Regulated On Activation Normal T Cell Expressed Research Articles

Vitamin D Modulation of the Innate Immune Response to Paediatric Respiratory Pathogens Associated with Acute Lower Respiratory Infections.

Vitamin D is an essential component of immune function and childhood deficiency is associated with an increased risk of acute lower respiratory infections (ALRIs). Globally, the leading childhood respiratory pathogens are Streptococcus pneumoniae, respiratory syncytial virus and the influenza virus. There is a growing body of evidence describing the innate immunomodulatory properties of vitamin D during challenge with respiratory pathogens, but recent systematic and unbiased synthesis of data is lacking, and future research directions are unclear. We therefore conducted a systematic PubMed literature search using the terms “vitamin D” and “Streptococcus pneumoniae” or “Respiratory Syncytial Virus” or “Influenza”. A priori inclusion criteria restricted the review to in vitro studies investigating the effect of vitamin D metabolites on human innate immune cells (primary, differentiated or immortalised) in response to stimulation with the specified respiratory pathogens. Eleven studies met our criteria. Despite some heterogeneity across pathogens and innate cell types, vitamin D modulated pathogen recognition receptor (PRRs: Toll-like receptor 2 (TLR2), TLR4, TLR7 and nucleotide-binding oligomerisation domain-containing protein 2 (NOD2)) expression; increased antimicrobial peptide expression (LL-37, human neutrophil peptide (HNP) 1-3 and β-defensin); modulated autophagosome production reducing apoptosis; and modulated production of inflammatory cytokines (Interleukin (IL) -1β, tumour necrosis factor-α (TNF-α), interferon-ɣ (IFN-ɣ), IL-12p70, IFN-β, Regulated on Activation, Normal T cell Expressed (RANTES), IL-10) and chemokines (IL-8 and C-X-C motif chemokine ligand 10 (CXCL10)). Differential modulation of PRRs and IL-1β was reported across immune cell types; however, this may be due to the experimental design. None of the studies specifically focused on immune responses in cells derived from children. In summary, vitamin D promotes a balanced immune response, potentially enhancing pathogen sensing and clearance and restricting pathogen induced inflammatory dysregulation. This is likely to be important in controlling both ALRIs and the immunopathology associated with poorer outcomes and progression to chronic lung diseases. Many unknowns remain and further investigation is required to clarify the nuances in vitamin D mediated immune responses by pathogen and immune cell type and to determine whether these in vitro findings translate into enhanced immunity and reduced ALRI in the paediatric clinical setting.

Wy14643, an agonist for PPARα, downregulates expression of TARC and RANTES in cultured human keratinocytes

Beneficial effects of Wy14643, an agonist for peroxisome proliferator-activated receptor (PPAR)α, on permeability barrier homeostasis-related functions of keratinocytes such as up-regulation of epidermal differentiation-related molecules and lipid synthesis, have been demonstrated. The present study demonstrated that Wy14643 reduced the expression of thymus and activation-related chemokine (TARC) and regulated on activation normal T cell expressed (RANTES) in both single- and 3D-cultured human keratinocytes. The combined data of the present and previous studies support the notion that Wy14643 could be a therapeutic agent that might simultaneously and directly modulate permeability barrier dysfunction and allergic inflammation in the pathogenesis of atopic dermatitis. As for the anti-microbial barrier function, the present study demonstrated that Wy14643 up-regulated expression of the anti-microbial peptide, human β defensin 3, in cultured human keratinocytes only in mRNA levels but not in protein ones, suggesting that Wy14643 might not directly account for the up-regulation of the anti-microbial peptide which has been reported in vivo.

