Capsaicin-enriched diet ameliorates autoimmune neuritis in rats.

BACKGROUND: Autoimmune neuropathies are common PNS disorders and effective treatment is challenging. Environmental influence and dietary components are known to affect the course of autoimmune diseases. Capsaicin as pungent component of chili-peppers is common in human nutrition. An influence of capsaicin on autoimmune diseases has been postulated. METHODS: We tested capsaicin in the animal model of experimental autoimmune neuritis (EAN) in Lewis rat. Rats were immunized with P2-peptide and were treated with capsaicin in different preventive settings. Electrophysiological, histological, and molecular biological analyses of the sciatic nerve were performed to analyze T-cell and macrophage cell count, TRPV1, and cytokine expression. Moreover, FACS analyses including the intestinal immune system were executed. RESULTS: We observed an immunomodulatory effect of an early preventive diet-concept, where a physiological dosage of oral capsaicin was given 10 days before immunization in EAN. A reduced inflammation of the sciatic nerve was significant detectable clinically, electrophysiologically (CMAPs reduced in control group p < 0.01; increase of nerve conduction blocks in control group p < 0.05), histologically (significant reduction of T-cells, macrophages and demyelination), and at cytokine level. In contrast, this therapeutic effect was missing with capsaicin given from the day of immunization onwards. As possible underlying mechanism, we were able to show changes in the expression of the capsaicin receptor in the sciatic nerve and the small intestine, as well as altered immune cell populations in the small intestine. CONCLUSION: This is the first report about the immunomodulatory effect of the common nutrient, capsaicin, in an experimental model for autoimmune neuropathies.

Tissue resident macrophages are sufficient for demyelination during peripheral nerve myelin induced experimental autoimmune neuritis?

The contribution of resident endoneurial tissue macrophages versus recruited monocyte derived macrophages to demyelination and disease during Experimental Autoimmune Neuritis (EAN) was investigated using passive transfer of peripheral nerve myelin (PNM) specific serum antibodies or adoptive co-transfer of PNM specific T and B cells from EAN donors to leukopenic and normal hosts. Passive transfer of PNM specific serum antibodies or adoptive co-transfer of myelin specific T and B cells into leukopenic recipients resulted in a moderate reduction in nerve conduction block or in the disease severity compared to the normal recipients. This was despite at least a 95% decrease in the number of circulating mononuclear cells during the development of nerve conduction block and disease and a 50% reduction in the number of infiltrating endoneurial macrophages in the nerve lesions of the leukopenic recipients. These observations suggest that during EAN in Lewis rats actively induced by immunization with peripheral nerve myelin, phagocytic macrophages originating from the resident endoneurial population may be sufficient to engage in demyelination initiated by anti-myelin antibodies in this model.

Tumor necrosis factor-α in Guillain-Barré syndrome, friend or foe?

INTRODUCTION: Guillain-Barré syndrome (GBS) is an immune-mediated disorder in the peripheral nervous system (PNS), and experimental autoimmune neuritis (EAN) serves as an animal model of GBS. TNF-α plays an important role in the pathogenesis of GBS and is a potential therapeutic target of GBS. Areas covered: 'TNF-α' and 'Guillain-Barré syndrome' were the keywords used to search for related publications on Pubmed. By binding to different TNF receptors, TNF-α bears distinct immune properties. TNF-α gene polymorphisms are associated with the features of GBS. The major role of TNF-α in GBS/EAN is to aggravate inflammation; however, data from several studies indicated a protective role of TNF-α. Multiple lines of evidence point to TNF-α as a potential therapeutic target for GBS. However, such clinical trials are scarce in that GBS per se is a probable side effect of anti-TNF-α treatment. Expert opinion: TNF-α plays a dual role in GBS and EAN, and is a potential therapeutic target on GBS/EAN.

Programmed Death Ligand 1 Plays a Neuroprotective Role in Experimental Autoimmune Neuritis by Controlling Peripheral Nervous System Inflammation of Rats.

