Costimulatory Molecule VSIG4 Exclusively Expressed On Macrophages Inhibit The Proliferation And Activation Of T Cells

Background and objective:Renal interstitial damage was a important cause of chronic renal failure (CRF). The main characteristics of tubulointerstitial damage was the inflammatory cells infiltration in renal interstitial, of which the infiltration and activation of T cells was the most critical. Costimulatory molecules was an important regulated mechanism for T cell activation. Costimulatory molecules was a group of Membrane surface molecules ,it can supplied necessary subsidiary signal for T,B cells to regulate the cell proliferation,differentiation and activation,and the lack of costimulatory signals to antigenic stimulation would cause T cell responses of anergy or apoptosis.In current study of kidney disease, T cell unresponsiveness was indirectly induced by inhibiting costimulatory pathway, thus achieve the purpose of reducing the renal tissue damage.These study have achieved some results. Some studies have shown that, through the inhibition of positive co-stimulatory pathways or enhanced negative costimulatory pathways to induction of effector T cell anergy, can effectively reduce the renal tissue injury.It had been confirmed that macrophage was another very important cell group in tubulointerstitial infiltration.Macrophages acted as a antigen presenting cell (APC), it constantly expressed MHC to provide the first signal of T cell activation and regulate T cell activation, phenotype and it play different immune effect because of its different phenotype; at the same time, macrophages also expressed various co-stimulatory molecules.In past studies it had shown that macrophages could secrete nitric oxide (NO),metalloproteinases,growth factors and other inflammatory mediators to stimulate the generation of renal interstitial fibrosis. However, recent studies confirmed that IL-4 and IL-13 activated macrophages (M2 macrophages) could reduce tubular damage in doxorubicin-induced SCID mice nephritis. Macrophage was not only defined as a primary factor of kidney inflammatory injury, it also possessed protective or regulatory action. Furthermore, Macrophages specifically located in the inflammatory tissue, can play local anti-inflammatory function mean while avoiding the systemic immune tolerance. So we supposed that if macrophages induce T cell unresponsivenes of renalinterstitial through expressing costimulatory molecules? This is our research focus.V-set and Ig domain–containing 4 (VSIG4),a new B7 family–related protein,also known as complement receptor of the immunoglobulin superfamily ( CRIg ) or Ig superfamily protein 39(Z39Ig),was a strong negative regulator of murine and human T cell proliferation and activation. VSIG4 was specifically expressed on resting tissue macrophages. In the study of renal biopsy specimens on renal disease patients,we found macrophages in the renal tissue infiltration area can exclusively express VSIG4,and the expression level was negatively related to the severity of the lesion.Then we study its role in animal experiments, we developed unilateral ureteral obstruction ( Unilateral ureteral occlusion, UUO ) nephropathy model in VSIG4 knockout ( VSIG4 - / - ) mice and normal wild type ( VSIG4 + / + ) mice , found the VSIG4 - / - mice postoperative renal interstitial inflammatory cell infiltration and tubular injury level were significantly heavier than the VSIG4 + / + mice, while in VSIG4 - / - group TGF beta 1, IL-2, IFN gamma, IL-10, TNF alpha expression level was significantly higher than that in VSIG4 + / + group, it mean that renal interstitial macrophages expressed VSIG4 to inhibit the infiltration and cytokines effect of CD3 +, CD4 +, CD8+T cells in renal interstitial, thus to inhibit macrophage mediated tubulointerstitial inflammation, relieving UUO mouse kidney pathological changes. In order to further study the inhibition effect and mechanism of VSIG4 in macrophages to T cell, we proceed experimental study in vitro on the basis of above experiments in vivo, First we proposed preparation and purification of VSIG4-Fc fusion protein to direct stimulate T cells in order to observe their effects on T cell proliferation and activation function; on the other hand, we proposed to acquire VSIG4 - / - mice and VSIG4 + / + mice sources macrophages, and respectively co-culture with T cells in vitro, compare T cell proliferation and activation in each group, to approach the effects and mechanisms of that macrophages specific express the VSIG4 to inhibitT cell proliferation and activation.Methods:1. VSIG4-Fc fusion protein preparation: pCEP_m VSIG4_Fc recombinant plasmid was sequenced in order to identify it .Then the plasamid was transfected into mammalian CHO cell by LipofectamineTM- 2000.The surpernatant of cultured cell was collected and analyzed by western blot to detect if there was the fusion protein,and the fusion protein was purified by Protein A affinity chromatography. The