains had similarly elevated concentrations and affinities of serum anti-AChR antibodies, and no difference between the two strains for frequencies of cells in lymphoid organs expressing mRNA of the B cell stimulating cytokine interleukin-4 was found. In contrast, T cell responses to AChR measured by proliferation and by enumeration of interferon-gamma-expressing cells at both mRNA and protein level were lower in the resistant WF rats. This strain showed, instead, an up-regulation of the anti-inflammatory transforming growth factor-beta. Strain-related differences in the susceptibility to actively induced EAMG are thus related to quantitative differences in distribution between pro-inflammatory and anti-inflammatory cytokines.

Topics: Animals; Antibody Affinity; Autoantibodies; Autoantigens; Base Sequence; Cytokines; Female; Immunity, Innate; In Situ Hybridization; Interferon-gamma; Interleukin-4; Lymphocyte Activation; Molecular Sequence Data; Myasthenia Gravis; Rats; Rats, Inbred Lew; Rats, Inbred WF; Receptors, Cholinergic; RNA, Messenger; T-Lymphocytes; Transforming Growth Factor beta

Nasal administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR plus Freund's complete adjuvant (FCA) resulted in prevention or marked decrease of the severity of EAMG, suppression of AChR-specific B cell responses and of AChR-reactive T cell functions. To examine the involvement of immunoregulatory cytokines and the underlying mechanisms involved in tolerance induction, in situ hybridization with radio-labelled synthetic oligonucleotide probes was adopted to enumerate mononuclear cells (MNC) expressing mRNA for the proinflammatory cytokine IFN-gamma, the B cell stimulating IL-4 and the immune response-down-regulating TGF-beta. Popliteal and inguinal lymph nodes from EAMG rats contained elevated numbers of AChR-reactive IFN-gamma, IL-4 and TGF-beta mRNA-expressing cells compared with control rats receiving PBS nasally and injected with FCA only. Nasal tolerance to EAMG was accompanied by decreased numbers of AChR-reactive IFN-gamma and IL-4 mRNA-expressing cells, and strong up-regulation of TGF-beta mRNA-positive cells in lymphoid organs compared with non-tolerized EAMG control rats. The relative affinity of anti-AChR antibodies was lower, but muscle AChR amounts were higher in nasally tolerized rats compared with non-tolerized EAMG control rats. The results suggest that IFN-gamma and IL-4 are central effector molecules in the development of EAMG, and that TGF-beta plays an important role in tolerance induction to EAMG.

Topics: Administration, Intranasal; Animals; Concanavalin A; DNA Probes; Electrophoresis, Polyacrylamide Gel; Female; Immune Tolerance; Immunoglobulin G; In Situ Hybridization; Interferon-gamma; Interleukin-4; Leukocytes, Mononuclear; Lymph Nodes; Muscles; Myasthenia Gravis; Myelin Basic Protein; Oligonucleotides; Rats; Rats, Inbred Lew; Receptors, Nicotinic; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation; Vaccination

Multiple sclerosis (MS) is associated with high levels of circulating T lymphocytes that respond to the myelin antigens myelin basic protein (MBP) and proteolipid protein (PLP) by producing various cytokines including interferon-gamma (IFN-gamma) that makes MS worse and transforming growth factor-beta (TGF-beta), an endogenously produced immunosuppressant that might act beneficially. To further define the role of TGF-beta in MS, we examined the effects of recombinant TGF-beta 1 (rTGF-beta 1) on autoantigen-mediated regulation of cytokines in MS and myasthenia gravis (MG). Blood mononuclear cells (MNC) were cultivated with or without rTGF-beta 1, and with or without autoantigen or the recall antigen PPD. MNC expressing cytokine mRNA were detected after in situ hybridization with radiolabeled cDNA oligonucleotide probes. Femtogram concentrations of rTGF-beta 1 suppressed MBP-, PLP- and PPD-induced upregulation of IFN-gamma, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha), TNF-beta and perforin in MS, and acetylcholine receptor (AChR)-induced augmentation of these pro-inflammatory cytokines in MG, but had no effects on autoantigen- or PPD-induced expression of IL-10 or TGF-beta itself. rTGF-beta 1 also suppressed numbers of myelin antigen-reactive IFN-gamma- and IL-4-secreting cells in MS and AChR-reactive IFN-gamma and IL-4 secreting cells in MG. The selective suppressive effects of TGF-beta 1 on autoantigen-induced upregulation of pro-inflammatory cytokines makes TGF-beta 1 attractive as a treatment alternative in MS and MG.