Programmed death 1 (PD-1; CD279), a member of the CD28 family, is an inhibitory receptor on T cells and is responsible for T cell dysfunction in infectious diseases and cancers. The ligand for PD-1, programmed death ligand 1 (PD-L1; also known as B7-H1, CD274), is a member of the B7 family. The engagement of PD-1 with programmed death ligand can downregulate autoreactive T cells that participate in multiple autoimmune diseases. Experimental autoimmune neuritis (EAN) is an animal model of Guillain-Barré syndrome, and the pathogenesis of EAN is mediated principally through T cells and macrophages. In this study, we investigated the effects of PD-L1 in EAN rats. For preventative and therapeutic management, we administered PD-L1, which successfully decreased the severity of EAN; it alleviated the neurologic course of EAN, as well as inhibited the infiltration of inflammatory cells and demyelination of sciatic nerves. Our data revealed that PD-L1 treatment inhibited lymphocyte proliferation and altered T cell differentiation by inducing decreases in IFN-γ+CD4+ Th1 cells and IL-17+CD4+ Th17 cells and increases in IL-4+CD4+ Th2 cells and Foxp3+CD4+ regulatory T cells. The expression levels of p-STAT3 and Foxp3 were significantly different in PD-L1-treated groups compared with the control group. Additionally, PD-L1 regulated the expression of Foxp3 and p-STAT3 in EAN, probably by inhibiting PI3K/AKT/mTOR signaling expression. In summary, PD-L1 is a potentially useful agent for the treatment of EAN because of its anti-inflammatory and neuroprotective effects.

Animal models of autoimmune neuropathy.

The peripheral nervous system (PNS) comprises the cranial nerves, the spinal nerves with their roots and rami, dorsal root ganglia neurons, the peripheral nerves, and peripheral components of the autonomic nervous system. Cell-mediated or antibody-mediated immune attack on the PNS results in distinct clinical syndromes, which are classified based on the tempo of illness, PNS component(s) involved, and the culprit antigen(s) identified. Insights into the pathogenesis of autoimmune neuropathy have been provided by ex vivo immunologic studies, biopsy materials, electrophysiologic studies, and experimental models. This review article summarizes earlier seminal observations and highlights the recent progress in our understanding of immunopathogenesis of autoimmune neuropathies based on data from animal models.

Membrane attack complex of complement is not essential for immune mediated demyelination in experimental autoimmune neuritis.

Antibody deposition and complement activation, especially membrane attack complex (MAC) formation are considered central for immune mediated demyelination. To examine the role of MAC in immune mediated demyelination, we studied experimental allergic neuritis (EAN) in Lewis rats deficient in complement component 6 (C6) that cannot form MAC. A C6 deficient Lewis (Lewis/C6-) strain of rats was bred by backcrossing the defective C6 gene, from PVG/C6- rats, onto the Lewis background. Lewis/C6- rats had the same C6 gene deletion as PVG/C6- rats and their sera did not support immune mediated haemolysis unless C6 was added. Active EAN was induced in Lewis and Lewis/C6- rats by immunization with bovine peripheral nerve myelin in complete Freund's adjuvant (CFA), and Lewis/C6- rats had delayed clinical EAN compared to the Lewis rats. Peripheral nerve demyelination in Lewis/C6- was also delayed but was similar in extent at the peak of disease. Compared to Lewis, Lewis/C6- nerves had no MAC deposition, reduced macrophage infiltrate and IL-17A, but similar T cell infiltrate and Th1 cytokine mRNA expression. ICAM-1 and P-selectin mRNA expression and immunostaining on vascular endothelium were delayed in Lewis C6- compared to Lewis rats' nerves. This study found that MAC was not required for immune mediated demyelination; but that MAC enhanced early symptoms and early demyelination in EAN, either by direct lysis or by sub-lytic induction of vascular endothelial expression of ICAM-1 and P-selectin.

