Effect of fasudil on experimental autoimmune neuritis and its mechanisms of action

This study aimed to investigate the therapeutic effect of fasudil on treating experimental autoimmune neuritis (EAN). Twenty-four EAN mice were randomly assigned to fasudil treatment (Fasudil group) or saline treatment (EAN model group) for 28 days. Clinical symptom score was evaluated every other day; inflammatory cell infiltration, demyelination, anti-myelin basic protein (MBP), inflammatory cytokines, inducible nitric oxide synthase (iNOS), and arginase-1 were detected in sciatic nerves at day 28. Th1, Th2, Th17, and Tregs proportions in splenocytes were detected at day 28. Clinical symptom score was found to be attenuated in the Fasudil group compared to the EAN model group from day 12 to day 28. Sciatic nerve inflammatory cell counts by HE staining and demyelination by luxol fast blue staining were both reduced, while MBP was increased in the Fasudil group compared to the EAN model group at day 28. Interferon γ (IFN-γ) and interleukin (IL)-17 were reduced, while IL-4 and IL-10 were elevated in the Fasudil group at day 28. Sciatic nerve M1 macrophages marker iNOS was decreased while M2 macrophages marker arginase-1 was increased in the Fasudil group at day 28. CD4+IFN-γ+ (Th1) and CD4+IL-17+ (Th17) cell proportions were both decreased, CD4+IL-4+ (Th2) cell proportion was similar, while CD25+FOXP3+ (Treg) cell proportion in splenocytes was increased in the Fasudil group. In summary, fasudil presented a good therapeutic effect for treating EAN by attenuating Th1/Th17 cells and promoting Tregs activation as well as M2 macrophages polarization.

Effect of fasudil on experimental autoimmune neuritis and its mechanisms of action.

This study aimed to investigate the therapeutic effect of fasudil on treating experimental autoimmune neuritis (EAN). Twenty-four EAN mice were randomly assigned to fasudil treatment (Fasudil group) or saline treatment (EAN model group) for 28 days. Clinical symptom score was evaluated every other day; inflammatory cell infiltration, demyelination, anti-myelin basic protein (MBP), inflammatory cytokines, inducible nitric oxide synthase (iNOS), and arginase-1 were detected in sciatic nerves at day 28. Th1, Th2, Th17, and Tregs proportions in splenocytes were detected at day 28. Clinical symptom score was found to be attenuated in the Fasudil group compared to the EAN model group from day 12 to day 28. Sciatic nerve inflammatory cell counts by HE staining and demyelination by luxol fast blue staining were both reduced, while MBP was increased in the Fasudil group compared to the EAN model group at day 28. Interferon γ (IFN-γ) and interleukin (IL)-17 were reduced, while IL-4 and IL-10 were elevated in the Fasudil group at day 28. Sciatic nerve M1 macrophages marker iNOS was decreased while M2 macrophages marker arginase-1 was increased in the Fasudil group at day 28. CD4+IFN-γ+ (Th1) and CD4+IL-17+ (Th17) cell proportions were both decreased, CD4+IL-4+ (Th2) cell proportion was similar, while CD25+FOXP3+ (Treg) cell proportion in splenocytes was increased in the Fasudil group. In summary, fasudil presented a good therapeutic effect for treating EAN by attenuating Th1/Th17 cells and promoting Tregs activation as well as M2 macrophages polarization.

The effects of vasoactive intestinal peptide in the rat model of experimental autoimmune neuritis and the implications for treatment of acute inflammatory demyelinating polyradiculoneuropathy or Guillain-Barré syndrome.

BACKGROUND: Guillain-Barré syndrome is an acute inflammatory demyelinating polyneuropathy that is characterized histologically by demyelination of peripheral nerves and nerve roots, infiltrates of T lymphocytes, and an inflammatory response that includes macrophage infiltrates. The aim of this study was to evaluate the effects of vasoactive intestinal peptide (VIP) in a rat model of experimental autoimmune neuritis (EAN). METHODS: Forty male Lewis rats were divided into a control group (N=10), an EAN group (N=10), an EAN group treated with 15 nmol of VIP (N=10), and an EAN group treated with 30 nmol of VIP (N=10). The rat model was created by subcutaneous injection of P2 polypeptide (200 µg P257-81) into the base of the tail. Intraperitoneal injection of VIP was given on day 7. Rats were weighed and functionally evaluated using an EAN score (0-10). On day 16, the rats were euthanized. The sciatic nerve was examined histologically and using immunohistochemistry with antibodies against CD8, CD68, and forkhead box p3 (Foxp3). Serum concentrations of IL-17 and interferon-α (IFN-α) were measured by ELISA on day 16 after creating the EAN model. RESULTS: The VIP-treated EAN groups had increased body weight and improved EAN scores compared with the untreated EAN group. CD8-positive and CD68-positive cells were significantly reduced in the EAN group treated with 30 nmol of VIP compared with 15 nmol of VIP. Foxp3-positive cells were significantly decreased in both EAN groups treated with VIP, and serum concentrations of IL-17 and IFN-α were significantly lower compared with the untreated EAN group (P<0.05). CONCLUSION: In a rat model of EAN, treatment with VIP resulted in functional improvement, reduced nerve inflammation, and decreased serum levels of inflammatory cytokines.

