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Macrophages but not Schwann cells express Ia antigen in experimental autoimmune neuritis.

This study was designed to identify which cells express major histocompatibility complex class II (Ia) antigen in experimental autoimmune neuritis and may therefore be antigen presenters. Serial 1-micron-thick cryosections of ventral roots of animals with experimental autoimmune neuritis were labeled with Ox6 antibody against rat Ia, the ED1 antibody to identify monocytes/macrophages and an antiserum against S100, a marker for Schwann cells. Ia-positive cells were predominantly present before overt clinical signs and demyelination (day 12). At later stages when many axons were demyelinated, their number was markedly reduced. Few Ia-positive cells that had extending long processes, which over some distance were in immediate contact with several myelin sheaths, were scattered in normal-appearing nerve roots at these later time points. Most of the Ia-positive cells could be identified as ED1-positive lean monocytes/macrophages, but in contrast most phagocytic macrophages in advanced stages of myelin degradation no longer expressed Ia. Ia-positive structures were invariably negative for S100 at early and late stages of experimental autoimmune neuritis, indicating that Schwann cells did not express identifiable Ia antigen. These findings contrast with reports of expression of major histocompatibility complex class II antigens by Schwann cells in human neuropathies. Furthermore they do not support the notion that aberrant Ia expression by Schwann cells plays a major pathogenic role in experimental autoimmune disease of the peripheral nervous system.

Animals↗

Regulation of experimental autoimmune neuritis by transforming growth factor-beta 1.

Experimental autoimmune neuritis (EAN) is a T-cell-mediated autoimmune disease characterized by demyelination and mononuclear cell infiltration of the peripheral nervous system. It is induced in Lewis rats by administration of myelin P2 protein or a synthetic peptide (SP-26) corresponding to amino acid residues 53-78 of bovine P2 protein. The effects of transforming growth factor-beta 1 (TGF-beta 1) on the clinical signs, histological changes, cell-mediated immune responses, and secretion of interferon-gamma (IFN-gamma) by lymphoid cells of rats with EAN were examined. Systemic administration of TGF-beta 1 markedly inhibited the clinical signs and histological changes of EAN when given intraperitoneally every other day for Days 0 through 18. In addition, it decreased proliferative responses and reduced the delayed-type hypersensitivity (DTH) response to SP-26 compared to control rats. The reduction in clinical severity correlated with skin test unresponsiveness (DTH) to the disease-inducing agent (SP-26) as well to decreased cellular responsiveness to the antigen in vitro. The decrease in cellular responsiveness extended to a decrease in at least one T cell lymphokine, IFN-gamma. The profound effect of TGF-beta on disease progression in EAN, a T-cell-mediated process, is consistent with a direct effect of this growth factor on T lymphocytes.

Amino Acid Sequence↗

Delayed-type hypersensitivity response in experimental autoimmune neuritis treated with peptide-coupled spleen cells.

Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune inflammatory disease of the peripheral nervous system that is characterized by demyelination and mononuclear cell infiltration. It is induced in Lewis rats by administration of myelin P2 protein or a synthetic peptide (SP-26) corresponding to amino acid residues 53-78 of bovine P2 protein. Recently, we showed that SP-26, when coupled to syngeneic spleen cells and administered intravenously, provided an effective means of inducing tolerance by inhibiting the clinical signs, decreased proliferative response of lymphoid cells to SP-26 and histological changes of EAN. However, our current data indicate that, despite tolerance induction in these Lewis rats, the antigen-specific delayed-type hypersensitivity (DTH) response to SP-26 remained intact. Furthermore, interferon (IFN)-gamma production by spleen cells of tolerized rats were unchanged as compared to EAN rats. The in vitro proliferation of T lymphocytes from tolerized rats stimulated by SP-26 was reduced as compared to EAN controls but was enhanced upon addition of exogenous interleukin-2. Thus, reduction in EAN clinical signs does not necessarily indicate a decrease in DTH response and IFN-gamma production in EAN Lewis rats. The implication of this finding in regard to immunoregulatory mechanism of DTH response is discussed.

Animals↗

Ciclosporin A prevents P2 T cell line-mediated experimental autoimmune neuritis (AT-EAN) in rat.

