L161982 Influences Macrophage Subtypes In Experimental Autoimmune Neuritis

1.ObjectiveGuillain Barre syndrome (Guillain-Barre syndrome, GBS) is one kind of autoimmune disease in peripheral nerve demyelination,charactered with the the pathological features of demyelinating polyneuropathy and inflammatory cell infiltration,the clinical manifestations is acute flaccid paralysis of limbs symmetry. Experimental autoimmune neuritis (experimental allergic, neuritis, EAN) is mediated by T cells of the peripheral nervous system inflammatory demyelinating autoimmune disease, and become the classical animal model for the study of human GBS for its similar clinical pathological features.In the pathogenesis of experimental autoimmune neuritis, as a classic of immune cells,macrophage participates in autoimmune disease including EAN. Researches have shown that macrophages mainly divided into two subtypes:the classical activation of M1 subtype macrophage and alternative activation of M2 subtype macrophages.Ml macrophages promote inflammation progress and phagocytosis.;While M2 subtype macrophages inhibit inflammation and promote nerve tissue repair.However, the mechanism of macrophages from M1 type to M2 type is not very clear.Prostaglandin (PG) as a medium in the process of inflammatory cells involved in the occurrence and development of EAN.PGE2 collaborates with EP2 and EP4 can inhibit inflammation. Studies have confirmed, the use of PGE2-EP4 receptor antagonists such as L161982, can promote recovery of inflammatory autoimmune disease and alleviation of the clinical symptoms including EAE.In this study, through the establishment of rat model of experimental autoimmune diseases, we use prostaglandin E2-EP4 receptor antagonist L161982 (EAN) to intervent the different stages of EAN, to observe the proportion of macrophage subsets’ expression and the variation of related cytokines,explore the mechanism of PGE2-EP4 sigal cell for M1 and M2 subtypes’ transformation,explore the contributing factors of recovery of EAN.2.Methods2.1 Preparation of antigenExtracte the peripheral nerve myelinfrom bovine nerve root fresh (bovine peripheral nerve myelin, BPM) by high speed centrifugation. Soluble BPM in incomplete Freund’s adjuvant containing H37Ra heat killed bacteria.Add physiological saline into suspension while the shock mixing. Each 100ul antigen emulsion containing BPM 5mg, Mycobacterium tuberculosis 1mg.2.2 Treatment of experimental animal2.2.1 The establishment of EAN modelTwenty-one female Lewis rats,6--8 weeks old, weighting 160-170g were used in the present study. All of the rats were maintained in specific—pathogen free condition.After 1 week adaptive feeding of Lewis rats,10%chloral hydrate was used for the animal anesthesia(0.3g/kg).EAN Was induced by subcutaneous injection into both hind footpads with 100ul antigen emulsion.2.2.2 Grouping of EAN rats and the intervention of L161982 in EAN ratsA total of 21 EAN rats were randomly divided into A, B, C 3 groups.Rats in A group were intraperitoneally injected with L161982 solution (5mg/kg), B group rats immune 1 days before to eighth days (Immune) by daily intraperitoneal injection of L161982 (5mg/kg), the rats in C group from fifth to 16 days (immune disease) daily intraperitoneal injection of L161982 (5mg/kg).2.3 Clinical scores of EAN ratsObserve the condition changes, daily body weight and clinical score for each group of EAN rats before and after immunization.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.2.4 Preperation of macrophagesThe EAN rats were sacrificed at the sixteeth day (the peak phase), injecte10mlDMEM to the rats’peritoneal on the sterile test bench,static 5min, open the abdominal cavity of rats.When the intestinal flattened, absorb the peritoneal lavage fluid by 2ml sterile disposable Straw, placed in the centrifuge tube. In the centrifuge after centrifugation, supernatant to leave, precipitae single cell suspension. Counting of cells, according to the cell density inoculated with 2 x 106/ml in 6 well plates, place the cells at 37℃, 5% CO2 incubator. Chage the liquid after 24h, remove the non-adherent cells, culture to the third days.2.5 Flow cytometric