Chemotactic Receptor Research Articles

Transcriptional Profiling of Antigen-Dependent Murine B Cell Differentiation and Memory Formation

Humoral immunity is characterized by the generation of Ab-secreting plasma cells and memory B cells that can more rapidly generate specific Abs upon Ag exposure than their naive counterparts. To determine the intrinsic differences that distinguish naive and memory B cells and to identify pathways that allow germinal center B cells to differentiate into memory B cells, we compared the transcriptional profiles of highly purified populations of these three cell types along with plasma cells isolated from mice immunized with a T-dependent Ag. The transcriptional profile of memory B cells is similar to that of naive B cells, yet displays several important differences, including increased expression of activation-induced deaminase and several antiapoptotic genes, chemotactic receptors, and costimulatory molecules. Retroviral expression of either Klf2 or Ski, two transcriptional regulators specifically enriched in memory B cells relative to their germinal center precursors, imparted a competitive advantage to Ag receptor and CD40-engaged B cells in vitro. These data suggest that humoral recall responses are more rapid than primary responses due to the expression of a unique transcriptional program by memory B cells that allows them to both be maintained at high frequencies and to detect and rapidly respond to antigenic re-exposure.

The trafficking of natural killer cells

Summary: Natural killer (NK) cells are large granular lymphocytes of the innate immune system that participate in the early control of microbial infections and cancer. NK cells can induce the death of autologous cells undergoing various forms of stress, recognizing and providing non‐microbial ‘danger’ signals to the immune system. NK cells are widely distributed in lymphoid and non‐lymphoid organs. NK cell precursors originate from the bone marrow and go through a complex maturation process that leads to the acquisition of their effector functions, to changes in their expression of integrins and chemotactic receptors, and to their redistribution from the bone marrow and lymph nodes to blood, spleen, liver, and lung. Here, we describe the tissue localization of NK cells, using NKp46 as an NK cell marker, and review the current knowledge on the mechanisms that govern their trafficking in humans and in mice.

Heme Induces Neutrophil Migration and Reactive Oxygen Species Generation through Signaling Pathways Characteristic of Chemotactic Receptors

Hemolysis or extensive cell damage can lead to high concentrations of free heme, causing oxidative stress and inflammation. Considering that heme induces neutrophil chemotaxis, we hypothesize that heme activates a G protein-coupled receptor. Here we show that similar to heme, several heme analogs were able to induce neutrophil migration in vitro and in vivo. Mesoporphyrins, molecules lacking the vinyl groups in their rings, were not chemotactic for neutrophils and selectively inhibited heme-induced migration. Moreover, migration of neutrophils induced by heme was abolished by pretreatment with pertussis toxin, an inhibitor of Galpha inhibitory protein, and with inhibitors of phosphoinositide 3-kinase, phospholipase Cbeta, mitogen-activated protein kinases, or Rho kinase. The induction of reactive oxygen species by heme was dependent of Galpha inhibitory protein and phosphoinositide 3-kinase and partially dependent of phospholipase Cbeta, protein kinase C, mitogen-activated protein kinases, and Rho kinase. Together, our results indicate that heme activates neutrophils through signaling pathways that are characteristic of chemoattractant molecules and suggest that mesoporphyrins might prove valuable in the treatment of the inflammatory consequences of hemorrhagic and hemolytic disorders.

Genome wide transcriptional analysis of resting and IL2 activated human natural killer cells: gene expression signatures indicative of novel molecular signaling pathways