Adoptive transfer experimental autoimmune neuritis (AT-EAN) produced in Lewis rats by injection of P2-reactive T lymphocyte line cells offers the unique possibility to study the exclusive contribution of cell-mediated immune responses to the pathogenesis of autoimmune disease of the peripheral nervous system (PNS). It further lends itself to the evaluation of novel therapeutic approaches that may bear relevance to the human acute Guillain-Barré syndrome. The effects of the immunosuppressive agent ciclosporin A on the clinical, electrophysiological and morphological expression of AT-EAN were examined. We found that ciclosporin A suppressed development of the disease. In view of the known actions of ciclosporin A on T cells, these results indicate the requirement of activation and clonal proliferation of T lymphocytes to produce myelin damage in autoimmune diseases of the PNS.

Animals↗

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.

Animals↗

Pain hypersensitivity in rats with experimental autoimmune neuritis, an animal model of human inflammatory demyelinating neuropathy.

Experimental autoimmune neuritis (EAN) is a T cell mediated autoimmune disease of the peripheral nervous system that serves as an animal model of the acute inflammatory demyelinating polyradiculoneuropathy in Guillain-Barre syndrome (GBS). Although pain is a common symptom of GBS occurring in 55-85% of cases, it is often overlooked and the underlying mechanisms are poorly understood. Here we examined whether animals with EAN exhibit signs of neuropathic pain including hyperalgesia and allodynia, and assessed their peripheral nerve autoimmune inflammation. We immunized Lewis rats with peripheral myelin P2 peptide (amino acids 57-81) emulsified with complete Freund's adjuvant, or with adjuvant only as control. P2-immunized rats developed mild to modest monophasic EAN with disease onset at day 8, peak at days 15-17, and full recovery by day 28 following immunization. Rats with EAN showed a significant decrease in withdrawal latency to thermal stimuli and withdrawal threshold to mechanical stimuli, in both hindpaws and forepaws, during the course of the disease. We observed a significant infiltration of T cells bearing alphabeta receptors, and a significant increase in antigen-presenting cells expressing MHC class II as well as macrophages, in EAN-affected rats. Our results demonstrate that animals with active EAN develop significant thermal hyperalgesia and mechanical allodynia, accompanied by pronounced autoimmune inflammation in peripheral nerves. These findings suggest that EAN is a useful model for the pain seen in many GBS patients, and may facilitate study of neuroimmune mechanisms underlying pain in autoimmune neuropathies.

Animals↗

Linomide-induced suppression of experimental autoimmune neuritis is associated with down-regulated macrophage functions.

Experimental autoimmune neuritis (EAN) is a T-cell mediated autoimmune disease of the peripheral nervous system, in which macrophages and T-cells feature prominently in nerve lesions. EAN represents a counterpart to Guillain-Barré syndrome in humans. In the present study, we investigated the in vitro and in vivo effects of Linomide (LS-2616, quinoline-3-carboxamide), a synthetic immunomodulatory compound, on macrophages in relation to EAN. Linomide strongly suppressed IFN-gamma and lipopolysaccharide (LPS)-induced IL-1 beta, TNF-alpha and IL-6 mRNA expression in macrophages in vitro as demonstrated by in situ hybridisation. Linomide administered daily subcutaneously from the day of inoculation completely prevented the development of clinical symptoms of EAN. Linomide administered from day 9 post immunisation (p.i.) significantly suppressed clinical EAN. Macrophages from Linomide-treated EAN rats showed decreased IL-1 beta, TNF-alpha and IL-6 mRNA expression in response to IFN-gamma and LPS. LPS-induced nitric oxide production by macrophages was also suppressed by Linomide in vitro. Linomide, however, does not affect macrophage death and release of lactate dehydrogenase. We conclude that Linomide may exert its actions in EAN and perhaps also in other autoimmune disease models, by suppressing macrophage functions.

Adjuvants, Immunologic↗

IL-10 suppresses experimental autoimmune neuritis and down-regulates TH1-type immune responses.

Experimental autoimmune neuritis (EAN) is a CD4+ T cell-mediated monophasic inflammatory disorder of the peripheral nervous system (PNS). Cellular mechanisms, including macrophage and T cell infiltration, and cytokines like IFN-gamma and TNF-alpha are intimately involved in the pathogenesis of EAN. Interleukin 10 (IL-10) is a TH2-type cytokine that suppresses monocyte and TH1 cell functions. We examined the effect of recombinant human IL-10 (rHuIL-10) in EAN. When administered from the start of immunization with bovine peripheral myelin emulsified in Freund's complete adjuvant, IL-10 effectively suppressed and shortened clinical EAN. Even when given after Day 12 post immunization (pi) after clinical EAN had been established, IL-10 also effectively suppressed the severity of EAN. Pheripheral nerve myelin antigen-reactive IFN-gamma-secreting TH1-like cells were decreased in lymph nodes from IL-10-treated compared to control EAN rats. PNS autoantigen-induced T cell proliferation and B cell responses were not affected. P2 protein-reactive IFN-gamma, TNF-alpha, IL-1 beta, and IL-6 mRNA-expressing lymph node cells were also downregulated in IL-10-treated compared to control EAN rats at Day 14 and 26 pi, while P2-reactive IL-4 mRNA-expressing cells were upregulated throughout treatment. Also, in IL-10-treated EAN rats, upregulated anti-P2 IgG1 and downregulated IgG2a were observed. Our results clearly show that rHuIL-10 can suppress clinical EAN, and this suppression is associated with downregulation of TH1 responses and macrophage function and upregulated TH2 responses.