Topics: Adult; Aged; Autoantigens; Cells, Cultured; Cytokines; Female; Gene Expression; Humans; In Situ Hybridization; Inflammation; Interferon-gamma; Interleukin-10; Interleukin-4; Leukocytes, Mononuclear; Male; Middle Aged; Multiple Sclerosis; Myasthenia Gravis; Myelin Basic Protein; Oligonucleotide Probes; Recombinant Proteins; RNA, Messenger; Transforming Growth Factor beta

CD8+ T cells can perform both Th1- and Th2-like functions by producing cytokines such as interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), as well as the immune response down-regulating transforming growth factor-beta (TGF-beta), which are all involved in the development of experimental autoimmune myasthenia gravis (EAMG), a model for human MG. We have reported that depletion of CD8+ T cells results in the suppression of EAMG accompanied by the down-regulation of AChR-specific B cell responses and AChR-reactive IFN-gamma secreting Th1-like cells. To identify the involvement of IFN-gamma, IL-4 and TGF-beta in the development of EAMG after CD8+ T cell depletion, the expression of mRNA for these cytokines was studied in mononuclear cells from popliteal, inguinal and mesenteric lymph nodes, spleen and thymus by adopting in situ hybridization with complementary DNA oligonucleotide probes. Depletion of CD8+ T cells resulted in decreased levels of IFN-gamma and IL-4 mRNA expressing cells in different lymphoid organs except thymus, but no change in the numbers of TGF-beta mRNA expressing cells. The results imply that the suppression of EAMG after depletion of CD8+ T cells is caused by decreasing the effector factors but not by increasing the suppressor factor(s).

Topics: Animals; Autoimmunity; CD8-Positive T-Lymphocytes; Cell Count; Disease Models, Animal; Down-Regulation; Female; Immunophenotyping; Interferon-gamma; Interleukin-4; Lymphoid Tissue; Myasthenia Gravis; Rats; Rats, Inbred Lew; RNA, Messenger; Transforming Growth Factor beta

Oral administration of acetylcholine receptor (AChR) or myelin basic protein (MBP) to Lewis rat prior to immunization with AChr or MBP and complete Freund's adjuvant (CFA) has previously been shown to prevent or delay the onset of experimental autoimmune myasthenia gravis (EAMG) or experimental allergic encephalomyelitis (EAE), which represent animal models of myasthenia gravis and multiple sclerosis, respectively. Here we show that Lewis rats immunized with AChr+MBP+CFA developed both signs of muscular weakness seen in EAMG and paresis characteristic for EAE. This disease was associated with high levels of anti-AChR and anti-MBP antibody secreting cells and of AChR- and MBP-reactive INF-gamma secreting Th1-like cells in lymph nodes. The diseased rats also showed upregulation of AChR- and MBP-induced mRNA expression of IFN-gamma in lymph node cells. Oral tolerization with AChR and MBP in combination prior to immunization with AChR+MBP+CFA alleviated clinical disease as well as AChR- and MBP-specific B cell node cells. The results implicate that oral tolerization simultaneously to more than one autoimmune disease-related autoantigen is feasible, and that suppression of autoantigen-induced IFN-gamma and augmentation of TGF-beta are pivotal in tolerance induction.

Topics: Administration, Oral; Animals; Encephalomyelitis, Autoimmune, Experimental; Female; Immune Tolerance; Interferon-gamma; Interleukin-4; Myasthenia Gravis; Myelin Basic Protein; Rats; Rats, Inbred Lew; Receptors, Cholinergic; RNA, Messenger; Transforming Growth Factor beta

