Studies On Mechanisms Of Monocytes Chemotaxis Regulated By TLR4 Signaling Pathway

Inflammation is a defensive response to activation of the innate immune system after a variety of traumatic stimulus such as infection and non-infectious irritatation 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.The one of the most important roles of inflammation is leukocyte migration,thus leukocyte migration is one of symptom of inflammation.Cell migration is that cells migrate along the concentration of some stimulations when they are stimulated,chemokine is one of the most important stimulations.The main role of the inflammatory chemokine is to make leukocytes migrate from circulation to the inflammation site.Based on the structure,chemokine can be divided into four different groups include:CC,CXC,CX3C and C.The receptor of chemokine is G protein coupled receptor.G protein coupled receptor(GPCR)has seven trans-membrane domain and constitute the largest known superfamily of cell-surface receptors.They control a variety of cellular and physiological processes,such as perception of light,pain,taste and smell,neurotransmission,digestion,and cardiovascular regulation.GPCRs are also important regulators of innate and acquired immunity.The human genome encodes more than 1,000 distinct GPCRs,which are classified into three main families and numerous subfamilies on the basis of sequence similarity.Following agonist stimulation,GPCRs activate heterotrimeric G proteins,which exchange bound GDP for GTP,leading to the dissociation of the G protein into activated Ga and G?? subunits.This dissociation promotes downstream signaling through specific effector proteins and second messengers.Upon agonist activation of receptors,a rapid attenuation of receptor responsiveness occurs through feedback mechanisms,which prevent acute and chronic overstimulation of the receptor,we call the process as desensitization.The rapid desensitization of receptor is mediated through the phosphorylation of receptors.During the process,GRKs play important role.In human,GRKs family has seven members can be classified into three subfamilies.GRK1 and GRK7 primarily regulate photoreceptors in the retina,GRK4 is expressed primarily in the testis,cerebellum and kidney,GRK2,GRK3,GRK5 and GRK6,both of which are widely expressed.Of them,GRK2 plays important role in regulation of leukocyte function.Activity of GRKs is tightly regulated by three mechanisms:(?)subcellular localization,(?)alterations in intrinsic kinase activity and(?)alterations in GRK expression[1].Regulation of GRK subcellular localization therefore represents a logical means of regulating monocyte migration.However,the regulation of subcellular localization of GRKs in monocytes and their effects on monocytes behavior remain unknown.During the inflammation and infection,there must exist multiple inflammation mediators include TNF,interleukin interferon and PAMP such as LPS,peptidoglycan and lipoprotein.LPS,a component of the outer membrane of Gram-negative bacteria,is a specific ligand for TLR4 and induces a range of inflammatory responses including production of pro-inflammatory mediators and induction of migration of innate immune cells to the site of infection.Monocytes are critical effector cells of the innate immune system that protect the host by migrating to inflammatory sites,differentiating to macrophages and dendritic cells,eliciting immune responses,and killing pathogenic microbes.Monocyte migration is tightly regulated by signaling mechanisms activated by chemokines,which act through chemokine receptors to direct monocyte migration along a concentration gradient.Monocyte chemoattractant protein 1(MCP-1),also known as CCL2,is a 13 kDa chemokine that plays an important role in monocyte activation and migration.MCP-1 is released from both hematopoietic and non-hematopoietic cells,including monocytes/macrophages,dendritic cells(DCs),astrocytes,endothelial cells and fibroblasts.MCP-1 derived from these MCP-1-produce cells contributes to the migration of monocytes into local tissue and organ during infection and inflammation.Physiological function of MCP-1 is mediated by binding to the CCR2 receptor,a member of the G protein-coupled receptor(GPCR)family.In this study,we investigated the role of,and the mechanism of,LPS-TLR4 signaling in regulating monocyte chemotaxis.We demonstrate that LPS augments MCP-1-induced monocyte migration.We also show that LPS,through p38 MAPK signaling,induces phosphorylation of GRK2 at Ser670,which in turn,suppresses GRK2 translocation to the membrane,thereby preventing GRK2-initiated internalization and desensitization of CCR2 in response to