Anti-cytokine autoantibodies in experimental autoimmune neuritis in Lewis rats

Experimental autoimmune neuritis (EAN) is a CD4+ T-cell-mediated, inflammatory demyelinating disease of the peripheral nervous system (PNS) that serves as a model for Guillain-Barre syndrome (GBS) in humans. Cytokine production has been suggested to act a pathogenic role for EAN. To study the potential role of cytokines in context with cytokine autoantibodies (Aabs) in EAN, we used in situ hybridization to detect mRNA expression of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, IL-4, IL-10, and transforming growth factor (TGF)-beta in lymph node mononuclear cell (MNC) and in sciatic nerve sections, as well as ELISA for detection of their autoantibodies in sera and cerebrospinal fluid (CSF) over the course of EAN. Increased mRNA expression for IFN-gamma and TNF-alpha was registered correlating with the peak of clinical signs of EAN, and high levels of mRNA expression for IL-4, IL-10, and TGF-beta were associated with EAN recovery. The levels of cytokine mRNAs were generally inversely correlated to their autoantibodies in serum and CSF, whereby the CSF levels were equal to or lower than the serum levels. Autoantibodies to IFN-gamma dose-dependently inhibited IFN-gamma-induced MHC expression by peritoneal macrophages proving a neutralizing biological effect of these autoantibodies. Our data demonstrate the existence of the anti-cytokine autoantibodies in the sera and CSF of rats with EAN; however, the role of anti-cytokine autoantibodies in the disease process of EAN remains to be resolved.     

丙戊酸对实验性自身免疫性神经炎大鼠保护作用的机制

目的观察丙戊酸(VAP)对实验性自身免疫性神经炎(EAN)大鼠的保护作用及其机制。方法实验大鼠随机分为VAP高剂量组、VAP低剂量组、EAN模型组、正常组,应用P2 57-81多肽与完全弗氏佐剂的混合液诱导EAN模型。VAP于免疫当天至第15d每天腹腔内注射。观察各组大鼠发病情况和坐骨神经组织病理学变化,检测外周血中Th17细胞和Foxp3+Treg细胞含量,检测淋巴结中TNF-α、IFN-γ、IL-17、TGF-βmRNA表达。结果 VAP高剂量组的最初发病时间迟于EAN组(P<0.05),其高峰期临床评分显著低于EAN组(P<0.05),坐骨神经炎性细胞浸润较EAN组明显减少;VAP高剂量组和低剂量组外周血中Th17细胞比例较EAN组显著减少(P<0.05),Foxp3+Treg细胞比例较EAN组显著增加(P<0.05),淋巴结中促炎细胞因子TNF-α、IFN-γ及IL-17mRNA表达与EAN组比较明显下降(P<0.05),VAP高剂量组抑炎细胞因子TGF-βmRNA表达与EAN组比较明显升高(P<0.05)。结论 VAP对EAN有治疗作用,这种作用可能与其能够增加Foxp3+Treg细胞和抑炎细胞因子TGF-β含量、减少TH17细胞含量和促炎细胞因子的表达有关。     

Resistance and susceptibility to experimental autoimmune neuritis in Sprague-Dawley and Lewis rats correlate with different levels of autoreactive T and B cell responses to myelin antigens

Experimental autoimmune neuritis (EAN) is a CD4⁺ T cell-mediated, inflammatory demyelinating disease of the peripheral nervous system (PNS) that serves as a model for Guillain-Barré syndrome (GBS) in humans. Various mouse and rat strains show different susceptibilities to EAN that can be induced by immunization with bovine PNS myelin (BPM) + Freund's complete adjuvant (FCA). We examined PNS-induced T and B cell responses and cytokine protein production as well as mRNA expression to study the mechanisms behind susceptibility to EAN in Lewis rats and resistance in Sprague-Dawley (SD) rats. Lewis rats with EAN have elevated PNS myelin-reactive interferon-γ (IFN-γ) production, TNF-α mRNA expression, and increased B cell responses to PNS myelin antigens, but low PNS myelin-reactive transforming growth factor-β (TGF-β) and interleukin (IL)-10 mRNA expression in lymph node mononuclear cells (MNC). In contrast, resistance to EAN in SD rats is associated with reduced BPM and P2 peptide-reactive IFN-γ production, TNF-α mRNA expression, and suppressed B cell responses to PNS myelin antigens as well as up-regulation of TGF-β and IL-10 mRNA expression. Resistance to EAN is also associated with low-grade inflammation or absence of histological evidence of EAN. These results suggest that differential autoreactive T and B cells responses to PNS myelin antigens are strain specific, and the susceptibility to EAN is related to quantitative rather than qualitative differences in distribution between pro-inflammatory and anti-inflammatory cytokines. J. Neurosci. Res. 54:373–381, 1998. © 1998 Wiley-Liss, Inc.     

