删除干扰素gamma对实验性自身免疫性神经炎的作用.doc

IFN- deficiency exacerbates experimental autoimmune neuritis in mice via upregulating Th17 cells despite a mitigated systemic Th1 immune response Hong-Liang Zhang1, Xiang-Yu Zheng1, Azimullan Sheikh2, Xiao-Ke Wang1,3, Naheed Amir2, Rayomand Press4, Abdu Adem2, Jie Zhu1,5,* 1Department of Neurobiology, Care Sciences and Society, Karolinska Institute, Stockholm, Sweden 2Department of Pharmacology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates 3Department of Neurosurgery, Second Hospital of Jilin University, Changchun, China4Division of Neurology, Department of Clinical Neuroscience, Karolinska Institute, Karolinska University Hospital Huddinge, Stockholm, Sweden5Department of Neurology, First Hospital of Jilin University, Changchun, China*Corresponding author: Dr Jie Zhu, Department of Neurobiology, Care Sciences and Society, Karolinska Institute, Karolinska University Hospital Huddinge, Novum, plan 5, SE141 86, Stockholm, Sweden (Tel. +46 8 58585494; Fax. +46 8 58585470; E-mail: Jie.Zhuki.se).AbstractPrevious studies have shown that interferon (IFN)-g is a proinflammatory cytokine that contributes to the pathogenesis of Guillain-Barr syndrome (GBS), an inflammatory demyelinating disease of the peripheral nervous system (PNS) in humans, and its animal model, experimental autoimmune neuritis (EAN). Treatments with anti-IFN-g antibodies can improve clinical outcome in GBS patients and EAN animals. Administration of IFN-g markedly worsened EAN. Paradoxically, the mice deficient in IFN- remain susceptible to experimental autoimmune encephalomyelitis, an analogous disease in the central nervous system. These observations raise a question whether IFN- might be protective in autoimmune demyelinating diseases. To clarify the role of IFN-g in the pathogenesis of autoimmune demyelinating disease, we used P0 protein peptide 180-199 to induce EAN in IFN-g knockout (KO) mice. After the acute phase of EAN, the clinical signs of IFN- KO mice were significantly more severe than those of wild type (WT) controls. After antigenic stimulation, the proliferation of splenic mononuclear cell was significantly higher in IFN- KO than WT mice with EAN. At the peak of EAN, the proportion of interleukin (IL)-17A expressing cells in cauda equina (CE) infiltrating cells, and the levels of IL-17A in sera were elevated in IFN- KO mice when compared with their WT counterparts. The proportions of MHC II, ED1, and IL-12 expressing cells, relative to total CE infiltrating cells were correspondingly higher in IFN- KO than WT mice with EAN. However, IFN- deficiency reduced the production of NO by cultured macrophages in response to proinflammatory stimuli and induced a systemic Th2-oriented immune response. In conclusion, IFN- deficiency exacerbates EAN via upregulating Th17 cells despite a mitigated systemic Th1 immune response. Key words: Interferon gamma; experimental autoimmune neuritis; Guillain-Barr syndrome; T helper 17 cell; interleukin 17A; nitric oxideIntroductionGuillain-Barr syndrome (GBS) is an inflammatory demyelinating disease of the peripheral nervous system (PNS) in humans. As a heterogeneous disease entity, GBS is currently defined as an organ specific immune mediated disorder resulting from a synergistic interaction between cellular and humoral immune responses to incompletely characterized antigens in the PNS (Zhang et al., 2010; van Doorn et al., 2008). The exact pathogenesis of GBS remains largely unknown. Experimental autoimmune neuritis (EAN) shares many clinical, histopathological, and electrophysiological features with acute inflammatory demyelinating polyneuropathy, a common clinical subtype of GBS and is therefore used as an animal model to explore the pathogenesis of GBS. Pathologically, EAN is characterized by breakdown of the blood-nerve barrier, robust accumulation of reactive T cells and macrophages in the PNS and demyelination of peripheral nerves (Zhang et al., 2008). As a typical marker for a Th1 response, interferon (IFN)-g is produced by T helper (Th)1 cells. IFN-g exerts its proinflammatory role by activating endothelial cells, macrophages and T cells, etc. IFN-g increases the expression of main histocompatibility complex (MHC) class II molecules thereby enhancing the antigen presenting capacity of macrophages. The potent proinflammatory activities of IFN-g combined with its