BackgroundHuman natural killer (NK) cells are the key contributors of innate immune response and the effector functions of these cells are enhanced by cytokines such as interleukine 2 (IL2). We utilized genome-wide transcriptional profiling to identify gene expression signatures and pathways in resting and IL2 activated NK cell isolated from peripheral blood of healthy donors.ResultsGene expression profiling of resting NK cells showed high expression of a number of cytotoxic factors, cytokines, chemokines and inhibitory and activating surface NK receptors. Resting NK cells expressed many genes associated with cellular quiescence and also appeared to have an active TGFβ (TGFB1) signaling pathway. IL2 stimulation induced rapid downregulation of quiescence associated genes and upregulation of genes associated with cell cycle progression and proliferation. Numerous genes that may enhance immune function and responsiveness including activating receptors (DNAM1, KLRC1 and KLRC3), death receptor ligand (TNFSF6 (FASL) and TRAIL), chemokine receptors (CX3CR1, CCR5 and CCR7), interleukin receptors (IL2RG, IL18RAB and IL27RA) and members of secretory pathways (DEGS1, FKBP11, SSR3, SEC61G and SLC3A2) were upregulated. The expression profile suggested PI3K/AKT activation and NF-κB activation through multiple pathways (TLR/IL1R, TNF receptor induced and TCR-like possibly involving BCL10). Activation of NFAT signaling was supported by increased expression of many pathway members and downstream target genes. The transcription factor GATA3 was expressed in resting cells while T-BET was upregulated on activation concurrent with the change in cytokine expression profile. The importance of NK cells in innate immune response was also reflected by late increased expression of inflammatory chemotactic factors and receptors and molecules involved in adhesion and lymphocyte trafficking or migration.ConclusionThis analysis allowed us to identify genes implicated in cellular quiescence and the cytokines and cytotoxic factors ready for immediate immune response. It also allowed us to observe the sequential immunostimulatory effects of IL2 on NK cells improving our understanding of the biology and molecular mediators behind NK cell activation.

Resveratrol inhibits expression and binding activity of the monocyte chemotactic protein-1 receptor, CCR2, on THP-1 monocytes

Monocyte chemotactic protein-1 and its receptor, CCR2, play a key role in atherosclerosis. We determined the effect of the polyphenol, resveratrol, on CCR2 and the mechanisms involved. Resveratrol treatment inhibited 125I-MCP-1 binding to THP-1 cells; 31, 56, 84% decrease for 10, 50 and 100 μM resveratrol, in the absence of any effect on receptor affinity. The inhibitory effect of resveratrol on 125I-MCP-1 binding to THP-1 cells and on CCR2 protein expression determined by FACS analysis was attenuated by treatment with L-NAME (NOS inhibitor), PD98059 (MAPK inhibitor) and LY294002 (PI3K inhibitor), whereas neither X/XO (reactive oxygen species generator) nor ICI182780 (estrogen receptor antagonist) had any effect. Concomitant with a decrease in CCR2 protein expression, resveratrol inhibited THP-1 CCR2 mRNA levels, in the absence of any effect on its stability; 26 and 45% inhibition at 10 and 50 μM resveratrol, respectively. This effect was not altered by co-treatment with L-NAME, PD98059 or ICI182780, but was potentiated by LY294002 and X/XO. Conclusions: Resveratrol inhibits monocyte CCR2 binding activity in an NO-, MAPK- and PI3K-dependent manner, whereas it inhibits CCR2 mRNA in an NO- and MAPK-independent, PI3K-dependent manner. These inhibitory effects of resveratrol on chemokine receptor binding and expression may contribute, in part, to its cardiovascular protective activity in vivo.

Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis

While studying the developmental functions of the Drosophila dopamine synthesis pathway genes, we noted interesting and unexpected mutant phenotypes in the developing trachea, a tubule network that has been studied as a model for branching morphogenesis. Specifically, Punch ( Pu) and pale ( ple) mutants with reduced dopamine synthesis show ectopic/aberrant migration, while Catecholamines up ( Catsup) mutants that over-express dopamine show a characteristic loss of migration phenotype. We also demonstrate expression of Punch, Ple, Catsup and dopamine in tracheal cells. The dopamine pathway mutant phenotypes can be reproduced by pharmacological treatments of dopamine and a pathway inhibitor 3-iodotyrosine (3-IT), implicating dopamine as a direct mediator of the regulatory function. Furthermore, we show that these mutants genetically interact with components of the endocytic pathway, namely shibire/dynamin and awd/nm23, that promote endocytosis of the chemotactic signaling receptor Btl/FGFR. Consistent with the genetic results, the surface and total cellular levels of a Btl-GFP fusion protein in the tracheal cells and in cultured S2 cells are reduced upon dopamine treatment, and increased in the presence of 3-IT. Moreover, the transducer of Btl signaling, MAP kinase, is hyper-activated throughout the tracheal tube in the Pu mutant. Finally we show that dopamine regulates endocytosis via controlling the dynamin protein level.