Toll-Like Receptor 2, Toll-Like Receptor 4, Myeloid Differentiation Response Gene 88, and Toll-IL-1 Receptor Domain-Containing Adaptor-Inducing Interferon-γ (TRIF) Selectively Regulate Susceptibility of P0106-125-Induced Murine Experimental Autoimmune Neuritis.

The functional relevance of the innate immune system has not yet been dissected in P0106-125-induced murine experimental autoimmune neuritis. Therefore, the role of Toll-like receptor (TLR) 2, TLR4, myeloid differentiation response gene 88, and Toll-IL-1 receptor domain-containing adaptor-inducing interferon-γ (TRIF), factors critically involved in the TLR signaling pathway, was studied in experimental autoimmune neuritis. In the absence of TLR2, TLR4, myeloid differentiation response gene 88, or TRIF, the clinical course was significantly attenuated compared to wild-type mice. This could be attributed to impaired NF-κB activation, as shown by the absence of nuclear translocation of RelA with a decreased expression of IL-6, IL-12p40, and IL-17A. Remarkably, P0106-125-immunized TLR20/0 mice exhibited a delayed recovery as compared to TLR40/0 mice, which was because of an impaired T helper cell 2 polarization. Immunized TLR20/0 mice were unable to induce OX40 and OX40L by matrix metalloproteinase-2 on splenic dendritic cells. Subsequently, M2 polarization was impaired and macrophages were unable to sufficiently induce T regulatory cells (Tregs). Thus, in the recovery phase, Tregs were significantly increased in TLR40/0 mice as compared to wild-type mice, whereas Tregs in immunized TLR20/0 mice were only slightly increased. Our data highlight the relevance of innate immunity and, especially, the tight interaction between the innate and the adaptive immune system, which should be considered for therapeutic approaches of autoimmune diseases.

Neurofascin as a target for autoantibodies in peripheral neuropathies.

OBJECTIVES: We asked whether autoantibodies against neurofascin (NF)186 or NF155, both localized at the nodes of Ranvier, are present in serum of patients with inflammatory neuropathy, and whether NF-specific monoclonal antibodies are pathogenic in vivo. METHODS: We cloned human NF155 and NF186, and developed an ELISA and cell-based assay to screen for antibodies to human NF in a total of 434 donors including 294 patients with Guillain-Barré syndrome variants acute inflammatory demyelinating polyneuropathy (AIDP), acute motor axonal neuropathy, and chronic inflammatory demyelinating polyneuropathy (CIDP). We characterized reactive samples by isotyping, tissue section staining, and epitope mapping. We also injected NF-specific monoclonal antibodies IV into rats with experimental autoimmune neuritis. RESULTS: We detected autoantibodies to NF by ELISA in 4% of patients with AIDP and CIDP, but not in controls. Most positive samples contained immunoglobulin G (IgG)1, IgG3, or IgG4 antibodies directed to only one isoform of NF. Two patients with CIDP showed particularly high (1:10,000 dilution) NF155-specific reactivity in both assays and stained paranodes. Two other patients with CIDP who benefited from plasma exchange exhibited antibodies to NF155 by ELISA, and upon affinity purification, antibodies to both isoforms were observed by both assays. Anti-NF monoclonal antibodies enhanced and prolonged induced neuritis in rats. CONCLUSIONS: Autoantibodies to NF are detected in a very small proportion of patients with AIDP and patients with CIDP, but may nevertheless be pathogenic in these cases.

Monocyte chemoattractant protein 1 and chemokine receptor CCR2 productions in Guillain-Barré syndrome and experimental autoimmune neuritis.