Early myelin lesions in experimental allergic neuritis

We examined the evolution of demyelination in spinal roots of Lewis rats immunized with whole nerve and complete Freund' adjuvant. Roots were morphologically normal until 11 days after immunization, when we found endoneurial edema and myelin vesiculation in the absence of mononuclear cell contacts. Macrophage-associated myelin stripping was not detected until day 12. Macrophage infiltrations were extensive by day 14, but lymphocytes were sparse. These observations indicate that in experimental allergic neuritis, myelin injury may occur before macrophage-mediated demyelination, and provide support for an early role of serum factors in the development of this disorder.     

T-cell receptor Vβ-element expression in peripheral nerves of Lewis rats suffering from experimental autoimmune neuritis

In experimental autoimmune neuritis (EAN), peripheral nerves are infiltrated by T-lymphocytes and macrophages. By RT-PCR and sequence analysis we characterized TCR Vβ-element usage in sciatic nerve tissue of Lewis rats suffering from EAN induced by immunization with peripheral myelin antigens. Several TCR Vβ-chain sequences were detected, which did not show homology to sequences of P2-reactive T cells published so far. In EAN induced with peripheral nerve myelin, but not with P2-protein or P2 peptide aa 53–78, TCR Vβ 8.2 sequences identical to sequences of encephalitogenic myelin basic protein (MBP) reactive T-cells were identified. These results provide further evidence for a contribution of MBP-directed T-cell reactivity to the pathogenesis of myelin induced EAN and may have implications for the pathogenesis of human demyelinating neuropathies.     

Major histocompatibility antigens and lymphocyte subsets during experimental allergic neuritis in the Lewis rat

Summary Major histocompatibility antigens were identified in frozen sections of normal Lewis rat peripheral nerve tissue with monoclonal antibodies and an avidin-biotin-peroxidase complex system. Class I antigen is normally required for cytotoxic/suppressor T lymphocyte function and class II antigen for activation of helper T lymphocytes. In the sciatic nerves class I antigen was expressed diffusely by most endoneurial and perineurial cells but class II antigen only by a minority. In the cauda equina class I antigen was expressed by all arachnoid and some endoneurial cells, while class II antigen was expressed by a smaller proportion of arachnoid cells in the endoneurium of spinal roots and interstitial cells surrounding dorsal root ganglion neurons. The endothelium of endoneurial, perineurial and meningeal vessels uniformly expressed class I but not class II antigen. Experimental allergic neuritis was induced in Lewis rats by immunisation with bovine intradural root myelin. Early lesions consisted of multifocal infiltration of the nerve roots by cells expressing leucocyte common antigen. Surrounding endoneurial cells showed markedly increased expression of major histocompatibility antigens. In inflammatory lesions about 10% of the cells were stained with pan T cell antibodies. T lymphocyte subsets were identified with antibody W3/25 for helper cells and MRC OX-8 for cytotoxic/suppressor cells. The W3/25 positive cells were usually slightly in excess of OX-8 positive cells and their relative proportions did not alter during the disease. The presence of class I antigen on normal endothelium and its increased expression on endoneurial cells in the early phase of inflammation suggest an important role for class I restricted lymphocytes in the pathogenesis of the early stages of experimental allergic neuritis.     

Differential susceptibility to experimental autoimmune neuritis in Lewis rat strains is associated with T-cell immunity to myelin antigens

Experimental autoimmune neuritis (EAN) is a CD4(+) T-cell-mediated inflammatory demyelinating disease of the peripheral nervous system (PNS) that serves as a model for Guillain-Barré syndrome (GBS) in humans. Various rat strains show different susceptibility to EAN. We examined PNS myelin-induced T- and B-cell responses and cytokine production in order to explore the mechanisms behind different EAN susceptibility in the three Lewis rat strains, Hannover, Charles River, and Taconic. Lewis rats of Hannover and Charles River strains exhibited a higher susceptibility to EAN than Lewis rats of the Taconic strain. The higher susceptibility was associated with increased inflammatory cell infiltrates and major histocompatibility class II expression as well as enhanced mitogenic (phytohemagglutinin-induced) and antigen-specific (P2 peptide 57-81-induced) lymphocyte proliferation compared with the Taconic strain. The Hannover strain also showed increased proinflammatory cytokine (interferon-γ and tumor necrosis factor-α) production in the PNS. Cross-cultures of T cells and macrophages from Hannover and Taconic rats revealed that the Hannover rats exerted the strongest priming function of T cells. In contract, the P2 peptide-induced antibody production was not different among the three Lewis rat strains. In conclusion, the differential susceptibility to EAN of Lewis rat strains is correlated primarily with T-cell immunity to myelin antigens.     

