The Role Of Agrin And Dystroglycan In Lymphocytes Activation

Growing evidence suggestes that T cells play a pivotal role in adaptive immunology including infection immunity, tumor immunity, autoimmunity, and transplant repulsion reaction, etc. In the past decades there have been much advances in the T cells activation and recognition between T cells and APCs, especially in very recent decade, following the immunological synapse be more broadly researched, the component involved in the lymphocytes activation and its mechanisms were deeply explored. The formation of immunological synapse is necessary step for the inter-molecular recognition of immunocytes, which was well known as the fact that it mainly depends on interaction and aggregation of some key molecules, i.e. TCR, CD3 and MHC. However, so far, it was unclear that the molecules initiate and trigger these events. The study on formation of neuromuscular junctions (NMJ), a sort of synapse, indicates that agrin, a heparin sulfate proteoglycan was required for the induction and regeneration of pre- and post-neurosynapse through its confirmed receptor dystroglycan. Since the development, formation and maintenance of the immunological synapse are thought to be similar to those in neurosynapse, In this study, we reasoned that agrin and dystroglycan might play an important role in the formation of immunological synapse.In the present study, we examined the expression and distribution of agrin and dystroglycan in some immune organs and immunocytes, and explored their role in the activation of lymphocytes. Then further studies were focused on the possible mechanisms of agrin and dystroglycan in the activation of lymphocytes before maturation and after maturation.Part I Expression of Agrin and Dystroglycan in Lymphocytes1.Distribution of agrin and dystroglycan in different immune organsIn order to study the expression of agrin and dystroglycan in immune system, first of all, RT-PCR and immunofluorescence histochemistry staining were used to analyze the expression and distribution of agrin and dystroglycan in spleen, thymus andmesenteric lymph nodes. It was demonstrated that both agrin and dystroglycan were expressed in all these immune tissues examined. These results suggested that agrin and dystroglycan might be expressed in various immunocytes. 2. Expression of agrin and dystroglycan in different immunocytesAs you know immune organs consistent of T cells, B cells, DCs etc. So it was necessary to further explore which type of immunocytes expressing agrin and dystroglycan. Purified T cells and B cells prepared by using negative selection magnetic beads were tested for purity as 97.77% and 95.46% respectively. Immature dentritic cells (imDC) and mature dentritic (mDC) cells were obtained by culturing bone marrow cells from the tibias and femurs. And identified according to their specific morphology. Macrophages were harvested by lavage 3 days after intraperitoneal injection of paraffin oil. All these prepared cells were used for RNA extractions and FACS analysis. RNA extractions from these cells were performed using the guanidinium isothiocyanate method. It was shown that agrin and dystroglycan were expressed in all the cells detected, not only at mRNA level but also at protein level.3.Agrin and Dystroglycan participating in the formation of capping-like structure on the lymphocytes membraneIn order to explore the function of agrin and dystroglycan in lymphocytes, we first identified the agrin and dystroglycan expression pattern. Western-blot was further conducted to identify whether agrin or dystroglycan were secreted into culture medium or expressed on the surface of lymphocytes. As a result, agrin was detected not in the lymphocytes membrane extract proteins but in the culture medium. And a clear 240kD protein band of agrin was observed in resting lymphocytes membrane extracts, but it was dim in activated lymphocytes membrane extracts. These results suggest that agrin might be deglycosylated after the lymphocytes are activated. However, in accordance with other reports in nervous system, dystroglycan was only detected in the resting or activated lymphocytes membrane extracts but not in the culture medium. In some concanavallin A (ConA) activated splenocytes, agrin and dystroglycan were observed being capped on the cell surface by confocal microscopy. These results suggested that agrin and dystroglycan might participate in the formation of immunological synapse and relate to lymphocytes activation.Part II The Effect of Agrin and Dystroglycan in the lymphocytesActivation1.Expression of Agrin and Dystroglycan in different activation statesIn order to explore the relationship between agrin or dystroglycan and immune activation, resting and activated lymphocytes harvested at different time points (12h, 24h, 48, 96h) (1X107) were prepared for RNA extractions. Real time PCR was conducted to analysis relative expression amounts of agrin and dystroglycan, and their amounts of mRNAs were normalized by GAPDH. Statistical significance was assessed using a one-way ANOVA (SSPS software 10.0). The LSD test was used for comparison between the different groups. Constitutively expression agrin and dystroglycan were up regulated and reached the summit after 48h and 24h, respectively. There was a significant difference between expression of agrin in resting and activated lymphocytes as well as dystroglycan (p<0.05). These results suggested that agrin and dystroglycan might be participating in lymphocytes activation through up-regulating the expression amounts.2.Construction and identification