inhibitory potential for the development of Th2 cells make IFN-g a central mediator of Th1 mediated autoimmune disorders. In addition to activation of macrophages and induction of MHC expression, IFN-g induces the differentiation of T cells to a Th1 phenotype, B cell class switching, apoptosis of T cell, and enhancement of production of other cytokine such as TNF-, interleukin (IL)-1 and IL-6. Most studies indicate that IFN- plays a crucial proinflammatory role in the pathogenesis of GBS and EAN. In patients with GBS, IFN- in serum was elevated and three times higher than the level of IL-4 during the acute phase (Hohnoki et al., 1998). Administration of neutralizing antibodies against IFN- was correlated with improved clinical outcome in patients with GBS (Elkarim et al., 1998). The level of IFN- producing cells in blood, lymph nodes and PNS tissue roughly paralleled clinical course of EAN. Moreover, IFN- receptor deficient mice showed milder symptoms of EAN than wild type mice (Zhu et al., 2001). Systemic administration of IFN- markedly worsened EAN (Hartung et al., 1990). Conversely, treatment with monoclonal antibodies (mAb) against IFN- ameliorated EAN (Hartung and Toyka, 1990). However, IFN- might be protective since some other autoimmune diseases are aggravated either in the IFN- blocking or gene knockout (KO) animal models such as experimental autoimmune encephalomyelitis (EAE) and collagen induced arthritis (CIA) (Chu et al., 2000; Kelchtermans et al., 2007). Also in patients with rheumatoid arthritis (RA), various studies have described protective effects for the use of recombinant IFN- (Machold et al., 1992).Recently, a new subset of Th cells was described and named Th17 based on production of IL-17 (Steinman, 2007). Th17 cells mediate tissue inflammation and autoimmune responses; hence they may help to explain various anomalies that contradicted the Th1/Th2 paradigm in many autoimmune disorders, such as GBS and EAN (Steinman, 2007). However, the role of Th17 cells in the pathogenesis of EAN remains largely unknown. Therefore, in the present study, we used IFN- KO mice to induce EAN, to further clarify the role of IFN- and Th17 cells in the pathogenesis of EAN. Materials and methodsAnimalsIFN- KO and WT mice were purchased from Taconic (Taconic, Ry, Demark) and housed at the animal facility of the Faculty of Medicine & Health Sciences, United Arab Emirates (UAE) University, Al Ain, UAE. Male mice, 5-6 weeks old were used for the study. All mice were housed on a 12/12 light-dark schedule with water and food available ad libitum. AntigenThe neuritogenic P0 peptide 180-199 (SSKRGRQTPVLYAMLDHSRS) of murine PNS myelin P0 protein, were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis (Chan and White, 2000), purified by HPLC using a Vydac reverse-phase column (Grace Vydac, Hesperia, CA, USA), and then analysed by Maldi-time of flight (TOF) mass spectrometry (Cambridge Research Biochemicals, Billingham, UK).Induction of the EAN model and assessment of the clinical courseEAN model was induced by immunizing twice (days 0 and 8) via subcutaneous injection of 150 g of P0 peptide 180-199 and 0.5 mg of Mycobacterium tuberculosis (strain H37 RA; Difco, Franklin Lakes, NJ, USA) in 25 l saline and 25 l Freunds incomplete adjuvant (FIA, ICN Biomedicals, Costa Mesa, CA, USA). Mycobacterium tuberculosis plus FIA is referred to as Freunds complete adjuvant (FCA). All mice received 400, 300, and 300 ng pertussis toxin (PTX, Merck, Whitehouse Station, NJ, USA) by intravenous injection on days -1, +1 and +3 post immunization (p.i.), respectively. Using a blinded protocol, two different examiners assessed clinical signs and body weight of mice immediately before immunization (day 0) and thereafter every two days until day 54 p.i. EAN was scored as follows: 0, normal; 1, less lively, reduced tone of the tail; 2, flaccid tail; 3, abnormal gait; 4, ataxia; 5, mild paraparesis; 6, moderate paraparesis; 7, severe paraparesis. Lymphocyte proliferation testBriefly, mice were sacrificed at the height of EAN (on day 28 p.i.) after perfusion with 0.9% saline, and the spleens were removed. Single cell suspensions of mononuclear cells (MNC) were prepared. Concanavalin A (ConA, Sigma-Aldrich, St. Louis, MO, USA), P0 peptide 180-199 (10 g/ml), and IL-23 (100 ng/ml, eBioscience, San Diego, CA, USA) were used as stimuli. The concentrations had maximum