vmip-ii and Sarcoma--Kaposi

Fractalkine (FKN/CX3CL1) is a cell surface-expressed chemokine involved in many aspects of leukocyte trafficking and activation. The various structural domains of FKN play distinct roles in its ability to bind and activate its receptor, CX3CR1. A human herpesvirus 8-encoded chemokine, termed viral macrophage inflammatory protein (vMIP)-II, is structurally similar to FKN; vMIP-II is a nonselective chemokine receptor antagonist (binding multiple chemokine receptors, including CX3CR1). The goal of this study was to identify FKN determinants of selectivity for its receptor and to further refine domains important in affinity and efficacy at CX3CR1. Chimeric and insertional mutagenesis was used to generate mutants of both vMIP-II and FKN, and the expressed proteins were evaluated for chemokine receptor binding affinities and efficacy at CX3CR1. Modification of the intervening amino acids between the first two conserved cysteine residues of FKN or vMIP-II indicated a role of the X3 bulge of FKN in affinity and selectivity for CX3CR1. Substitution of the vMIP-II N terminus with that of FKN created an agonist that was just as potent and efficacious as FKN for binding and stimulating CX3CR1, whereas replacement of the FKN N terminus with the cognate domain of vMIP-II disrupted the ability of FKN to bind CX3CR1. Furthermore, the entire N terminus of FKN was necessary for the high-affinity and full agonist properties of FKN at CX3CR1. These results refine the pharmacophore for chemokine binding to and activation of CX3CR1 and demonstrate the usefulness of modified virally encoded chemokines as templates for the development of selective chemokine receptor antagonists.

Topics: Amino Acid Sequence; Antiviral Agents; Binding Sites; Binding, Competitive; Cell Line; Chemokine CX3CL1; Chemokines; Chemokines, CX3C; CX3C Chemokine Receptor 1; Humans; Kinetics; Membrane Proteins; Molecular Sequence Data; Phosphorylation; Receptors, Chemokine; Recombinant Fusion Proteins; Sarcoma, Kaposi; Transfection

The viral CC chemokine macrophage inhibitory protein-II (vMIP-II) encoded by human herpes virus 8 (HHV-8) binds to multiple chemokine receptors, however, its ability to control the initial recruitment of specific leukocyte subtypes from the peripheral circulation has not been fully clarified. Here we show that vMIP-II blocks the firm arrest and transmigration of monocytes or Th1-like T lymphocytes triggered by RANTES immobilized on activated human microvascular endothelium (HMVEC) under flow conditions. The internalization of the receptors CCR1 and CCR5 that mediate arrest and transmigration of these cells in response to RANTES was prevented by vMIP-II, supporting its role as an antagonist of CCR1 and CCR5. In contrast, vMIP-II triggered the firm arrest of eosinophils and Th2-like T cells by engaging CCR3, as confirmed by its down-regulation. Immunohistochemical analysis of HHV-8-associated Kaposi's sarcoma lesions marked by vMIP-II expression and mononuclear cell infiltration revealed a predominance of Th2-type CCR3(+) lymphocytes over Th1-type CXCR3(+)/CCR5(+) leukocytes, indicating that as a CCR3 agonist vMIP-II can drive a Th2-type immune response in vivo. Thus, our data provide evidence for a immunomodulatory role of vMIP-II in directing inflammatory cell recruitment away from a Th1-type towards a Th2-type response and thereby facilitating evasion from cytotoxic reactions.

Topics: CCR5 Receptor Antagonists; Cell Adhesion; Cell Division; Cell Line; Chemokine CCL5; Chemokines; Chemotaxis, Leukocyte; Cytotoxicity, Immunologic; Endocytosis; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Eosinophils; Herpesvirus 8, Human; Humans; Immunohistochemistry; Interleukin-1; Lymphocyte Activation; Monocytes; Receptors, CCR1; Receptors, CCR3; Receptors, CCR5; Receptors, Chemokine; Sarcoma, Kaposi; Th1 Cells; Th2 Cells