MCP-1.This therefore results in enhanced monocyte migration.These findings reveal a novel function for TLR4 signaling in promoting innate immune cell migration.Our researches found that:(1)LPS-TLR4 augments MCP-1-induced monocytes migration.Monocyte migration to the site of infection is an essential part of first line immune defenses.MCP-1 plays an important role in initiating monocyte activation and migration.LPS,however,promotes innate immune cell migration through an as yet unclear mechanism.In order to address the influence of LPS on chemokine-induced monocyte migration,we used a Boyden chamber assay,in which monocyte migration was driven by MCP-1.As shown in Fig.1A,within a MCP-1 concentration range of 0 to 20 ng/ml,MCP-1 induced monocyte migration in a dose-dependent manner,whereas,a higher concentration of 200 ng/ml of MCP-1 decreased monocyte migration,possibly due to a rapid induction of chemokine receptor desensitization.Importantly,while LPS alone failed to induce monocyte migration,LPS significantly augmented monocyte migration in response to MCP-1 in a concentration rage of 2-200ng/ml.This effect of LPS was mediated through TLR4,since TLR4-deficiency completely prevented the LPS-enhanced monocyte migration in response to MCP-1.The synergistic effects of LPS on MCP-1-induced chemotaxis shown in vitro were also shown in vivo in mice.Intratracheal(i.t.)instillation of MCP-1 in WT mice,induced monocyte infiltration into the lungs in a time-dependent manner,and LPS given i.t.significantly enhanced this MCP-1-induced monocyte infiltration in the lungs at 6 h after MCP-1 stimulation.Also,consistent with the in vitro findings,genetic deletion of TLR4 prevented LPS-enhanced monocyte migration into the lungs.In addition,we observed using light microscopy that in WT mice LPS alone did not induce monocyte infiltration in the lungs,but instead induced neutrophil sequestration in the lungs.Taken together,these results indicate that LPS signals via TLR4 to augment,but not directly induce,monocyte migration in response to chemoattractants through a mechanism that is different from LPS-induced neutrophil migration.(2)LPS-TLR4 augments MCP-1-induced monocyte migration by modulating cell surface expression of CCR2 receptors.Upon agonist activation of receptors,a rapid attenuation of receptor responsiveness,called desensitization,occurs through feedback mechanisms,which prevent acute and chronic over-stimulation of the receptor.Receptor internalization is a major mechanism of receptor desensitization.It has been reported that MCP-1 induces CCR2 internalization,and therefore regulates the desensitization of its receptor in monocytes.In order to elucidate the effect of LPS on MCP-1-induced CCR2 internalization,we detected cell surface CCR2 expression on monocytes following MCP-1 and/or LPS treatment using flow cytometry.As shown in Fig.2A,MCP-1 induced decrease in cell surface expression of CCR2 in a time-dependent manner.However,LPS prevented MCP-1-induced CCR2 internalization as shown in Fig.2B.This effect of LPS was blocked by TLR4 deficiency.The data suggest that LPS-TLR4 augments MCP-1 induced monocyte migration by modulating cell surface expression of CCR2.(3)GRK2 is a key molecule regulating MCP-1-induced CCR2 internalization.GRK2 is known to promote CCR2 desensitization through phosphorylation.To determine the role of GRK2 in mediating MCP-1-induced CCR2 internalization in monocytes/macrophages,we used an RNA interference approach to knockdown GRK2 in RAW264.7 cells,and analyzed the influence of GRK2 knockdown on cell surface expression of CCR2 and the ability of monocytes to migrate.Transfection of Accell small interfering RNA to GRK2 resulted in 85%decrease in GRK2 protein expression in RAW264.7 cells as shown in Fig.3A.In these cells the MCP-1-induced decrease of cell surface expression of CCR2 was markedly suppressed,which was associated with increased cell migration in response to MCP-1.Noteworthy,GRK2 knockdown also diminished the influence of LPS on cell surface CCR2 expression as well as prevented the enhancing effect of LPS on the cell migration.Aggregately,the results demonstrate a critical role of GRK2 in mediating LPS/MCP-1 regulation of monocytes migration.