Inflammation and proinflammatory cytokine production,but no demyelination of facial nerves,in experimental autoimmune neuritis in Lewis rats

Experimental autoimmune neuritis (EAN) is a CD4⁺ T cell-mediated, inflammatory demyelinating disease of the peripheral nervous system (PNS) that serves as a model for Guillain–Barré syndrome (GBS) in humans. The facial nerve paralysis is relatively commonly found in GBS patients. Here, EAN was established in Lewis rats by immunization with P2 peptide 57–81, a purified component of peripheral nerve myelin, and Freund's complete adjuvant (FCA). To study whether the facial nerves are involved in the pathogenic process during the EAN course, we observed the clinical and pathological changes as well as cytokine production in facial nerves on Day 14 postimmunization (p.i.), i.e. at height of clinical EAN. As a result, all rats immunized with P2 peptide 57–81 developed severe EAN on Day 14 p.i., but none of the rats manifested clinical signs of facial nerve paralysis. Additionally, only mild inflammatory cell infiltration and proinflammatory cytokine, interferon-γ (IFN-γ) and tumour necrosis factor (TNF-) production as well as devoid demyelination were seen in facial nerves of the EAN rats. On the contrary, severe inflammation and demyelination as well as upregulated IFN-γ and TNF- production were observed in sciatic nerves of the same EAN rats. The underlying mechanism for the difference of the local manifestation of the disease process of EAN remains to be resolved.     

P0 myelin protein produces experimental allergic neuritis in Lewis rats

P0 protein was prepared from bovine spinal root myelin. The purity was shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunostaining with affinity purified antisera. P0 in the presence of lysophosphatidylcholine induced paralysis and histological lesions resembling experimental allergic neuritis in Lewis rats. Lysophosphatidylcholine also enhanced the ability of P2 to produce neuritis. The conformation of these proteins may be important in determining their ability to induce experimental allergic neuritis. P0 deserves consideration as an antigen relevant to Guillain-Barré syndrome.     

Th1-like cell responses to peripheral nerve myelin components over the course of experimental allergic neuritis in Lewis rats

Experimental allergic neuritis (EAN) is a T cell-mediated animal model of Guillain-Barré syndrome characterized by inflammation and demyelination of peripheral nerves. EAN can be induced by immunization of rats with bovine peripheral nerve myelin (BPM) or the myelin proteins P2 or PO, but the extent of T cell responses over the course of EAN is incompletely defined. We studied the T cell responses to these proteins and the glycolipid GM1 by enumerating T helper type 1 (Th1)-like cells secreting interferon-γ (IFN-γ) after short-term culture of mononuclear cells (MNC) in presence of antigen. Already 7 days post immunization (p.i.) with BPM and before onset of clinical EAN, lymph nodes contained elevated levels of P2 responsive T cells. At the height of EAN on day 14 p.i. and during recovery, T cell levels responding to BPM, PO and GM1 were also elevated. The same temporal profiles and specificities were registered for antigen reactive spleen MNC. The results implicate that Th1-like cells with multiple specificities including the glycolipid GM1 occur at increased levels in lymphoid organs in EAN rats, and that IFN-γ may be an important effector molecule in the induction of nerve damage.     

Chronic relapsing experimental allergic neuritis in Lewis rats: Effects of thymectomy and splenectomy

Summary Experimental allergic neuritis (EAN) was induced in Lewis rats aged 4 months by the inoculation of whole bovine dorsal root with Freund's complete adjuvant. Prolonged follow-up demonstrated that a relapsing course is a regular feature of the disorder in animals at this age. Although the initial disease episode was the most severe, clinical recovery from subsequent relapses was less satisfactory, this probably being related to persistent morphological abnormalities in the peripheral nervous system. Antecedent thymectomy, splenectomy, or the two combined, had little effect on the clinical course of the disorder, apart from reducing the duration of relapses. This was only statistically significant following combined thymectomy/splenectomy. Histological abnormalities, however, tended to be less severe in the operated as compared with normal control or sham-operated animals with EAN. The animals must have attained an immunocompetent state at the time of thymectomy and/or splenectomy. The capacity to develop EAN presumably resides in the draining lymph nodes and the occurrence of relapses is due to the continuing presence of antigen at the injection sites.Supported by the National Fund for Research into Crippling Diseases and the Medical Research Council     