expression product was identified bySDS-PAGE and immunoblotting.2. The immunological effect of VSIG4-Fc fusion protein: T cells were stimulated with PMA in vitro. Then VSIG4-Fc and mouse IgG1 control were respectively added to cell culture system. T cell proliferation was measured by CCK8, T cell surface Fas/FasL expression was detected by FACS,protein expression of IL-2, IFN-γwas detected by ELISA.3. T cells,VSIG4-/- macrophage and VSIG4+/+ macrophage preparation:Purified CD4+T cells were purified from normal mice spleen lymphocyte cell suspension using CD4- specific MicroBeads (miltenyi Biotec) according to instructions. Macrophages were collected from the peritoneal cavity of VSIG4-/- and normal C57B6 mice by washing the peritoneum.Purified T cells were stimulated by indicated concentrations of mouse anti–CD3e antibody (BD Biosciences Pharmingen).4. The experiment grouping of VSIG4-/- macrophage or VSIG4+/+ macrophage with T cells co-culture experiment: Group 1(blank group): T cells was cultured alone without CD3 antibody. Group1( negative control group ):T cells was cultured alone witn CD3 antibody. Group 3 (VSIG4-/-/T group), macrophages were collected from the peritoneal cavity of VSIG4-/- mice by washing the peritoneum, then cultured with purified T cells on the stimulation of anti–CD3e antibody (BD Biosciences Pharmingen). In the Group 4(positive control group), macrophages from wide type C57B6 mouse was co-cultured with T cells. .5.The proliferation and activation of T cells in the co-culture experiment: T cell proliferation was assaied by 3H-TdR incorporation, the activity of CD69 was tested by FACS; The mRNA expression of IL-2,IFN-γwas determined by reverse transcription polymerase chain reaction (RT-PCR). The protein expression of IL-2,IFN-γwas determined by enzyme-linked immunosorbent assay。Results:1. The preparation of VSIG4-Fc: PCEP_m VSIG4_Fc recombinant plasmid was successfully transfected to CHO cells by positive liposome Lipofect amineTM2000 .And in transfection cell and supernatant we detected the expression of VSIG4-Fc protein. transfection product was purified by protein A affinity chromatography,purified product was identified as VSIG4 protein by Western blottiong.2. Effect of VSIG4-Fc fusion protein on the proliferation and activation of T cell: CCK8 assay detected T cell proliferation, in the control group T cell proliferation increased significantly, while in experimental group which added the VSIG4-Fc fusion protein the OD value was lower than that of the control group, and there is a significant difference (P <0.05). Fas/FasL expression on T-cell surface detected by flow cytometry showed that Fas / FasL was significantly increased after cultrued with VSIG4-Fc fusion protein,(P <0.05). ELISA assay detected IL-2, INF-γprotein expression levels found IL-2, INF-γlevels were significantly decreased that after added the VSIG4-Fc fusion protein (P <0.05). Results as above suggested that co-cultureed with VSIG4-Fc fusion protein can inhibite the proliferation and activation of T cell.3. VSIG4-/- macrophages and VSIG4 +/+ macrophage was co-cultured with T cell: 3H-TdR incorporation detedted T cell proliferation, the results showed that 3H incorporation volume of VSIG4-/-/T group and VSIG4 +/+/ T group was both significantly lower than that of the negative control, and 3H incorporation of VSIG4-/-/T group was higher than that of VSIG4 +/+/T group.It suggested that the inhibition role of macrophages on T cell proliferation was significantly reduced after VSIG4 gene knockout. Flow cytometric detected T-cell surface activation molecule CD69 showed in VSIG4-/-/T group and VSIG4 +/+/ T group CD69 positive cells rat was significantly reduced compared with the negative control group (p<0.01).And the experimental group was significantly higher than the positive control group (P<0.01). It suggested that after VSIG4 gene knockout macrophages's inhibition capacity on T cells activation diminished. RT-PCR, ELISA assay detected IL-2, IFN-γprotein and gene expression from T cells, the results showed that: compared with negative control group, the expression of IL-2, IFN-γin VSIG4-/-/T group, VSIG4 +/+/ T group were significantly decreased (p <0.01), and in VSIG4-/-/T group the expression of IL-2 and IFN-γwere significantly higher than that of VSIG4 +/+/ T group (p <0.01, <0.05). We can found that after VSIG4 knockout, the degree of T cell activation increased in the co-culture system.Conclusion:1 we transfected VSIG4-Fc-pCI-neo plasmid to CHO cells,and successfully prepared VSIG4-Fc fusion protein. And we confirmed that VSIG4-Fc fusion protein could inhibit T cell proliferation and activation in vitro confirmed.2 .VSIG4-/- macrophages and VSIG4+/+macrophages was co-cultured with T cells,the reslut showed after VSIG4 gene knockout, macrophages's inhibition to T-cell proliferation and activation was diminished, it suggested that VSIG4 play an important role in the process of macrophages inhibiting T cells proliferator-activated .