(4)LPS-induced phosphorylation of GRK2 at serine 670 suppresses GRK2 translocation to cell membrane.It has been reported that GRK2 translocation to the cell membrane upon MCP-1 stimulation is a determinant for desensitization of the CCR2 receptor in monocytes.We hypothesized that LPS may act to preventing GRK2 subcellular translocation and so suppress CCR2 internalization and subsequent desensitization in response to MCP-1,and therefore enhance monocyte migration.To test this hypothesis,we treated monocytes with MCP-1 and/or LPS for up to 60 min,and then extracted membrane proteins from the monocytes for detection of membrane-bound GRK2.As shown in Fig.4A,MCP-1 increased the membrane-bound GRK2 in a time-dependent manner,which suggests induction of translocation of GRK2 to the cell membrane.LPS,however,prevented this increase in membrane-bound GRK2 in response to MCP-1 stimulation.Again,TLR4-deficiency prevented LPS-mediated effects on MCP-1-mediated translocation of GRK2.These results suggest a critical inhibitory role for LPS in GRK2 translocation to cell membrane,which is an important step promoting CCR2 desensitization.Mitogen-activated protein kinases(MAPKs)have been reported to regulate GRK2 activity.In vitro and in situ experiments have shown that ERK1 is able to phosphorylate recombinant GRK2 at serine 670(Ser670),which lies within the G??binding domain of GRK2.Another study showed that MAPK phosphorylation at this site impairs the GRK2-G?? interaction,thereby inhibiting GRK2 translocation to the cell membrane,and subsequent induction of kinase activity and GPCR regulation.To elucidate whether LPS-TLR4,through modification of GRK2 phosphorylation,regulates GRK2 mobility,we detected specific phosphorylation of Ser670 in GRK2 in monocytes following stimulation of bone marrow-derived monocytes(BMDM)with MCP-1 and/or LPS.Western blotting for phospho-Ser670 on GRK2 showed that LPS,or LPS plus MCP-1,induced the phosphorylation of GRK2 at Ser670,whereas,MCP-1 alone failed to increase the phosphorylation level of Ser670.Furthermore,LPS or LPS plus MCP-1 were unable to induce the phosphorylation of GRK2 on Ser670 in TLR4-deficient monocytes.These data demonstrate that LPS-TLR4 signaling modifies GRK2 phosphorylation at Ser670.(5)Opposite roles of p38 MAPK and ERK in regulating monocyte migration induced by LPS and MCP-1.MAPK,including ERK,JNK and p38,are important regulatory kinases involved in cell migration.MCP-1-induced monocyte migration has been reported to be dependent on p38 MAPK.However,the role of MAPK in LPS-enhanced monocyte migration has not yet been elucidated.We therefore investigated the role of ERK,JNK and p38 in LPS-enhanced monocyte migration in response to MCP-1 using pharmacological inhibitor approaches.We found,using the Boyden chamber migration assay,that ERK inhibitor PD98059 significantly increased monocyte migration induced by LPS/MCP-1.In contrast,p38 inhibitor SB203580 abrogated the LPS/MCP-1-induced monocyte migration,and JNK inhibitor SP600125 had no effect on the LPS/MCP-1-induced monocyte migration.To further address the mechanism by which ERK and p3 8 regulate monocyte migration,we determined the effect of ERK and p38 on the phosphorylation level of GRK2 at Ser670.Monocytes were pretreated with MAPK inhibitor for 30 min,followed by MCP-1 and/or LPS stimulation for additional 30 min.As shown in Fig.6B,Western blotting demonstrated that p38 inhibitor SB203580 prevented LPS-induced phosphorylation of GRK2 at Ser670.However,ERK inhibitor PD98059 appeared to increase phosphorylation of GRK2 at Ser670.These data suggest that p38 enhances LPS-induced Ser670 phosphorylation,whereas ERK signaling negatively regulates Ser670 phosphorylation.These alterations in the phosphorylation of GRK2 at Ser670 are associated with consistent changes in levels of cell membrane-bound GRK2 as well as cell surface expression of CCR2.In monocytes pretreated with p38 inhibitor SB203580,which decreased the phosphorylation of GRK2 at Ser670,the membrane-bound level of GRK2 was increased and cell surface expression of CCR2 was decreased.In contrast,in the cells pretreated with ERK inhibitor PD98059,which increased the Ser670 phosphorylation,the membrane-bound level of GRK2 was reduced and cell surface expression of CCR2 was elevated.Considered as a whole,phosphorylation of Ser670 of GRK2 is a critical regulatory target for LPS-TLR4 regulation of GRK2 subcellular translocation,in which,p38 mediates LPS-TLR4 signaling and this is negatively regulated by ERK.In summary,we have identified a novel interaction between TLR4 and chemokine receptors that can control monocyte migration,an essential component of the innate immune response.The results support a new model,as shown in Fig.7,in which LPS-TLR4 signaling augments monocyte migration by modulating the cell surface expression of chemokine receptors in a GRK2-dependent manner.Intervention in the specific signaling pathway that involving in the regulation of GRK2 subcellular translocation may represent a therapeutic strategy for controlling inflammation during bacterial infection.