ABSTRACT
Background Experimental autoimmune uveitis (EAU) is a common animal model of uveitis.Natural killer (NK) cells have been confirmed to be a type of strong inflammation-causing cells,but its role in EAU is still studing.Objective This study was designed to explore the role and mechanism of NK cells in the pathogenesis of EAU.Methods Thirty-six SPF Lewis rats were randomly divided into expeimental control group and EAU 6-,9-,12-,16-,and 21-day groups (6 rats for each group).Rats in EAU group received subcutaneous injection interphotoreceptor retinoid binding protein (IRBP) combining 5 mg/ml tubercle bacillus with complete Freund's adjuvant (CFA) emulsion in foot pads,and then 400 ng pertussis toxin was intraperitoneally injected to extablish EAU models in the EAU 6-,9-,12-,16-,and 21-day group,and normal saline solution combined with CFA and 400 ng pertussis toxin was used in the same way in the experimental control group.The inflammatory response was observed by slit lamp daily after modeling and scored based on Caspi criteria.The eyeballs were extracted in 6,9,12,16 and 21 days after modeling for retinal histopathological examination,Immunofluorescent double-staining was employed to detect and locate the expression of NK cells in the retina.In addition,25 model rats were divided into EAU 0-,3-,6-,9-and 12-day groups,with 5 rats for each group,and eyeballs were extracted to prepare tissue homogenate.The expression of CXCL10 mRNA,and CXCL12 mRNA NK cell chemokines,in the tissue homogenate was assayed by real-time quantitative PCR.The use and care of the rats followed Regulations for the Administration of Affair Concerning Experimental Animal by State Science and Technology Commission.Results No inflammatory sign in ocular anterior segment of the rats was seen in the experimental control group.The expansion of rat iris vessels was found in the EAU 6-day group,and exudes and hypopyon of the anterior chamber occurred in the EAU 9-day group and the inflammation peaked in the EAU 12-day gorup.The rat retinal structure was normal in the experimental control group,and the arrangement disorder of retinal structure,the cell separation in outer nuclear layer and damage of photoreceptors were found under the optical microscope in different degree in various EAU groups,with the most serious change in the EAU 12-day group.Immunofluorescent double staining showed normally arranged nucleus in the experimental control group,and a lot of NK infiltration was seen in the EAU 6-day group and peaked in the EAU 9-day group.The expression level of CXCL10 mRNA in the EAU 9-day group was 34.298 ± 16.689,which was significantly higher than that in the EAU 3-,6-and 12-day group,respectively (1.390 ± 0.660,3.359 ± 2.581,4.711 ±1.387) (all at P＜0.01).No significant differences were found in the relative expression of CXCL12 mRNA among different EAU groups (F=2.851,P＞0.05).Conclusions Retinal NK cell infiltration occurs in the early stage of EAU,and the severity of NK cell infiltration is consistent with the inflammatory process and CXCL10 expression,suggesting NK cells play an important role in the early stage of EAU,and CXCL10 is an important chemokine of NK cells in EAU rats.
ABSTRACT
Background Chrysin has many biological activities,and its anti-inflammatory effect has been confirmed.However,whether it can treat experimental autoimmune uveitis (EAU) is still not elucidated.Objective This study was to investigate the therapeutic effect of chrysin on EAU and explore the potential mechanism.Methods EAU animal models were established in 30 SPF C57BL/6J mice by the subcutaneous injection and ball pad injection of interphotoreceptor retinoid binding protein1-20 (IRBP1-20)/complete Freund adjuvant (CFA),and then the models were randomized into the model control group and chrysin-treated group.Chrysin solution dissolved by 10 μl dimethyl sulfoxide (DMSO)+140 μl PBS was administrated by gavaging in the mice with the dlose 25 mg/kg from 3 days before immunization through 21 days everyday in the chrysin group,and equal volume of solvent was used in the same way in the model control group.Retinas were examined by indirect ophthalmoscope once per day,and inflammation and pathological scores of retina were performed based on the criteria of Caspi on the 21st day after injection.The apoptosis of retinal cells was assayed by TUNEL staining,and the relative expressions of proinflammatory cytokines interferon-γ (IFN-γ),interleukin-17 A (IL-17A),IL-6 and tumor necrosis factor-α (TNF-α) and signal transducer and activator of transcription 1 (STAT1),STAT3,p-STAT1,p-STAT3 in mouse retinas were detected by Western blot.Results Compared with the model control group,the inflammation scores and pathological scores of retinal inflammation were significantly reduced in the chrysin group (inflammation scores:0.50± 0.45 vs 1.58±0.92,t=2.600,P=0.026;pathologic scores:0.58±0.38 vs 1.83±0.75,t=3.638,P=0.005).The infiltration of a large number of inflammatory cells,retinal vasculitis and granulomatous lesions were found in mouse retinas in the model control group,however,the morphology of mouse retinas in the chrysin group was normal based on hematoxylin-eosin staining.The number of apoptotic cells was remarkable lessened in the chrysin group compared with the model control group under the fluorescence microscope.Western blot assay resolved that significantly downregulation in the expressions of IFN-γ,IL-17A,IL-6 and TNF-α was seen in the chrysin group in comparison with the model control group (t =7.802,P =0.001;t =14.906,P =0.000;t =10.241,P =0.001;t =3.304,P =0.030),and the relative expression levels of STAT1,STAT3,p-STAT1 and p-STAT3 were considerably lower in the chrysin group than those in the model control group (t=8.965,P=0.001;t=8.358,P=0.001;t=4.864,P=0.031;t=4.730,P=0.009).ConclusionsChrysin or chrysin-like flavonoids ameliorate intraocular inflammatory symptoms in EAU mice by inhibiting the activity of STAT signal pathway.
