Studies On The Signaling Mechanism And Biological Consequence Of IP-10 Induced By MRP8/14

Inflammation is a defensive response to activation of the innate immune system after a variety of traumatic stimulus such as infection and non-infectious irritataion,and manifested as redness,swelling,heat,pain and organ dysfunction. Inflammation is a double-edged sword. Moderate inflammatory response to the body is an effective preventive measure which can function as killing pathogenic microorganisms and limiting the spread of pathogens; However, excessive inflammation will lead to the release of a large number of inflammatory mediators such as cytokines into the blood,and result in out-of control inflammation and further systemic inflammatory response syndrome (SIRS), or even multiple system organ failure (MSOF) which can endanger the lives of patients. Sepsis is characterized by SIRS plus a documented infection,which has been confirmed by clinical the presence of bacteria or highly suspicious foci,and according to the severity,sepsis can be divided into sepsis,severe sepsis and septic shock. Because of the high incidence and high mortality, sepsis has become an leading cause of death of non-cardiac patients in Intensive Care Unit (ICU) and seriously affect the quality of human life.Compensatory anti-inflammatory response syndrome (CARS) is the anti-inflammatory runaway theory which is proposed by Roger Bone in 1997. During the development of SIRS,endogenous anti-inflammatory mediators such as interleukin 4 (IL-4), interleukin 10 (IL-10) were produced in vivo accompanied by the release of massive inflammatory mediators which were used to suppress and downregulate the production of inflammatory mediators so as to keep the balance between pro- and anti-inflammation, further control the inflammation and maintain the body's homeostasis. However, CARS appeares due to the excessive release and flood into the blood of anti-inflammatory mediators which caused by excessive anti-inflammatory response. CARS is characterized by immunosuppression which may be benefical or deleterious. CARS can reduce inflammatory response-induced damage to the body to a certain extent in early stage, however, it caused uncontrolled infection because of severe suppression of immune function later. For decades,researchers focus on the research of important pro- or anti-inflammatory mediators during SIRS and CARS, however, current research has just known a bit of the complex regulatory network.Pathogen associated molecular pattern (PAMP), such as lipopolysaccharide(LPS), peptidoglycan, lipoproteins, invade the body and establish the first line of defense against pathogenic microorganisms by promoting the innate immunity swiftly through recognizing and binding with the cell surface pattern recognition receptors.The major innate immune cells include: phagocytes, dendritic cells, natural killer cells and so on,while phagocytes,such as monocytes,macrophages and polynuclear neutrophilic leukocytes which are characterized as the major innate immune cells play important roles in the process of phagocytosis and killing of pathogenic microorganisms. Adaptive immunity response is specific and efficient defense mechanisms aimed at distinctive pathogenic microorganisms (antigens), and lymphocytes, including T lymphocytes and B lymphocytes are the most important adaptive immunity effectors. A bridge is built for the transition between innate and adaptive immune respond just after monocytes, macrophages, and dendritic cells capture antigen and present the antigen information to'T lymphocytes. Both processes not only have definite stages and steps, but also keep close contact with each other and integrity. So the regulatory functions and mechanisms of the innate immunity on the adaptive immunity have been a hot research field of pathology and immunology.Myeloid-related protein 8 (MRP8) and myeloid-related protein 14 (MRP 14) are newly identified important inflammatory mediators. MRP8/14 exert affluent biological functions include: protein phosphorylation, cytoskeletal remodeling, cell migration, regulation of calcium homeostasis, promote apoptosis, metabolism of arachidonic acid and regulation of neutrophils NADPH oxidase. However, their roles in the inflammation and related diseases have only recently been taken seriously. In 2007, MRP8/14 were found to function importantly during the sepsis development when Johannes Roth laboratory identified new components that regulate the inflammatory cascade during sepsis. MRP8/14 oligomers can be protected from endotoxin-induced lethal shock and Escherichia coli-induced abdominal sepsis.MRP8 and MRP 14 were synthesized and secreted into the extracellular upon stimulation and activation of macrophages. And inflammatory response induced by LPS were ampilified by MRP8/14 oligomers. MRP8/14 belong to the calcium binding protein S100 family member which normally express constitutively on neutrophils and monocytes , and they are abundant in neutrophils which account for 50% of the soluble cytoplasmic components; MRP8/14 can be released into the intracellular by macrophages upon stimulated by inflammation and keep high levels in the blood. Under pathological conditions, MRP8/14 can also be release into intracellular actively or passively by necrotic or activated cells which exert immunomodelatory effects as important endogenous damage associated molecular patterns (DAMPs). At the same time, the significance of MRP8 and MRP14 proteins in a variety of inflammatory autoimmune diseases also has been reported, such as rheumatoid arthritis, systemic lupus