Tubular cell dedifferentiation and peritubular inflammation are coupled by the transcription regulator Id1 in renal fibrogenesis

During renal fibrogenesis, tubular epithelial-mesenchymal transition is closely associated with peritubular inflammation; however, it is not clear whether these two processes are connected. We previously identified the inhibitor of differentiation-1 (Id1), a dominant negative antagonist of basic helix-loop-helix transcription factors, as a major trigger of tubular cell dedifferentiation after injury. Id1 was induced selectively in degenerated proximal tubule and collecting duct epithelia after injury and was present in both the cytoplasm and nucleus, suggesting shuttling between these two compartments. Interestingly, the upregulation of Id1 was associated with peritubular inflammation in mouse and human nephropathies. In vitro, Id1 potentiated NF-κB signaling and augmented RANTES expression in kidney epithelial cells, which led to an enhanced recruitment of inflammatory cells. Id1 also induced Snail1 expression and triggered tubular epithelial dedifferentiation. In vivo, genetic ablation of Id1 in mice reduced peritubular inflammation and decreased tubular expression of RANTES following ureteral obstruction. Mice lacking Id1 were also protected against myofibroblast activation and matrix expression, leading to a reduced total collagen deposition in obstructive nephropathy. Thus, these results indicate that Id1 shuttles between nucleus and cytoplasm and promotes peritubular inflammation and tubular epithelial dedifferentiation, suggesting that these two events are intrinsically coupled during renal fibrogenesis.

The X-Ray Structure of RANTES

The biological activity of chemokines requires interactions with cell surface proteoglycans. We have determined the structure of the chemokine RANTES (regulated on activation normal T cell expressed) in the presence of heparin-derived disaccharide analogs by X-ray crystallography. These structures confirm the essential role of the BBXB motif in the interaction between the chemokine and the disaccharide. Unexpected interactions were observed in the 30s loop and at the amino terminus. Mutant RANTES molecules were designed to abrogate these interactions and their biological activity examined in vivo. The K45E mutant within the BBXB motif lost the capacity to bind heparin and the ability to elicit cellular recruitment. The Y3A mutant maintained its capacity to bind heparin but was unable to elicit cellular recruitment. Finally, a tetrasaccharide is the smallest oligosaccharide which effectively abolishes the ability of RANTES to recruit cells in vivo. These crystallographic structures provide a description of the molecular interaction of a chemokine with glycosaminoglycans.

RANTES, eotaxin and eotaxin‐2 expression and production in patients with aspirin triad

Polyposis, asthma, aspirin-intolerance and aspirin-triad are mostly accompanied with eosinophilia of mucosal airways. Chemotactic cytokines, the CC-chemokines regulated on activation, normal T-cell expressed (RANTES), eotaxin, and eotaxin-2 activate and attract eosinophilic leukocytes to the site of inflammation. This points to the implication of CC-chemokines in eosinophilia of nasal tissue of these diseases. Therefore, nasal polypous tissue specimens of patients suffering from chronic nasal polypous sinusitis (NP), intrinsic asthma (ATA), aspirin-intolerance (AINA), and aspirin-triad (TRIAD) were investigated. The amount of mRNA and protein of CC-chemokines was analyzed using semi-quantitative reverse transcriptase polymerase chain reaction and chemokine-specific enzyme-immuno-assays. The patterns of CC-chemokines were compared. The mRNA-expression as well as protein synthesis of CC-chemokines was quantified in all tissues investigated. The expression of RANTES-mRNA in NP, ATA, AINA, and TRIAD (averaging 148-324% D-glyceraldehyde-3-phosphate dehydrogenase) and protein synthesis (0.13-0.15 ng/mg tissue weight) did not differ significantly. But the protein synthesis of eotaxin- and eotaxin-2-mRNA was significantly (P < 0.05) higher in TRIAD (3.3 pg/mg and 3.4 ng/mg tissue weight) (4 ng/mg tissue weight), than in NP, ATA, or AINA (1.8 pg/mg and 2.1 ng/mg, 2.1 pg/mg and 1.6 ng/mg, or 1.7 pg/mg and 2.2 ng/mg tissue weight, respectively). Patients suffering from TRIAD in association with tissue eosinophilia were characterized by elevated eotaxin and eotaxin-2 mRNA-expression as well as protein-synthesis. This pointed to the implication of eotaxins and RANTES in eosinophilia-associated diseases. Further studies will have to prove, whether the analysis of these chemokines might improve the diagnosis of eosinophilia associated polyposis and initiate the development of new therapeutic strategies.