Selective expression and cellular localization of pro-inflammatory chemokine ligand/receptor pairs in the sciatic nerves of a severe murine experimental autoimmune neuritis model of Guillain-Barré syndrome.

AIMS: To determine if specific pro-inflammatory chemokine ligand/receptor pairs expressed in the peripheral nerves of Guillain-Barré syndrome patients are expressed in a severe murine experimental autoimmune neuritis (sm-EAN) model and to determine their cellular localization as a prerequisite to designing potentially therapeutic interventions in vivo. METHODS: Sm-EAN was induced in 8-12-week-old female SJL/J mice using bovine peripheral nerve myelin emulsified in complete Freund adjuvant with pertussis toxin and recombinant mouse interleukin-12 acting as co-adjuvants, with appropriate controls. Mice were evaluated for neuromuscular weakness and weighed daily. Dorsal caudal tail and sciatic nerve motor electrophysiological studies were performed at expected maximal severity. Sciatic nerves were harvested and specific chemokine ligand/receptor expression was determined using real-time quantitative reverse transcriptase polymerase chain reaction and indirect fluorescent immunohistochemistry. RESULTS: CCL2/CCR2, CXCL10/CXCR3 and CCL5/CCR1, CCR5 expression was significantly increased in the sciatic nerves of sm-EAN mice compared with controls. CCL2 was expressed on Schwann cells with CCR2 expressed on F4/80+ macrophages and CD3+ T cells. CXCL10 was expressed on endoneurial endothelial cells and within the endoneurial interstitium, with CXCR3 expressed on CD3+ T-lymphocytes. CCL5 co-localized to axons, with CCR1 and CCR5 expression on F4/80+ macrophages and rare CD3+ T cells. CONCLUSIONS: This study suggests that CCL2 expressed by Schwann cells and CXCL10 expressed by endoneurial endothelial cells may induce F4/80+ macrophage and CD3+ T cell-mediated inflammation and demyelination in sm-EAN. CCL2-CCR2 and CXCL10-CXCR3 signalling pathways are potential targets for therapeutic intervention in peripheral nerve inflammation.

Distribution of inducible nitric oxide synthase and tumor necrosis factor-alpha in the peripheral nervous system of Lewis rats during ascending paresis and spontaneous recovery from experimental autoimmune neuritis.

BACKGROUND: Inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) are pleiotropic molecules with widespread action in autoimmune diseases. OBJECTIVE: This study characterizes the distribution of iNOS and TNF-alpha in the spinal nerve roots, dorsal root ganglia and sciatic nerve of Lewis rats during experimental autoimmune neuritis (EAN). METHODS: Macrophages and neutrophils were identified by immunofluorescence as cellular sources of iNOS and TNF-alpha at various stages of EAN induced by synthetic peptide 26. RESULTS: As the disease progressed, iNOS- and TNF-alpha-bearing cells gradually infiltrated the cauda equina, dorsal root ganglia, Th12-L3 spinal roots, and the sciatic nerve. A severer EAN profile developed when more iNOS- and TNF-alpha-bearing cells were present, and the recovery from EAN was related to the disappearance of these cells and the regeneration of nerve fibers. CONCLUSIONS: This is the first report to show iNOS- and TNF-alpha-immunoreactive cells in dorsal root ganglia during EAN, suggesting an underlying pathology for the neuropathic pain behavior in EAN. Our results suggest that the cells bearing iNOS and TNF-alpha in the different parts of the peripheral nervous system are involved in the development of the clinical signs observed at each stage of EAN.

Clinical, electrophysiological and pathologic correlations in a severe murine experimental autoimmune neuritis model of Guillain-Barré syndrome.

