Inhibitory Effect Of CD147and Integrin Antagonist On Th17Cell Differentiation In Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic chronic inflammation autoimmunedisease characterized by destruction of joint cartilage and bone.Though theetiopathogenisis is still unclear. Th17cells are implicated in pathogenesis ofarthritis, especially in local inflammation and bone destruction. The in vitrocytokine requirements for Th17development are reasonably well established,however, it is less clear what their in vivo immune requirements are. There arenumerous macrophages which migrate from bone marrow in the inflamedsynovial membrane and excessed synovial fluid. The activated monocytes can beused as antigen presenting cells to interact with T lymphocytes, and further leadto the activation of T lymphocytes in situ. There is little research before about theeffect of monocytes on Th17. One recent study has reported that activatedmonocytes derived from RA patients spontaneously and specifically promoteTh17, but not Th1or Th2responses. Surprisingly, Th17intracellular IL-17expression was induced by monocytes not in a TNF-and IL-1β dependentfashion but a cell-contact dependent manner.CD147is a widely expressed plasma membrane protein, which has beenimplicated in T cell activation and immunological synapse formation.Immunosuppressant Cyclosporin A which can inhibit immunological synapseformation, pacitpates in CD147–cyclophilin interactions.CD147as a number ofadhesion molecules can interacte with molecules of integrin family including3β1、6β1、4β1、4β7. In this study, we investigated whether these antagonist, including CD147antagonist---5A12, HAb18(mAbs against CD147), integrinRGD mimetic peptide and CsA, could effect the ability of activate monocytesinducing Th17.【Objective】1. To explore the optimal induction of Th17in the contact of activatedmonocytes and establish stable cocultrue system to induce Th17in vitro.2. To test the effects of cell-to-cell contact with actived monocytes on Th17differentiation.3. To observe the difference between RA and healthy control in proportion ofTh17, IL-17and relative cytokines.4. To detect the regulation effect of mAbs against CD147, cRGD and CsA on Th17differentiation, surface markers and IL-1β and IL-6.【Methods】(three parts):1. CD4~+T cells and CD14~+monocytes were purificated by magnetic cell sortingand cultured (the ratio of CD4~+T cells and CD14~+monocytes was1:1)together. Cultures were stimulated with LPS alone or anti-CD3mAb alone orLPS plus anti-CD3mAb for3days. In the anti-CD3mAb stimulation,different concentrations of LPS were added. Coculture cells were activatedunder LPS/anti-CD3costimulation for3,6, or10days. The percentage ofIL-17+T cells and INF-γ+T cells was determined by fow cytometry.2. Purificated CD4~+T cells and CD14~+monocytes were separated into CD4~+Tcells alone-cultrue, two kind of cells cell-to-cell cocultrue and transwellinserted cocultrue. The percentage of IL-17+T cells was determined by fowcytometry after3days.3.20healthy controls and20confirmed cases of active RA patients werecollected. Purificated CD4~+T cells and CD14~+monocytes were cultured together under optimal stimulation.5A12, HAb18, cRGD and CsA wereadded12h in advance.3days later the percentage of IL-17+, CCR4~+, CCR6+and IL-23R+cells was determined by fow cytometry and IL-1β, IL-6, IL-17were detected by ELISA.【Results】1. LPS or anti-CD3mAb alone induced only very low percentage of IL-17+Tcells,(1.30±0.19)%or (1.10±0.21)%, respectively. The percentage,（2.01±0.46）%, was substantially higher by the LPS and anti-CD3mAbcostimulation. In the presence of0.1μg/ml CD3mAb and low-concentrationLPS (0.1μg/mL) stimulation favored Th17differentiation. The highestproportion of IL-17+T cells was found at day3（2.13%±0.32%）, with levelsdeclining at day6and day10, while, the highest proportion of IFN-γ+T cellswas found at day6（17.45%±3.04%）, declining at day10.2. The proportion of IL-17+,(2.39±0.13)%, was observed when cocultrue wasstumilated compaired with control (0.46±0.11)%, CD4~+T cultured alone andtranswell inserted cocultrue couled not significantly increace the proportion ofTh17after stimulation.3. IL-17and IL-23R expression, as well as IL-1β and IL-6secretion, wassignificantly increased in RA，in comparison of HC. Both CsA and CD147mAbs inhibited the production of cytokines (including IL-17, IL-1β and IL-6)and decreased the frequency of the IL-17+and IL-23R+cells. cRGD onlyinhibited the production of IL-17and decreased the frequency of the IL-23R+cells.【Conclusion】1. Low-concentration LPS stimulation plus anti-CD3mAb in short term supportedthe optimal Th17generation. Th17differentiation required direct contact with actived monocytes. This model might closely mimic the environment ofrheumatoid arthritis in vivo and propose an effective way for the generation ofhuman Th17cells.2. CsA, CD147antogonist and cRGD suppressed the IL-17production andinhibited the Th17cells differentiation from both healthy individuals and patientswith RA, which indicated that CD147and integrin might be implicated in thedevelopment of RA by effecting the Th17cells.