Heparin-aggregated RANTES can be crystallised

Crystals of RANTES (regulated on activation normal T-cell expressed) have been grown in the presence of heparin-derived sulphated oligosaccharides which cause RANTES to aggregate severely. The crystals have a tendency to be polycrystalline but diffract to 3.8 - 1.8 A resolution. Oligosaccharides length and stoichiometry influence aggregation, nucleation and crystal growth and quality. Surprising, rather than inhibiting crystallisation, aggregation appears to stimulate nucleation. We have co-crystallised RANTES in the presence of oligosaccharides ranging in size from 1 to 12 sugar moieties. The best crystals, both according to size and diffraction quality have been obtained with a six moiety sugar. Crystals grow in the same space group with similar cell parameters as previously reported. We find no homogeneous binding of the sulphated sugars to RANTES even after eliminating sulphate from the crystallisation conditions to avoid competition with sulphates from the sulphated sugars. There is no electron density at the sulphate positions characterised in the original structure and the residues involved in sulphate binding adopt a different side chain orientation. Either heterogeneous binding of the sulphated sugars or an active ratchet-like mechanism by which the sulphated sugars are eliminated from the crystal lattice as the crystals grow may be responsible for the absence of sugars in the structure. The fact that aggregated proteins can be crystallised is important, since it is generally accepted that proteins that are polydisperse by light dynamic scattering are poor candidates for crystallisation trials.

Multiple Regulatory Domains Control IRF-7 Activity in Response to Virus Infection

Recent studies implicate the interferon regulatory factors (IRF), IRF-3 and IRF-7, as key activators of Type 1 interferon genes, as well as the RANTES (regulated on activation normal T cell expressed) chemokine gene. Both IRF-3 and IRF-7 are regulated in part by virus-induced C-terminal phosphorylation, leading to nuclear translocation, stimulation of DNA binding, and transcriptional activities. Structure-function studies with IRF-7 suggested a complex organization of the C-terminal region, with a constitutive activation domain located between amino acids 150-246, an accessory inducibility region at the very end of IRF-7 between amino acids 467 and 503, and an inhibitory region (amino acids 341-467) adjacent to the C-terminal end that interferes with transactivation. Furthermore, an element that increases basal and virus-inducible activity is located between amino acids 278 and 305. A transcriptionally active form of IRF-7 was also generated by substitution of Ser-477 and Ser-479 residues with the phosphomimetic Asp. IRF-7, particularly IRF-7(S477D/S479D), was a strong transactivator of type I interferon and RANTES chemokine gene expression. Unlike wild type IRF-3, IRF-7 overexpression was able to stimulate inteferon gene expression in the absence of virus infection. Using tagged versions of IRF-7 and IRF-3, the formation of homo- and heterodimers was detected by co-immunoprecipitation. These results demonstrate that IRF-3 and IRF-7 transcription factors possess distinct structural characteristics that impart complementary rather than redundant functional roles in cytokine gene activation.

Unique Role of the Chemokine Domain of Fractalkine in Cell Capture: KINETICS OF RECEPTOR DISSOCIATION CORRELATE WITH CELL ADHESION

The chemokine fractalkine (FK) has two structural features that make it unique in the chemokine family: a CX(3)C motif and an extended carboxyl terminus that anchors it to the cell surface. This mucin-like stalk or an equivalent spacer is required for FK to mediate the adhesion of cells expressing its receptor, CX(3)CR1. To determine whether the ability of FK to act as a cell adhesion molecule is due to the unique presentation of a chemokine domain on a stalk or to properties of the chemokine domain itself, we created a series of chimeras in which other soluble chemokines (RANTES (regulated on activation normal T cell expressed), monocyte chemoattractant protein 1, macrophage inflammatory protein 1 beta, secondary lymphoid tissue chemokine, and interleukin 8) were fused to the mucin stalk. When tested in a static-cell adhesion assay, many of these chemokine chimeras demonstrated activity equivalent to that of FK. In flow assays, however, none of the chimeras captured cells as efficiently as FK. Interestingly, FK captured cells expressing either CX(3)CR1 or the viral receptor US28. Cells bound to FK without rolling or detaching, whereas the interleukin 8 and monocyte chemoattractant protein 1 chimeras induced primarily cell rolling and detaching, respectively. In binding studies, FK has a significantly slower off-rate from its receptors than any of the other chemokine chimeras had for their cognate receptors. We conclude that presentation of a chemokine atop a mucin-like stalk is not, in and of itself, sufficient to capture cells. The unique ability of FK to mediate adhesion under flow may be a function of its slow receptor off-rate.

