| As a neuropeptide and endocrine hormone, alpha-melanocyte stimulating hormone (α-MSH) is an effective anti-inflammatory immune regulator which plays a vital role in the modulation of immune reaction. There are five receptors forα-MSH, named melanocortin receptor 15 (MC15R). MCRs are type I G protein-coupled receptors (GPCRs), also known as seven transmembrane receptors, among which MC1R, MC3R, MC5R take part in the regulation of immune system. MCRs protein share the common sequence between 42% and 67%, are know to be the smallest G proteins coupled receptors up to now, and functionally coupled with the adenylate cyclase (AC). Upon binding with the MCRs,α-MSH activates the Gs, a subunit of G proteins. Then Gs activates AC, which converts ATP to cAMP. The increased cAMP in the cytoplasm activates protein kinase A (PKA). PKA takes part in the regulation of other signal transduction pathway through phosphorylating the involved proteins. Much work has been done to show that the increased concentration of cAMP in cytoplasm inhibits the T cell activation mediated by TCR signaling transduction, so cAMP plays a vital role in the immune regulation.Upon the activation of T cells contacted with the antigen presenting cells (APCs), the binding of molecular on the surface of both cells is needed to form stable, tight contact structure, also known as immunological synapse (IS). Immunological synapse is important to the sustaining signal transduction. The molecular involved in this structure includes TCR/CD3-MHC/Ag, CD28/CD80 and LFA-1/ICAM-1, etc. The formation of effective immunological synapse is the key step to initiate the TCR signaling.Lipid rafts are membrane microdomains enriched with cholesterol and sphingolipid. Lipid rafts serve as a signaling platform in the membrane by the way of recruiting some molecular (TCR, CD3, LAT, ZAP-70, Lck, etc.) to and excluding some molecular (such as CD43, CD45, etc.) out of the contact zone between T cells and APCs, the results of this phenomena is the formation of effective immunological synapse and sustained signal transduction. Lipid rafts are randomly distributed in the membrane of resting T cells. After TCR or other surface molecular cross-linked, lipid rafts assembled into larger patches and responsible for the recruiting signaling molecular into the contact sites, initiate the effective TCR signaling and transfer the activation signals to the cytoplasm from outside.During the accumulation of lipid raft and the formation of immunological synapse, the molecular movement in the plasma membrane depends on the reorganization and rearrangement of the cytoskeleton actin, which in favor of the formation of IS and effective combining between signal molecules, and the production of the effective activation signaling.In view of the role ofα-MSH in the process of anti-inflammatory and immune regulation, it is significance to elucidate the mechanism of the its participation in the TCR signal transduction. Then to further illuminate its regulation effect in the neuron-endocrine-immunity network, and provide new theory for the prevention of inflammatory mediated by the immune response and the induction of the immune tolerance at the same time, which will also provide targets for selection and design new drugs against the inflammatory or in favor of immune suppression.As the best known anti-inflammatory function ofα-MSH and the key roles of T lymphocytes activation status in the adaptive immune reaction, also for the presence of the unknown precise mechanism ofα-MSH in the immune regulation, so this research project will start with the initial step, the lipid raft accumulation and the formation of immunological synapse, to the T cell activation. The molecular mechanism of T cell activation regulated byα-MSH were elucidated through investigating the movement of lipid rafts, the arrangement of cytoskeleton actin, the expression of related signal molecular and the secretion of cytokines. Then it will further illuminate the regulation mechanism of neuron-immunity-endocrine network, enrich the theory of inflammation mediated by the immune response, and provide a new target for the selection and design of new drugs for anti-inflammatory or immune suppression.Part I. The regulatory effect ofα-MSH on the cytokine secretion and signal protein expression of the activated T lymphocytesJurkat T cells of lymphoma as the research object, after activation (by SEB 100ng/ml, PMA 50ng/ml, CD3/CD28 mAb 1μg/ml, respectively) with the presence ofα-MSH (10-7mol/L) or not, a series of cytokines quantified by enzyme linked immunosorbent assay. These cytokines include interleukin 1(IL-1), IL-2, IL-4, IL-5, IL-10, TNF-αand IFN-γ. The expression of CD3, LFA-1, CD45, CD69 (T cell early activation marker) were assayed with