Met-RANTES (human)

Eosinophils are predominant effector cells not only in allergic diseases but also in connective tissue diseases. The recruitment of eosinophils to the site of inflammation and release of reactive oxygen species leading to tissue damage and propagation of the inflammatory response are mediated by chemokines. Thus, agents that would be able to inhibit or antagonize chemokine-induced eosinophil activation are interesting as therapeutical agents. We describe the effect of a chemokine receptor antagonist, Met-RANTES, on human eosinophil effector functions in response to RANTES, monocyte chemoattractant protein (MCP)-3 and eotaxin. Met-RANTES was able to inhibit dose-dependently [Ca2+]i transients in eosinophils following stimulation with RANTES, MCP-3 and eotaxin. Whereas maximal and half-maximal inhibitory effect of Met-RANTES following stimulation with RANTES and MCP-3 were observed at 2 micrograms/ml and 1 microgram/ml, respectively, maximal and half-maximal inhibitory effects of Met-RANTES in response to eotaxin were detected at 10 micrograms/ml and 3 micrograms/ml. Moreover, eotaxin-induced [Ca2+]i transients were only half reduced at a Met-RANTES concentration at which RANTES and MCP-3 were completely blocked. Besides its effect on [Ca2+]i transients, Met-RANTES dose-dependently inhibited actin polymerization in eosinophils following chemokine stimulation. Whereas Met-RANTES totally inhibited RANTES- and MCP-3-induced actin polymerization at 5 micrograms/ml, the eotaxin-induced response was only reduced by 50%. However, Met-RANTES inhibited dose-dependently the release of reactive oxygen species in response to RANTES, MCP-3 and eotaxin. Again, eotaxin-induced release of reactive oxygen species, however, was only half reduced at a Met-RANTES concentration (10 micrograms/ml) at which RANTES and MCP-3 were completely blocked. The results of this study show that (1) Met-RANTES is an effective and powerful antagonist of effector functions of human eosinophils following stimulation with RANTES, MCP-3 and eotaxin; (2) Met-RANTES seems to be able to antagonize the response of eosinophils through chemokine receptor 1 (CCR1) preferentially to CCR3; (3) Met-RANTES antagonizes eosinophil but not neutrophil effector functions and might be therefore of interest for a new therapeutical approach to prevent the invasion and destructive power of eosinophils in diseases that are accompanied by eosinophil infiltration such as allergic asthma and connective tissue diseases.

CC chemokine receptor 5 (CCR5) is a pro-inflammatory chemokine receptor that is expressed on cells of the immune system, and specializes in cell migration in response to inflammation and tissue damage. Due to its key role in cell communication and migration, this receptor is involved in various inflammatory and autoimmune diseases, in addition to HIV infection. Met-RANTES is a modified CCR5 ligand that has previously been shown to antagonize CCR5 activation and function in response to its natural ligands in vitro. In vivo, Met-RANTES is able to reduce inflammation in models of induced inflammatory and autoimmune diseases. However, due to the fact that Met-RANTES is also capable of partial agonist activity regarding receptor signaling and internalization, it is clear that Met-RANTES does not function as a conventional receptor antagonist. To further elucidate the effect of Met-RANTES on CCR5, receptor trafficking was investigated in a CHO-CCR5-GFP cell line using the Opera confocal plate reader. The internalization response of CCR5 was quantified, and showed that Met-RANTES internalized CCR5 in a slower, less potent manner than the agonists CCL3 and CCL5. Fluorescent organelle labeling and live cell imaging showed CCL3 and CCL5 caused CCR5 to traffic through sorting endosomes, recycling endosomes and the Golgi apparatus. In contrast, Met-RANTES caused CCR5 to traffic through sorting endosomes and the Golgi apparatus in a manner that was independent of recycling endosomes. As receptor trafficking impacts on cell surface expression and the ability of the receptor to respond to more ligand, this information may indicate an alternative regulation of CCR5 by Met-RANTES that allows the modified ligand to reduce inflammation through stimulation of a pro-inflammatory receptor.

The effects of the recombinant chemokine human RANTES (hRANTES) on the release of glutamate from human neocortex glutamatergic nerve endings were investigated. hRANTES facilitated the spontaneous release of d [(3)H]D-aspartate ([(3)H]DASP-) by binding Pertussis toxin-sensitive G-protein-coupled receptors (GPCRs), whose activation caused Ca(2+) mobilization from inositol trisphosphate-sensitive stores and cytosolic tyrosine kinase-mediated phosphorylations. Facilitation of release switched to inhibition when the effects of hRANTES on the 12 mM K(+)-evoked [(3)H]D-ASP exocytosis were studied. Inhibition of exocytosis relied on activation of Pertussis toxin-sensitive GPCRs negatively coupled to adenylyl cyclase. Both hRANTES effects were prevented by met-RANTES, an antagonist at the chemokine receptors (CCRs) of the CCR1, CCR3, and CCR5 subtypes. Interestingly, human neocortex glutamatergic nerve endings seem to possess all three receptor subtypes. Blockade of CCR1 and CCR5 by antibodies against the extracellular domain of CCRs prevented both the hRANTES effect on [(3)H]D-ASP release, whereas blockade of CCR3 prevented inhibition, but not facilitation, of release. The effects of RANTES on the spontaneous and the evoked release of [(3)H]D-ASP were also observed in experiments with mouse cortical synaptosomes, which may therefore represent an appropriate animal model to study RANTES-induced effects on neurotransmission. It is concluded that glutamate transmission can be modulated in opposite directions by RANTES acting at distinct CCR receptor subtypes coupled to different transduction pathways, consistent with the multiple and sometimes contrasting effects of the chemokine.