Examination of the Function of RANTES, MIP-1α, and MIP-1β following Interaction with Heparin-like Glycosaminoglycans

Chemokines are a group of small proteins that have a variety of functions, including the activation and recruitment of immune cells during episodes of inflammation. In common with many cytokines, it has been observed that chemokines have the potential to bind heparin-like glycosaminoglycan molecules, which are normally expressed on proteoglycan components of the cell surface and extracellular matrix. The significance of this interaction for chemokine activity remains a subject of debate. In this study, Chinese hamster ovary cells were transfected separately with the human chemokine receptors CCR1 and CCR5, and these receptors were shown to induce an intracytoplasmic Ca(2+) flux and cellular chemotaxis following stimulation with the natural CC chemokine ligands (MIP-1alpha, RANTES (regulated on activation normal T cell expressed), and MIP-1beta). In further experiments, mutant CHO cells, with a defect in normal glycosaminoglycan (GAG) expression, were also transfected with, and shown to express similar levels of, CCR1 and CCR5. Although these receptors were functional, it was found that the mutant cells required exposure to higher concentrations of ligands than the wild-type cells in order to produce the same intracytoplasmic Ca(2+) flux. Radioligand binding experiments demonstrated that specific chemokine receptors expressed by wild-type cells had a significantly greater affinity for MIP-1alpha than similar receptors expressed by GAG-deficient mutants. However, there was no significant difference between these cells in their affinity for RANTES or MIP-1beta. In conclusion, it has been demonstrated clearly that GAG expression is not necessary for the biological activity of the chemokines MIP-1alpha, RANTES, or MIP-1beta. However, the presence of cell surface GAGs does enhance the activity of low concentrations of these chemokines by a mechanism that appears to involve sequestration onto the cell surface.

Differential Activation of CC Chemokine Receptors by AOP-RANTES

RANTES (regulated on activation normal T cell expressed) has been found at elevated levels in biological fluids from patients with a wide range of allergic and autoimmune diseases and is able to attract several subtypes of leukocytes including eosinophils and monocytes into inflamed tissue. Amino-terminal modifications of RANTES produce receptor antagonists which are candidates for blocking this cellular recruitment. Met-RANTES has been shown to modulate inflammation in vivo, while AOP-RANTES is a potent inhibitor of R5 human immunodeficiency virus type 1 (HIV-1) strains and has been shown to down-modulate CCR5 and prevent recycling of the receptor. We have studied the effect of AOP-RANTES in eosinophil activation and have found that it is able to efficiently elicit eosinophil effector functions through CCR3, as measured by the release of reactive oxygen species and calcium mobilization, whereas Met-RANTES is inactive in these assays. AOP-RANTES is found to inhibit CCR3-mediated HIV-1 infection with moderate potency, in contrast to its potent inhibition of CCR5-mediated HIV-1 infection. Furthermore, we have investigated the abilities of these modified proteins to down-modulate CCR1 and CCR3 from the surface of monocytes and eosinophils. We show here that AOP-RANTES is much less effective than RANTES in down-modulation of CCR1. Surprisingly, recycling of CCR1 was minimal after incubation with RANTES while there was complete recycling with AOP-RANTES. In the case of CCR3, no significant difference was found between RANTES and AOP-RANTES in down-modulation and recycling. It therefore appears that trafficking of RANTES receptors follows different patterns, which opens up potential new targets for therapeutic intervention.