FACS. The highly fluorescent Ca2+ -sensitive indicator Fluo-3/AM was used to measure the [Ca2+ ]i in the T cells with FACS. After the incubation of T cells with Fluo-3-AM (5μM), [Ca2+ ]i were continuously measured in 300 seconds during the period of T cell activation with the presence ofα-MSH or not. After activation, nuclear transcript factor NF-κB and p38MAPK in TCR signal transduction were measured with sodium dodecylsulfate polyacrylamide gel electriphoresis (SDS-PAGE) and Western blotting.From the research, it was found thatα-MSH could inhibit the production of IL-2,INF-γ,TNF-αby Jurkat T cells stimulated with PMA, SEB and CD3/CD28 mAb, respectively, in 12 hours. And the results showed thatα-MSH prevented the declining production of IL-4 and IL-5 from Jurkat T cells activated with PMA or CD3/CD28 mAb. The cytokine profile data indicate thatα-MSH inhibits the Th1 differentiation and promotes the Th2 differentiation from Th0 cells.The expression of SLP-76, CD28 and LAF-1 by activated Jukat T cells treated with CD3/CD28 mAb were down-regulated byα-MSH. CD69, the early activation maker, expressed on the surface of activated Jurkat T cells treated with PMA or CD3/CD28 mAb was also down-regulated byα-MSH. While theα-MSH increased the CD45 expression on the surface of Jurkat T cells activated with CD3/CD28 mAb. The expression of CD69 was gradually increased on the Jurkat T cells activated by CD3/CD28 mAb in 22 hours. This kind of increased CD69 expression was significantly down-regulated byα-MSH (P<0.05). While in the group of PMA as a activator of Jurkat T cells, the CD69 expression increased rapidly in 8 hours, and remained high expression in 32 hours. There was no effect of inhibition on the increased CD69 expression of the Jurkat T cells activated by PMA in the presence ofα-MSH. But there was a decreased CD69 expression in the Jurkat T cells stimulated with Phytohemagglutinine (PHA) 7 hours later in the presence ofα-MSH.Intracellular free Ca2+ concentration ([Ca2+ ]i ) as a second messenger plays an important role in the TCR signal transduction. Using fluo-3-AM as an indicator, the [Ca2+ ]i was detected by FACS.[Ca2+ ]i elevated upon the activation of T cells stimulated by CD3/CD28 mAb or PMA. [Ca2+ ]i decreased after the administration of CD3/CD28 mAb or PMA withα-MSH in 5 min during the investigation.After activation, nuclear transcript factor NF-κB and p38MAPK were also decreased in the cytoplasm of Jurkat T cells after the administration ofα-MSH and activator.PartⅡ. The regulatory effect ofα-MSH on the amount of lipid rafts and the distribution of signaling protein in lipid rafts from activated T cellsA fast and sensitive method was adopted for isolation of detergent-resistant membrane (DRMs, also known as lipid rafts) from T cells by sucrose density gradient ultra centrifugation. The fractions with different density were collected. Proteins in different fractions were precipitated and purified with trichloroacetic acid (TCA). Signaling proteins in factions of lipid rafts were analyed by Western blotting. CD3 and CD28 in lipid rafts were analyzed with flow cytometric measurements combined with the methods that T cells were pretreated with methyl-β-cyclodextrin (MBCD) to deplete the cholesterol. The B subunit of cholera toxin (CTB), which specifically binds to ganglioside GM1 (a main component and marker of lipid rafts) labeled with florescence and used to visualize the lipid rafts under the laser scanning confocal microscopy (LSCM). mRNA levels of GM3 synthase were measured with real time polymerase chain reaction.The results show thatα-MSH reduces the amount of total protein in lipid rafts from activated Jurkat T cells. The proteins (such as LTA, ZAP-70, Lck and Talin, etc.) in lipid rafts were also decreased in activated Jurkat T cells after the administration withα-MSH. The distribution of CD3 and CD28 in lipid rafts from activated Jurkat T cells was down-regulated byα-MSH. FACS measurements showed that the expression of GM1 was also down-regulated byα-MSH. Real-time PCR analysis showed that the mRNA levels of GM3 synthase were inhibited byα-MSH in activated Jurkat T cells. It was observed under the LSCM thatα-MSH prevented the lipid rafts accumulation on Jurkat T cells activated by CD3/CD28 mAb.PartⅢ. The regulatory effect ofα-MSH on the formation of immunological synapse between T cells and APCs and its molecular mechanismRaji B cells from lymphoma were used as antigen presenting cells (APCs). Pulsed and loaded with staphylococcal enterotoxin B (SEB), Raji B cells conjugate with Jurkat T cells to form immunological synapse. The distribution of surface antigen, cytoskeleton actin rearrangement and IS were visualized with laser confocal scanning microscopy. The cytoskeleton F-actin polymerization and the number