The herpesvirus 8 encoded chemokines vCCL2 (vMIP-II) and vCCL3 (vMIP-III) target the human but not the murine lymphotactin receptor
© Lüttichau; licensee BioMed Central Ltd. 2008
Received: 16 November 2007
Accepted: 21 April 2008
Published: 21 April 2008
Large DNA-viruses such as herpesvirus and poxvirus encode proteins that target and exploit the chemokine system of their host. The Kaposi sarcoma- associated herpes virus (KSHV) encodes three chemokines. Two of these, vCCL2 and vCCL3, target the human lymphotactin receptor as an antagonist and a selective agonist, respectively. Therefore these virally endcoded chemokines have the potential to be used as tools in the study of lymphotactin receptor pathways in murine models.
The activities of vCCL2, vCCL3, human lymphotactin (XCL1) and murine lymphotactin (mXCL1) were probed in parallel on the human and murine lymphotactin receptor (XCR1 and mXCR1) using a phosphatidyl-inositol assay. On the human XCR1, vCCL3, mXCL1 and XCL1 acted as agonists. In contrast, only mXCL1 was able to activate the murine lymphotactin receptor. Using the same assay, vCCL2 was able to block the response using any of the three agonists on the humane lymphotactin receptor with IC50s of 2–3 nM. However, vCCL2 was unable to block the response of mXCL1 through the murine lymphotactin receptor.
This study shows that vCCL2 and vCCL3 cannot be used to investigate lymphotactin receptor pathways in murine models. These results also add vCCL2 and vCCL3 to a growing list of viral chemokines with known human chemokine receptor targets, which do not target the corresponding murine receptors. This fits with the observation that viral and endogenous ligands for the same human chemokine receptor tend to have relatively divergent amino-acid sequences, suggesting that these viruses have fine-tuned the design of their chemokines such that the action of the viral encoded chemokines cannot be expected to cross species barriers.
During the last 15 years, more than 40 chemokines have been identified in the human genome and nearly all have been characterized pharmacologically as agonists and led to the identification of 18 signaling 7TM chemokine receptors [1, 2].
Chemokines are 70–80 amino acid proteins with a characteric three-dimensional fold, which are involved in guiding and activating distinct leukocyte subsets. Chemokines can be divided into four sub-families on the basis of the pattern and number of the conserved cysteine residues located near their N-terminus, which are involved in disulfide binding formation; the CC-, CXC-, CX3C and XC family, respectively. The XC-chemokines have only one cysteine in the N-terminus. Chemokines act through 7TM GPCRs of which we today know ten CC-chemokine receptors (CCR1-10), six CXC-receptors (CXCR1-6), one CX3C-receptor (CX3CR1) and one XC-receptor (XCR1). The role played by the lymphotactin receptor (XCR1) in the immune system is poorly understood.
Chemokines encoded by human viruses and their human and murine chemokine receptor targets.
human chemokine receptor targets
also targeting the corresponding murine chemokine receptor
Selective CXCR2 agonist
Selective CCR2 agonist
Selective CCR8 agonist
Broadspectrum chemokine receptor
Selective XCR1 agonist
Selective CCR8 antagonist
Obviously viral encoded chemokines are important in the study of viral pathogenesis, but they can also be used as tools in the investigation of specific chemokine receptors. Blocking of chemokine receptor action is important in several assays and immunological models studying the chemokine system. One example is the selective CCR8 antagonist MC148 encoded by the Molluscuum Contagiosum Virus . Another example is the broad-spectrum chemokine antagonist vCCL2 encoded by HHV8, which blocks a number of chemokine receptors such as CCR1, CCR2, CCR5, CX3CR1, CXCR4 and the lymphotactin receptor XCR1 [6, 7]. Thus vCCL2 has been shown to reduce the inflammatory response in small animal models models [12–15]. However, viral-encoded agonists are also useful in the investigation of the role of a chemokine receptor even when the endogenous human ligand has been identified, because they can have greater potency and be more stable than their human counterparts. This is the case for another HHV8 encoded chemokine vCCL3, which was recently found to have a 10-fold higher potency than lymphotactin on the human lymphotactin receptor .