Infiltration of activated lymphocytes and monocytes is a key phenomenon in the pathogenesis of Guillain-Barré syndrome (GBS) and experimental autoimmune neuritis (EAN). To investigate the role of chemokines, we determined the blood and nerve tissue expression of monocyte chemoattractant protein 1 (MCP-1), a major chemoattractant of monocytes and activated lymphocytes, and its receptor CCR2 in GBS and EAN. MCP-1 circulating levels (ng/ml) in GBS were increased at the time of progression, peaked at the time of plateau and normalized with recovery. MCP-1 circulating levels were the highest in the most disabled patients. The number of circulating CCR2 positive cells was lower in patients with GBS than in healthy subjects (p<0.004). In GBS, MCP-1 expression was observed in epineurial and endoneurial vessels, on infiltrating cells, Schwann cells and in the endoneurial extracellular matrix. Some CCR2 positive cells were observed in nerve biopsies of GBS patients. In EAN, a slight positivity for MCP-1 was observed in the sciatic nerve. There was no circulating CCR2 positive cells. However, at the time of plateau, a conspicuous infiltration of CCR2 positive cells was observed in the sciatic nerve that was no longer observed at the time of recovery. These results suggest that MCP-1 and CCR2 may participate to the recruitment of circulating mononuclear cells in nerve tissue in EAN and GBS.

Detection and quantification of antibodies to the extracellular domain of P0 during experimental allergic neuritis.

Quantification of the peripheral nerve myelin glycoprotein P0 and antibodies to P0 is difficult due to insolubility of P0 in physiological solutions. We have overcome this problem by using the water-soluble recombinant form of the extracellular domain of P0 (P0-ED) and describe newly developed assays which allow detection and quantitation of P0 and antibodies to P0, in serum and cerebrospinal fluid (CSF). These sensitive and specific assays based on the ELISA technique were used to study humoral immune responses to P0 during experimental autoimmune ("allergic") neuritis (EAN). In order to establish these tests, monoclonal antibodies to different epitopes of rodent and human P0-ED were produced. A two-antibody sandwich-ELISA allowing quantitation of P0 (lower detection limit of 0.5 ng/ml or 30 fmol/ml) and an antibody-capture ELISA (lower detection limit 1 ng specific antibody/ml) to detect antibodies to P0 in serum and CSF were developed. EAN was induced in rats by active immunization with bovine myelin or the neuritogenic protein P2 or by adoptive transfer using P2 specific CD4 positive T cells. Serum and CSF were assayed for the presence of P0-ED and antibodies to P0-ED or P2. Antibodies to P0-ED were detected during active myelin-induced EAN, but not during P2-induced or adoptive transfer EAN. The anti-P0-ED antibodies in the CSF showed a correlation with disease activity. In contrast, in the same model antibodies to P2 persisted long after the disease ceased. No soluble P0-like fragments could be found in serum or CSF during any of the three types of EAN. We conclude that P0 may be a B-cell epitope in EAN. These findings warrant a screen for antibodies to P0-ED in human immune neuropathies.

Peripheral nervous system demyelination from systemic transfer of experimental allergic neuritis serum.

The ability of systemically transferred experimental allergic neuritis (EAN) serum to produce EAN lesions in recipient animals was studied. Seventeen Lewis rats received five daily 1-ml intraperitoneal (i.p.) injections of sera from rabbits with EAN induced with bovine myelin/complete Freund's adjuvant (CFA). Another 17 rats received similar injections of sera from rabbits inoculated with CFA alone. On day 0 (the first day of i.p. injections), all rats were injected in the proximal tibial branch of the right sciatic nerve with a single 10-microliters injection of 0.03 M 5-hydroxytryptamine (5-HT) in sterile 0.15 M saline. Proximal tibial branches of left sciatic nerves received similar single injections of saline alone. Animals were then studied using electrophysiological and histological techniques. In all animals, intraneural saline injection had no significant effect upon nerve conduction. In the presence of circulating CFA serum, 5-HT injection caused a mild gradual decrease in amplitude ratio becoming maximal by day 17 (P < 0.005) and partially resolving by day 28. In contrast, in the presence of circulating EAN serum, 5-HT injection caused a more rapid and severe decrease in amplitude ratio becoming maximal by days 6-10 (P < 0.001 day 6; P < 0.0001 day 10) and completely resolving by day 28. Histological analysis of nerves injected with 5-HT in CFA serum-treated animals showed areas of mild demyelination, axonal degeneration and some fibre loss consistent with needle trauma. In contrast, 5-HT-injected nerves in animals administered EAN serum showed areas of marked cellular infiltration and severe demyelination in association with numerous debris-filled infiltrating cells.(ABSTRACT TRUNCATED AT 250 WORDS)

[Disturbance of immunoregulation in experimental allergic neuritis in rabbits: II. The relationship between the changes in CSF and serum serotonin concentrations and immune responses].