Animals↗

Glucocorticosteroids modulate antigen-induced T cell apoptosis in experimental autoimmune neuritis and cause T cell proliferation in situ.

Treatment of experimental autoimmune disorders of the nervous system with high doses of glucocorticosteroids (GC) or with administration of the specific antigen is effective and associated with marked T cell apoptosis in situ. Here we investigated in adoptive transfer-experimental autoimmune neuritis (AT-EAN) of the Lewis rat whether induction of T cell apoptosis resulting from T cell activation by antigen therapy can be further augmented by glucocorticosteroids (GC). AT-EAN was induced by intravenous injection of P2-specific T cell blasts. At the maximum of disease two pulses of the antigen recombinant human P2 (rhP2) were given within 12 h. Methylprednisolone was administered simultaneously or 2 h after the antigen and animals were killed 6 h after the second antigen injection. Using an in situ tailing technique followed by immunocytochemical analysis, the presence of DNA fragmentation in T lymphocytes was confirmed. The bromodeoxyuridine (BrdU) technique was employed to detect in situ proliferating cells. T cell apoptosis in sciatic nerve was enhanced after monotherapy with either antigen or GC compared to the control group receiving an irrelevant myelin protein, recombinant human P0. In combination therapy, a synergistic effect on T cell apoptosis in sciatic nerve was obtained when methylprednisolone was injected sequentially, 2 h after rhP2 protein. BrdU incorporation in the sciatic nerve as well as in the spleen, a major lymphoid organ, was significantly enhanced in animals treated with antigen followed by GC 2 h later as compared to rats receiving rhP2 only, speaking for T cell proliferation in situ associated with T cells undergoing apoptosis. Our findings underscore that different proapoptotic stimuli may act synergistically, depending on the timing of the second treatment. In this scenario even local T cell proliferation in the inflamed nervous system occurs. These results support the paradigm of antigen presentation in the nervous system.

Adrenal Cortex Hormones↗

T cell vaccination does not induce resistance to experimental autoimmune neuritis.

The effectiveness of T cell vaccination was analyzed in experimental autoimmune neuritis (EAN) that can be induced by immunization with bovine P2 protein or a peptide representing the amino acids 53-78 of P2 (P2 53-78). Lewis rats were vaccinated with glutaraldehyde-fixed lymph node cells which had been primed in vivo with P2 protein or P2 53-78 and had been activated in vitro with concanavalin A. Vaccinated animals were not protected from EAN induced by immunization with P2 protein in complete Freund's adjuvant (CFA). In a second set of experiments Lewis rats were vaccinated with irradiated or fixed P2-specific T cell lines of different specificity and neuritogenicity and were subsequently challenged with P2 53-78 in CFA. Likewise, severity of P2 53-78-induced EAN was not different between naive and T line-vaccinated groups. In spleens of vaccinated animals a substantial suppressive activity was demonstrated which was positively correlated with a weak anti-ergotypic response of these spleen cells. The fact that development of actively induced EAN was not prevented or even mitigated by T cell vaccination, in spite of an apparent vaccination-induced response to and on T lymphocytes, suggests that protection from disease is not readily induced in every autoimmune disease model.

Animals↗

Modulation of experimental autoimmune neuritis in Lewis rats by oral application of myelin antigens.