Elucidating The Role Of Insulin Receptor In Type-I Diabetes By Developing An IR Transgenic Mouse Model (130.37)

Abstract In the nonobese diabetic (NOD) mouse, type1 diabetes (T1D) is an autoimmune disease characterized by insulitis and T cell-mediated destruction of pancreatic islet β cells. The importance of insulin receptor (IR) expression in the pathogenesis of diabetes was examined since it was shown that IR is a chemotactic receptor capable of directing cell movement in response to insulin. Our published data shows that IR High/+ T cells aggressively transfer insulitis and diabetes while IR Low/− T cells are capable of neither. Therefore, IR may function to deliver T cells to the islet. Recently, it has been shown in NOD mice that benign insulitis can occur in the absence of T cell-MHC/Ag interactions. The object of this work is to test whether T cells overexpressing IR, can move into the pancreas based on insulin chemotaxis alone, without being targeted to the pancreas by antigenic specificity. To that end a transgenic mouse will be made wherein FLAG tagged IR expression will be targeted to all T cells. If IR expression is a mechanism to move T cells into the islet then down regulation of IR expression or blocking of chemotactic IR activity specifically in T cells, will provide a new therapeutic target to block cell movement into the pancreas, thus preventing diabetes.

Stable Overexpression of Smad7 in Human Melanoma Cells Impairs Bone Metastasis

Melanoma has a propensity to metastasize to bone, where it is exposed to high concentrations of transforming growth factor-beta (TGF-beta). Because TGF-beta promotes bone metastases from other solid tumors, such as breast cancer, we tested the role of TGF-beta in melanoma metastases to bone. 1205Lu melanoma cells, stably transfected to overexpress the natural TGF-beta/Smad signaling inhibitor Smad7, were studied in an experimental model of bone metastasis whereby tumor cells are inoculated into the left cardiac ventricle of nude mice. All mice bearing parental and mock-transfected 1205Lu cells developed osteolytic bone metastases 5 weeks post-tumor inoculation. Mice bearing 1205Lu-Smad7 tumors had significantly less osteolysis on radiographs and longer survival compared with parental and mock-transfected 1205Lu mice. To determine if the reduced bone metastases observed in mice bearing 1205Lu-Smad7 clones was due to reduced expression of TGF-beta target genes known to enhance metastases to bone from breast cancer cells, we analyzed gene expression of osteolytic factors, parathyroid hormone-related protein (PTHrP) and interleukin-11 (IL-11), the chemotactic receptor CXCR4, and osteopontin in 1205Lu cells. Quantitative reverse transcription-PCR analysis indicated that PTHrP, IL-11, CXCR4, and osteopontin mRNA steady-state levels were robustly increased in response to TGF-beta and that Smad7 and the TbetaRI small-molecule inhibitor, SB431542, prevented such induction. In addition, 1205Lu-Smad7 bone metastases expressed significantly lower levels of IL-11, connective tissue growth factor, and PTHrP. These data suggest that TGF-beta promotes osteolytic bone metastases due to melanoma by stimulating the expression of prometastatic factors via the Smad pathway. Blockade of TGF-beta signaling may be an effective treatment for melanoma metastasis to bone.