Results and discussion
Recently, we reported that the HHV8 encoded chemokine vCCL3 is a selective agonist of the human lymphotactin receptor XCR1, while vCCL2, encoded by the same virus, acts as an antagonist on this same receptor .
Agonistic activity on XCR1 and mXCR1
In contrast, only mXCL1 was able to activate the murine lymphotactin receptor, while vCCL3 and XCL1 were unable to do so (Figure 2). It should be noted that only a relatively high concentration of mXCL1 (100 nM) consistently generated an IP3 response through the murine lymphotactin receptor. This observation could be explained if mXCL1 was not properly folded or was partly proteolyzed. However, this is unlikely as 1 nM of mXCL1 in all assays was able to activate the human lymphotactin receptor. To rule out a vector-specific effect, we performed similar assays using the mXCR1 gene inserted into pcDNA3.1. However, no difference in results was found using the two vector constructs (data not shown). The low potency of mXCL1 on the mXCR1 in the IP3 assay could also be related to the cell-line used, but again this explanation seems unlikely as we also had difficulties in generating calcium mobilization responses to mXCL1 in single clones of L1.2- and 300.19- cells transfected with murine lymphotactin receptor cDNA, which suggested that mXCL1 indeed had low potency for the lymphotactin receptor. A more likely explanation of the poor potency of mXCL1 for mXCR1 could be that either the extra N-terminal methionine or the lack of glycosylation of the E. Coli-produced recombinant murine lymphotactin prevented proper activation of the murine but not the human lymphotactin receptor.
Antagonistic activity on XCR1 and mXCR1
Virally encoded chemokines and species barriers
This sequence divergence between viral and human chemokines may explain why human and not viral encoded chemokines in general are able to cross a species barrier. Human encoded chemokines are very similar to their murine counterparts but very dissimilar to the viral encoded chemokines they share receptor targets with. Thus one can imagine that a virus during evolution has picked up an ancestral chemokine gene and optimized it in a unique way to suit the virus, while retaining the ability to target the corresponding chemokine receptor of its host. However, during this process the hijacked chemokine has changed so much that it has lost the ability to cross species barriers. As seen in Table 1, the CMV encoded chemokine vCXCL1, the pox virus encoded chemokine MC148 and, as shown in this paper, the KSHV encoded chemokines vCCL2 and vCCL3 are not able to target their corresponding murine receptors, while only vCCL1 encoded by KSHV has retained this ability [3, 5–7, 10, 11, 17–20].
vCCL2 has been used in several in vivo models, especially in rodent models, as a broad-spectrum chemokine receptor blocker. For example vCCL2 has been shown to reduce T-cell mediated inflammation in a murine lymphocytic choriomeningitis model , to protect the brain of mice against cerebral ischemia , to reduce inflammation in a rat model of spinal cord contusion  and in a rat model of glomerulonephritis . The results from this study suggest that the reduction of inflammation by vCCL2 in these experiments is not due to inhibition of the lymphotactin receptor, but must be due to inhibition of one or more of the CCR1, CCR2, CCR5, CX3CR1 and CXCR4 receptors.
The chemokines vCCL2 and vCCL3 encoded by KSHV do not target the murine lymphotactin receptor, although they act as a high potency antagonist and agonist respectively, on the human lymphotactin receptor. So the only tool left, for investigating the role of the lymphotactin receptor pathways in murine models, is the commercially available recombinant form of murine lymphotactin with a rather low potency on the mXCR1 receptor. Furthermore, when the findings of this paper are combined with the results from other studies on the ability of chemokines encoded by human viruses to target the murine counterparts to their human chemokine receptor targets, it can be concluded that they rarely do so. In contrast, human encoded chemokines in general are able to cross the species barrier and target their corresponding murine chemokine receptors.
mXCL1, XCL1, vCCL2 were purchased from R&D (Minneapolis, MN). Recombinant vCCL3 (GenBank accession number U93872) was produced as described previously , briefly cell media collected from COS7 cells transfected with the K4.1 gene from HHV8 was collected and purified on a cation-exchange column followed by reverse phase HPLC. The elution position of the recombinant vCCL3 protein as well as the purity was identified with mass-spectroscopy and NH2-terminal sequence analysis on an ABI 494 protein sequencer (Perkin-Elmer, CA).