Experimental autoimmune neuritis (EAN) in Lewis rats is a T cell-mediated disease and serves as an animal model of human inflammatory demyelinating neuropathies. EAN can be induced by immunization with complete bovine peripheral nerve myelin (BPM), the myelin protein P2 or its neuritogenic peptide, each emulsified in complete Freund's adjuvant (CFA). The present study evaluates the effect of oral tolerization with BPM or P2 protein on the development of actively induced EAN. Oral administration of BPM strongly suppressed clinical and histological signs of EAN subsequently induced by BPM/CFA, but feeding of P2 protein alone did not affect its course. In contrast, feeding of BPM did not mitigate the course of EAN subsequently induced by immunization with neuritogenic P2 peptide/CFA. Oral therapy with BPM after onset of myelin-induced EAN only slightly ameliorated the further course of disease, but significantly reduced lethality of this severe form of disease. The findings suggest that immunogenicity of the antigens fed determine strength of tolerance, that downregulation of EAN occurs at the site of immunization and not in the nerve, and that active suppression rather than specific anergization is operative in mediating resistance to EAN. However, only partial tolerance to myelin-induced EAN was achieved in naive animals by transfer of spleen/LN cells from rats orally tolerized with BPM. Although methodic factors may have limited the effect of the cells, the result is suggestive of some contribution of anergy to oral tolerance in the present model. Cholera toxin and LPS were identified as oral adjuvants for BPM and prolonged the state of tolerance. However, LPS exhibited proinflammatory properties if EAN was induced early after BPM/LPS-feeding. Thus, oral application of a mixture of myelin components in combination with cholera toxin may be a useful treatment for chronic inflammatory neuropathies considered autoimmune in nature.

Adjuvants, Immunologic↗

Prevention of experimental autoimmune neuritis by nasal administration of P2 protein peptide 57-81.

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. Both EAN and GBS are associated with upregulated T and B cells responses to PNS myelin proteins including P2 protein, and by changes of the Th1/Th2 cell balance in favor of Th1. Here we report that EAN can be prevented by the dominant neuritogenic peptide 57-81 of the PNS P2 protein when given nasally before immunization of Lewis rats with bovine PNS myelin (BPM) + Freund's complete adjuvant (FCA). P2 peptide-tolerized rats were also resistant to EAN relapse after challenge with BPM. Tolerance to EAN in rats receiving high dose (60 microg/day/rat) P2 peptide nasally was associated with specific T and B cell anergy. This was characterized by the failure of T cells to proliferate in response to PNS myelin antigens, while responsiveness to phytohemagglutinin was retained. Numbers of BPM- and P2 peptide-reactive interferon-gamma mRNA expressing lymph node cells were reduced, while levels of P2 peptide-reactive interleukin 4 and transforming growth factor-beta mRNA-expressing cells were markedly upregulated on day 18 post immunization in the rats receiving high dose P2 peptide nasally. Tolerance to EAN was also associated with lower CD4+ cell infiltration, low-grade inflammation, or the absence of histological evidence of EAN, as well as with low IL-2 receptor and MHC class II molecule expression within the PNS. This is the first study showing that mucosal tolerance is applicable to EAN and, as an extension, could be considered in GBS.

Administration, Intranasal↗

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.

Action Potentials↗

Peptide 53-78 of myelin P2 protein is a T cell epitope for the induction of experimental autoimmune neuritis.

We have recently described the clinical and pathological features of experimental autoimmune neuritis (EAN) in Lewis rats inoculated with varying doses of a synthetic peptide corresponding to the amino acid residues 53-78 of bovine P2 protein (SP-26). Immunization with this synthetic peptide was able to induce severe clinical and pathological characteristics of EAN. We are now reporting that, SP-26 T cell lines derived from spleen and lymph node cell populations of such immunized rats, upon being triggered by SP-26, can adoptively transfer severe clinical and histological signs of EAN to naive syngeneic recipients. The disease appears 7-8 days postinoculation of the cells and persist 5-10 days. The pathological features were indistinguishable from SP-26-induced active EAN which appears 12-15 days after sensitization. Examination of the surface phenotype of the cells that were used for the passive transfer of EAN by FACS analysis, showed majority of the cells to be CD4+, Ia+ cells.

Animals↗

The role of macrophages in experimental autoimmune neuritis induced by a P2-specific T-cell line.

A P2-specific T-cell line with a helper/inducer phenotype (W3/25+) mediates experimental autoimmune neuritis in the Lewis rat after adoptive transfer to naive recipients. Moderately severe disease was induced in these experiments by the injection of 1 x 10(7) T cells. Motor and mixed afferent nerve conduction, F responses, H reflexes, and lumbar somatosensory evoked potentials were monitored, and morphological alterations were scored semiquantitatively at the end of the experiments. The role of macrophages and macrophage-derived inflammatory mediators in the effector phase of the disease was investigated by administering different inhibitors of macrophage metabolism, including silica, dexamethasone, and a variety of cyclooxygenase and lipoxygenase blockers. Silica and dexamethasone suppressed the clinical, electrophysiological, and morphological manifestations of the disease almost completely, indicating that macrophages are essential for the generation of inflammatory lesions. The inhibitors of arachidonic acid conversion failed to mitigate the severity of the disease. This is in contrast to observations in actively induced experimental autoimmune neuritis in which eicosanoid biosynthesis seems to play a decisive role in the pathogenesis of the disease.