stimulatory effects as assessed in pilot experiments. After 60 hrs incubation, the proliferation was assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI, USA) according to the manufacturers instructions. Briefly, assays were performed by adding a small amount of the reagent directly to culture wells. The CellTiter 96 AQueous One Solution Cell Proliferation Assay contains a novel tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS(a). The MTS tetrazolium compound is bioreduced into a colored formazan product soluble in cell culture medium by NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells. After incubation for 4 hrs, the absorbance at 490nm was recorded with an ELISA reader (Tecan, Mnnedorf, Switzerland).Macrophage cultures and NO detectionMice were sacrificed and standard lavage of the peritoneal exudates with 10 ml serum-free culture medium DMEM/F12 (Invitrogen, Carlsbad, CA, USA) was performed. The lavage fluid contained peritoneal exudate mononuclear cells (PEMs), which represented mainly macrophages as tested by labeling with the macrophage marker ED1. The single cell suspension was centrifuged at 300 g for 10 min. The pellets were re-suspended with DMEM/F12 supplemented with 5% fetal bovine serum (FBS, Sigma), 100 IU/ml penicillin and 100 g/ml streptomycin (both from Gibco-Invitrogen, Grand Island, NY, USA). Macrophages were seeded in 5.3 cm Petri-dishes (Becton Dickinson, San Jose, CA, USA) at a concentration of 2 106/ml. The cultured cells were stimulated with lipopolysaccharide (LPS, Sigma), recombinant mouse IFN- (Hycult Biotechnology, Uden, The Netherlands), tumor necrosis factor- (TNF-, Sigma), polyinosinic-polycytidylic acid (PIC, Sigma), IL-12 (Sigma), and combinations thereof. After 24 hrs incubation, the supernatants were collected and snap-frozen for cytokine and nitric oxide (NO) detection. After removal of the supernatants, 5 ml culture media with Brefeldin A (3 g/ml, eBioscience) was added. Cells were harvested for subsequent flow cytometric analysis after 6 hrs. NO production was measured by the supernatant levels of nitrite, the stable biological oxidation product of NO, by using the modified Griess reagent (Sigma). The detecting procedure was performed according to the manufacturers instructions. The concentrations of nitrite were quantified by extrapolation from the standard curve obtained by using sodium nitrite (Sigma) solutions at concentrations of 9, 3, 1, 0.33, 0.11, 0.033, 0 mg/ml.Isolation of infiltrating cells in cauda equina (CE)Infiltrating cells in CE were isolated according to the method described by Duan (Duan et al., 2004) in our group. Briefly, CE fragments were carefully removed from phosphate-buffered saline (PBS) perfused mice, transferred to RPMI-1640, ground and passed through a 70 m cell nylon mesh (Becton Dickinson, Franklin Lakes, NJ, USA). The collected cells were suspended in 27% Percoll in PBS and centrifuged at 1000 g for 30 min and at 4C. The pellets were harvested.Flow cytometry FITC-, PE-, APC- and PerCP-conjugated antibodies were purchased from commercial suppliers. Specifically, anti-mouse-ED1 monoclonal antibodies were from AbD Serotec; anti-mouse-CD4, CD8, CD25, CD16/32, IL-4, IL-10, IL-12, IL-17A, IL-17F, FoxP3, IFN-, TNF-, MCP-1, CXCL9, TCR and TCR monoclonal antibodies were from eBioscience; anti-mouse-CD45 monoclonal antibodies were from BD Biosciences (San Jose, CA, USA); monoclonal antibodies against mouse MHC-II and CD86 were from Abcam (Cambridge, UK). Cells were harvested and washed with 1% bovine serum albumin (BSA, Sigma) in PBS. For staining of molecules with extracellular expression, cells were incubated with FITC-, PE-, APC, and/or PerCP-conjugated antibodies for 15 min at room temperature (RT). For staining of molecules with intracellular expression, cells were fixed with 2% paraformaldehyde (Merck) for 15 min at RT and permeabilized with 0.5% saponin (Sigma) in PBS containing 1% BSA. The permeabilized cells were incubated with FITC-, PE-, APC, and/or PerCP-conjugated antibodies for 15 min at RT. FITC-, PE-, APC- and PerCP-conjugated isotype antibodies (from BD Biosciences) were used as negative controls. After fixation and permeabilization, cells were washed twice, resuspended in 1% paraformaldehyde in PBS and stored at 4C until flow cytometric analysis by an FACSCantoTM II cytometer (Becton Dickinson) using FACSDiva (Becton