Cloning of mXCR1
The mXCR1 gene was amplified by PCR from a murine cDNA library and inserted into the pcDNA3.1 vector and the pTEJ8 vector. Start- and end-primers were designed from the GenBank accession number NM 011798. Nucleotide sequence analysis was performed on an ABI 310 sequence system (Perkin-Elmer, CA) in-house or by MWG Biotech (Ebersberg, Germany). The human XCR1 gene inserted in the pcDNA3.1 vector was purchased from the UMR cDNA Resource Center (Rolla, MO).
Stable cell lines
mXCR1 in pTej was transfected into the murine pre-B cell line L1.2 and hXCR1 in pcDNA 3.1 was transfected into the murine pre-B cell line 300.19. Stable transfectants were obtained after limiting dilution and chemical selection with G418 and functional clones were selected based upon their calcium responses to mXCL1 and XCL1, respectively.
COS-7 cells were transiently transfected by a calcium phosphate precipitate method with addition of chloroquine. Briefly, 2 × 106 COS-7 cells were transfected with 30 ug of cDNA encoding the promiscuous chimeric G-protein, GαΔ6qi4myr (abbreviated as Gqi4myr), which allows the Gαi-coupled receptor to couple to the Gαq pathway (phospholipase C activation measured as PI-turnover) , with or without 20 ug receptor (mXCR1 or XCR1) cDNA. After transfection, COS-7 cells (2.5 × 104 cells/well) were incubated for 24 hours with 2 μCi of 3H-myo-inositol in 0.4 ml growth medium per well in 24-multiwells tissue culture plates. Cells were washed twice in 20 mM Hepes, pH 7.4, supplemented with 140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 10 mM glucose and 0.05% (w/v) bovine serum albumin and were incubated in 0.4 ml of the same buffer supplemented with 10 mM LiCl at 37°C for 15 minutes. The ligands were subsequently added and incubated for 90 min. In the antagonist assay vCCL2 was added 10 min before the agonist to ensure proper interaction of the receptors with vCCL2. Cells were extracted by addition of 1 ml 10 mM Formic acid to each well followed by incubation on ice for 30–60 min. The generated [3H]-inositol phosphates were purified on AG1-X8 anion-exchange resin (Bio-Rad Laboratories, Hercules, CA). Determinations were made in duplicate.
Calcium mobilization experiments
L1.2 cells stably transfected with mXCR1 were loaded with Fura-2AM (Molecular Probes, Eugene, OR) in RPMI with 1% FCS for 20–30 min. and washed in the same buffer. Aliqouts were made of 1 × 106 cells, each aliqout was pelleted and resuspended in 500 ul PBS 1% FCS with 10 mM EGTA. Flourescence was measured on a Jobin Yvon FlouroMax-2 (Jobin Yvon Spex, Cedex, France) as the ratio of emission at 490 nm when excited at 340 nm and 380 nm respectively.
Kaposi sarcoma-associated herpes virus
human herpesvirus 8
viral macrophage inflammatory protein
I thank Kirsten Culmsee for excellent technical assistance.