Lipoxin Receptors

Lipoxins (LXs) represent a class of arachidonic acid (AA) metabolites that carry potent immunoregulatory and anti-inflammatory properties, LXA4 and LXB4 being the main components of this series. LXs are generated by cooperation between 5-lipoxygenase (LO) and 12- or 15-LO during cell-cell interactions or by single cell types. LX epimers at carbon 15, the 15-epi-LXs, are formed by aspirin-acetylated cyclooxygenase-2 (COX-2) in cooperation with 5-LO. 15-epi-LXA4 is also termed aspirin-triggered LX (ATL). In vivo studies with stable LX and ATL analogs have established that these eicosanoids possess potent anti-inflammatory activities. A LXA4 receptor has been cloned. It belongs to the family of chemotactic receptors and clusters with formyl peptide receptors on chromosome 19. Therefore, it was initially denominated formyl peptide receptor like 1 (FPRL1). This receptor binds with high affinity and stereoselectivity LXA4 and ATL. It also recognizes a variety of peptides, synthetic, endogenously generated, or disease associated, but with lower affinity compared to LXA4. For this reason, this receptor has been renamed ALX. This review summarizes the current knowledge on ALX expression, signaling, and potential pathophysiological role. The involvement of additional recognition sites in LX bioactions is also discussed.

2P202 バクテリア走化性受容体の共有結合修飾によるシグナル増幅への影響(細胞生物的課題(接着・運動・骨格・伝達・膜),口頭発表,第45回日本生物物理学会年会)

2P202 バクテリア走化性受容体の共有結合修飾によるシグナル増幅への影響(細胞生物的課題(接着・運動・骨格・伝達・膜),口頭発表,第45回日本生物物理学会年会)

Eosinophilic esophagitis: New insights into an emerging disease

Eosinophilic esophagitis: New insights into an emerging disease

Differential Signaling of G Protein-Coupled Chemotactic Receptors (CXCR4, cysLT1 and S1P1) Expressed in Hematopoietic Progenitor Cells.

We have previously demonstrated consistent and high expression of the G protein-coupled receptors (GPCR) CXCR4, cysLT1 and S1P1 in human hematopoetic progenitor and stem cells (HPC). All of these receptors induced chemotactic responses in mobilized CD34+ HPC. As their ligands are present in the hematopoietic microenvironment, these GPCR may be involved in HPC homing and/or proliferation. Ligands for CXCR4 (SDF-1) and cysLT1 (cys-leukotrienes) are both produced by stromal cells, while the S1P1 ligand (sphingosine 1-phosphate, S1P) is released by megakaryocytes and platelets. As GPCR may activate multiple signaling pathways resulting in different functional effects, we compared the signaling capacities of CXCR4, cysLT1 and S1P1 in CD34+ HPC and CD34+ progenitor cell lines. At optimal ligand concentrations, cysLT1 mediated the strongest intracellular calcium fluxes, in contrast to CXCR4 (intermediate response) and S1P1 (almost absent calcium fluxes). Pertussis toxin (PTX) only partially inhibited cysLT1-mediated calcium signaling, indicating coupling of the receptor to both Gi and Gq proteins, while CXCR4-mediated responses were exclusively PTX-sensitive and therefore only Gi-dependent. Both cysLT1 and CXCR4 induced phosphorylation of the proliferation-related p44/42 (Erk) MAP kinase. Again, much stronger responses were mediated by cysLT1, which again were only partially PTX-sensitive. No Erk/MAP kinase activation was mediated by S1P1. We could demonstrate that pyk2, a kinase that links calcium signaling with Erk/MAP kinase activation, was involved in Gq-mediated signaling of cysLT1, as its phosphorylation was not inhibited by PTX. The fact that cysLT1 employs both Gi and Gq-mediated signaling pathways, while CXCR4 signaling was exclusively Gi-dependent, may explain why enhanced proliferation of CD34+ hematopoietic progenitor cells in cytokine-supplemented serum-free liquid cultures was only seen in response to ligands of cysLT1, but not in response to the CXCR4 ligand SDF-1. Activation of CXCR4 resulted in phosphorylation of Akt, which was not observed in response to ligands of cysLT1, while both GPCR induced translocation of NFkappaB to the nucleus. We conclude that in CD34+ HPC, Gi protein-mediated signaling of CXCR4 is mainly involved in cell migration, while Gi and Gq-mediated signaling of cysLT1 also affects cell proliferation predominantly by combined and profound activation of the Erk/MAP kinase pathway. The signaling capacity of S1P1 however was limited compared to the other GPCRs.

