Defining the anti-inflammatory activity of a potent myxomaviral chemokine modulating protein,M-T7, through site directed mutagenesis

Viral chemokine modulating proteins provide new and extensive sources for therapeutics. Purified M-T7, a poxvirus-derived secreted immunomodulatory protein, reduces mononuclear cell invasion and atheroma in rodent models of angioplasty injury as well as aortic and renal transplant, improving renal allograft survival. M-T7 is a rabbit species-specific interferon gamma receptor (IFNγR) homolog, but also inhibits chemokine/glycosaminoglycan (GAG) interactions for C, CC and CXC chemokines, with cross-species specific inhibitory activity. M-T7 anti-atheroma activity is blunted in GAG deficient mouse aortic transplants, but not in CC chemokine receptor deficient transplants, supporting M-T7 interference in chemokine/GAG interactions as the basis of the atheroma-inhibitory activity. We have assessed point mutants of M-T7 both in vivo in a mouse angioplasty model and in vitro in tissue culture and binding assays, in order to better define the primary mechanism of anti-atheroma activity. Of these M-T7 mutants, the R¹⁷¹E and E²⁰⁹I M-T7 mutants lost inhibitory activity for plaque growth in hyperlipidemic ApoE^−/− mice after angioplasty injury and R¹⁷¹E, moreover, greatly exacerbated plaque growth and inflammation. F¹³⁷D retained some inhibitory activity for plaque growth. In contrast, for cell migration assays, M-T7-His_6X, F¹³⁷D, R¹⁷¹E, and E²⁰⁹I all inhibited CC chemokine (RANTES) mediated cell migration. For the ligand binding assays, R¹⁷¹E and E²⁰⁹I had significantly reduced binding to RANTES and IFNγ, whereas F¹³⁷D retained wild-type binding activity. Heparin treatment further reduced RANTES binding of all three M-T7 mutants. In summary, point mutations of M-T7, R¹⁷¹E and E²⁰⁹I, exhibited reduced anti-inflammatory properties in vivo after mouse angioplasty with a loss of in vitro binding to RANTES and IFNγ, indicating these point mutations partially disrupt M-T7 ligand-binding activities. Unexpectedly, the M-T7 mutants all retained inhibitory activity for human monocyte THP-1 cell migration ex vivo, suggesting additional inhibitory properties against human monocyte THP-1 cells that are independent of chemokine inhibition.     

Glycosaminoglycan binding properties of the myxoma virus CC-chemokine inhibitor, M-T1

Poxviruses encode a number of secreted virulence factors that function to mitigate or modulate the host immune response. M-T1 is a secreted 43-kDa glycoprotein produced by the myxoma virus, a poxvirus pathogen of rabbits, that binds CC-chemokines with high affinity, blocks binding to their cognate G-protein coupled receptors, and thereby inhibits chemokine-induced leukocyte chemotaxis. The present study indicates that M-T1, but not the related vaccinia virus 35-kDa CC-chemokine-binding protein, can localize to cell surfaces through an interaction with glycosaminoglycan molecules. In addition to biochemically characterizing the nature of this interaction, we demonstrate that M-T1 can also simultaneously interact with CC-chemokines while bound to heparin, suggesting that the binding sites on M-T1 for chemokines and heparin are distinct. Furthermore, using recombinant baculovirus-expressed M-T1 truncation and internal deletion mutants, we localize the heparin-binding region of M-T1 to the C terminus of the protein, a region that contains a high abundance of basic residues and includes two clusters of basic amino acid residues that resemble Cardin and Weintraub heparin-binding consensus sequences. The ability of M-T1 to simultaneously interact with chemokines and glycosaminoglycans may enable M-T1 to tether to endothelial surfaces or extracellular matrix and capture host chemokines that are expressed close to sites of virus infection.     