This study was supported by the by grants from the Foundation of A.P.Møller and Chastine Mc-Kinney Møller, the Foundation of Arvid Nilsson, the Augustinus Foundation, the Beckett-Foundation, the Foundation of Bent Bøgh and wife, the Foundation of Carl and Ellen Hertz, the Foundation of Christian Larsen and Ellen Larsen, the Foundation of Frode V. Nyegaard and wife, the Foundation of E. Danielsen and wife, the Foundation of Einar Hansen and wife, the Foundation of Michael Hermann Nielsen, the Foundation of Else and Mogens Wedell-Wedellsborg, the Foundation of Karl G Andersen, the Harboe-Foundation, the Illum-Foundation, the Foundation of Johan Boserup and Lise Boserup, the Foundation of Niels Hansen and wife, the Foundation of Werner Richter and wife, the Foundation of Ove William Buhl Olesen and wife, the Foundation of Meta and Håkon Bagger and the Foundation of Jakob Madsen and wife.
- Murphy PM, Baggiolini M, Charo IF, Hebert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, Power CA: International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev 2000, 52: 145-176.PubMedGoogle Scholar
- Rollins BJ: Chemokines. Blood 1997, 90: 909-928.PubMedGoogle Scholar
- Dairaghi DJ, Fan RA, McMaster BE, Hanley MR, Schall TJ: HHV8-encoded vMIP-I selectively engages chemokine receptor CCR8. Agonist and antagonist profiles of viral chemokines. J Biol Chem 1999, 274: 21569-21574. 10.1074/jbc.274.31.21569View ArticlePubMedGoogle Scholar
- Dewin DR, Catusse J, Gompels UA: Identification and characterization of U83A viral chemokine, a broad and potent beta-chemokine agonist for human CCRs with unique selectivity and inhibition by spliced isoform. J Immunol 2006, 176: 544-556.View ArticlePubMedGoogle Scholar
- Endres MJ, Garlisi CG, Xiao H, Shan L, Hedrick JA: The Kaposi's sarcoma-related herpesvirus (KSHV)-encoded chemokine vMIP-I is a specific agonist for the CC chemokine receptor (CCR)8. J Exp Med 1999, 189: 1993-1998. 10.1084/jem.189.12.1993PubMed CentralView ArticlePubMedGoogle Scholar
- Kledal TN, Rosenkilde MM, Coulin F, Simmons G, Johnsen AH, Alouani S, Power CA, Luttichau HR, Gerstoft J, Clapham PR, Clark-Lewis I, Wells TN, Schwartz TW: A broad-spectrum chemokine antagonist encoded by Kaposi's sarcoma-associated herpesvirus. Science 1997, 277: 1656-1659. 10.1126/science.277.5332.1656View ArticlePubMedGoogle Scholar
- Luttichau HR, Stine J, Boesen TP, Johnsen AH, Chantry D, Gerstoft J, Schwartz TW: A highly selective CC chemokine receptor (CCR)8 antagonist encoded by the poxvirus molluscum contagiosum. J Exp Med 2000, 191: 171-180. 10.1084/jem.191.1.171PubMed CentralView ArticlePubMedGoogle Scholar
- Luttichau HR, Lewis IC, Gerstoft J, Schwartz TW: The herpesvirus 8-encoded chemokine vMIP-II, but not the poxvirus-encoded chemokine MC148, inhibits the CCR10 receptor. Eur J Immunol 2001, 31: 1217-1220. 10.1002/1521-4141(200104)31:4<1217::AID-IMMU1217>3.0.CO;2-SView ArticlePubMedGoogle Scholar
- Luttichau HR, Clark-Lewis I, Jensen PO, Moser C, Gerstoft J, Schwartz TW: A highly selective CCR2 chemokine agonist encoded by human herpesvirus 6. J Biol Chem 2003, 278: 10928-10933. 10.1074/jbc.M211329200View ArticlePubMedGoogle Scholar