Severe murine experimental autoimmune neuritis (sm-EAN) in SJL/J mice is a recently described, but incompletely characterized mouse model of Guillain-Barré syndrome (GBS). Electrophysiological and pathologic characterization during the disease course is a necessary prerequisite to designing mechanistic studies that may be relevant to GBS pathogenesis. Sm-EAN is a monophasic disorder with electrophysiological evidence for a diffuse demyelinating polyneuropathy with axonal loss at peak severity. Regression analyses demonstrated strong correlations between neuromuscular severity scores and electrophysiological parameters during the disease course. Progressive multi-focal or diffuse demyelination with axonal loss was observed pathologically in sciatic nerves in association with mononuclear cell infiltrates (F4/80+ macrophages>CD3+ T-lymphocytes>CD19+ B-lymphocytes), peaking at maximal severity. Regression analyses demonstrated strong correlations between severity scores and inflammatory cell counts. The correlative data imply that mononuclear infiltration, as well as demyelination and axonal loss are directly related to the observed neuromuscular weakness in sm-EAN. The high induction rates, as well as pathologic similarities with AIDP make sm-EAN a robust model to study the pathogenesis of human peripheral nerve inflammation using objective outcome measures.

FTY720 ameliorates experimental autoimmune neuritis by inhibition of lymphocyte and monocyte infiltration into peripheral nerves.

Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune demyelinating inflammatory disease of the peripheral nervous system (PNS). T cells and macrophages are essential for the initiation and development of EAN. FTY720 acts as an agonist of sphingosine-1-phosphate receptors, resulting in inhibition of lymphocyte egress from secondary lymphoid tissues and thymocytes from thymus. This investigation describes the immunosuppressive effects of FTY720 in EAN, the animal model of autoimmune neuropathies. FTY720 (1 mg/kg body weight) completely suppressed paraparesis if administrated from the day of immunization. Furthermore, FTY720 greatly reduced the severity and duration of EAN even when administrated after the appearance of the first neurological sign. T cell, B cell, and macrophage infiltration and demyelination of sciatic nerves were significantly decreased in FTY720-treated EAN. Therefore, FTY720 might be a potential candidate for treatment of inflammatory neuropathies.

Aggravation of experimental autoimmune neuritis in TNF-alpha receptor 1 deficient mice.

The role of tumor necrosis factor (TNF)-alpha and its receptors in the pathogenesis of experimental autoimmune neuritis (EAN) induced by P0 peptide 180-199 in TNFR1 (p55) deficient (TNFR1-/-) mice was investigated. Compared to wild type EAN mice, TNFR1-/- EAN mice developed significantly more severe clinical signs, in parallel with enhanced numbers of inflammatory infiltrating cells in peripheral nerves and splenic P0-reactive T cell proliferation, as well as increased obviously MHC class II and CCR3 expression on the macrophages in the cauda equina. Our data indicated that TNF-alpha might have anti-inflammatory effect preventing the development of EAN in this mouse model.

Apolipoprotein E deficiency enhances the antigen-presenting capacity of Schwann cells.

Apolipoprotein E (apoE) has immunomodulatory properties and has been implicated in the pathogenic mechanism of autoimmune diseases. Previously, the authors found that apoE deficiency increased the susceptibility to experimental autoimmune neuritis (EAN), an animal model for human Guillain-Barré syndrome. To further elucidate the mechanism behind apoE deficiency exacerbating EAN, the authors investigated the role of major target and important antigen-presenting cells of the peripheral nerve system, Schwann cells (SCs), in apoE knockout mice. Treatment of apoE deficient SCs with recombinant mouse interferon-gamma and lipopolysaccharide resulted in higher MHC-II and CD40 expression as compared with normal SCs derived from wild-type mice. The increased MHC-II and CD40 expression on SCs was accompanied by lower levels of intracellular IL-6 production within SCs of apoE deficiency, which is confirmed by the neutralization with anti IL-6 antibody. The increased antigen-presenting capacity of apoE deficient SCs was further explored by enhancement of T cell proliferation co-cultured with P0 peptide 180-199 specific T cells derived from EAN mice immunized with the P0 peptide. In conclusion, apoE may protect mice from EAN and probably also from chronic inflammatory demyelinating polyneuropathy by affecting the antigen-presenting function of SCs via influence of IL-6 production.

