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Short Communication |
Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, New Zealand
Correspondence
Stephen B. Fleming
stephen.fleming{at}stonebow.otago.ac.nz
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ABSTRACT |
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A supplementary table and three figures are available with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). The benign lesions resolve in approximately 4–6 weeks. Virus replication is specific to keratinocytes (Haig & Mercer, 1998) and there is no evidence of systemic infection. Despite an apparent potent inflammatory response and normal cell-mediated response, ORFV is able to reinfect its host (Haig et al., 2002). The discovery of several secreted immunomodulators encoded by the virus may explain this phenomenon. These include a homologue of interleukin (IL)-10 (Fleming et al., 1997), a vascular endothelial growth factor (Lyttle et al., 1994), a dual-specificity granulocyte–macrophage colony-stimulating factor IL-2-binding protein (Deane et al., 2000) and a chemokine-binding protein (CBP) (Seet et al., 2003). These factors are thought to work in concert to suppress inflammation and innate immunity and delay the development of adaptive immunity (reviewed by Fleming & Mercer, 2007).
Chemokines are a large family of secreted chemotactic proteins that regulate inflammation-induced leukocyte recruitment to sites of infection, as well as homeostatic migration of leukocytes through lymphoid organs (Baggiolini, 1998; Cyster, 1999). Chemokines bind and transduce signals through distinct members of the G protein-coupled receptor superfamily (Rossi & Zlotnik, 2000). Members of the chemokine family are classified as CC, CXC, CX3C and C based on the arrangement of cysteine residues at the N terminus (Rollins, 1997). In general, CC chemokines are chemoattractants for monocytes (Uguccioni et al., 1995) whereas CXC chemokines are potent chemoattractants of neutrophils or lymphocytes (Larsen et al., 1989). Lymphotactin (C chemokine) is a chemoattractant for lymphocytes, NK cells, neutrophils and B cells (Huang et al., 2001). In innate immunity, chemokines establish concentration gradients by attaching to the extracellular matrix through glycosaminoglycan binding domains, by which leukocytes are recruited to sites of inflammation (Yu et al., 2005).
ORFV strain NZ2 CBP has no mammalian homologue, shares low sequence identity (<17 %) to other poxvirus and herpesvirus CBPs and has a unique chemokine binding profile (Seet et al., 2003). Surface plasmon resonance revealed that ORFVNZ2–CBP binds a wide range of human CC chemokines and the C chemokine lymphotactin with high affinity, but does not bind CXC chemokines. We predict that the ORFV CBP secreted into the skin epithelium by the virus competes with chemokines for their cognate receptors and thereby sets up a blockade around the site of the lesion to inhibit the recruitment of leukocytes. Here, we report the ability of the ORFVNZ2–CBP to inhibit the migration of murine monocytes in response to specific inflammatory chemokines, using chemotaxis assays, and its ability to impair the recruitment of these cells in a murine skin inflammation model. Monocytes are derived from bone marrow progenitors and are central players in both innate and adaptive immunity because of their endocytic activity and their ability to differentiate into antigen-presenting cells (Imhof & Aurrand-Lions, 2004).
The predominant chemokines produced during damage and inflammation of epithelial tissues are CCL2 (MCP-1), CCL3 (MIP-1
) and CCL5 (RANTES) (Kopydlowski et al., 1999; Wetzler et al., 2000). We investigated the binding affinity kinetics of ORFVNZ2–CBP for these murine chemokines using surface plasmon resonance (Biacore). ORFVNZ2–CBP–FLAG was expressed and purified as previously described (Seet et al., 2003). The surface of a CM-5 chip was coated with approximately 300–400 response units (pg mm–2) of CBP–FLAG by standard amine coupling. All experiments were performed at 25 °C with HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005 % polysorbate 20, pH 7.4). The coupled CBP was exposed to murine chemokines at a flow rate of 100 µl min–1, at concentrations ranging from 3 to 300 nM for the duration of 60 s. Chemokines were injected over the blank control and the coupled CBP–FLAG flow cells. Following an association period of 30 s, HBS-EP was injected over both surfaces to monitor the dissociation phase of binding. Chemokines were allowed to dissociate for 200 s before the surface was regenerated using a mixture of ionic and acidic solutions. The data were analysed using BIAevaluation software using the 1 : 1 protocol for binding with mass transfer. The binding affinities of CBP–FLAG to murine chemokines were as follows: CCL2, 0.406 nM; CCL3, 0.14 nM; CCL5, 0.03 nM. The high affinity interactions are the result of very fast association kinetics (Kon>107 M–1 s–1) and slow dissociation kinetics (Koff<10–3 s–1) (see Supplementary Table S1, available in JGV Online). The binding affinity of CBP–FLAG for the murine inflammatory chemokines tested was similar to that found for human chemokines (Seet et al., 2003).
Next, we investigated the ability of CBP–FLAG to block the migration of monocytes in chemotaxis assays in response to murine CCL2, CCL3 and CCL5. Monocytes were generated from C57BL/6 mouse bone marrow and cultured in the presence of 10 % M-CSF for 5 days (Schuetze et al., 2005). By this time, monocytes were 90 % positive for CD11b (allophycocyanin-conjugated rat anti-mouse integrin/CD11b, clone M1/70, isotype rat IgG2b; R&D Systems) and expressed low levels of MHC-II (phycoerythrin-conjugated rat anti-mouse I-A/I-E, clone M5/114.15.2, isotype rat IgG2b, 
; BD Biosciences) (Supplementary Fig. S1, available in JGV Online).