Multiple Charged and Aromatic Residues in CCR5 Amino-terminal Domain Are Involved in High Affinity Binding of Both Chemokines and HIV-1 Env Protein

CCR5 is a functional receptor for MIP-1alpha, MIP-1beta, RANTES (regulated on activation normal T cell expressed), MCP-2, and MCP-4 and constitutes the main coreceptor for macrophage tropic human and simian immunodeficiency viruses. By using CCR5-CCR2b chimeras, we have shown previously that the second extracellular loop of CCR5 is the major determinant for chemokine binding specificity, whereas the amino-terminal domain plays a major role for human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus coreceptor function. In the present work, by using a panel of truncation and alanine-scanning mutants, we investigated the role of specific residues in the CCR5 amino-terminal domain for chemokine binding, functional response to chemokines, HIV-1 gp120 binding, and coreceptor function. Truncation of the amino-terminal domain resulted in a progressive decrease of the binding affinity for chemokines, which correlated with a similar drop in functional responsiveness. Mutants lacking residues 2-13 exhibited fairly weak responses to high concentrations (500 nM) of RANTES or MIP-1beta. Truncated mutants also exhibited a reduction in the binding affinity for R5 Env proteins and coreceptor activity. Deletion of 4 or 12 residues resulted in a 50 or 80% decrease in coreceptor function, respectively. Alanine-scanning mutagenesis identified several charged and aromatic residues (Asp-2, Tyr-3, Tyr-10, Asp-11, and Glu-18) that played an important role in both chemokine and Env high affinity binding. The overlapping binding site of chemokines and gp120 on the CCR5 amino terminus, as well as the involvement of these residues in the epitopes of monoclonal antibodies, suggests that these regions are particularly exposed at the receptor surface.

Aggregation of RANTES Is Responsible for Its Inflammatory Properties: CHARACTERIZATION OF NONAGGREGATING, NONINFLAMMATORY RANTES MUTANTS

The biology of RANTES (regulated on activation normal T cell expressed) aggregation has been investigated using RANTES and disaggregated variants, enabling comparison of aggregated, tetrameric, and dimeric RANTES forms. Disaggregated variants retain their G(i)-type G protein-coupled receptor-mediated biological activities. A correlation between RANTES aggregation and cellular activation has been demonstrated. Aggregated RANTES, but not disaggregated RANTES, activates human T cells, monocytes, and neutrophils. Dimeric RANTES has lost its cellular activating activity, rendering it noninflammatory. Macrophage inflammatory protein 1alpha, macrophage inflammatory protein-1beta, and erythrocytes are potent natural antagonists of aggregated RANTES-induced cellular activation. There is a clear difference in the signaling properties of aggregated and disaggregated RANTES forms, separating the dual signaling pathways of RANTES and the enhancing and suppressive effects of RANTES on human immunodeficiency virus infection. Disaggregated RANTES will be a valuable tool to explore the biology of RANTES action in human immunodeficiency virus infection and in inflammatory disease.

Identification of Amino Acid Residues Critical for Aggregation of Human CC Chemokines Macrophage Inflammatory Protein (MIP)-1α, MIP-1β, and RANTES: CHARACTERIZATION OF ACTIVE DISAGGREGATED CHEMOKINE VARIANTS

Human CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation normal T cell expressed) self-associate to form high-molecular mass aggregates. To explore the biological significance of chemokine aggregation, nonaggregating variants were sought. The phenotypes of 105 hMIP-1alpha variants generated by systematic mutagenesis and expression in yeast were determined. hMIP-1alpha residues Asp26 and Glu66 were critical to the self-association process. Substitution at either residue resulted in the formation of essentially homogenous tetramers at 0.5 mg/ml. Substitution of identical or analogous residues in homologous positions in both hMIP-1beta and RANTES demonstrated that they were also critical to aggregation. Our analysis suggests that a single charged residue at either position 26 or 66 is insufficient to support extensive aggregation and that two charged residues must be present. Solution of the three-dimensional NMR structure of hMIP-1alpha has enabled comparison of these residues in hMIP-1beta and RANTES. Aggregated and disaggregated forms of hMIP-1alpha, hMIP-1beta, and RANTES generally have equivalent G-protein-coupled receptor-mediated biological potencies. We have therefore generated novel reagents to evaluate the role of hMIP-1alpha, hMIP-1beta, and RANTES aggregation in vitro and in vivo. The disaggregated chemokines retained their human immunodeficiency virus (HIV) inhibitory activities. Surprisingly, high concentrations of RANTES, but not disaggregated RANTES variants, enhanced infection of cells by both M- and T-tropic HIV isolates/strains. This observation has important implications for potential therapeutic uses of chemokines implying that disaggregated forms may be necessary for safe clinical investigation.