analysis of peritoneal macrophages of EAN ratsSingle cell suspension was prepared after 3 days incubation.6 test tubes(named 1、2、 3、4、5、6) were prepared.Put 106/ml cells into tube 1 to5,tube 6 is blank control.Mark Ml, M2 macrophages and totle macrophages subtypes respectively with CD68 and CD86 or CD 163 fluorescent antibody combination.Tubel is CD86 antibody marked with PE,tube2 is CD 163 marked with Alexa Flour,tube3 is CD68 antibody marked with FITC,tube4 isCD86 antibody、CD68 antibody and FITC antibody,tube5 is CD 163 marked with Alexa Flour and CD68 antibody marked with FITC,tube6 is blank control.After fully mixed, dark incubation at 4℃ temperature after 30min, after washing with the 2mLBSA,1500r/min 5min centrifugation, collect the supernatant. Each tube with 400 L PBS suspension cells, the sieve, adding 1% paraformaldehyde 100ul to be fixed. Flow cytometry was used to detect 3 groups of rats two macrophage subsets.Preperation of Spleen mononuclear cells2.6Determination IL-12 and IL-10 of Spleen mononuclear cells culture supernatant by ELISA3 groups of EAN rats were killed by cervical dislocation, under sterile conditions, removal of the spleen of EAN rats were placed on the 5ml DMEM containing medium, centrifugal tube, cut the spleen in cell screen in a sterile test bench collected grinding fluid of spleen mononuclear cells. Place in the centrifuge tube, 1200 rpm/min. Each tube with 2 ml red blood cell lysis, put it at room temperature after 5min until the cell suspension become colorless, each tube with 1ml fetal bovine serum to terminate the broken red cells.1200 rpm/min,5 min after centrifugation supernatant, adding 2mlDMEM medium and mixed. After fully mixed, count cells. Place the cells according to the density of 2 x 106/ml inoculated in 6 well plates, at 37 ℃,5% CO2 incubator. The supernatant was collected after 3 days of culture, cell culture plate, placed in 2ml centrifuge tube, the content of spleen mononuclear cells in 3 groups were detected in cultured rat IL-12 and IL-10 in the supernatant by ELISA method.2.7 Statistical analysisThe SPSS 19.0 computer program was used for all calculations and statistical evaluations. Differences among different groups were tested by one factor analysis of variance. Results were presented as means ± SD and a level of p<0.05 was considered significant.3.Results3.1 The incidence and clinical score of rats in each groupFrom sixth days to fifteenth days after immunization,the symptoms of EAN rats were showed in each group. Compared with group A, group B,and group C was significantly delayed the onset of EAN (P<0.05), the rats in group B than in group C was significantly delayed the onset of EAN (P<0.05); compared with A group, B group, C group, the peak incidence of clinical score decreased significantly (P<0.05); compared with C group, B group rats were significantly lower clinical scores (P<0.05)3.2 Detect two macrophage subsets proportion with flow cytometry.Compared with A group, L161982 decreased the proportion of CD86 cells in peritoneal lavage cells (P<0.05), reduce the cell proportion of CD86 CD68 cells (P<0.05), CD 163 cells increased the proportion of CD68 cells in group B and group C (P<0.05); compared with C group, the changes of the proportion in B group is more significant (P<0.05)3.3 Determination IL-12 and IL-10 of Spleen mononuclear cells culture supernatant by ELISACompared with A group,L161982 decreased the concentration of IL-12(P<0.05) and increased the concentration of IL-10 (P<0.05) in groupB and groupC in spleen mononuclear cell culture supernatant;compared with C group, the changes of the concentration in B group is more significant (P<0.05)4.Conclusions:4.1 L161982 decreased the percentages of M1 macrophages in totle macrophahges and increased the percentages of CD163+cells in totle macrophage of EAN rats,the immunization phase is more significant.4.2L161982decreased the level of IL-12 associated with M1 macrophages and increased the level of IL-10 associated with M2 macrophages,the immunization phase is more significant.4.3L161982 reduced EAN rats clinical score, delayed the onset time of EAN rats, the immunization phase is more significant.