- Luttichau HR, Johnsen AH, Jurlander J, Rosenkilde MM, Schwartz TW: Kaposi sarcoma-associated herpes virus targets the lymphotactin receptor with both a broad spectrum antagonist vCCL2 and a highly selective and potent agonist vCCL3. J Biol Chem 2007, 282: 17794-17805. 10.1074/jbc.M702001200View ArticlePubMedGoogle Scholar
- Penfold ME, Dairaghi DJ, Duke GM, Saederup N, Mocarski ES, Kemble GW, Schall TJ: Cytomegalovirus encodes a potent alpha chemokine. Proc Natl Acad Sci USA 1999, 96: 9839-9844. 10.1073/pnas.96.17.9839PubMed CentralView ArticlePubMedGoogle Scholar
- Chen S, Bacon KB, Li L, Garcia GE, Xia Y, Lo D, Thompson DA, Siani MA, Yamamoto T, Harrison JK, Feng L: In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar-Kyoto (WKY) rats by vMIP-II. J Exp Med 1998, 188: 193-198. 10.1084/jem.188.1.193PubMed CentralView ArticlePubMedGoogle Scholar
- Ghirnikar RS, Lee YL, Eng LF: Chemokine antagonist infusion attenuates cellular infiltration following spinal cord contusion injury in rat. J Neurosci Res 2000, 59: 63-73. 10.1002/(SICI)1097-4547(20000101)59:1<63::AID-JNR8>3.0.CO;2-WView ArticlePubMedGoogle Scholar
- Lindow M, Nansen A, Bartholdy C, Stryhn A, Hansen NJ, Boesen TP, Wells TN, Schwartz TW, Thomsen AR: The virus-encoded chemokine vMIP-II inhibits virus-induced Tc1-driven inflammation. J Virol 2003, 77: 7393-7400. 10.1128/JVI.77.13.7393-7400.2003PubMed CentralView ArticlePubMedGoogle Scholar
- Takami S, Minami M, Nagata I, Namura S, Satoh M: Chemokine receptor antagonist peptide, viral MIP-II, protects the brain against focal cerebral ischemia in mice. J Cereb Blood Flow Metab 2001, 21: 1430-1435. 10.1097/00004647-200112000-00007View ArticlePubMedGoogle Scholar
- Kostenis E: Is Galpha16 the optimal tool for fishing ligands of orphan G-protein-coupled receptors? Trends Pharmacol Sci 2001, 22: 560-564. 10.1016/S0165-6147(00)01810-1View ArticlePubMedGoogle Scholar
- Boshoff C, Endo Y, Collins PD, Takeuchi Y, Reeves JD, Schweickart VL, Siani MA, Sasaki T, Williams TJ, Gray PW, Moore PS, Chang Y, Weiss RA: Angiogenic and HIV-inhibitory functions of KSHV-encoded chemokines. Science 1997, 278: 290-294. 10.1126/science.278.5336.290View ArticlePubMedGoogle Scholar
- Luttichau HR, Gerstoft J, Schwartz TW: MC148 encoded by human molluscum contagiosum poxvirus is an antagonist for human but not murine CCR8. J Leukoc Biol 2001, 70: 277-282.PubMedGoogle Scholar
- Rubant S, Ludwig RJ, Pfeffer J, Schulze-Johann P, Kaufmann R, Pfeilschifter JM, Boehncke WH, Radeke HH: Eukaryotic expression of the broad-spectrum chemokine receptor antagonist vMIP-II and its effects on T-cell function in vitro and in vivo. Exp Dermatol 2006, 15: 634-642. 10.1111/j.1600-0625.2006.00455.xView ArticlePubMedGoogle Scholar
- Sparer TE, Gosling J, Schall TJ, Mocarski ES: Expression of human CXCR2 in murine neutrophils as a model for assessing cytomegalovirus chemokine vCXCL-1 function in vivo. J Interferon Cytokine Res 2004, 24: 611-620.View ArticlePubMedGoogle Scholar
- Kuloglu ES, McCaslin DR, Kitabwalla M, Pauza CD, Markley JL, Volkman BF: Monomeric solution structure of the prototypical 'C' chemokine lymphotactin. Biochemistry 2001, 40: 12486-12496. 10.1021/bi011106pPubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.