Naturally Occurring CCR5 Extracellular and Transmembrane Domain Variants Affect HIV-1 Co-receptor and Ligand Binding Function

Analysis of CCR5 variants in human immunodeficiency virus, type 1 (HIV-1), high risk cohorts led to the identification of multiple single amino acid substitutions in the amino-terminal third of the HIV-1 co-receptor CCR5 suggesting the possibility of protective and permissive genotypes; unfortunately, the low frequency of these mutations did not led to correlation with function. Therefore, we used analytical methods to assess the functional and structural significance of six of these variant receptors in vitro. These studies showed three categories of effects on CCR5 function. 1) Mutations in the first extracellular domain of CCR5 severely reduce specific ligand binding and chemokine-induced chemotaxis. 2) An extracellular domain variant, A29S, when co-expressed with CD4, supported HIV-1 infection whereas the others do not. 3) The transmembrane region variants of CCR5 support monotropic HIV-1 infection that is blocked by addition of some receptor agonists. Mutations in the first and second transmembrane domains increase RANTES (regulated on activation normal T-cell expressed) binding affinity but did not affect MIP1beta binding affinity presumably based on differences in ligand-receptor interaction sites. Furthermore, the CCR5 transmembrane mutants do not respond to RANTES with the classical bell-shaped chemotactic response curve, suggesting that they are resistant to RANTES-induced desensitization. These data demonstrate that single amino acid changes in the extracellular domains of CCR5 can have profound effects on both HIV-1 co-receptor and specific ligand-induced functions, whereas mutations in the transmembrane domain only affect the response to chemokine ligands.

Identification and Characterization of Small Molecule Functional Antagonists of the CCR1 Chemokine Receptor

The CC chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES (regulated on activation normal T cell expressed) have been implicated in rheumatoid arthritis and multiple sclerosis. Since their effects are mediated through the CCR1 chemokine receptor, we set up a small molecule CCR1 antagonist program to search for inhibitors. Through high capacity screening we discovered a number of 4-hydroxypiperidine compounds with CCR1 antagonist activity and report their synthesis and in vitro pharmacology here. Scatchard analysis of the competition binding data revealed that the compounds had Ki values ranging from 40 to 4000 nM. The pharmacological profile of the most potent member of this series, compound 1 (2-2-diphenyl-5-(4-chlorophenyl)piperidin-lyl)valeronitri te), was further evaluated. Compound 1 showed concentration-dependent inhibition of MIP-1alpha-induced extracellular acidification and Ca2+ mobilization demonstrating functional antagonism. When given alone, the compound did not elicit any responses, indicating the absence of intrinsic agonist activity. Compound 1 inhibited MIP-1alpha- and RANTES-induced migration in peripheral blood mononuclear cells in a dose-responsive manner. Selectivity testing against a panel of seven transmembrane domain receptors indicated that compound 1 is inactive on a number of receptors at concentrations up to 10 microM. This is the first description of CCR1 receptor antagonists that may be useful in the treatment of chronic inflammatory diseases involving MIP-1alpha, RANTES, and CCR1.