Animals↗

Inhibition of experimental autoimmune neuritis by an antibody to the lymphocyte function-associated antigen-1.

BACKGROUND: Experimental autoimmune neuritis (EAN) is an animal model of Guillain-Barré syndrome. The mechanisms underlying cellular trafficking and homing of autoreactive immune cells to the peripheral nervous system during EAN and Guillain-Barré syndrome are unknown. We investigated the role of the adhesion molecule lymphocyte function-associated antigen-1 in the pathogenesis of EAN. EXPERIMENTAL DESIGN: EAN was induced in Lewis rats either by immunization with bovine spinal root myelin or by adoptive transfer of P2-specific T cells. Animals were treated intraperitoneally with a monoclonal antibody to lymphocyte function-associated antigen-1 (WT-1) or phosphate-buffered saline and scored for clinical signs. Histology was performed on sciatic nerve and cauda equina and assessed for infiltration and demyelination. Severity of EAN and the corresponding histologic alterations were compared in the different treatment groups. The in vitro effect of WT-1 on T cell proliferation was evaluated. RESULTS: Treatment with WT-1 prevented or efficiently suppressed myelin-induced EAN. In contrast, sham treatment of animals failed to alter the clinical course of EAN. Histologic examination of the peripheral nervous system showed a marked reduction of inflammatory infiltration and perivascular demyelination in animals treated with WT-1. Adoptive transfer EAN was not affected by the administration of WT-1. The differential action in the two models suggests that WT-1 appears to act primarily on the induction phase of the immune response but has no significant impact on the effector phase. In vitro studies with WT-1 revealed that the antibody inhibits the concanavalin A-dependent proliferation of neuritogenic P2-specific T cells. CONCLUSIONS: Our data indicate that lymphocyte function-associated antigen-1 is critically involved in the pathogenesis of EAN. Further analysis of this model may provide insight into the process of immune cell recruitment from the circulation into the peripheral nervous system in immune-mediated neuropathies.

Animals↗

Intranasal administration of recombinant mouse interleukin-12 increases inflammation and demyelination in chronic experimental autoimmune neuritis in Lewis rats.

To examine whether interleukin (IL)-12 modulates ongoing chronic experimental autoimmune neuritis (EAN), we evaluated the effects of recombinant mouse IL-12 (rmIL-12) in Lewis rats with chronic EAN, induced by immunization with P0 peptide (180-199) plus complete Freund's adjuvant. Rats were treated intranasally with either 0.1 or 1 microg/rat/day rmIL-12 for 6 days from the onset of clinical chronic EAN, on days 5-10 postimmunization (p.i.). Only high-dose rmIL-12 exacerbated chronic EAN. This clinical effect was associated with higher numbers of inflammatory cells and more severe demyelination in sciatic nerve sections on days 15 and 80 p.i. compared with low-dose rmIL-12-treated rats and phosphate-buffered saline (PBS)-treated control rats. High-dose rmIL-12 increased significantly the lymph node mononuclear cell proliferation in response to P0 peptide 180-199 and IFN-gamma production in the sciatic nerves. These data indicate that intranasally administered IL-12 acts as a proinflammatory cytokine in chronic EAN. Effective inhibition of IL-12 in vivo could be considered for therapeutic use in chronic inflammatory demyelinating polyradiculoneuropathy.

Administration, Intranasal↗

Increased expression of phospholipase D1 in the sciatic nerve of rats with experimental autoimmune neuritis.

Phospholipase D1 (PLD1) expression in the sciatic nerve was studied in induced experimental autoimmune neuritis (EAN) in Lewis rats. PLD1 immunoreactivity was seen in some Schwann cells in the sciatic nerves of normal rats. In parallel with the progression of EAN, PLD1-positive Schwann cells significantly increased in number and showed intense immunoreactivity. PLD1 was also detected in some ED1+ macrophages in EAN lesions. These results suggest that PLD1 in macrophages and Schwann cells plays an important role in the activation of these cells in the pathogenesis of EAN, an animal model of human peripheral demyelinating disease.

Amino Acid Sequence↗