Immunohistochemical study of caveolin-1 in the sciatic nerves of Lewis rats with experimental autoimmune neuritis.

The expression of caveolin-1 and the related molecule endothelial nitric oxide synthase (eNOS) was analyzed in the sciatic nerves of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that caveolin-1 significantly increased in the sciatic nerves with EAN upon initiation of cell infiltration during the early and peak stages (days 10 and 14 post-immunization, p.i.) and declined thereafter. The pattern of eNOS expression over the course of EAN largely matched that of caveolin-1. Immunohistochemistry showed that in EAN lesions, intense caveolin-1 immunostaining occurred in ED1-positive macrophages as well as in vessels, while the caveolin-1 immunoreaction was reduced in Schwann cells in the inflammatory lesions. Consequently, we postulated that caveolin-1 expression increased in the sciatic nerves with EAN; this possibly mediated either molecular trafficking or nitric oxide generation partly through the activation of eNOS in vascular endothelial cells, as well as in inflammatory macrophages in EAN and/or cellular apoptosis of inflammatory cells.

Contribution of resident endoneurial macrophages to the local cellular response in experimental autoimmune neuritis.

Macrophages are intimately involved in the pathogenesis of inflammatory neuropathies. The contribution of resident endoneurial macrophages is unknown since their differentiation from infiltrating macrophages is difficult due to missing cellular markers. Previous studies demonstrated the participation of resident macrophages in Wallerian degeneration and the pathogenesis of hereditary neuropathies. The question arises whether resident macrophages are involved in experimental autoimmune neuritis (EAN) where they could contribute to immunosurveillance and antigen presentation. To address this question we used bone marrow chimeric rats, allowing the differentiation between resident and hematogenous cells. Immunohistochemistry and in situ hybridization were applied on to identify and characterize resident macrophages in terms of morphological features, expression of activation markers, proliferation, phagocytosis, and MHC-II expression. Endoneurial macrophages of resident origin were detectable at all stages of disease with a contribution of at least 27% of the total macrophages. They appeared activated by morphological and immunohistochemical criteria and proliferated early. MHC-II-positive resident macrophages were observed that had phagocytosed myelin. These results demonstrate that the macrophage response in EAN is partly of intrinsic origin. The rapid activation and proliferation of resident endoneurial macrophages points toward an active role of these cells in inflammatory peripheral nerve disease, especially early in disease.

Axonal protection in experimental autoimmune neuritis by the sodium channel blocking agent flecainide.

Inflammatory demyelinating neuropathies such as Guillain-Barre syndrome (GBS) and its animal model, experimental autoimmune neuritis (EAN), are typically acute monophasic diseases of the PNS that can leave affected individuals with permanent disability due primarily to axonal degeneration. The mechanisms underlying the degeneration are not understood, but we have previously shown in vitro and in vivo that axons can degenerate when exposed to the inflammatory mediator nitric oxide, and that axons can be protected by application of the sodium channel-blocking agent, flecainide. Here we examine whether flecainide administration can similarly reduce axonal degeneration in the periphery in animals with EAN. EAN was induced in Lewis rats (n = 116, in three independent trials), and rats received either flecainide (Flec) (30 mg/kg/day) or vehicle (Veh) from the onset of disease expression. Flecainide administration significantly reduced the mean (SD) scores for neurological deficit at both the peak of disease (Flec: 5.7 (2.7), Veh: 8.0 (3.6), P < 0.001) and at the termination of the trials 25-29 days post-inoculation (Flec: 2.2 (2.4), Veh: 4.2 (4.2), P < 0.001). Histological examination of the tibial nerve of EAN animals revealed that flecainide provided significant protection against axonal degeneration so that 80.0% of the normal number of axons survived in flecainide-treated rats compared with 62.8% in vehicle-treated rats (P < 0.01). These findings may indicate a novel avenue for axonal protection in GBS and other inflammatory demyelinating neuropathies.