HIV and Other Lentiviral Infections Cause Defects in Neutrophil Chemotaxis, Recruitment, and Cell Structure: Immunorestorative Effects of Granulocyte-Macrophage Colony-Stimulating Factor

Patients with HIV infection exhibit deficits in bacterial and fungal clearance, and possibly depressed innate immunity. In this study, we observed that neutrophils from HIV-infected patients have a profound defect in chemotaxis in response to endogenous (IL-8) and bacterial (fMLP) chemoattractants, which was directly correlated with peripheral CD4(+) lymphocyte levels but not plasma viral load. A similar chemotactic defect was observed in the feline immunodeficiency virus (FIV) model of HIV infection. Intravital microscopy of FIV-infected animals revealed marked impairment in the in vivo recruitment of leukocytes; specifically integrin-dependent neutrophil adhesion and emigration induced by bacterial products. Treatment of FIV-infected animals with GM-CSF re-established both neutrophil recruitment (rolling, adhesion, and emigration) and in vitro chemotaxis to the levels seen in uninfected animals. This restoration of neutrophil responses was not due to GM-CSF-mediated priming. Rather, HIV and FIV infections resulted in defective neutrophil development, with an ensuing reduction in neutrophil granularity and chemotactic receptor expression. GM-CSF therapy restored neutrophil granularity, implying restoration of normal neutrophil development. Together, our findings underscore the fundamental defects in innate immunity caused by lentivirus infections, while also indicating that GM-CSF may be a potential immunorestorative therapy for HIV-infected patients.

Plasminogen Activator Inhibitor-1 in Chronic Kidney Disease

In 1984, Loskutoff et al. ([1][1]) purified plasminogen activator inhibitor-1 (PAI-1) from conditioned media of cultured endothelial cells. This 50-kd glycoprotein is the primary physiologic inhibitor of the serine proteases tissue-type and urokinase-type plasminogen activators (tPA and uPA,

Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context

Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Antibody reactivity was lost by antigen treatment with sulfatase or preincubation with soluble tyrosine sulfate, indicating its specificity. The isolation of this antibody signals the potential of phage antibody libraries in the derivation of reagents specific for post-translational modifications, although the extensive screening required indicates that such antibodies are extremely rare.

CD4 Expression on Activated NK Cells: Ligation of CD4 Induces Cytokine Expression and Cell Migration

NK cells play an important role in the innate immune response. We have isolated NK cells from human lymphoid tissues and found that these cells express the CD4 molecule on their surface at levels higher than those found on peripheral blood NK cells. To study the functional role of the CD4 molecule on NK cells, we developed an in vitro system by which we are able to obtain robust CD4 expression on NK cells derived from blood. CD4+ NK cells efficiently mediate NK cell cytotoxicity, and CD4 expression does not appear to alter lytic function. CD4+ NK cells are more likely to produce the cytokines gamma-IFN and TNF-alpha than are CD4- NK cells. Ligation of CD4 further increases the number of NK cells producing these cytokines. NK cells expressing CD4 are also capable of migrating toward the CD4-specific chemotactic factor IL-16, providing another function for the CD4 molecule on NK cells. Thus, the CD4 molecule is present and functional on NK cells and plays a role in innate immune responses as a chemotactic receptor and by increasing cytokine production, in addition to its well-described function on T cells as a coreceptor for Ag responsive cell activation.