erythematosus, giant cell arteritis, multiple sclerosis, cystic fibrosis, autoimmune diseases and chronic enteritis. MRP8 and MRP 14 proteins positive myeloid cells are the first infiltrating cells in inflammatory injury and both two proteins are elevated significantly in the patients' serum,suggesting that they may have very important functions during the development of inflammation and autoimmune diseases.LPS and pro-inflammatory cytokines such as tumor necrosis factor a (TNF-a),IL-1, can upregulate the expression of MRP8 and MRP 14 in human myeloid cells in vitro. MRP8/14 functions as cytokine in certain regions and it's most prominent role is to attract leukocytes to the sites of inflammation. In vitro studies suggest that MRP 8/14 can promote the expression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1),which thereby contribute to the recruitment of inflammatory cells through regulating the transendothelial migration of myeloid cells by enhancing the binding of ICAM-1 with CD11b/CD18(Mac-1). At the same time, MRP8/14 can further enhance the expression and release of proinflammatory cytokines which can then promote the expression and release of MRP8/14 so as to amplify the inflammatory signals with a positive feedback way. Although we have known something about the mechanisms which MRP8/14 be invovled in the inflammation,but there are still many problems unclear, in particular, how the extracellular MRP8/14 be secreated acting on and responding the cells. We have known that extracellular MRP8/14 oligomer functions depending on binding with cell surface specific receptor located on the target cells.Studies have shown that advanced glycation end products receptor (RAGE), CD36,and Toll-like receptor 4 (TLR4) are probably the cell membrane receptor for MRP 8/14, however, they are not the single way for transmitting signals, there still exist other receptors or signal transduction pathways for MRP8/14. Thus,it's very complicated for MRP8/14 to transmit signals, MRP protein may mediate different biological effect through binding with various receptors by different domains in different cells.TLR4 is is an important pattern recognition receptor which belongs to the Toll-like receptors (TLR) family and expresses mainly in the cell membrane. TLR4 plays privitol roles in the transition between innate and adaptive immunity through sensoring the stimulation from extrinsic PAMPs and damaged/departed DAMPs, and then recruit the downstream adaptor proteins and further results in the signal cascade of the cells which eventually mediates the innate and adaptive immune system activation. TLR4 is the the most extensively and hottest studied family of PRRs and it's ligands include bacteria, virus or LPS and a variety of important DAMPs molecules such as HMGB1, MRP8/14. TLR4 is mainly expressed in a variety of immune cells which are characterized as the first defense line of innate immune system, including macrophages, DC, B cells, specific types of T cells and epithelial and endothelial cells. Studies show that TLR4 can adjust adaptive immunity through the activation of DC and further cytokines secretion. TLR4 mediates the activation of T cells through capturing pathogenic microorganisms antigens by DC and then migrating to the secondary lymphoid tissue and presenting the antigens to naive T cells. Researchers notify that whether DC can effectively present the antigens to T and B cells after capturing antigens depends on the existence of TLR ligand of the antigens. DC selectively presents pathogenic microbial antigens rather than apoptotic cell antigens without TLR ligand. Thus, TLR play essential roles in mediating the adaptive immune response in inflammation, however, we have known little about the mechanisms of the connection between innate and adaptive immunity by TLR,researches should still go on.Interferon-y inducible protein 10 (IP-10) which belongs to the CXC chemokine subfamily members and mainly produced by activated mononuclear phagocytes and further act on the mononuclear phagocytes or T lymphocytes in an autocrine or paracrine manner. During the process of inflammation, monocytes, T lymphocytes and NK cells induce the synthesis of IP-10 after exposuring to vascular endothelial cells when hemorheology changes, and further induced IP-10 play significant roles in the migration and penetrating of cells from bloos vessels to the the sites of inflammtion through enhancing the expression of cell surface selectins and integrins.In additon,IP- 10 plays an important role in mediating adaptive immune response through the chemotaxis of activated T lymphocytes, NK, and NKT cells by binding with its specific receptor CXCR3.We found that IP-10 was dramatically elevated among the cytokines released by monocytes and macrophages stimulated by MRP8/14. MRP8/14 was abundant in neutrophils while extracellular MRP8/14 can induce IP-10 expression by acting on the monocytes and macrophages which further attract lymphocytes to the inflammatory sites. So we suppose that the cross-talk between cells and cells may contribute much to the transition between innate and adaptive immunity during inflammation. Based on above theoretical background, we deeply studied the regulatory mechanisms of IP-10 expression stimulated by MRP8/14 in monocytes and macrophages and further explore it's biological significance.Human THP-1 monocytes and mouse RAW264.7 macrophages were cultured in vitro, while alveolar macrophages(AM?) and bone marrow macrophages (BMM)were also isolated and cultured, Luminex, RT-PCR and Real-time PCR techniques were used to observe the time- and dose manner, the