The core domain of chemokines binds CCR5 extracellular domains while their amino terminus interacts with the transmembrane helix bundle

CCR5 is a functional receptor for various inflammatory CC-chemokines, including macrophage inflammatory protein (MIP)-1alpha and RANTES (regulated on activation normal T cell expressed and secreted), and is the main coreceptor of human immunodeficiency viruses. The second extracellular loop and amino-terminal domain of CCR5 are critical for chemokine binding, whereas the transmembrane helix bundle is involved in receptor activation. Chemokine domains and residues important for CCR5 binding and/or activation have also been identified. However, the precise way by which chemokines interact with and activate CCR5 is presently unknown. In this study, we have compared the binding and functional properties of chemokine variants onto wild-type CCR5 and CCR5 point mutants. Several mutations in CCR5 extracellular domains (E172A, R168A, K191A, and D276A) strongly affected MIP-1alpha binding but had little effect on RANTES binding. However, a MIP/RANTES chimera, containing the MIP-1alpha N terminus and the RANTES core, bound to these mutants with an affinity similar to that of RANTES. Several CCR5 mutants affecting transmembrane helices 2 and 3 (L104F, L104F/F109H/F112Y, F85L/L104F) reduced the potency of MIP-1alpha by 10-100 fold with little effect on activation by RANTES. However, the MIP/RANTES chimera activated these mutants with a potency similar to that of MIP-1alpha. In contrast, LD78beta, a natural MIP-1alpha variant, which, like RANTES, contains a proline at position 2, activated these mutants as well as RANTES. Altogether, these results suggest that the core domains of MIP-1alpha and RANTES bind distinct residues in CCR5 extracellular domains, whereas the N terminus of chemokines mediates receptor activation by interacting with the transmembrane helix bundle.     

Dengue virus nonstructural protein NS5 induces interleukin-8 transcription and secretion

Elevated circulating levels of chemokines have been reported in patients with dengue fever and are proposed to contribute to the pathogenesis of dengue disease. To establish in vitro models for chemokine induction by dengue 2 virus (DEN2V), we studied a variety of human cell lines and primary cells. DEN2V infection of HepG2 and primary dendritic cells induced the production of interleukin-8 (IL-8), RANTES, MIP-1alpha, and MIP-1beta, whereas only IL-8 and RANTES were induced following dengue virus infection of HEK293 cells. Chemokine secretion was accompanied by an increase in steady-state mRNA levels. No chemokine induction was observed in HEK293 cells treated with poly(I:C) or alpha interferon, suggesting a direct effect of virus infection. To determine the mechanism(s) involved in the induction of chemokine production by DEN2V, individual dengue virus genes were cloned into plasmids and expressed in HEK293 cells. Transfection of a plasmid expressing NS5 or a dengue virus replicon induced IL-8 gene expression and secretion. RANTES expression was not induced under these conditions, however. Reporter assays showed that IL-8 induction by NS5 was principally through CAAT/enhancer binding protein, whereas DEN2V infection also induced NF-kappaB. These results indicate a role for the dengue virus NS5 protein in the induction of IL-8 by DEN2V infection. Recruitment and activation of potential target cells to sites of DEN2V replication by virus-induced chemokine production may contribute to viral replication as well as to the inflammatory components of dengue virus disease.     

The BBXB motif of RANTES is the principal site for heparin binding and controls receptor selectivity

The chemokine RANTES (regulated on activation normal T cell expressed and secreted; CCL5) binds selectively to glycosaminoglycans (GAGs) such as heparin, chondroitin sulfate, and dermatan sulfate. The primary sequence of RANTES contains two clusters of basic residues, (44)RKNR(47) and (55)KKWVR(59). The first is a BBXB motif common in heparin-binding proteins, and the second is located in the loop directly preceding the C-terminal helix. We have mutated these residues to alanine, both as point mutations as well as triple mutations of the 40s and 50s clusters. Using a binding assay to heparin beads with radiolabeled proteins, the (44)AANA(47) mutant demonstrated an 80% reduction in its capacity to bind heparin, whereas the (55)AAWVA(59) mutant retained full binding capacity. Mutation of the (44)RKNR(47) site reduced the selectivity of RANTES binding to different GAGs. The mutants were tested for their integrity by receptor binding assays on CCR1 and CCR5 as well as their ability to induce chemotaxis in vitro. In all assays the single point mutations and the triple 50s cluster mutation caused no significant difference in activity compared with the wild type sequence. However, the triple 40s mutant showed a 80-fold reduction in affinity for CCR1, despite normal binding to CCR5. It was only able to induce monocyte chemotaxis at micromolar concentrations. The triple 40s mutant was also able to inhibit HIV-1 infectivity, but consistent with its abrogated GAG binding capacity, it no longer induced enhanced infectivity at high concentrations.     

