Guang-Xian Zhang

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.

Suppression of acute and protracted-relapsing experimental allergic encephalomyelitis by nasal administration of low-dose IL-10 in rats

In this study we report for the first time that nasal administration of the Th2 cell-related cytokine interleukin-10 (IL-10), at concentrations of 1.5 microg/rat and 15 microg/rat, suppressed clinical signs of acute experimental allergic encephalomyelitis (EAE) in Lewis rats and prevented the development and relapse of protracted-relapsing EAE (PR-EAE) in DA rats. In contrast, subcutaneous injection of IL-10 (15 microg/rat) did not inhibit acute EAE. The IL-10-mediated suppression of EAE was associated with decreased myelin antigen-specific T-cell proliferative responses and IFN-gamma secretion in both acute and PR-EAE. In sections of spinal cords derived from rats nasally pretreated with IL-10, there were no infiltrating CD4+ T cells or macrophages, which are considered as major encephalitogenic or inflammatory cells. Most interestingly, nasally administered IL-10 also inhibited MHC class II expression in microglia, indicating that IL-10 administration by the nasal route prevents the activation of microglia. Administration of cytokines via the nasal route offers an exciting alternative in the prevention and treatment of autoimmune diseases.

Transforming growth factor-β1 induces apoptosis of rat microglia without relation to bcl-2 oncoprotein expression

Transforming growth factor-beta (TGF-beta) plays a role as an immunosuppressive cytokine within the central nervous system (CNS). The CNS cells targeted by action of TGF-beta1 have not been defined. In this study, we tested the effect of TGF-beta1 on microglia, astrocytes and oligodendrocytes from newborn rats. TGF-beta1 selectively induced apoptosis of microglia, and not of astrocytes or oligodendrocytes. To study the apoptotic mechanism, bcl-2 oncoprotein expression in microglia, astrocytes and oligodendrocytes was measured. Bcl-2 was mainly expressed in microglia, indicating that microglial bcl-2 does not prevent TGF-beta1-mediated microglial apoptosis. The relative protein expression of bcl-2 in microglia was not related to frequency of microglial apoptosis. Thus, TGF-beta1-induced microglial apoptosis was regulated by a bcl-2-independent mechanism. Expression of cytokine (IL-1beta, IL-6, IL-12, IFN-gamma and TNF-alpha) mRNA on microglia was not influenced by treatment with TGF-beta1.

Interleukin 10 aggravates experimental autoimmune myasthenia gravis through inducing Th2 and B cell responses to AChR.

The damage of acetylcholine receptor (AChR) at neuromuscular junctions of experimental autoimmune myasthenia gravis (EAMG), an animal model of human MG, is mediated by B cells which require T cell help. The Th2 associated cytokine IL-10 suppresses production of cytokines released by Th1 cells and is considered for treatment of human autoimmune diseases. To evaluate the role of IL-10 in EAMG, rhIL-10 was administered daily to Lewis rats by the subcutaneous route starting at the day of immunization and continued for 7 weeks. IL-10 failed to abrogate EAMG at low dose (0.1 or 1 microg/day) and at the dose of 3 microg/day caused earlier onset and aggravated clinical signs of EAMG when compared to EAMG rats injected with PBS only. Although Th1 responses reflected by AChR-induced lymphocyte proliferation and levels of IFN-gamma secreting cells, as well as AChR-induced Th1 cytokine mRNA expression was suppressed, augmented IL-4 mRNA expression and AChR-specific B cell responses may play an important role in the failure of IL-10 to abrogate EAMG. This study implicates a critical precaution in planning immunotherapy of IL-10 in antibody-mediated autoimmune diseases, e.g. 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.

Linomide suppresses both Th1 and Th2 cytokines in experimental autoimmune myasthenia gravis

Suppressive effects of the synthetic immunomodulatory drug Linomide have been shown in several autoimmune models, including antibody-mediated experimental autoimmune myasthenia gravis (EAMG), a model for human myasthenia gravis (MG). To define the mechanisms underlying EAMG suppression, we injected Linomide subcutaneously at different doses into Lewis rats immunized with Torpedo acetylcholine receptor (AChR) in complete Freunds adjuvant (CFA), and investigated AChR-specific T and B cell responses, and the levels of lymph node cells expressing mRNA of different cytokines after AChR stimulation in vitro. Both 160 and 16, but not 1.6, mg/kg/day of Linomide effectively suppressed clinical muscle weakness, accompanied by decreased AChR-induced T and B cell responses. Linomide also suppressed the mRNA expression of the Th1 cytokines IFN-gamma, IL-12 and TNF-alpha as well as the Th2 cytokines IL-4 and IL-10, which are important in the immunopathogenesis of EAMG by promoting antibody production. There were no differences for IL-1beta, IL-6, lymphotoxin or TGF-beta expression in Linomide-treated vs nontreated control EAMG rats. We conclude that Linomide suppresses clinical EAMG as well as B and T cell responses to AChR by counteracting the production of AChR-induced Th1 and Th2 cytokines.

