Joanne Link

Suppression of experimental autoimmune myasthenia gravis by nasal administration of acetylcholine receptor.

Experimental autoimmune myasthenia gravis (EAMG) is a well established animal model, which can be induced in various animal species and strains with acetylcholine receptor (AChR) and represents an experimental counterpart of human myasthenia gravis (MG). Current immunotherapies of both EAMG and MG are non-specific and limited by their toxicity. Tolerance to EAMG has been achieved by oral administration of milligram quantities of Torpedo AChR. In the present report we demonstrate that nasal administration of microgram doses of Torpedo AChR to female Lewis rats prior to immunization with Torpedo AChR and complete Freunds adjuvant resulted in the prevention of subsequently induced EAMG, the suppression of serum anti-AChR antibody levels, the decrease of delayed-type hypersensitivity responses to AChR, as well as the suppression of AChR-specific immunoglobulin G-secreting cells, AChR-reactive interferon-gamma-secreting cells and T cell proliferation in peripheral lymphoid organs, particularly in popliteal and inguinal lymph nodes regional to immunization. We conclude that clinical signs of EAMG can be efficiently prevented by nasal administration of AChR in parallel with the downregulation of both B and T cell responses specific to AChR.

Increased levels of interferon‐gamma (IFN‐γ), IL‐4 and transforming growth factor‐beta (TGF‐β) mRNA expressing blood mononuclear cells in human HIV infection

Evidence has been presented for the involvement of IFN‐γ, IL‐4 and TGF‐β in AIDS. Measured plasma levels may, however, poorly reflect in vivo production, since cytokines act auto‐ and paracrinally and have very short half life in plasma. In situ hybridization with complementary DNA oligonucleotide probes was used lo enumerate blood mononuclear cells expressing cytokine messenger RNA(mRNA). HIV‐infected patients had elevated blood levels of cells expressing each of the cytokines, with predominance for cells expressing TGF‐β mRNA. All AIDS patients included had elevated numbers of IL‐4 mRNA‐expressing cells, and levels of cells expressing this cytokine correlated inversely with counts of CD4+ cells in blood, reflecting the involvement of Th2‐like cells in later stages of HIV infection. The described approach should be useful in further studies of cytokines in HIV infection and other diseases.

Transforming growth factor-β1 suppresses autoantigen-induced expression of pro-inflammatory cytokines but not of interleukin-10 in multiple sclerosis and myasthenia gravis

Abstract 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-γ (IFN-γ) that makes MS worse and transforming growth factor-β (TGF-β), an endogenously produced immunosuppressant that might act beneficially. To further define the role of TGF-β in MS, we examined the effects of recombinant TGF-β1 (rTGF-β1) on autoantigen-mediated regulation of cytokines in MS and myasthenia gravis (MG). Blood mononuclear cells (MNC) were cultivated with or without rTGF-β1, 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-β1 suppressed MBP-, PLP- and PPD-induced upregulation of IFN-γ, IL-4, IL-6, tumor necrosis factor-α (TNF-α), TNF-α 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-β itself. rTGF-β1 also suppressed numbers of myelin antigen-reactive IFN-γ- and IL-4-secreting cells in MS and AChR-reactive IFN-γ and IL-4 secreting cells in MG. The selective suppressive effects of TGF-β1 on autoantigen-induced upregulation of pro-inflammatory cytokines makes TGF-β1 attractive as a treatment alternative in MS and MG.

Mucosal Tolerance to Experimental Autoimmune Myasthenia Gravis Is Associated with Down‐regulation of AChR‐specific IFN‐γ‐expressing Th1‐like Cells and Up‐regulation of TGF‐β mRNA in Mononuclear Cells

Oral and nasal administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR and complete Freunds 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.

Augmented interferon-γ, interleukin-4 and transforming growth factor-β mRNA expression in blood mononuclear cells in myasthenia gravis

Abstract 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-γ (IFN-γ) that promotes cell-mediated immunity, the Th2 cell-related interleukin-4 (IL-4) that augments B cell immunity, and transforming growth factor-β (TGF-β) 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-γ, IL-4 and TGF-β. MG patients had elevated numbers of cells expressing IFN-γ 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-β-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.

Optic neuritis is associated with myelin basic protein and proteolipid protein reactive cells producing interferon-γ, interleukin-4 and transforming growth factor-β

Abstract Studies on patients with monosymptomatic optic neuritis (ON) should give opportunities to identify features typical for early multiple sclerosis (MS). There are increased T and B cell responses to the myelin components myelin basic protein (MBP) and proteolipid protein (PLP) in both ON and MS, but there is little information on the types of cytokines produced by such cells. We describe the use of in situ hybridization with complementary DNA oligonucleotide probes to detect and enumerate mononuclear cells expressing mRNA for the cytokines interferon-γ (IFN-γ) which augments cell-mediated immunity; interleukin-4 (IL-4) which promotes the B cell response; and transforming growth factor β (TGF-β) that in many cases downregulates immune responses. Expression of these cytokines was studied in mononuclear cells from peripheral blood and cerebrospinal fluid (CSF) from patients with ON and MS after in vitro exposure to MBP and PLP, and in absence of antigen. There were elevated levels of cells that in response to MBP and PLP expressed IFN-γ, IL-4 and TGF-β mRNA in blood and further enriched in CSF in both ON and MS, compared to patients with other neurological diseases. The results suggest that IFN-γ, IL-4 as well as TGF-β are involved in both ON and MS, and that the cytokine profile in early MS as reflected by ON is not different from that in clinically definite MS.

High numbers of autoantigen‐reactive mononuclear cells expressing interferon‐gamma (IFN‐γ), IL‐4 and transforming growth factor‐beta (TGF‐β) are present in cord blood

Umbilical cord blood of neonates and peripheral blood of healthy adults were analysed by in situ hybridization for numbers of mononuclear cells (MNC) expressing the cytokines IFN‐γ, TGF‐β and IL‐4 mRNA without culture and after culture in the presence of acetylcholine receptor (AChR), myelin basic protein (MBP) and peripheral myelin protein P2. These antigens were chosen since they represent autoantigens in putatively immune‐mediated neurological diseases, The numbers of cells expressing cytokine mRNA after 72 h culture in the presence of AChR, MBP and P2 were higher in cord blood than in peripheral blood of healthy adults. IFN‐γ, TGF‐β and IL‐4 were always elevated in parallel. In cord blood there was a pronounced reactivity to several of the tested antigens, while such broad reactivity was not found in peripheral blood of healthy adults. No differences in cytokine mRNA expression were found between cord blood and peripheral blood of adults when cells were analysed without culture. The results show a capacity of cord blood cells to react to several autoantigens by the up‐regulation of cytokine mRNA expression.