Interference with heparin binding and oligomerization creates a novel anti-inflammatory strategy targeting the chemokine system

A hallmark of autoimmunity and other chronic diseases is the overexpression of chemokines resulting in a detrimental local accumulation of proinflammatory immune cells. Chemokines play a pivotal role in cellular recruitment through interactions with both cell surface receptors and glycosaminoglycans (GAGs). Anti-inflammatory strategies aimed at neutralizing the chemokine system have to-date targeted inhibition of the receptor-ligand interaction with receptor antagonists. In this study, we describe a novel strategy to modulate the inflammatory process in vivo through mutation of the essential heparin-binding site of a proinflammatory chemokine, which abrogates the ability of the protein to form higher-order oligomers, but retains receptor activation. Using well-established protocols to induce inflammatory cell recruitment into the peritoneal cavity, bronchoalveolar air spaces, and CNS in mice, this non-GAG binding variant of RANTES/CCL5 designated [44AANA47]-RANTES demonstrated potent inhibitory capacity. Through a combination of techniques in vitro and in vivo, [44AANA47]-RANTES appears to act as a dominant-negative inhibitor for endogenous RANTES, thereby impairing cellular recruitment, not through a mechanism of desensitization. [44AANA47]-RANTES is unable to form higher-order oligomers (necessary for the biological activity of RANTES in vivo) and importantly forms nonfunctional heterodimers with the parent chemokine, RANTES. Therefore, although retaining receptor-binding capacity, altering the GAG-associated interactive site of a proinflammatory chemokine renders it a dominant-negative inhibitor, suggesting a powerful novel approach to generate disease-modifying anti-inflammatory reagents.     

Oxidant tone regulates RANTES gene expression in airway epithelial cells infected with respiratory syncytial virus. Role in viral-induced interferon regulatory factor activation

Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part, through its ability to induce chemokine synthesis in infected airway epithelial cells. RANTES (regulated upon activation, normal T-cells expressed and secreted) is a CC chemokine which recruits and activates monocytes, lymphocytes, and eosinophils, all cell types present in the lung inflammatory infiltrate induced by RSV infection. In this study we investigated the role of reactive oxygen species in the induction of RANTES gene expression in human type II alveolar epithelial cells (A549), following RSV infection. Our results indicate that RSV infection of airway epithelial cells rapidly induces reactive oxygen species production, prior to RANTES expression, as measured by oxidation of 2',7'-dichlorofluorescein. Pretreatment of airway epithelial cells with the antioxidant butylated hydroxyanisol (BHA), as well a panel of chemically unrelated antioxidants, blocks RSV-induced RANTES gene expression and protein secretion. This effect is mediated through the ability of BHA to inhibit RSV-induced interferon regulatory factor binding to the RANTES promoter interferon-stimulated responsive element, that is absolutely required for inducible RANTES promoter activation. BHA inhibits de novo interferon regulator factor (IRF)-1 and -7 gene expression and protein synthesis, and IRF-3 nuclear translocation. Together, these data indicates that a redox-sensitive pathway is involved in RSV-induced IRF activation, an event necessary for RANTES gene expression.     

Identification of the Pharmacophore of the CC Chemokine-binding Proteins Evasin-1 and -4 Using Phage Display

To elucidate the ligand-binding surface of the CC chemokine-binding proteins Evasin-1 and Evasin-4, produced by the tick Rhipicephalus sanguineus, we sought to identify the key determinants responsible for their different chemokine selectivities by expressing Evasin mutants using phage display. We first designed alanine mutants based on the Evasin-1·CCL3 complex structure and an in silico model of Evasin-4 bound to CCL3. The mutants were displayed on M13 phage particles, and binding to chemokine was assessed by ELISA. Selected variants were then produced as purified proteins and characterized by surface plasmon resonance analysis and inhibition of chemotaxis. The method was validated by confirming the importance of Phe-14 and Trp-89 to the inhibitory properties of Evasin-1 and led to the identification of a third crucial residue, Asn-88. Two amino acids, Glu-16 and Tyr-19, were identified as key residues for binding and inhibition of Evasin-4. In a parallel approach, we identified one clone (Y28Q/N60D) that showed a clear reduction in binding to CCL3, CCL5, and CCL8. It therefore appears that Evasin-1 and -4 use different pharmacophores to bind CC chemokines, with the principal binding occurring through the C terminus of Evasin-1, but through the N-terminal region of Evasin-4. However, both proteins appear to target chemokine N termini, presumably because these domains are key to receptor signaling. The results also suggest that phage display may offer a useful approach for rapid investigation of the pharmacophores of small inhibitory binding proteins.     

