| ObjectiveUveitis is one of the most common causes of human visual disability and blindness. The etiology and mechanism of uveitis are complicated. Recurrent chronic uveitis often results in severe clinical complications and the treatment is tricky. Recurrent experimental autoimmune uveitis (rEAU) has similar characteristics as human recurrent chronic uveitis, providing us with an important animal model to explore the pathogenesis and new therapy of uveitis. Mensenchymal stem cells (MSCs) are a kind of multipotent adult stem cells that possess immunosuppressive properties. Studies have shown that MSCs can inhibit the proliferation and function of various immune cells in vitro and in vivo. This study was to investigate the therapeutic effect of MSCs on rEAU, and the effect of MSC therapy on R16specific T lymphocyte proliferation and helper T lymphocyte (Th)17in rEAU were also analyzed, so as to provide the theoretical basis for clinical application of MSCs in uveitis.Methods1. Culture of MSCs:MSCs were seperated from bone marrow of Wistar rats, cultured and amplified in vitro. The phenotypes of cultured cells were characterized by flow cytometry.2. Induction of rEAU model:To induce EAU, the interphotoreceptor retinoid-binding protein (IRBP) R16segment was emulsified1:1(vol/vol) with Complete Freund’s adjuvant (CFA), containing Mycobacterium tuberculosis H37RA(2.5g/l).The antigen was injected subcutaneously into one hind footpad of Lewis rats. To induce rEAU. on the day10after immunization, T lymphocytes from the spleen and draining lymph nodes of EAU rats were seperated and stimulated with R16in vitro for48hours.1×107activated T lymphocytes were transferred into Lewis rats intravenously.3.MSC treatment:24rEAU rats were randomly divided into the MSC treatment group and the PBS control group. During the day4to day6after T lymphocyte transfer, the rEAU rats were treated intravenously with5×106MSCs diluted in lml PBS or with an equal volume of PBS once a day.4. Clinical observation:From the day after T lymphocyte transfer, the clinical situations of anterior chamber in rEAU rats were examined with a slit lamp biomicroscope regularly.The inflammatory responses of eyes were scored according to Caspi criteria until the day50.5. Histopathological observation:On the day50after T lymphocyte transfer, histopathological changes of retina were examined by hematoxylin and eosin staining.6. Assay of spleen T lymphocyte proliferation by5-bromo-2-deoxyuridine (Brdu):On the day10after T lymphocyte transfer, spleen T lymphocytes were seperated and cultured in vitro for48hours and labeled with Brdu. The Brdu incorporation was assessed at450nm using an enzyme-linked immunosorbent assay reader. The proliferative response was expressed as stimulation index.7. Analysis of CD4+IL-17+T lymphocytes by flow cytometry:On the day10after T lymphocyte transfer. T lymphocytes from the spleen, cervical draining lymph nodes and eyes were seperated and stained by mouse anti-rat mAbs against CD4and interleukin (IL)-17. The proportions of CD4+IL-17+T lymphocytes were analyzed by flow cytometry.Results1. MSCs were successfully separated and identified.2. Rat models of rEAU were successfully induced.3. Clinical observation:MSC treatment could reduce average clinical scores and incidences of inflammation during the50days after T lymphocyte transfer (P<0.05) MSC-treated rats showed reduced signs of uveitis in the first episode of the disease (P<0.05)4. Histopathological observation:On day50after T lymphocyte transfer, chronic inflammation of eye was observed. MSC-treated rats showed remissive inflammatory cell infiltration and retinal damages.5. Assay of spleen T lymphocyte proliferation by Brdu:The proliferation of spleen T lymphocytes in rEAU was depended on the concentration of R16. MSC treatment could inhibit the proliferation of R16specific T lymphocytes from the spleen (P<0.05)6. Analysis of CD4+IL-17-T lymphocytes by flow cytometry:MSC treatment could decrease the proportions of CD4+IL-17+T lymphocytes from the spleen and cervical draining lymph nodes as well as reduce the infiltration of CD4+IL-17+T lymphocytes into eyes (P<0.05)ConclusionThe development of rEAU is mediated by activated R16antigen specific T lymphocytes. Th17and its primary cytokine IL-17play an important role in rEAU. MSC treatment could ameliorate rEAU by inhibiting the proliferation of T lymphocytes and down-regulating Th17. |
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