Multiple sclerosis (MS) is characterized by patchy accumulations of inflammatory cells combined with demyelination. There are mononuclear cells in blood and cerebrospinal fluid of patients with MS that produce interferon-gamma and interleukin-4 in response to myelin basic protein (MBP) and proteolipid protein (PLP). Here we describe autoantigen-induced production of transforming growth factor-beta (TGF-beta). This multifunctional cytokine has inhibitory effects on the growth, differentiation, and effector functions of activated T cells. Blood and cerebrospinal fluid cells were exposed in short-term cultures to MBP and PLP and, after hybridization with complementary DNA oligonucleotide probes, they were evaluated for TGF-beta mRNA expression. Patients with MS had higher numbers of MBP- and PLP-responsive TGF-beta mRNA expressing cells in blood compared with control patients with other neurological diseases or myasthenia gravis and a five- and threefold further increment in their cerebrospinal fluid. In blood of patients with myasthenia gravis, where the acetylcholine receptor (AChR) is a target for autoaggressive immunity, there were increased levels of AChR-responsive TGF-beta mRNA expressing cells. Thymectomized myasthenia gravis patients showed higher levels of TGF-beta mRNA expressing cells compared with patients not thymectomized. Numbers of cells responding to AChR in MS and MBP in myasthenia gravis did not differ from numbers found in absence of antigen. Patients with other neurological diseases showed infrequent and low responses to MBP, PLP, and AChR. Diseases with presumed autoimmune pathogenesis are associated with organ-specific autoantigen-induced TGF-beta production, which is increased after thymectomy.

Topics: Adult; Autoantigens; Cerebrospinal Fluid; Female; Gene Expression; Humans; Male; Middle Aged; Multiple Sclerosis; Myasthenia Gravis; Myelin Proteins; Myelin Proteolipid Protein; Receptors, Cholinergic; RNA, Messenger; Transforming Growth Factor beta

Oral administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with Torpedo AChR+complete Freund's adjuvant (CFA) results in the prevention of experimental autoimmune myasthenia gravis (EAMG) and the suppression of AChR-specific B cell responses and counteracts the development of AChR-reactive interferon-gamma (IFN-gamma) secreting T cells. To study the involvement of the T helper type 1 (Th1) cell-related lymphokine IFN-gamma, the Th2 cell-related interleukin-4 (IL-4), and transforming growth factor beta (TGF-beta) that suppresses the synthesis of IFN-gamma and IL-4, we used in situ hybridization with complementary DNA oligonucleotide probes to enumerate mononuclear cells (MNC) expressing mRNA for the cytokines IFN-gamma, IL-4, and TGF-beta. Upon in vivo recognition of AChR, popliteal, inguinal, and mesenteric lymph nodes, spleen and thymus of rats with EAMG contained higher levels of IFN-gamma, IL-4, and TGF-beta mRNA-expressing cells compared to CFA-injected control rats, implicating the involvement in EAMG of AChR-reactive Th1 and Th2 cells in parallel. TGF-beta was also upregulated in EAMG. Oral tolerance to EAMG was characterized by suppression of the levels of MNC expressing IFN-gamma and IL-4, but augmentation of cells expressing TGF-beta. The results suggest that IFN-gamma, IL-4, and TGF-beta are involved in the development of EAMG, and that TGF-beta is important in the induction of oral tolerance to EAMG.

Topics: Animals; Gene Expression; Immune Tolerance; Interferon-gamma; Interleukin-4; Lymphoid Tissue; Myasthenia Gravis; Myelin Basic Protein; Rats; Rats, Inbred Lew; Receptors, Nicotinic; RNA, Messenger; T-Lymphocytes, Helper-Inducer; Time Factors; Tissue Distribution; Transforming Growth Factor beta

