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. 2000 Jan 3;191(1):171-80.
doi: 10.1084/jem.191.1.171.

A highly selective CC chemokine receptor (CCR)8 antagonist encoded by the poxvirus molluscum contagiosum

H R Lüttichau  1 , J Stine, T P Boesen, A H Johnsen, D Chantry, J Gerstoft, T W Schwartz
Affiliations

A highly selective CC chemokine receptor (CCR)8 antagonist encoded by the poxvirus molluscum contagiosum

H R Lüttichau et al. J Exp Med. .

Abstract

The MC148 CC chemokine from the human poxvirus molluscum contagiosum (MCV) was probed in parallel with viral macrophage inflammatory protein (vMIP)-II encoded by human herpesvirus 8 (HHV8) in 16 classified human chemokine receptors. In competition binding using radiolabeled endogenous chemokines as well as radiolabeled MC148, MC148 bound with high affinity only to CCR8. In calcium mobilization assays, MC148 had no effect on its own on any of the chemokine receptors, but in a dose-dependent manner blocked the stimulatory effect of the endogenous I-309 chemokine on CCR8 without affecting chemokine-induced signaling of any other receptor. In contrast, vMIP-II acted as an antagonist on 10 of the 16 chemokine receptors, covering all four classes: XCR, CCR, CXCR, and CX(3)CR. In chemotaxis assays, MC148 specifically blocked the I-309-induced response but, for example, not stromal cell-derived factor 1alpha, monocyte chemoattractant protein 1, or interleukin 8-induced chemotaxis. We thus concluded that the two viruses choose two different ways to block the chemokine system: HHV8 encodes the broad-spectrum chemokine antagonist vMIP-II, whereas MCV encodes a highly selective CCR8 antagonist, MC148, conceivably to interfere with monocyte invasion and dendritic cell function. Because of its pharmacological selectivity, the MC148 protein could be a useful tool in the delineation of the role played by CCR8 and its endogenous ligand, I-309.

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Figures

Figure 1
Figure 1
Purification of recombinant MC148 expressed in COS cells. The elution profile of MC148 (black box) on reverse-phase HPLC is shown. mAU, milliabsorbance units.
Figure 2
Figure 2
Alignment of known CCR8 ligands to MC148. vMIP-II, vMIP-I, MC148, and I-309 were aligned using CLUSTALW 1.7. Identical amino acids are shown white on black, whereas similar amino acids are shown white on gray. The line above the alignment shows the secondary structure of vMIP-II as determined by NMR 51. *Cysteine residues.
Figure 4
Figure 4
Effect of recombinant MC148 and vMIP-II on calcium mobilization on a panel of cell lines stably transfected with humane chemokine receptors. 10−7 M MC148, vMIP-II, or vehicle was added to the cells at 50 s, followed by a submaximal dose of an appropriate endogenous human chemokine at 150 s. The height of the response with the endogenous ligand was measured, and the heights in the experiments with MC148 and vMIP-II were expressed as percent of the height in the experiment with the vehicle. A representative example of each experiment is shown to the left, whereas the box diagram to the right shows the average inhibition with SEM as indicated. Asterisks (*) indicate statistically significant inhibition (P < 0.05). The chemokine concentrations used were: CCR1, 10−10 M RANTES; CCR2, 10−8 M MCP-1; CCR3, 10−9 M MCP-3; CCR4, 10−9 M macrophage-derived chemokine; CCR5, 10−9 M RANTES; CCR6, 10−8 M liver-activated and -regulated chemokine; CCR7, 10−9 M secondary lymphoid tissue chemokine; CCR8, 10−8.5 M I-309; CCR9, 10−7 M thymus-expressed chemokine; CXCR1, 10−9 M IL-8; CXCR2, 10−9 M IL-8; CXCR3, 10−10 M I-TAC; CXCR4, 10−10 M SDF-1α; CXCR5, 10−10 M B cell–attracting chemokine 1; XCR1, 10−7 M murine lymphotactin; and CX3CR1, 10−9 M fractalkine.
Figure 3
Figure 3
Heterologous and homologous competition binding experiments with recombinant MC148 with various chemokine receptors. Heterologous competition binding curves for MC148 are shown to the left; human chemokine corresponding to the radioactively labeled human chemokine used (▪), MC148 (▵), and vMIP-II (○). Homologous binding curves for MC148 are shown to the right; MC148 (▴), I-309 (□), and vMIP-II (○). For every homologous binding experiment with 125I–MC148, a homologous control curve was made with the human chemokine known to bind the receptor to make sure that the receptor was expressed on the surface of the cell.
Figure 6
Figure 6
Effect of MC148 on chemotactic activity on THP-1 cells (monocytic cell line), PBLs, neutrophils, and CCR8-transfected L12 cells. To the left, dose–response curves for MC148 (▵). Dose–response curves (□) for MCP-1, SDF-1α, IL-8, and I-309 are shown for comparison. The bottom panel includes a dose–response curve for vMIP-II (○). To the right, dose–response curves for inhibition by MC148 (▴) of chemotaxis induced by 5 nM MCP-1 on THP-1 cells, 10 nM SDF-1α on PBLs, 10 nM IL-8 on neutrophils, and 10 nM I-309 on CCR8-transfected L12 cells. The bottom panel includes a dose–response curve for inhibition by vMIP-II (•). One representative assay out of three is shown for THP-1 cells, out of four for PBLs, out of three for neutrophils, and out of five for CCR8-transfected L12 cells.
Figure 5
Figure 5
Dose–response curve of recombinant MC148 on I-309–induced calcium mobilization in stably CCR8-transfected HEK293 cells. 10−7, 10−8, 10−9, and 10−10 M MC148 or vehicle was added to the cells at 50 s, followed by 10−8.5 M I-309 at 150 s. The height of the response with I-309 was measured, and the heights in the experiments with the different concentrations of MC148 were expressed as percent of the height in the experiment with the vehicle. A representative example is shown to the left; the box diagram to the right shows the average inhibition with SEM as indicated.
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