Dickinson) software. Surface and intracellular molecule expressions were assessed by determining the mean fluorescence intensities (MFI) or positive cell percentages. Cells from all groups were collected and analyzed at each time point on the same day with the same cytometer settings. Flow cytometric data were analyzed with the CellQuest Pro software. ELISA for measuring IL-6, IL-10, IL-12, IL-17A and TNF- in sera and cell culture supernatants Standardized procedure for the sandwich ELISA was established after optimization of experimental parameters. Coating and detecting antibodies against mouse IL-6, IL-10, IL-12, IL-17A, and TNF-, and recombinant mouse IL-6, IL-10, IL-12, IL-17A and TNF- proteins as standards, respectively, were purchased from eBioscience. Briefly, monoclonal antibodies were coated onto standard ELISA plates (Nalge Nunc, Roskilde, Denmark) in a volume of 100 ml/well overnight at 4C. After three washings with phosphate-buffered saline (PBS), uncoated sites were blocked with 100 l 10% FBS in PBS for 1 hr at RT. Duplicates of samples or of recombinant standard were added and the plates were incubated for 1 hr at RT. After washing, the plates were incubated with biotinylated detecting antibody against IL-6, IL-10, IL-12, IL-17A and TNF- (eBioscience), respectively, for 2 hrs at RT. Then avidin-conjugated horseradish peroxidase (HRP, eBioscience) was added for 30 min. Color reaction was performed with 100 l of tetramethylbenzidine (TMB, Sigma) for 30 min and the reaction was terminated by adding 2 M sulfuric acid (Sigma). The plates were immediately read at 450 nm with an ELISA reader. The concentrations of proteins were quantified by extrapolation from the standard curve. ELISA for measurement of anti-P0 peptide 180-199 antibodies (IgM, IgG, IgG1, IgG2a and IgG2b) in seraSerum samples were obtained from mice at the peak of EAN disease (day 28 p.i.). Samples of groups of 8-10 mice were pooled. P0 peptide 180-199 was coated at 4 C overnight by adding 10 mg/ml in 100 ml per well onto ELISA plates. After three washings with PBS, uncoated sites were blocked with 100 l 10% FBS in PBS for 2 hrs at RT. After three washings, serum samples were diluted to 1:100 with 1% BSA in PBS, applied to plate wells and incubated for 1.5 hrs at RT. Then plates were incubated for 1 hr with peroxidase-conjugated affinipure rabbit anti-mouse IgM, IgG, IgG1, IgG2a and IgG2b (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) after dilution to 1:5000 with 1% BSA in PBS. After three washings, peroxidase substrate TMB was added and 15-30 min later the reaction was terminated by adding 2 M sulfuric acid. The plates were read at 450 nm using an ELISA reader (Tecan).StatisticsData were presented as mean values standard deviations (SD). The statistical program for social sciences 14.0 (SPSS, IBM, West Grove, PA, USA) was used for analysis all data. One-way analysis of variance (ANOVA) and Kruskal-Wallis test were used to compare values among groups followed by Students t-test or Mann-Whitney u-test to compare values between groups. All tests were two-tailed, with the level of significance set to p 0.05.ResultsIFN- deficiency exacerbates the clinical signs after the acute phase of EANAll mice immunized with P0 peptide 180-199 in combination with PTX and FCA acquired EAN. On about day 28 p.i., EAN reached the nadir of in both groups. After about 10 days, mice with EAN gradually recovered. On day 59 p.i., almost all mice recovered from the disease. No difference was seen between the two groups with regard to the onset day after immunization. Nor were seen any differences between the two groups as regards the clinical signs during the acute phase. From day 14 p.i., the clinical signs of IFN- KO mice were significantly more severe than those of WT controls (ANOVA, p 0.01, Figure 1).IFN- deficiency enhances MNC priming responsesAt the nadir of EAN (on day 28 p.i.), splenic MNC were cultured and stimulated with P0 peptide 180-199 and ConA for antigenic and mitogenic proliferation. For both sets of stimulation, the proliferation was higher in mice with EAN than in naive mice (p 0.05 for all comparisons, Figure 2). After antigenic stimulation, the proliferation of splenic MNC was significantly higher in IFN- KO than WT mice with EAN (p 0.05, Figure 2). IFN- deficiency increases IL-17A and IL-12+ infiltrating cells in CEMice were sacrificed at