of IS formation were analyzed with FACS during T cell activation or not. The proteins, such as ezrin-radix-moesin (ERM), Vav, ERM binding protein 50KDa (EBP50), Talin, and CDC42, etc. related to the cytoskeleton were analyzed by Western blotting.LASM data show thatα-MSH reduced the number of IS formation, inhibited the accumulation of lipid rafts, prevented the CD3 capping, restrained the polymerization of cytoskeleton F-actin during T cell activation. The number of CD3 capping was increased after T cell activation by PMA (P<0.01, n=4), andα-MSH inhibited the CD3 capping (P<0.05, n=4). The ratio of IS against all cells gated in FACS from resting T and B cells was 5.16%. T cells stimulated by CD3/CD28 mAb boosted the IS ratio to 18.02%, while this ratio was decreased to 12.3% in the presence ofα-MSH.FACS data show that the actin polymerization attained in 10 min in Jurkat T cells stimulated with CD3/CD28 mAb, whileα-MSH suppressed the actin polymerization in 40 min.The expression of EBP50, an adaptor protein linking the membrane and cytoskeleton actin through binding ERM proteins, was not changed in Jurkat T cells stimulated with CD3/CD28 mAb in 12 hours, butα-MSH inhibits the expression of EBP50 in activated Jurkat T cells. In Jurkat T cells activated by PMA, EBP50 expression was down-regulated after 10 hours of stimulation, andα-MSH up-regulated it in 1 hour. For the important role of EBP50 in the cell motility, high level of EPB50 reduces the mobility and enhance the rigidity of cell membranes, which is not prone to form the immunological synapse.The expression of ERM protein was not altered by CD3/CD28 mAb,α-MSH also had no effect on ERM protein expression in Jurkat T cells activated by CD3/CD28 mAb. Butα-MSH down-regulated the high expression of ERM protein in the Jurkat T cells stimulated by PMA.α-MSH was in favor of the phosphorylation of ERM proteins in 60 min. The phosphorylated ERM protein increases the rigidity of cell membrane, which is not benefit for the formation of immunological synapse.α-MSH suppressed the increased expression of Talin protein in Jurkat T cells stimulated by CD3/CD28 mAb in 2 hours. For Talin is an adaptor protein linking the intergrin (such as CD28, LFA-1, etc. ) and cytoskeleton, and takes part in the out-in signaling transduction, soα-MSH interferes the out-in signaling mediated by intergrin which is also important in the full T cell activation. PMA had no effect on the expression of Talin protein in Jurkat T cells, so did theα-MSH. This phenomenon maybe the results of the un-relationship between PMA and intergin mediated signal pathways. The Rho GTPase, Cdc42, expression level increased in Jurkat T cells administrated with CD3/CD28 mAb, and theα-MSH reduced this increased expression in 12 hours.α-MSH suppressed the phosphorylated Vav level in activated Jurkat T cells.Conclusionα-MSH suppresses the expression and secretion of IL-2, TNF-αand IFN-γ, which is in favor of Th0 cell differentiation into Th1. Andα-MSH facilitates the expression and secretion of IL-4, IL-5, which takes part in the Th2 reaction. The effect ofα-MSH on IL-10 expression and secretion remains to be elucidated in the future research.α-MSH inhibits the expression of CD69, CD28, LFA-1 and SLP-76 upon T cell activation.α-MSH down-regulates the levels of [Ca2+]i, NF-κB and p38 MAPK of the activated T cells.α-MSH reduces the capability of activated T cells to form mature immunological synapse with APCs, and inhibits the cytoskeleton F-actin polymerization of activated T cells.α-MSH prevents the accumulation of lipid rafts during T cell activation, suppresses the syntheses of GM1 and GM3, the marker and main components of lipid rafts.α-MSH reduces the amount of proteins in lipid rafts, including signal (CD3, CD28 and Lck) and adaptor protein (LAT and ZAP-70) for signal transduction, and prevents the formation of signal platform from lipid rafts in stimulated T cells, then inhibits the T cell activation.α-MSH inhibits the formation of immunological synapse, reduces the number of IS formed between activated T cells and APCs.α-MSH prevents the CD3 capping in the activated T cells, which is the initial step to the T cell activation through TCR signal transduction.In conclusion,α-MSH facilitates the T cell differentiation into Th2 cells, inhibit the synthesis and accumulation of lipid rafts during T cell activation, prevents the formation of immunological synapse, prohibits the signal proteins in the membrane surface move to the lipid rafts and the contact sites between T cells and APCs. All the data acquired shows thatα-MSH play a key role in the regulation during T cell activation, which is vital to the treatment of inflammatory reaction mediated by immune response.
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