Differential Activation of CC Chemokine Receptors by AOP-RANTES

Abstract

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.Chemokines are chemotactic proteins that play a central role in immune and inflammatory responses by the attraction and activation of leukocytes. They can be divided into two major classes on the basis of the arrangement of the amino acid cysteine in the amino-terminal region: the CXC and CC chemokines, and two minor subclasses, each comprising a single member, the C and CX3C subclasses (1-3). Initially, it was generally accepted that the chemokine subclasses differ in their biological activity to stimulate different kinds of leukocytes, so that CXC chemokines are mediators of acute inflammation through neutrophil activation while the CC chemokines mediate chronic inflammation by attracting leukocytes such as eosinophils, monocytes, lymphocytes, basophils, and dendritic cells. However, this paradigm has recently been shown to have exceptions; for example, CXCR3 is expressed on activated T cells (4) and neutrophils can be activated by CC chemokines following stimulation with interferon-␥ (5).Chemokines mediate their effects by binding to cell-surface receptors that belong to the seven-transmembrane domain G protein-coupled receptor superfamily (1). More recently, chemokine receptors have been subject to intense scrutiny following the discovery that several of them are co-receptors for HIV 1 cell entr...

Intracellular Domains of CXCR3 That Mediate CXCL9, CXCL10, and CXCL11 Function

“…However, this apparent redundancy has not been supported by in vivo studies that have instead demonstrated that individual chemokines that activate the same receptor can have unique functions in vivo (2). This may be related to differential chemokine expression in vivo and/or differential receptor activation by different chemokine ligands (3).…”

Inhibition of CD26/Dipeptidyl Peptidase IV Enhances CCL11/Eotaxin-Mediated Recruitment of Eosinophils In Vivo

“…CCR3 internalization in eosinophils and flow cytometric measurements were performed as described elsewhere (13,17). Quantification of CD26 gene expression on keratinocytes using real-time PCR was performed as described previously (18).…”

IL-3 Induces Down-Regulation of CCR3 Protein and mRNA in Human Eosinophils

“…Human granulocytes were isolated from heparin-anticoagulated venous blood from normal nonatopic healthy or atopic donors using Ficoll (Pharmacia, Erlangen, Germany) density gradient centrifugation as described previously (23). For further purification, granulocytes were resuspended in HEPES-buffered HBSS (Life Technologies, Grand Island, N.Y.), pH 7.4, containing 1 mg/ml BSA (HBSS plus BSA).…”

“…RANTES induction of CCR5 down-regulation allows its recycling to the cell surface, whereas the modified RANTES protein AOP-RANTES prevents the re-expression of cell surface CCR5 (22). On the other hand, CCR1 does not recycle after RANTES stimulation (23). CXCR2 has been shown to be degraded in the lysosomal compartment (24) whereas CCR3 appears to follow both fates; it is partially degraded in the lysosomes, but ϳ70% of the receptors recycle (23,25).…”

“…On the other hand, CCR1 does not recycle after RANTES stimulation (23). CXCR2 has been shown to be degraded in the lysosomal compartment (24) whereas CCR3 appears to follow both fates; it is partially degraded in the lysosomes, but ϳ70% of the receptors recycle (23,25).…”