of antisense cDNA plasmids against agrin or dystroglycan (pcDNA3-As-AG, pcDNA3-As-DG)In order to down regulate the expression of agrin or dystroglycan to study the functions of these two molecules in lymphocytes activation, the PCR products for agrin and dystroglycan were linked to plasmid pcDNA3 digested with enzymes EcoRV and BamHI. Plasmids were proved to be correct by PCR with specific primers for agrin or dystroglycan, and sequencing. The antisense plasmids were successfully constructed and nominated with pcDNA-As-AG against agrin, pcDNA-As-DG against dystroglycan.3.Effect of antisense plasmids pcDNA-As-AG and pcDNA-As-DG on inhibiting the expression of agrin or dystroglycanThe antisense plasmids pcDNA-As-AG and pcDNA-As-DG were successfully transferred into lymphocytes with Iipofectamine2000 respectively, and then lymphocytes were activated with ConA (5u.g/ml) followed by staining with specific antibodies against agrin or dystroglycan in 36hours. FACS was performed to analyse the decrease degrees of agrin or dystroglycan in these transfected cells. The expression of both of agrin and dystroglycan were notably down regulated to nearly 50% to 60%. 4.Effect of agrin and dystroglycan on the non-specific activationFirst of all, constructed anti-sense plasmids pcDNA-As-AG and pcDNA-As-DG were used to study the functions of agrin and dystroglycan in the non-specific activation. The 3H-TdR incorporation assays were performed to evaluate the specific proliferation of activated lymphocytes. CPM values were significantly decreased in lymphocytes either transfected with antisense plasmids pcDNA-As-DG or with pcDNA-As-AG after activation with non-specific stimulator ConA. The control lymphocytes were tranfected with pcDNA3 and the other one not with any plasmids transfected lymophcytes. These results suggested that agrin and dystroglycan participate in the proliferation of non-specific activated lymphocytes. S.Effect of agrin or dystroglycan on the antigen-specific activationSpecific lymphocytes activation plays a very important role in immune response; the 3H-TdR incorporation assays were performed to evaluate the proliferation of lymphocytes activated with BSA in vitro. CPM values of these cultures were significantly decreased when transfected with either antisense plasmids pcDNA-As-DG or pcDNA-As-AG after activation with specific anigen BSA compared to those tranfected with pcDNA3 or non any plasmids transfected lymophcytes. these lymphocytes were pre-immunized with BSA (100(ag/mI). These data revealed that agrin and dystroglycan also participated in the antigen specific activation of lymphocytes.6.The effect of antibody against agrin or dystroglycan on the activation of lymphocytesTo further study the role of agrin and dystroglycan in the proliferation of lymphocytes activation, the neutralizing antibodies against agrin (10|ag/ml) and dystroglycan (10p.g/ml) were added to lymphocytes suspension in the presence or absence of ConA (5(^g/ml). After 56h, 3H-TdR was added (0.5|aG per well) to each well. It was demonstrated that ligation of dystroglycan could enhanced the proliferation of lymphocytes in the present of ConA (10(ig/ml). However, the proliferation of lymphocytes was slightly decreased after the neutralizing antibody against secreted protein agrin was added to the medium containing ConA. However, solely anti-dystroglycan antibody or agrin antibody could not initiate the proliferation of lymphocytes. Further, anti-dsytroglycan antibody was added to the CD3 coated plate to explore the costimulatory function of dystroglycan. It was demonstrated that dystroglycan antibody accompany with anti-CD3 antibody pre-coated could initiate the proliferation of lymphocytes, and synergetic effect of antibody againstdystroglycan was detected in the present with antibody against CD28 and CD3. These results suggested that dystroglycan might be a costimulator in the activation of lymphocytes.Part III The Study on Mechanisms of Agrin and Dystroglycan in the Activation of Lymphocytes1. Dystroglycan might be the only one receptor of agrin in lymphocytesIn order to further study the mechanisms of agrin and dystroglycan in the activation of lymphocytes. At first, RT-PCR was performed to identify the existence of two supposed receptors dystroglycan and MuSK (muscle specific receptor kinase) in lymphocytes, which were putative receptors in nervous system. To explore the possible mechanism of agrin in lymphocytes activation, we confirmed that whether dystroglycan or MuSK existed in the lymphocytes. Consistence with the part I results; dystroglycan was expressed in lymphocytes, but not MuSK which was expressed in muscle cells. Also the other dystrophin-glycoproteins complex components including syntrophin, utrophin, dystrophin, dystrobrevin, sarcospan, sarcoglycan a, sarcoglycan P, sarcoglycan y, sarcoglycan 6, and sarcoglycan C, were expressed in lymphocytes as well as muscle cells. These results indicated that dystroglycan was likely the putative receptor of agrin in lymphocytes.2. Agrin participates in the proliferation and formation of immunological synapse of lymphocytes mediated by dystroglycanIn nervous system, agrin is considered to participate in the aggregation and rearrangement of acetycholine receptors mediated by its putative receptors dystroglycan and MuSK. The research above showed us that in lymphocytes MuSK was absent, but dystroglycan was broadly expressed, and all of other components in DGC (dystrophin-glycoprotein complex) were detected in the lymphocytes. These data strongly suggested that dystroglycan could be an