Curcumin ameliorates rat experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is a helper T cell‐mediated autoimmune demyelinating inflammatory disease of the peripheral nervous system that serves as an animal model for human Guillain‐Barre syndrome. Curcumin, a naturally occurring polyphenolic phytochemical isolated from the medicinal plant Curcuma longa, has anti‐inflammatory activities. Here we investigated the therapeutic effects and potential mechanisms of curcumin in EAN rats. Exogenous curcumin treatment (100 mg/kg/day) significantly delayed the onset of EAN neurological signs, ameliorated EAN neurological severity, and reduced body weight loss of EAN rats. In EAN sciatic nerves, curcumin treatment suppressed the inflammatory cell accumulation and the expression of interferon (IFN)‐γ, tumor necrosis factor‐α, interleukin (IL)‐1β, and IL‐17. Furthermore, curcumin treatment significantly decreased the percentage of CD4⁺ T helper cells in EAN spleen and suppressed concanavalin A‐induced lymphocyte proliferation in vitro. In addition, curcumin altered helper T cell differentiation by decreasing IFN‐γ⁺CD4⁺ Th1 cells in EAN lymph node and spleen. In summary, our data demonstrate that curcumin could effectively suppress EAN by attenuating inflammation, indicating that curcumin might be a candidate for treatment of autoimmune neuropathies. © 2014 Wiley Periodicals, Inc.     

Complement depletion suppresses Lewis rat experimental allergic neuritis

Lewis rats immunized with myelin and complete Freund's adjuvant were treated with cobra venom factor (CVF) which depletes the C3 component of complement. CVF given at day 9 delayed the onset of experimental allergic neuritis (EAN) by 2-3 days and when given at days 9 and 12 delayed the onset of EAN by 4-5 days. Lumbar nerve roots of CVF-treated rats had significantly less demyelination than those from control EAN rats.     

Neurophysiological changes in demyelinating and axonal forms of acute experimental autoimmune neuritis in the Lewis rat

Myelin-sensitized T- and B-cells (lymph node cells) induced experimental autoimmune neuritis (EAN) in Lewis rats after passive transfer to naive recipients. After 6 days, all recipient rats developed tail paresis that progressed to limb paresis within 12-72 h. Progressive nerve conduction changes consistent with demyelination in the sciatic nerve (conduction block and prolongation of the distal motor latencies) and lumbar nerve roots (initial low F-wave frequencies followed by later prolongation in F-wave latencies) were observed during the disease. For comparison, adoptive transfer experimental autoimmune neuritis (AT-EAN) of differing disease severity was induced by titrating the dose of P2-specific T-cells. In contrast to EAN induced by myelin-sensitized T- and B-cells, AT-EAN was predominantly associated with rapid nerve conduction changes consistent with axonal dysfunction and degeneration. These findings demonstrate that distinct forms of EAN with different pathophysiological mechanisms are induced by the passive transfer of P2-specific T-cell lines or myelin-specific T-cells and B-cells. The electrophysiological changes in EAN induced by myelin-specific T- and B-cells are very similar to those seen clinically during acute inflammatory demyelinating polyneuropathy, whereas AT-EAN has less resemblance to axonal forms of Guillain-Barré syndrome.     

Glial cell line‐derived neurotrophic factor is expressed by inflammatory cells in the sciatic nerves of Lewis rats with experimental autoimmune neuritis

Neurotrophic factors, including glial cell line‐derived neurotrophic factor (GDNF), have been known to play a role in neuroprotection in the injured peripheral nervous system (PNS). To evaluate the involvement of GDNF in experimental autoimmune neuritis (EAN) pathogenesis, the expression of GDNF in rat sciatic nerves with EAN was studied. Western blot analysis showed that the level of GDNF protein significantly increased 1.8‐fold at the paralytic stage of EAN at day 12 post‐immunization (PI) (p < 0.01), and its level further increased approximately 2.5‐fold at day 21 PI (p < 0.001) in the sciatic nerves of EAN‐affected rats compared with those of control rats, and then declined thereafter at day 28 PI. Immunohistochemical analysis showed that axons and Schwann cells constitutively contained GDNF in normal controls. In sciatic nerves with EAN at day 12 PI, GDNF was immunostained in infiltrating inflammatory cells including macrophages and T cells. Collectively, we postulate that GDNF plays a regulatory role in EAN paralysis. A paradoxical role of inflammatory cells to ameliorate PNS inflammation remains to be further studied in EAN, an animal model of human demyelinating disease.     