First identification of a chemotactic receptor in an invertebrate species: Structural and functional characterization of Ciona intestinalis C3a receptor

First identification of a chemotactic receptor in an invertebrate species: Structural and functional characterization of Ciona intestinalis C3a receptor

The Multiple Personalities of the Chemokine Receptor CCR7 in Dendritic Cells

CCR7 was described initially as a potent leukocyte chemotactic receptor that was later shown to be responsible of directing the migration of dendritic cells (DCs) to the lymph nodes where these cells play an important role in the initiation of the immune response. Recently, a variety of reports have indicated that, apart from chemotaxis, CCR7 controls the cytoarchitecture, the rate of endocytosis, the survival, the migratory speed, and the maturation of the DCs. Some of these functions of CCR7 and additional ones also have been described in other cell types. Herein we discuss how this receptor may contribute to modulate the immune response by regulating different functions in DCs. Finally, we also suggest a possible mechanism whereby CCR7 may control its multiple tasks in these cells.

Endocytic Function of von Hippel-Lindau Tumor Suppressor Protein Regulates Surface Localization of Fibroblast Growth Factor Receptor 1 and Cell Motility

The tumor suppressor VHL (von Hippel-Lindau protein) serves as a negative regulator of hypoxia-inducible factor-alpha subunits. However, accumulated evidence indicates that VHL may play additional roles in other cellular functions. We report here a novel hypoxia-inducible factor-independent function of VHL in cell motility control via regulation of fibroblast growth factor receptor 1 (FGFR1) endocytosis. In VHL null tumor cells or VHL knock-down cells, FGFR1 internalization is defective, leading to surface accumulation and abnormal activation of FGFR1. The enhanced FGFR1 activity directly correlates with increased cell migration. VHL disease mutants, in two of the mutation hot spots favoring development of renal cell carcinoma, failed to rescue the above phenotype. Interestingly, surface accumulation of the chemotactic receptor appears to be selective in VHL mutant cells, since other surface proteins such as epidermal growth factor receptor, platelet-derived growth factor receptor, IGFR1, and c-Met are not affected. We demonstrate that 1) FGFR1 endocytosis is defective in the VHL mutant and is rescued by reexpression of wild-type VHL, 2) VHL is recruited to FGFR1-containing, but not EGFR-containing, endosomal vesicles, 3) VHL exhibits a functional relationship with Rab5a and dynamin 2 in FGFR1 internalization, and 4) the endocytic function of VHL is mediated through the metastasis suppressor Nm23, a protein known to regulate dynamin-dependent endocytosis.

Dynamics of protein kinase C mediated phosphorylation of the C5a receptor on Serine‐334

Phosphorylation of Ser-334, one out of three key serine phosphorylation sites within the C5aR carboxyl terminus, was shown to be required for the C5a-stimulated internalization of this chemotactic leukocyte receptor (Braun et al., J Biol Chem 278:4277, 2003). Because of the functional significance of this phosphorylation site for C5aR endocytosis we generated phosphosite-specific monoclonal antibodies which specifically react with phosphoserine-334 in ligand-activated C5aR. Treatment of db-cAMP stimulated U937 cells, which endogenously express C5aR, with activators (PMA) or inhibitors (bisindolylmaleimide) of protein kinase C (PKC) revealed that both homologous, C5a-induced and heterologous, C3a-induced phosphorylation of Ser-334 was mediated by PKC. A C5aR synthetic peptide was found to be a substrate for recombinant conventional (α,β I, β II, γ) and novel (δ) PKC isozymes in an in vitro phosphorylation assay. However, in intact myeloid cells only a PKCβ I/β II-selective inhibitor (LY333531), but not a PKCδ inhibitor efficiently blocked Ser-334 phosphorylation. Consistent with our previous observation in the related CCR5 receptor system, PKC phosphorylated C5aR at low C5a concentrations in a very rapid (t½ ~ 20 sec), albeit transient manner. Surprisingly, at high concentrations (>10nM) of C5a the PKC-mediated phosphorylation of serine-334 was essentially blocked. This could be attributed to the even faster (t½ < 5 sec) GRK-mediated phosphorylation of C5aR and consecutive firm binding of ß-arrestin. Our findings indicate that β-arrestin modulates PKC-mediated receptor phosphorylation and thus define a new regulatory role for this important adaptor protein.