The serotonin (5-HT) concentrations in the CSF and serum of the rabbits with experimental allergic neuritis (EAN) were determined. The relationships between the 5-HT concentrations and the rate of the specific lymphocyte transformation as well as that of the formation of the antimyelin antibody were investigated. The results indicated that after the rabbits were sensitized with myelin basic protein (MBP), the 5-HT levels in the CSF and serum of the rabbits with EAN increased. It was, therefore, considered that 5-HT might play a role in the inhibition of the lymphocyte transformation and the antibody production.

Plasma serotonergic activation in rats during the course of experimental allergic neuritis.

The plasma serotonin (5-HT) levels in Lewis rats were measured during the course of experimental allergic neuritis (EAN) induced by use of purified bovine myelin. The plasma 5-HT levels in EAN rats were significantly higher than those in control rats 15 days (day 15) and 20 days (day 20) after the inoculation. On day 15 and day 20, the 5-hydroxytryptophan levels in the EAN rats were higher than those in the control rats. It is suggested that 5-HT plays a role in the clinical course of EAN.

Spontaneous chemiluminescence activity of peripheral blood cells during experimental allergic neuritis (EAN).

In 30 Lewis rats the time course of spontaneous monocyte and granulocyte chemiluminescence activity (CLA) during myelin-induced experimental allergic neuritis (EAN) was studied. CLA of blood and peritoneal cells, and clinical, histopathological and electrophysiological findings were compared with data from 14 animals immunized with complete Freund's adjuvant and ten normal Lewis rats. About 12 days post-immunization there was a significant (p less than 0.003) rise in monocyte, but not in granulocyte CLA in EAN animals. CLA correlated significantly (p less than 0.02) with infiltrate formation in nerve roots, preceding clinical or electrophysiological signs of disease by 1-2 days. There was no increase in peritoneal monocyte CLA, which was consistently higher than blood monocyte CLA (p less than 0.002).

In vivo monoclonal antibody treatment with Ox19 (anti-rat CD5) causes disease relapse and terminates P2-induced immunospecific tolerance in experimental allergic neuritis.

The role of CD5+ lymphocytes in the recovery phase and on immunospecific protection against experimental allergic neuritis (EAN) was examined in Lewis rats by in vivo treatment with Ox19, a mouse anti-rat CD5 monoclonal antibody. Animals pretreated with the peripheral nerve basic protein P2 and thereby rendered resistant to the disease showed clinical signs of EAN after intraperitoneal (i.p.) Ox19 injection given at the same time as the rechallenge with neuritogenic doses of myelin in Freund's complete adjuvant. Non-pretreated rats recovered from signs of EAN developed a clinical relapse after i.p. Ox19 injections. Taken together, these data suggest an important regulatory role of the CD5 receptor in the immune response.

Lack of effect of fish oil-enriched diet on experimental allergic neuritis in Lewis rats.

Lewis rats were maintained on a diet enriched with menhaden oil, a rich source of the arachidonic acid (AA) substitute eicosapentaenoic acid (EPA). After dietary manipulation the blood neutrophils showed normal leukotriene (LT)B4 but increased LTB5 concentrations, and the CD4+/CD8+T lymphocyte subset ratio was temporarily perturbed. The dietary manipulation did not affect the severity of actively induced experimental allergic neuritis (EAN).

Serum-induced demyelination: an electrophysiological and histological study.

Serum from rabbits with EAN in the acute phase of the disease has been injected into rat sciatic nerve, and compared to control rabbit serum and serum from patients with demyelinating neuropathy and to normal human control serum. Electrophysiological studies were performed on all rat sciatic nerves so injected, and the nerve was then removed and examined histologically. Control rabbit and human serum and neuropathic human serum, when injected in a 20 microL quantity through a 30 gauge needle, did not produce significant electrophysiological abnormalities. EAN serum, however, produced significant dispersion of the muscle action potential and conduction block. All serum produced some histological evidence of demyelination but that seen with EAN serum was quite profound compared to all other sera. It is concluded that humoral factors are present within animals with EAN which has potent demyelinating potential. We were not able to demonstrate the same effect from patients with demyelinating neuropathy in this test system.