Prednisolone therapy of experimental allergic neuritis in Lewis rats does not induce relapsing or chronic disease

The effects of therapeutic prednisolone treatment on experimental allergic neuritis (EAN) in Lewis rats were evaluated in a controlled clinical and electrophysiological study. Since steroid therapy has been suspected to cause relapsing or chronic disease, monitoring was extended over 200 days. Short-term steroid treatment (5 days of 15 mg/kg prednisolone, n = 8) with sudden steroid withdrawal was compared with long-term application (30 days, beginning at 7.5 mg/kg) in descending dosage (n = 8). The experiment included saline-injected controls (n = 8) and controls for stress possibly exerted by the handling of the animals. Treatment was begun at the onset of clinical signs. The clinical and electrophysiological data indicated that deterioration, recovery and mild (insignificant) relapse (after day 30 and day 108) occurred in all groups at the same time. Both steroid application schemes significantly (p less than 0.03) attenuated the severity and shortened the duration of EAN. Relapse was not aggravated after steroid treatment. The clinical course and electrophysiological findings were unaltered by the experimental procedures and by mild experimental stress.     

Assessment of lesion evolution in experimental autoimmune neuritis by gadofluorine M-enhanced MR neurography

Experimental autoimmune neuritis (EAN) represents an animal model of acute inflammatory nerve injury mirroring pathophysiological aspects of the human Guillain-Barré syndrome. In the present study, we for the first time visualized the spatiotemporal evolution of autoimmune nerve injury and recovery by magnetic resonance imaging (MRI) by use of the novel micellar magnetic resonance (MR) contrast agent gadofluorine M (Gf). EAN was induced in Lewis rats by T-cell transfer (AT-EAN) leading to severe axonal damage, and Gf was applied intravenously at various disease stages mostly 24 h before MRI. In naive rats, Gf enhancement was present solely in the vascular compartment. In AT-EAN, clinically asymptomatic rats already showed consistent Gf uptake in spinal nerves on day 3, while sciatic nerves were spared. The cauda equina correspondingly exhibited massive T-cell infiltration. Gf enhancement further extended to the plexus lumbosacralis on day 4. On days 5 and 6, the entire peripheral neuraxis from the cauda equina, along the sciatic down to the tibial and peroneal nerves, showed strong Gf enhancement. Spinal and peripheral nerves now exhibited massive inflammation and axonal injury on parallel histological analysis. Gf enhancement persisted in the afflicted nerves until complete recovery and disappeared with a proximodistal gradient. In conclusion, Gf-enhanced MR neurography opens a new avenue for monitoring nerve damage in-vivo during an immune attack.     

Neuropathological findings in chronic relapsing experimental allergic neuritis induced in the Lewis rat by inoculation with intradural root myelin and treatment with low dose cyclosporin A

Experimental allergic neuritis (EAN) was induced in Lewis rats by inoculation with bovine intradural root myelin and adjuvants. Rats treated with subcutaneous cyclosporin A (CsA) (4 mg/kg on 3 days per week from the day of inoculation until day 29) developed a chronic relapsing course. Tissues from the spinal cord, nerve roots, dorsal root ganglia and sciatic nerve of CsA-treated rats sampled during relapses and remissions were studied by light and electron microscopy. Control rats that were not treated with CsA were studied during or after episodes of acute EAN. Both control and CsA-treated animals studied in the first episode of EAN had evidence of inflammation and primary demyelination of the nerve roots and dorsal root ganglia. In control and CsA-treated animals that had recovered from the first episode there was evidence of remyelination. In CsA-treated animals in the second episode there was severe inflammation and demyelination and remyelination of the nerve roots and dorsal root ganglia, and in addition there was significant demyelination and remyelination in the spinal nerves and sciatic nerves and dorsal columns of the spinal cord, particularly in later stages of disease. In later episodes there was less inflammation, but there was continuing demyelination and onion bulbs were present. In animals sampled after recovery from chronic relapsing EAN onion bulbs were present. Occasional small onion bulbs were also observed in control animals that were inoculated with higher doses of myelin. Plasma cells were present in the inflammatory lesions of later episodes. Mast cells were also observed at different stages of the disease. We conclude that the CsA form of chronic relapsing EAN has clinical and pathological similarities with the human disease, chronic inflammatory demyelinating polyradiculoneuropathy.