Multiple sclerosis (MS) is characterized by perivascular inflammation and high levels of circulating T and B lymphocytes that respond to the myelin antigens myelin basic protein (MBP) and proteolipid protein (PLP), thereby suggesting a role for immunoregulatory cytokines.. Blood mononuclear cells (MNC) were prepared from patients with MS, optic neuritis (ON), myasthenia gravis (MG), other inflammatory (OIND) and non-inflammatory neurological diseases (OND), and from patients with HIV infection and healthy controls. MNC expressing cytokine mRNA were detected by in situ hybridization with radiolabelled cDNA oligonucleotide probes. Numbers of cytokine mRNA expressing cells were presented per standard numbers of MNC.. MS patients had elevated numbers of MNC in blood expressing T helper type 1 (Th1) cell related interferon-gamma (IFN-gamma), Th2 cell associated interleukin-4 (IL-4) and the endogenously produced immunosuppressant transforming growth factor-beta (TGF-beta). IFN-gamma and TGF-beta correlated with MS disability: EDSS score < 3 was associated with high numbers of TGF-beta mRNA positive cells while IFN-gamma mRNA positive cells tended to be low. The reverse was seen in patients with EDSS > or = 3. Cultures of MNC in presence and absence of antigen revealed that MBP and PLP induced strong responses in MS reflected by high levels of IFN-gamma, IL-4 and TGF-beta mRNA expressing cells. Recombinant (r) TGF-beta 1 dose-dependently suppressed MBP-induced upregulation of the proinflammatory cytokines IFN-gamma, IL-4, IL-6, tumor necrosis factor-alpha, (TNF-alpha), TNF-beta and perforin, but not of the immunosuppressive and probably advantageous IL-10. Cytokine mRNA expressing cells were enriched in the MS patients' cerebrospinal fluid, as were the cytokine mRNA positive cells detected after culture in presence of MBP and PLP, reflecting an autonomy of the immune response in this compartment. ON, in many instances representing early MS, did not differ from clinically definite MS regarding profiles of IFN-gamma, IL-4 and TGF-beta. Also patients with MG had elevated numbers of IFN-gamma, IL-4 and TGF-beta mRNA expressing blood MNC. They were further augmented upon culture of the MG patients' MNC in presence of acetylcholine receptor (AChR). An upregulation of AChR-induced TGF-beta was observed in thymectomized patients. rTGF-beta suppressed AChR-induced upregulation of proinflammatory cytokines but not IL-10. Elevated numbers of IFN-gamma, IL-4 and TGF-beta mRNA expressing blood MNC were also found in patients with OIND (aseptic meningo-encephalitis, chronic inflammatory demyelinating polyneuropathy, polymyositis, Eaton-Lambert syndrome) and in HIV-infected patients. In HIV infection, numbers of IL-4 mRNA positive cells correlated inversely with CD4+ cell counts, reflecting the involvement of IL-4 in later stages of the disease. Patients with non-inflammatory neurological diseases and healthy subjects had either no or low numbers of IFN-gamma, IL-4 and TGF-beta mRNA expressing cells when blood MNC were examined without previous culture, and after culture in presence and absence of MBP, PLP and AChR as antigens. An exception was a healthy pregnant lady who showed high le. High numbers of in vivo activated and of organ-specific antigen-responsive Th1 and Th2 like cells expressing IFN-gamma and IL-4 mRNA are characteristic for MS and MG. Upregulation of TGF-beta in MS patients with little disability and in MG after thymectomy implicates that TGF-beta has desirable effects in human diseases with autoimmune background.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; DNA, Complementary; Female; Gene Expression Regulation; HIV Infections; Humans; Interferon-gamma; Interleukin-4; Lambert-Eaton Myasthenic Syndrome; Male; Middle Aged; Monocytes; Multiple Sclerosis; Myasthenia Gravis; Oligonucleotide Probes; Optic Neuritis; Polymyositis; Polyradiculoneuropathy; RNA, Messenger; Transforming Growth Factor beta

The abnormal T lymphocyte-dependent production of antibodies to the nicotinic acetylcholine receptor (AChR) in myasthenia gravis (MG) suggests a role for immunoregulatory cytokines. We examined the T helper type 1 (Th1) cell-associated interferon-gamma (IFN-gamma) that promotes cell-mediated immunity, the Th2 cell-related interleukin-4 (IL-4) that augments B cell immunity, and transforming growth factor-beta (TGF-beta) that downregulates immune responses but enhances isotype switching. Blood mononuclear cells (MNC) expressing cytokine mRNA were enumerated after in situ hybridization with labelled complementary DNA oligonucleotide probes for IFN-gamma, IL-4 and TGF-beta. MG patients had elevated numbers of cells expressing IFN-gamma and IL-4 compared to patients with non-inflammatory neurological diseases and healthy controls, implying that both Th1- and Th2-like cells are involved in MG. TGF-beta-positive cells were also elevated in MG. The levels of cytokine-positive MNC were similar in MG and in control patients with other inflammatory neurological diseases. There were no associations between numbers of cytokine-positive blood MNC and clinical variables of MG, but individual patients need to be studied over the course of MG to clarify a relation between the cytokines under study and clinical or laboratory variables of MG.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Gene Expression Regulation; Humans; Interferon-gamma; Interleukin-4; Leukocytes, Mononuclear; Middle Aged; Myasthenia Gravis; RNA, Messenger; Transforming Growth Factor beta