Transforming growth factor‐beta 1 (TGF‐β1)‐mediated inhibition of glial cell proliferation and down‐regulation of intercellular adhesion molecule‐1 (ICAM‐1) are interrupted by interferon‐gamma (IFN‐γ)

We utilized a model of myelin basic protein (MBP) activation in vivo and MBP‐stimulated cultures in vitro to study the influence of TGF‐β1 on glial cell proliferation and ICAM‐1/leucocyte function‐associated antigen‐1 (LFA‐1) expression, and to observe the antagonistic effects of TGF‐β1 and IFN‐γ. TGF‐β1 inhibited MBP‐stimulated and MBP‐activated glial cell proliferation, especially in MBP‐stimulated separated microglia and astrocytes, and down‐regulated the expression of ICAM‐1 on MBP‐stimulated glial cells and separated microglia. ICAM‐1 expression on MBP‐activated glial cells was intensely suppressed, whereas its expression on MBP‐stimulated astrocytes was not influenced. TGF‐β1 had no effect on LFA‐1 expression. In contrast, IFN‐γ up‐regulated ICAM‐1 expression, but inhibited proliferative response on MBP‐stimulated glial cells when cultured without TGF‐β1. Examination of TGF‐β1 and IFN‐γ interactions revealed that TGF‐β1‐mediated inhibition of proliferation and down‐regulation of ICAM‐1 on glial cells were prevented by IFN‐γ. The suppressive effect was re‐established with high doses of TGF‐β1 in cultures, indicating that biological effects of TGF‐β1 vary depending on nitric oxide (NO) production, its concentration in the microenvironment and regulation of the cytokine network.

The cerebrospinal fluid from patients with multiple sclerosis promotes neuronal and oligodendrocyte damage by delayed production of nitric oxide in vitro

Nitric oxide (NO) may be involved in myelin and oligodendrocyte injury associated with multiple sclerosis (MS), a demyelinating disease of unknown etiology. The cerebrospinal fluid (CSF) from MS patients may provide an important signal inducing a pathologic process within the central nervous system (CNS). To investigate this question, CSF-induced NO production by glial cells was studied in 38 patients with multiple sclerosis (MS), 30 patients with other CNS inflammatory diseases (ID) and 20 with tension headache (TH) as control. Neuron damage was estimated by release of lactate dehydrogenase (LDH), whereas oligodendrocyte damage was estimated by a percentage of viable cells in primary oligodendrocyte cultures. Here we show that CSFs from 13/38 (34%) patients with MS stimulate glial cells to produce NO, compared to 2/20 (10%) of patients with ID and 1/30 (3%) with tension headache. The levels of NO production correlated positively with the amounts of LDH released and negatively with percentage of viable oligodendrocytes, suggesting that NO may represent a mechanism for oligodendrocyte losses in affected tissues and play a role in lesion formation in MS and its animal model experimental allergic encephalomyelitis (EAE).

Cellular mRNA expression of interferon-gamma (IFN-γ), IL-4 and transforming growth factor-beta (TGF-β) in rats nasally tolerized against experimental autoimmune myasthenia gravis (EAMG)

Nasal administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR plus Freunds 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 radiolabelled synthetic oligonucleotide probes was adopted to enumerate mononuclear cells (MNC) expressing mRNA for the proinflammatory cytokine IFN‐γ, the B cell stimulating IL‐4 and the immune response‐down‐regulating TGF‐β. Popliteal and inguinal lymph nodes from EAMG rats contained elevated numbers of AChR‐reactive IFN‐γ, IL‐4 and TGF‐β 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‐γ and IL‐4 mRNA‐expressing cells, and strong up‐regulation of TGF‐β 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‐γ and IL‐4 are central effector molecules in the development of EAMG, and that TGF‐β plays an important role in tolerance induction to EAMG.

Shift from anti-to proinflammatory cytokine profiles in microglia through Lps-or Ifn-γ-mediated pathways

In this study, cytokine mRNA profiles in microglia from newborn rats were detected by in situ hybridization. Under natural culture conditions, microglia expressed the immunosuppressive transforming growth factor-beta 1 (TGF-beta 1) and interleukin (IL) 10 to a greater degree than the pro-inflammatory cytokines IL-1 beta, IL-6, IL-12, interferon-gamma (IFN-gamma) and TNF-alpha. High TGF-beta 1 and IL-10 levels could reflect one mechanism for immune privilege within the CNS under physiological conditions. Stimulation of microglia with LPS or IFN gamma resulted in strong up-regulation of proinflammatory cytokines, while TGF-beta 1 and IL-10 were down-regulated. These effects of LPS or IFN-gamma are anticipated to reflect immunopathogenic processes within the CNS.