Close Association of the First and Fourth Extracellular Domains of the Duffy Antigen/Receptor for Chemokines by a Disulfide Bond Is Required for Ligand Binding

It has been demonstrated that the promiscuous chemokine binding profile of the Duffy antigen/receptor for chemokines (DARC) is given by its extracellular NH2-terminal region. However, the relationship among the Fy6, Fya/b, and Fy3 epitopes, localized in the first and fourth extracellular domains of DARC, respectively, and the chemokine binding sites remained a matter of controversy. Here, we performed cross-displacement and cross-inhibition experiments indicating that all anti-Fy6, anti-Fya, and anti-Fy3 monoclonal antibodies and interleukin 8 are antagonists for binding to red cells. Biopanning of phage peptide libraries with an anti-Fy6 monoclonal antibody led to the identification of the motif Phe22-Glu23, the mutation of which altered the binding of both anti-Fy6 and chemokines (interleukin 8, MGSA, RANTES (regulated on activation normal T cell expressed)) to DARC transfectants. These results characterized the core of the Fy6 epitope and provided definitive proof of the tight relationship between Fy6 and the chemokine receptor site. Analysis of red cells treated by sulfhydryl group-modifying reagents suggested that the chemokine receptor function of DARC required the integrity of disulfide bond(s) but not that of free sulfhydryl group(s). Accordingly, mutation of cysteines 51 and 276 abolished chemokine binding to DARC transfectants. Altogether, our results suggested that the chemokine binding pocket of DARC included sequences located in the first and fourth extracellular domains which are brought into close vicinity by a disulfide bridge.

Molecular Cloning and Functional Characterization of a Novel Human CC Chemokine Receptor (CCR5) for RANTES, MIP-1β, and MIP-1α

Chemokines affect leukocyte chemotactic and activation activities through specific G protein-coupled receptors. In an effort to map the closely linked CC chemokine receptor genes, we identified a novel chemokine receptor encoded 18 kilobase pairs downstream of the monocyte chemoattractant protein-1 (MCP-1) receptor (CCR2) gene on human chromosome 3p21. The deduced amino acid sequence of this novel receptor, designated CCR5, is most similar to CCR2B, sharing 71% identical residues. Transfected cells expressing the receptor bind RANTES (regulated on activation normal T cell expressed), MIP-1beta, and MIP-1alpha with high affinity and generate inositol phosphates in response to these chemokines. This same combination of chemokines has recently been shown to potently inhibit human immunodeficiency virus replication in human peripheral blood leukocytes (Cocchi, F., DeVico, A. L., Garzino-Demo, A., Arya, S. K., Gallo, R. C., and Lusso, P.(1995) Science 270, 1811-1815). CCR5 is expressed in lymphoid organs such as thymus and spleen, as well as in peripheral blood leukocytes, including macrophages and T cells, and is the first example of a human chemokine receptor that signals in response to MIP-1beta.

Mechanisms of Murine RANTES Chemokine Gene Induction by Newcastle Disease Virus

We have previously defined the lipopolysaccharide (LPS)-responsive element (LRE) in the promoters of murine RANTES (regulated on activation normal T-cell expressed) (MuRantes) and murine IP-10/crg-2, chemokines which have potent chemotactic properties for inflammatory cells including monocytes and T lymphocytes. In the present work, we studied the transcriptional mechanism of MuRantes gene induction by virus and compared it with that of LPS in an effort to understand the host responses to virus and bacterial toxins at the molecular level. MuRantes mRNA expression is induced by Newcastle disease virus (NDV) and LPS in the RAW 264.7 macrophage cell line and peritoneal macrophages of LPS-responsive C3HeB/FeJ mice. In LPS-hyporesponsive C3H/HeJ mice, only NDV induces this chemokine gene, indicating that the pathways of transcriptional activation by NDV and LPS are not identical. Using a transient transfection assay, the minimal virus-responsive element (VRE) was localized between nt -175 and -116. The VRE contains previously defined LRE motif 1 (TCAYRCTT) and motif 3 ((T/A)GRTTTCA(G/C)TTT), which were shown to also be important for initiation of transcription by virus. NDV-stimulated nuclear extracts were tested for trans-activating factors able to bind the VRE. The chromosomal protein HMG-I(C) was shown to bind the 3'-A.T-rich domains of the VRE, and the presence of HMG-I(C) was demonstrated in the VRE-protein complex formed with nuclear extracts from NDV-stimulated, but not unstimulated cells. These findings demonstrate the role of HMG-I(C) in activation of MuRantes promoter by NDV.