Identification of a gammaherpesvirus selective chemokine binding protein that inhibits chemokine action

Chemokines are involved in recruitment and activation of hematopoietic cells at sites of infection and inflammation. The M3 gene of gammaHV68, a gamma-2 herpesvirus that infects and establishes a lifelong latent infection and chronic vasculitis in mice, encodes an abundant secreted protein during productive infection. The M3 gene is located in a region of the genome that is transcribed during latency. We report here that the M3 protein is a high-affinity broad-spectrum chemokine scavenger. The M3 protein bound the CC chemokines human regulated upon activation of normal T-cell expressed and secreted (RANTES), murine macrophage inflammatory protein 1alpha (MIP-1alpha), and murine monocyte chemoattractant protein 1 (MCP-1), as well as the human CXC chemokine interleukin-8, the murine C chemokine lymphotactin, and the murine CX(3)C chemokine fractalkine with high affinity (K(d) = 1. 6 to 18.7 nM). M3 protein chemokine binding was selective, since the protein did not bind seven other CXC chemokines (K(d) > 1 microM). Furthermore, the M3 protein abolished calcium signaling in response to murine MIP-1alpha and murine MCP-1 and not to murine KC or human stromal cell-derived factor 1 (SDF-1), consistent with the binding data. The M3 protein was also capable of blocking the function of human CC and CXC chemokines, indicating the potential for therapeutic applications. Since the M3 protein lacks homology to known chemokines, chemokine receptors, or chemokine binding proteins, these studies suggest a novel herpesvirus mechanism of immune evasion.     

Structure of a CXC chemokine-receptor fragment in complex with interleukin-8.

BACKGROUND: Interactions between CXC chemokines (e.g. interleukin-8, IL-8) and their receptors (e.g. CXCR-1) have a key role in host defense and disease by attracting and upregulating neutrophils to sites of inflammation. The transmembrane nature of the receptor impedes structure-based understanding of ligand interactions. Linear peptides based on the N-terminal, extracellular portion of the receptor CXCR-1 do bind to IL-8, however, and inhibit the binding of IL-8 to the full-length receptor. RESULTS: The NMR solution structure of the complex formed between IL-8 and one such receptor-based peptide indicates that a cleft between a loop and a beta hairpin constitute part of the receptor interaction surface on IL-8. Nine residues from the C terminus of the receptor peptide (corresponding to Pro21-Pro29 of CXCR-1) occupy the cleft in an extended fashion. Intermolecular contacts are mostly hydrophobic and sidechain mediated. CONCLUSIONS: The results offer the first details at an atomic level of the interaction between a chemokine and its receptor. Consideration of other biochemical data allow extrapolation to a model for the interaction of IL-8 with the full-length receptor. In this model, the heparin-binding residues of IL-8 are exposed, thereby allowing presentation of the chemokine from endothelial cell-surface glycosaminoglycans. This first glimpse of how IL-8 binds to its receptor provides a foundation for the structure-based design of chemokine antagonists.     

Chemokines as mediators of tumor angiogenesis and neovascularization

Chemokines are a superfamily of structurally homologous heparin-binding proteins that influence tumor growth and metastasis. Several members of the CXC and CC chemokine families are potent inducers of neovascularization, whereas a subset of the CXC chemokines are potent inhibitors. In this paper, we review the current literature regarding the role of chemokines as mediators of tumor angiogenesis and neovascularization.     

