The CC-chemokine CCL11 (eotaxin) is an antagonist of CCR4

Giovanna Nalesso, Cathryn Weston, Robert Slack, David Hall

GlaxoSmithKline, Stevenage, UK

CC-chemokine receptor 4 (CCR4) is expressed on Th2 cells which have been suggested to be a key cell type involved in the development of chronic inflammation in allergic diseases. The chemokine agonists for CCR4 are CCL22 (MDC) and CCL17 (TARC) which are chemoattractants of these cells. CCL11 (eotaxin) is an agonist at CCR3 and is primarily thought of as an eosinophil chemoattractant, although it has been reported to have weaker activity at CCR2 and CCR5. In this report, we present data indicating that eotaxin, at high concentrations, acts as an antagonist of the effects of MDC and TARC on human CCR4+ T cells.

Agonist-induced increases in the filamentous (F)-actin content of human CCR4+ T cells were measured by a slight modification of the method of Pilette et al (2004). Binding of [125I]TARC (0.1 nM) was measured with a scintillation proximity assay (SPA) using membranes (25 μg/ml) from CHO cells transfected with human CCR4. Non-specific binding (NSB) was defined in the presence of MDC (10 nM). The SPA bead concentration was 2.5 mg/ml. The assay buffer contained 20 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 10 μg/ml saponin and 0.1 % bovine serum albumin.

TARC and MDC induced concentration-dependent increases in the F-actin content of human T cells with pEC50s of 10.03 ± 0.04 (n = 25) and 9.98 ± 0.02 (n = 49), respectively. Incubation of the cells with eotaxin (0.3, 1 or 3 μM) for 30 min induced surmountable rightward shifts in the concentration-response curve to TARC or MDC. Schild plots of these data were linear with slope 0.94 ± 0.07 (TARC) and 1.12 ± 0.09 (MDC). Neither was significantly different from unity (p > 0.05, Student’s t-test; n = 3). The pA2 values derived from these Schild plots were 6.71 ± 0.13 (TARC) and 6.12 ± 0.07 (MDC). These values were significantly different from each other (p < 0.01, Student’s t-test), an observation which is incompatible with a simple competitive interaction and which has also been seen with small molecule antagonists (Hall et al, this meeting). When eotaxin was added to the cells simultaneously with the agonist or pre-incubated for only 5 min, the pA2 was not significantly different from those obtained after 30 min incubation indicating that the eotaxin was not broken down markedly during the incubation. The binding of [125I]TARC was inhibited by eotaxin to the NSB level. The pKi was 6.31 ± 0.06 (n = 3). The slope of the inhibition curve was 0.85 ± 0.05 which was not significantly different from unity.

It has been suggested (Sebastiani et al, 2005) that a number of chemokines can bind to TARC and MDC and modulate their activity at CCR4. This could explain the differing affinity estimates against the two agonists for this protein since such an interaction would not have the same affinity for the two chemokines. However, eotaxin had a similar apparent affinity when incubated with the agonists for > 30 min or for 15 sec which would require very rapid equilibration of this interaction. Thus, eotaxin is an antagonist of the effects of TARC and MDC at CCR4 and inhibits the binding of [125I]TARC. It is unclear whether this is due to sequestration of the chemokine or an interaction with CCR4.

Pilette et al (2004) Eur. Resp. J. 23, 876 – 884

Sebastiani et al (2005) Eur. J. Immunol. 35, 746-756