effective receptor of agrin in lymphocytes. In order to test this hypothesis, we first co-transfected antisense plasmids pcDNA-As-AG and pcDNA-As-DG into lymphocytes. Dramatically, there were no difference between pcDNA-As-AG transfected group, pcDNA-As-DG transfected group, and co-transfected pcDNA-As-AG plus pcDNA-As-DG group. We primarily proposed that agrin and dystroglycan could share a common activation pathway in lymphocytes. Further, pEGFP-DG that containing full-length dystroglycancoding gene and green fluorescence protein were transfected into lymphocytes followed by staining with Cy3-labeled antibody against mouse IgG, we observed that agrin binded the over-expressed dystroglycan with the confocal microscopy. Combined with our previous results, the present study indicated agrin and dystroglycan were co-localized in the immunological synapse formation between macrophages and antigen specific T cells, or between naive T cells and DCs, or co-capping in the ConA activated lymphocytes, we concluded that agrin participated in the formation and proliferation mediated by dystroglycan.3.Both agrin and dystroglycan participate in the formation of immunological synapseThe activation of T cells are leaded by muti-complex factor. Generally, T cells become activated after stimulated by polyclonal stimulators such as ConA, PHA etc, or when they meet the APC presenting specific antigen peptide or contact with dentritic cells. Aim to explore whether agrin or/and dystroglycan participate in the formation of immunological synapse, the confocal microscopy were conducted to observe the location of agrin and dystroglycan in the resting or activation lymphocytes followed by staining with specific antibody against agrin or dystroglycan, respectively: We observed that CD3 molecules could be aggregated to form the capping-like structure after 30min to one hour upon ConA stimulation. Agrin and dystroglycan were both uniformly distributed on the surface of resting lymphocytes, but they were both co-localized with CD3 in the capping-like structure. In other way, agrin and dystroglycan were co-localized not only in the ConA activated lymphocytes capping-like structure, but also in the synapses formation between APCs and naive T cells, and macrophages and antigen specific T cells. In APCs, dystroglycan and agrin were also observed on the surface of lymphocytes; Capping-like structure was not formed, although they were distributed on APCs. 4.Dystroglycan was expressed in different development stage thymocytesImmunofluorescence staining was performed with the FITC-labled CD4 antibody and PE-labled-CD8 antibody for thymocytes obtained from fetal mouse thymus day during day 15 to day 20, followed by FACS analysis. On day 15, all thymocytes were double negative for CD4 and CD8. After that, CD8 mono-positive cells were first detected and then CD4+CD8+, CD4+CD8-, and CD4-CD8+ were sequentially observed. FACS and RT-PCR were performed to analyse the expression of dystroglycan in fetal thymus lobes. Dystroglycan was expressed in fetal thymus atdifferent development stages and all different kinds of cells including CD4-CD8-, CD4+CD8+, CD4+CD8- and CD4-CD8+ cells. It was high expressed in CD4+CD8+, CD4+CD8- and CD4-CD8+ cells than in CD4-CD8- thymocytes. In order to explore the effect of dystroglycan on the activation of early stage thymocytes, FTOC method must be established. Mouse fetal thymus lobes were taken out from pregnant mice at day 15 and then placed onto the filter membrane in a 24-well plate culture well. Culture medium immersed sponges were placed in the wells. The thymus lobs were well developed in such organ culture system in vitro, thymuses were smoothly developed from CD4CD8 double negative stage to double positive stage and end to mono-positive stage. The ratio of CD4 or CD8 cells in different development stage was similar in vivo. However, in the later stages of thymus lobes development in vitro culture system, some mature T cells including CD4+CD8- or CD4-CD8+ cells, could migrate to peripheral blood or peripheral immune organs, but not to other sites. Because of their stay at the lobes, a high proportion of mono-positive cells were determined. 5.Dystroglycan participated in the thymus developmentIn vitro culture system, The number of CD4+CD8+ T cells were obviously decreased at the cultured day 3 after antibody against dystroglycan were added. Further, the development of thymus lobes were dramticaly inhibited, following with higher double negative (CD4-CD8-, 71.6%) cells, lower double positive cells (CD4+CD8+, 7.5%) and lower mono-positive cells (CD4-CD8+, 6.84%), compared to non-treated thymus lobes with 26.53% double negative CD4-CD8- cells, 39.81% double positive CD4+CD8+ cells and 20.66% CD4-CD8+ mono-positive cells. However, the ratios of CD4+CD8- cells were not found significantly changed. 6.DystrogIycan participated in thymus development partly depend on the thymocytes early stage activation and apoptosisFITC-labled anti-CD69 antibody and PE-labled anti-CD95 antibody were used to stain the thymocytes single cells suspension prepared by treated with type IV collagenase. Compared to non-treated and isotype control groups, after the neutralization antibody against dystroglycan were added on the lobes the expression of CD69 molecules, a very early activation mark for T cells, on the surface of thymocytes were obviously increased (at day 3 with non-treated group 6.18%, isotype control group 7.7%, and dystroglycan antibody group 12.08%; at day6 with non-treated group 5.4%, isotype control 8.5%, and 14.55% in dystroglycan antibody...