A kinetics and modeling study of RANTES(9-68) binding to heparin reveals a mechanism of cooperative oligomerization

Heparan sulfate (HS) and heparin bind to virtually all chemokines and have been shown to play critical roles in the regulation of their activities. However, both binding mechanisms and structural features involved in chemokine-HS interactions remain poorly defined. In the study presented here, we analyzed the binding of heparin to RANTES(9-68), a N-terminally truncated form of the CC-chemokine RANTES. Using biochemical and surface plasmon resonance (BIAcore system) approaches, we showed that the RANTES(9-68)-heparin interaction was characterized by a complex binding model that involved dimerization of the chemokine through a mechanism of positive cooperativity. Since RANTES(9-68) remains monomeric in solution, we concluded that heparin induced chemokine dimerization. The structure of a complex involving a RANTES dimer and a heparin heptadecasaccharide was proposed by molecular modeling. This model was used to design a dimer of "head to head" coupled octasaccharides that would fit the internal symmetry of the chemokine dimer. This engineered oligosaccharide bound RANTES(9-68) much better than a natural heparin fragment of the same length, further supporting the interaction process and the proposed structural model. Altogether, the data reported here provide a basis for understanding the mechanisms by which HS modulates RANTES functions.     

Reovirus triggers cell type-specific proinflammatory responses dependent on the autocrine action of IFN-beta

Resident cells of the respiratory and gastrointestinal tracts, including epithelial and fibroblast cells, are the initial sites of entry for many viral pathogens. We investigated the role that these cells play in the inflammatory process in response to infection with reovirus 1/L. In A549 human bronchial or HT-29 human colonic epithelial cells, interferon (IFN)-beta, regulated on activation T cell expressed and secreted (RANTES), IFN-gamma-inducible protein (IP)-10, and interleukin-8 were upregulated regardless of whether cells were infected with replication-competent or replication-deficient reovirus 1/L. However, in CCD-34Lu human lung fibroblast cells, IFN-beta, IP-10, and RANTES were expressed only after infection with replication-competent reovirus 1/L. Expression of interleukin-8 in CCD-34Lu fibroblast cells was viral replication independent. This differential expression of IFN-beta, RANTES, and IP-10 was shown to be due to the lack of induction of IFN regulatory factor-1 and -2 in CCD-34Lu fibroblast cells treated with replication-deficient reovirus 1/L. We have shown that cytokine and/or chemokine expression may not be dependent on viral replication. Therefore, treatment of viral infections with inhibitors of replication may not effectively alleviate inflammatory mediators because most viral infections result in the generation of replication-competent and replication-deficient virions in vivo.     

Differential Ligand Binding to a Human Cytomegalovirus Chemokine Receptor Determines Cell Type–Specific Motility

While most chemokine receptors fail to cross the chemokine class boundary with respect to the ligands that they bind, the human cytomegalovirus (HCMV)-encoded chemokine receptor US28 binds multiple CC-chemokines and the CX₃C-chemokine Fractalkine. US28 binding to CC-chemokines is both necessary and sufficient to induce vascular smooth muscle cell (SMC) migration in response to HCMV infection. However, the function of Fractalkine binding to US28 is unknown. In this report, we demonstrate that Fractalkine binding to US28 not only induces migration of macrophages but also acts to inhibit RANTES-mediated SMC migration. Similarly, RANTES inhibits Fractalkine-mediated US28 migration in macrophages. While US28 binding of both RANTES and Fractalkine activate FAK and ERK-1/2, RANTES signals through Gα12 and Fractalkine through Gαq. These findings represent the first example of differential chemotactic signaling via a multiple chemokine family binding receptor that results in migration of two different cell types. Additionally, the demonstration that US28-mediated chemotaxis is both ligand-specific and cell type–specific has important implications in the role of US28 in HCMV pathogenesis.     