ABSTRACT
Human autoimmune uveitis encompasses a diverse group of common ocular inflammatory diseases.Unknown factors trigger the breakdown of self-tolerance and subsequently lead to the irreversible vision impairment.So the explore and research has never ceased for a long time.Previous study indicated that T helper lymphocyte 1 (Th1) plays a key role in the pathogenesis and development of human autoimmune uveitis.But recently accumulating evidences from both animal and clinical studies suggest that Th17 participates in the pathological mechanism of human autoimmune uveitis.Studies showed that Th1 and Th17 determine the pathological process by the regulation signal of specific cytokines and transcription factors.More and more evidences revealed that Th1 and Th17 are the predominant influences in induction of uveitis.Unveiling the roles of Th1/Th17-related cytokines will help to elucidate the mechanism and progression of uveitis.Recent findings of Th1/Th17-related cytokines regarding pathogenesis and regulation attributed in uveitis were summarized and the novel viewpoint of treatments was discussed.
ABSTRACT
Experimental autoimmune uveitis is an organ-specific T-cell mediated autoimmune disease characterized by inflammation and consequent destruction of the neural retina and adjacent tissues. Inflammation in experimental autoimmune uveitis may be induced in rodents by immunization with retinal antigens, such as interphotoreceptor retinoid-binding protein. We present a review of experimental studies that correlate primary immunobiological functions with this chronic disease and the possible use of molecules for the treatment of autoimmune uveitis.
A uveíte autoimune experimental é uma doença autoimune mediada por células T, órgão-específica e caracterizada por inflamação e subsequente destruição da retina neural e tecidos adjacentes. A inflamação na uveíte autoimune experimental pode ser induzida em roedores pela imunização com antígenos retinianos, tais como a proteína interfotorreceptora ligante de retinoide. Apresentamos aqui uma revisão de estudos experimentais que correlacionam as principais funções imunobiológicas com esta doença crônica e o possível uso de moléculas para o tratamento da uveíte autoimune.
ABSTRACT
Objective To observe the efficacy of the anti-tumor necrosis factor-α monoclonal antibody(TNF-α MCAb)in the treatment of experimental autoimmune uveoretinitis(EAU). Methods EAU animal models were induced by interphotoreceptor retinoid-binding protein(IRBP)R16 peptide with immunization.The rats were divided into 2 groups according to the injection times.TNF-α MCAh was administered intravenously on day 6 or 4,6 and 8 post-immunization respectively,and then to observe the clinical expression by slit-lamp microscope.Meanwhile,take the rats which did not accept TNF-α MCAb as conttol group.Delayed type hypersensitivity(DTH)responses were measured on day 13 postimmunization of IRBP R16;the rats were killed on day 14 post-immunization of IRBP R16,and then enucleated the eyes for histopathological examination.To detect the cytokine level of IFN-γ,IL-4 in serum and IFN-γ in aqueous humor by enzyme-liked immunosorbent assay(ELISA)on day 14 postinjection.The hyperplasia responses of antigen specific lymphocyte of draining lymph node cells were detected. Results The TNF-αMCAb group had mitigated ocular inflammation and decreased pathological grades compared with the control group;the IFN-γ concentrations in aqueous humor and serum were decreased,IL-4 was increased in serum;DTH responses were decreased;the hyperplasia responses of draining lymphocytes to IRBP R16 peptide were decreased,all the differences were statistically significant(P＜0.01).The rats accepted TNF-α MCAb thrice had much better curative effect than the rats injected once(P＜0.05). Conclusions Injection of TNF-α MCAb can inhibit ocular inflammation and specific immune cells of EAU remarkably and change the Th1/Th2 balance.Many times injections of TNF-α MCAb were more effective than once.