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.

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-gamma (IFN-gamma) and tumour necrosis factor (TNF-alpha) 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-gamma and TNF-alpha 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.

Accumulation of immunoglobulin across the 'blood-nerve barrier' in spinal roots in adoptive transfer experimental autoimmune neuritis.

At the onset of Guillain-Barré syndrome, disruption of diffusion barriers, such as the blood-nerve barrier, probably increases the exposure of spinal roots and peripheral nerves to macromolecules, some of which might be pathogenic. As a measure of such disruption, we measured the accumulation in the endoneurium of spinal roots and sciatic nerve of systemically administered 125I-labelled immunoglobulin in adoptive transfer experimental autoimmune neuritis (AT-EAN) in the rat. AT-EAN is a model of Guillain-Barré syndrome, induced by injection of activated T lymphocytes sensitized to myelin P2 protein. Immunoglobulin accumulation was expressed as counts/min/mg in fixative-perfused roots as a percentage of that in serum, measured 24 h after intraperitoneal injection of 0.1 micro Ci 125I-labelled immunoglobulin. Immunoglobulin accumulation in the roots of normal rats was 3 +/- 1% (mean +/- SE), but this first increased 3(1/2) days after cell injection, peaked at 22 +/- 2% on day 4(1/2), and declined to normal by day 8. T lymphocytes and polymorphonuclear leucocytes first appeared within the endoneurium at day 3(1/2), and macrophages and a few erythrocytes at day 4. Neurological deficit appeared on day 4 and was maximal on day 6. Demyelination and axonal degeneration began at day 5. The first abnormality detected in AT-EAN was a rapid increase in the passage of immunoglobulin into spinal roots, together with endoneurial infiltration of T lymphocytes and polymorphonuclear leucocytes. Accumulation of immunoglobulin was maximal during the worsening of neurological deficit, and declined rapidly before the onset of neurological recovery.

Efficacy of leukemia inhibitory factor in experimental autoimmune neuritis.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that exerts neurotrophic and myotrophic actions. We have investigated the effect of LIF in experimental autoimmune neuritis (EAN), an animal model of Guillain-Barré syndrome (GBS). Treatment with LIF at the onset of the disease showed a slight, but not significant, improvement in the clinical course but no effect on nerve histology.

Initiation and development of experimental autoimmune neuritis in Lewis rats is independent of the cytotoxic capacity of NKR-P1A+ cells.

Natural killer (NK) cells are implicated in T cell-mediated autoimmune diseases. Experimental autoimmune neuritis (EAN) is a CD4+ T cell-mediated animal model of the Guillain-Barré syndrome in human. The role of NK cells in the initiation and development of EAN remains unclear. In the present study, we demonstrate that anti-NKR-P1A monoclonal antibody (mAb) treatment in vivo did not affect the initiation and development of clinical EAN in Lewis rats induced by immunization with peripheral nerve myelin P0 protein peptide 180-199 and Freund's complete adjuvant, as well as the proportion of NKR-P1A+ cells (including NK cells and NKT cells) in the spleen. Furthermore, inflammatory cell infiltrations and demyelination in the peripheral nervous system (PNS) and in vitro P0 peptide 180-199-specific splenocyte proliferation were not different in anti-NKR-P1A mAb-treated rats compared to the control antibody-treated rats. The cytotoxic activity of NKR-P1A+ cells, determined by NK cell-sensitive K562 cells as target cells, decreased markedly in anti-NKR-P1A mAb-treated rats, suggesting that decrease of the cytotoxic activities of NKR-P1A+ cells is not sufficient to alter clinical EAN, although NKR-P1A+ cells may participate in the pathogenesis of T cell-mediated autoimmune diseases, such as EAN, by the mechanisms that involve the release of cytokines.