Ligand-independent homomeric and heteromeric complexes between interleukin-2 or -9 receptor subunits and the gamma chain

Signaling via interleukin-2 (IL-2) and interleukin-9 receptors (IL-2R and IL-9R) involves heteromeric interactions between specific interleukin receptor subunits, which bind Janus kinase 1 (JAK1) and the JAK3 binding common gamma chain (gamma c). The potential existence and roles of homomeric and heteromeric complexes before ligand binding and their modulation by ligand and JAK3 are unclear. Using computerized antibody-mediated immunofluorescence co-patching of epitope-tagged receptors at the surface of live cells, we demonstrate that IL-2Rbeta, IL-9Ralpha, and gamma c each display a significant fraction of ligand-independent homomeric complexes (24-28% co-patching), whereas control co-patching levels with unrelated receptors are very low (7%). Heteromeric complex formation of IL2-Rbeta or IL-9Ralpha with gamma c is also observed in the absence of ligand (15-30%). Ligand binding increases this hetero-oligomerization 2-fold but does not affect homo-oligomerization. Co-expression of IL-2Ralpha does not affect the hetero-oligomerization of IL-2Rbeta and gamma c. Recruitment of gamma c into heterocomplexes is partly at the expense of its homo-oligomerization, suggesting that a functional role of the latter may be to keep the receptors inactive in the absence of ligand. At the same time, the preformed complexes between gamma c and IL-2Rbeta or IL-9Ralpha promote signaling by the JAK3 A572V mutant without ligand, supporting a pathophysiological role for the constitutive oligomerization in triggering ligand-independent activation of JAK3 (and perhaps other JAK mutants) mutants identified in several human cancers.     

Glycosaminoglycans interact selectively with chemokines and modulate receptor binding and cellular responses.

Chemokines selectively recruit and activate a variety of cells during inflammation. Interactions between cell surface glycosaminoglycans (GAGs) and chemokines drive the formation of haptotactic or immobilized gradients of chemokines at the site of inflammation, directing this recruitment. Chemokines bind to glycosaminoglycans on human umbilical vein endothelial cells (HUVECs) with affinities in the micromolar range: RANTES > MCP-1 > IL-8 > MIP-1alpha. This binding can be competed with by soluble glycosaminoglycans: heparin, heparin sulfate, chondroitin sulfate, and dermatan sulfate. RANTES binding showed the widest discrimination between glycosaminoglycans (700-fold), whereas MIP-1alpha was the least selective. Almost identical results were obtained in an assay using heparin sulfate beads as the source of immobilized glycosaminoglycan. The binding of chemokines to glycosaminoglycan fragments has a strong length dependence, and optimally requires both N- and O-sulfation. Isothermal titration calorimetry data confirm these results; IL-8 binds heparin fragments with a K(d) of 0.39-2.63 microM, and requires five saccharide units to bind each monomer of chemokine. In membranes from cells expressing the G-protein-coupled chemokine receptors CXCR1, CXCR2, and CCR1, soluble GAGs inhibit the binding of chemokine ligands to their receptors. Consistent with this, heparin and heparin sulfate could inhibit IL-8-induced neutrophil calcium flux. Chemokines can therefore form complexes with both cell surface and soluble GAGs; these interactions have different functions. Soluble GAG chemokines complexes are unable to bind the receptor, resulting in a block of the biological activity. Previously, we have shown that cell surface GAGs present chemokines to the G-protein-coupled receptors, by increasing the local concentration of protein. A model is presented which brings together all of these data. The selectivity in the chemokine-GAG interaction suggests selective disruption of the haptotactic gradient may be an achievable therapeutic approach in inflammatory disease.     

Dissection of the interferon gamma-MHC class II signal transduction pathway reveals that type I and type II interferon systems share common signalling component(s).

We have used a herpes virus thymidine kinase (HSV-TK) based metabolic selection system to isolate mutants defective in the interferon gamma mediated induction of the MHC class II promoter. All the mutations act in trans and result in no detectable induction of MHC and invariant chain (Ii) gene expression. Scatchard analysis indicates that the mutants have a normal number of surface IFN gamma receptors with the same affinity constant. The mutants fall into two broad categories. One class of mutants is still able to induce MHC class I, IRF-1, 9-27, 1-8 and GBP genes by IFN gamma. A second class of mutants is defective for the IFN gamma induction of all the genes tested; surprisingly, the IFN alpha/beta induction of MHC class I, 9-27, ISG54 and ISG15 genes is also defective in these mutants, although different members of this class can be discriminated by the response of the GBP and IRF-1 genes to type I interferons. These data demonstrate that the signalling pathways of both type I and type II interferon systems share common signal transduction component(s). These mutants will be useful for the study of IFN gamma regulation of class II genes and Ii chain, and to elucidate molecular components of type I and type II interferon signal transduction.