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Objective To observe the interferon-γ(IFN-γ),interhukin-2(IL-2)levels of Th1 cytokine and IL-4、IL-10 levels of Th2 cytokine in serum and culture supernatants of splenic cells of the rats in the prevention of experimentaI autoimmune uveoretinitis(EAU)by oral tolerance. Methods 72 Lewis rats were randomly divided into EAU group,oral tolerance group(which including 10 μg、100 μg、1 mg、10 mg of S antigen group respectively)and control group,12 rats in each group.The animal model of EAU was induced by immunization with S antigen(50 μg)and Freund's complete adjuvant.Oral tolerance 10 μg、100μg、1 mg and 10 mg group were fed with 1 ml mixture of 10 μg、100μg、l mg、10 mg S antigen and 1 mg trypsin inhibitor respectively by intuhation,once the other day,totally 7 times,and then induced EAU according to above methods;control group was fed with 1 ml mixture of phosphate buffered saline and 1 mg trypsin inhibitor,once the other day,totally 7 times,and then induced EAU.The clinical manifestation of EAU in the eye were recorded,the eyeballs were enucleated at the peak of EAU,followed by pathological grading.Meanwhile the serum was colleced;splentic cells were separated and cultured to collect the supernatant.Cytokine levels of IFN-γ,IL-2,IL-4 and IL-10 in serum.cultured supernatant of splenic cells were determined by enzyme-linked immunosorbent assay(ELISA). Results Compared with EAU and control group,the levels of IFN-γ and IL-2(Th1 eytokine)in the serum in 100 μg and 1 mg group were decreased while the levels of IL-4 and IL-10(Th2 eytokine)were inereased,the diffefences were statistically significant(F=51.9,68.8,35.7,7.5,P＜0.01).Compared the levels of Th1 and Th2 cytokines in the serum in 10 μg,10 mg group with EAU and control group.the differences were not statistically significant.In 100 gg、1 mg group,the levels of IFN-γ and IL-2(Th1 cytokine)in the culture supernatant of splenic cells were decreased while the Ievels of IL-4 and IL-10(Th2 cytokine)were increased,compared with EAU and control group,the differences were statistically significant(F=57.1,15.6,33.1,167.7,P＜0.01).Compared the levels of Th1 and Th2 eytokine in the culture supernatant of splenic cells in 10 μg、10 mg groups with EAU and control group,the difference are not statistically significant. Conclusions In the process to prevent EAU by oral intake,the levels of IFN-γ and IL-2 (Th1 cytokine)were decrease while the levels of IL-4 and IL-10(Th2 cytokine).Oral administration with tOO high or low dose of the antigen can not prevent EAU as well as the cytokine levels do not change obviously.Cytokines has played an important role in the prevention of EAU.
ABSTRACT
Objective To investigate the expression and significance of inducible co-stimulator (ICOS) in experimental autoimmune uveoretinitis (EAU). Methods EAU was induced in 24 Lewis rats (immune group) by immunization with retinal S-antigen (50 ?g) and complete Freund′s adjuvant, and another 4 rats were in the control group. Anterior segment of the rats′ eyes were observed by split microscope every day. Immunohistochemical staining was performed using polyclonal antibodies to ICOS on the sections of the spleen which were obtained from the rats in immune group at the 7th, 12th, 15th and 21st days after immunisation respectively. Western blotting was performed to investigate the dynamic expression of ICOS protein in the spleen. The same procedures were made at the corresponding time points in the rats in control group. Results A few ICOS positive cells were observed in the normal spleen. The number of ICOS positive cells in immune group increased obviously at the 7th and 12th days after immunization, reached the peak at the 15th day, and decreased at the 21st day which was still higher than that in the control group. The result of Western blotting showed that the dynamic changes of ICOS protein was identical with the changes of positive-cell number detected by immunohistochemistry. Conclusions The enhanced expression of ICOS happens before EAU occurs, which increases when the inflammation occurs and deteriorates, and decreases at the alleviative stage of EAU. It suggests that ICOS participates in the formation, development and disappearance of EAU and plays an important role in the incidence of EAU.