connection with IFN-? and possible regulatory mechanisms of IP-10 expression stimulated by MRP8/14; At the same time, we constructed different domain fusion proteins of MRP 14 to screen the domains that mediated the inductive effect. Further gene knock-out mouse, kinases inhibitors, Western blot and EMSA were used to select and verify the cell membrane receptor and singalling pathways involved in the IP-10 expression induced by MRP8/14; at last, we used Boyden Chamber experiments in vitro to verify MRP8/14-IP10 axis role in the chemotaxis of activated lymphocytes which were activated and highly expressed CXCR3 in vitro. Further we used Air pouch experiment to detect the chemotaxis of MRP8/14-IP10 for activated lymphocytes in septic shock model.Our research indicated that:(1) MRP8/14 induces IP-10 protein and mRNA expression in a time and dose-dependent manner; IP-10 protein was elevated in the cell culture supemant after 6h stimulated by MRP8/14, and gradually increased with the extension of simulation time, difference is significant (F=316.9000, P=0.000) . IP-10 mRNA expression enhanced after lh stimulated by MRP8/14, kept rising and reached peak at 6h, began to decline at 12h and returned to normal level at 24h. 0.75 ?g/ml MRP8/14 protein can significantly induce IP-10 production, with the increase of stimulated concentration, IP-10 production were further enhanced, difference is significant(F=55.560, P=0.000)(2) MRP8/14 induces IP-10 expression in THP-1 cells in an IFN-y independent manner. IFN-y can not be induced by MRP8/14 (F=1.205, P=0.364) and IFN-?neutralizing antibody had no effect on IP-10 expression induced by MRP8/14 (P>0.05).(3) MRP8/14 treatment increases IP-10 mRNA expression mainly at the transcriptional level; MRP 14 protein which contains calcium-binding domain has the ability to induce IP-10 expression. The effect of transcriptional inhibitor ActD and new protein synthesis inhibitor Cyclo on the production of IP-10 is different(F=229.822, P=0.000) , IP-10 mRNA can only be inhibited by transcriptional inhibitor ActD (P<0.01) rather than new protein synthesis inhibitor Cyclo (P>0.05),suggesting that MRP8/14 regulate IP-10 expression mainly at the transcriptional level.Also different Domains of MRP 14 protein has different activities (F=251.061,P=0.000) , which contain calcium binding motif such as EF hand-1, the EF hand-2,EF hand-1+2 had the biological activity in inducing IP-10 expression(P<0.01),however CT terminal domain had no activity (P>0.05).(4) MRP8/14 induces IP-10 expression through binding with TLR4 receptor in THP-1 cells. IP-10 protein and mRNA expression were dramatically inhibited in TLR4-/- mouse AM? compared with normal mouse AM? after stimulated by MRP8/14, however, its protein and mRNA expression can not be affected in RAGE-/-mouse AM?.(5) JNK, JAK1/3-STAT1 and NF-?B signaling pathways are involved in the IP-10 expression stimualted by MRP8/14 (F=589.655, P=0.000; F=1183.000,P=0.000) ; First kinases inhibitors experiment showed that JNK kinase inhibitor SP600125 can partially inhibit IP-10 expression, JAK1 , JAK3 kinase inhibitor JAK inhibitor I,JAK3 inhibitor can fully inhibit IP-10 expression (P<0.01),also its expression can partially be inhibited by NF-?B inhibitors including PDTC,Bay 11-7082 and STAT1 inhibitor MTA (P<0.05) . Further we used Western blot and EMSA to detect the phosphorylation of JNK, JAK1/3-STAT1 kinase and the NF-?B DNA transcriptional activity.(6) MRP8/14-IP10 axis can induce the chemotaxis of activated lymphocytes;and the effect can be blocked by IP-10 neutralized Ab?kinase inhibitors?TLR4-/-(F=225.748, P=0.000); lymphocytes were activated and expressed highly CXCR3 and CD69 with PHA synergistically with IL-2 in vitro. The normal BMM supernant stimulated by MRP8/14 can dramatically induce the chemotaxis of activated lymphocytes, while IP-10 neutralizing antibody can inhibit the chemotaxis (P<0.01).At the same time, JAK inhibitor I and JAK3 in inhibitor can fully inhibit the chemotaxis (P<0.01), however SP600125 and Bay11-7082 can only partially inhibit the chemotaxis (P<0.01) . The TLR4-/- BMM supernant stimulated by MRP8/14 had no chemotatic effect (P<0.01).(7) MRP8/14-IP 10 axis can induce the chemotaxis of activated lymphocytes in septic shock model(F=167.448, P=0.000); In septic shock mouse model, splenocytes were activated only after 8 h of LPS stimulation, also CXCR3 and CD69 expression were dramatically enhanced, and reached peack at 16 h, declined at 24 h; The supernant stimulated by MRP8/14 can dramatically induce the chemotaxis of acticated lymphocytes (P<0.01).Our research put forward the signaling pathway model of MRP8/14-TLR4-IP10 axis by the studies on signal transduction pathways and biological effects; During the process of inflammation, inflammatory mediator MRP8/14 was released by necrotic neutroohils that inflitrated into the inflammatory sites and further induced IP-10 expression through activating JAK1/3-STAT1 NF-?B and JNK signaling pathways by binding with antigen-presenting cells-momocytes and macrophages membrane receptor TLR4. So concentration gradient of IP-10 was constructed in and outside the vessle at sites of inflammation, adaptive immune response was then activated through the chemotaxis and infiltration into the inflammatory sited of activated T, B lymphocytes under the chemotatic effect of the concentration gradient. This study have far-reaching significance in our understanding of the transition between innate and adaptive immunity during the sepsis, we believe that we can provide better ideas and the starting point for further clinical studies.