Roles Of Th17 Interrelated Cytokines In Experimental Allergic Neuritis

ObjectiveGuillain-Barre syndrome (GBS) is a common autoimmune disease, characterized with the demyelination of peripheral nerve and inflammatory cells infiltration. Now the pathogenesis of GBS is still unknown. Experimental autoimmune neuritis (EAN), a T cell-mediated, acute, autoimmune demyelination disease of the PNS, is publicly known as a classic animal model for studying pathogenic mechanisms of GBS.Recently, a new linage of Th cells was identified, termed T helper cell 17(Th17), which character is different from Thl and Th2.They produce cytokines include IL-17,IL-17F and IL-22, etc. Th17 cells are characterized by their production of proinflammatory cytokines IL-17. Interleukin 23(IL-23) is a newly identified cytokine belonging to the IL-12 family. It has been shown that IL-23 plays a role in maintaining or stabilizing the Th17 phenotype, or in survival of Th17 cells. Accumulating evidence suggests Th17 cells play an important role in the pathology of various autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), rheumatoid arthritis (RA), inflammatory bowel disease(IBD)and systemic lupus erythematosus (SLE).This pathological role might largely be fulfilled through the secretion of IL-17 and related cytokines. Whether Th17 cell has relation with EAN is not clear. To explore the roles of Th17 in experimental allergic neuritis (EAN), We establish the Guillain-Barre syndrome(GBS) animal models with EAN and observed the expression of Th17 interrelated cytokines IL-17 and IL-23 in EAN, which may provide a new understanding of the pathogenesis and therapy of GBS.Methods1. Antigens:Bovine peripheral nerve myelin (BPM) was prepared from intra-and extradural spinal nerve roots by ultracentrifugation.2. Animals:Thirty-two female Lewis rats,7-8 weeks old, weighting 160-180g were used in the present study. All of the rats were maintained in specific-pathogen free condition.32 female Lewis rats were randomly divided into EAN group (n=24) and the control group (n=8) groups.3. Induction and clinical evaluation of EAN:10% chloral hydrate was used for the animal anesthesia(300mg/Kg). EAN was induced by subcutaneous (s.c) injection into both hind footpads with 100μL of inoculum containing 5 mg of bovine peripheral myelin (BPM) and 1 mg of mycobacterium tuberculosis and 50μL Freund's incomplete adjuvant. In addition to immunization, Freund's incomplete adjuvant and mycobacterium tuberculosis were injected subcutaneously in the control group. Clinical scores were assessed immediately before immunization and thereafter every day until day 21 post immunization (p.i). Severity of paresis was graded as follows: 0= no illness; 1= flaccid tail; 2= dragging both hind limbs; 3= paralysis of both hind limbs; 4= paralysis of four limbs or death; intermediate scores of 0.5 increment were given to rats with intermediate signs.4.Rats sciatic nerves collection:The EAN rats were sacrificed at the onset, peak and recovery phase respectively and the control rats were killed at d21 after experiment. Under aseptic conditions, sciatic nerve segments were excised. Some were fixed in 4% paraformaldehyde for histopathological assessment and immunohistochemistry, the other were stored at -80℃for RT-PCR.5. Histopathological assessment:Sciatic nerve segments were fixed in 4% paraformaldehyde, and embedded in paraffin. Multiple longitudinal sections (5-6μm slices) of sciatic nerves were stained with hematoxylin and eosin to observe the histopathology.6. Paraffin tissue sections (5μm) were deparaffinized, hydrated and treated for blocking endogenous peroxidase activity, repairing antigen. The sections were incubated with polyclonal rabbit anti-rat IL-17,IL-23 antibody for 1h and then stained according to the avidin-biotin technique. The numbers of positive cells were counted at×40 magnification in the entire section area. The average results were expressed as cells per field of high magnification.7. Total RNA was isolated from the sciatic nerves using TRIZOL according to the manufacturer's instruction, was quantified using absorption of light at 260 and 280 nm (A260/280) and reverse transcribed into cDNA using the TIAN script RT Kit.PCR analysis using primers specific for IL-17,IL-23 and the housekeeping gene (3-actin. Results were calculated as levels of target mRNAs relative to those of the house-keeping gene P-actin using Quantity One system.The SPSS 13.0 computer program was used for all calculations and statistical evaluations. Results were expressed as means±standard deviation (SD). Differences between groups were evaluated by one-factor analysis of variance (ANOVA) or rank-sum test. The level of significance was set to p<0.05.Results1. The symptoms of the EAN rats showed on day 9 post immunization (p.i) developed to the climax on day 15p.i, recovered on day 21 p.i. No abnormality is found in the control group.2. Peak numbers of IL-17 positive cells and quantity of IL-17 mRNA in the sciatic nerves were detected on day 15p.i, correlating with development of severe clinical signs.3. Peak numbers of IL-23 positive cells and quantity of IL-23 mRNA in the sciatic nerves were detected on day 9p.i. Conclusions1. The IL-17 might plays an important role in the development of EAN.2. IL-23 maybe correlates with the induction of the disease.