Chemotaxis assays were performed with transwells containing 5 µm membrane inserts (Costar) with recombinant murine chemokines CCL2, CCL3 and CCL5 (R&D Systems). Monocytes were added to the top chamber of the transwell assay system at 1x105 cells ml–1 and chemokines were added to the bottom chamber. To ensure maximal cell migration, the assay was optimized for chemokine concentration and incubation time. We noted that some cells adhered to the bottom of the membrane while other cells migrated entirely through the membrane into the medium in the lower chamber. Others have reported similar findings using this assay system (Dieu-Nosjean et al., 1999). Cells on the underside of membranes were counted over four fields and averaged as described previously (Sozzani et al., 1997). Cells that migrated through the membrane into the medium were counted using flow cytometry (FACSCalibur, BD BioSciences; Cellquest software). We observed a dose–response relationship for all chemokines tested. The concentrations of CCL2, CCL3 and CCL5 that induced a four- to sixfold increase in monocyte migration were 50, 12.5 and 25 ng ml–1, respectively (Supplementary Fig. S2, available in JGV Online). The optimal migration time of 3 h was consistent with published data (Wang et al., 1993).
We then tested the ability of CBP–FLAG to block migration in response to the inflammatory chemokines. Titrations of CBP–FLAG were added simultaneously with the optimal amount of chemokine (determined above) added to the bottom chamber. After 3 h incubation, migrant cells adhered to membranes or in medium in the bottom chamber were quantified as described above. CBP–FLAG reduced chemokine-induced migration of monocytes in a dose-dependent manner (Fig. 1). For all three chemokines, inhibition of monocyte migration by CBP–FLAG was significant for a chemokine : CBP–FLAG molar ratio of 1 : 1 (P<0.05), with the exception of CCL2 (adherent cells only). The highest inhibition was observed with a CBP–FLAG : chemokine molar ratio of 4 : 1 that reduced migration to background levels (cells only control).
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; Haberstroh et al., 2002). In preliminary experiments, we determined the optimal levels of LPS (in 20 µl) to inject into the dermis of the shaved abdomen of C57BL/6 mice and the time at which maximal monocyte recruitment was evident. Evans blue dye (2 µl of 1 % solution) was added to each sample to visualize the diffusion of injected material and to identify the site of injection of LPS 24 h post-treatment, when animals were sacrificed and approximately equal areas of skin were excised. Cells were isolated from the skin by treatment with collagenase and dispase, phenotyped for the cell surface markers Gr-1 (anti-Gr-1 allophycocyanin-conjugated, clone RB6-8C5, isotype IgG2b; R&D Systems) and CD11b (biotinylated anti-mouse CD11b, clone M1/70, isotype IgG2b; BD Biosciences), which together identify inflammatory monocytes (Geissmann et al., 2003), and were analysed by flow cytometry. The results showed a sixfold increase in monocyte recruitment using 1 µg LPS and the response was maximal at 24 h post-treatment (Supplementary Fig. S3, available in JGV Online).
Animals were co-injected with 1 µg LPS with or without various amounts of CBP–FLAG (total volume 20 µl). After 24 h, skin was excised from the injection sites and weighed; cells were isolated and stained as described above. Total cells were determined by haemocytometer enumeration. From the flow cytometric analysis of 10 000 cells and the total cell count, the number of monocytes (mg skin)–1 was determined. Fig. 2(a) shows that 100 and 1 ng of CBP–FLAG significantly inhibited recruitment for Gr-1+/CD11b+ monocytes (P<0.01, paired Student's t test), whereas with 0.01 ng of CBP–FLAG, the cell numbers were similar to the PBS control. From phenotype marker analysis, a population of Gr-1+/CD11b– cells was identified. These cells represent granulocytes or neutrophils (Ishikawa & Miyazaki, 2005). CBP–FLAG did not have an effect on the recruitment of these cell types, which remained constant regardless of the dose of CBP–FLAG (Fig. 2b).
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).
Finally, histology was performed to confirm the above findings. Mouse experiments were carried out as above; skin samples were treated with zinc salt fixative (González et al., 2001) and embedded in paraffin wax for cellular analysis. Multiple 5 µm sections were cut at 40 µm intervals. Adjacent sections were stained with haematoxylin and eosin (H & E) (Anderson et al., 2001), CD11b marker [biotin-labelled CD11b, used at 1/200 dilution, with streptavidin-conjugated Alexa488 (1/50 dilution); R&D systems] and 3,3'-diaminobenzidine (Sigma) for peroxidase granules (neutrophils). The H & E-stained sections show an accumulation of leukocytes in the thickened dermis of LPS-injected skin (Fig. 3a). In contrast, skin co-injected with CBP–FLAG or CBP–FLAG+LPS showed no dermal thickening and fewer leukocytes. The LPS-injected skin also showed an increased accumulation of CD11b+ cells but this was not seen when CBP was co-injected (Fig. 3a, c). The numbers of neutrophils were consistent between the LPS only and CBP–FLAG+LPS sections (Fig. 3a and d).
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C57Bl/6 mice produce a Th1 dominant inflammatory response to LPS (Reiner & Locksley, 1993; Takeda et al., 2003) typical of a viral infection. Nevertheless, the dynamics of the cutaneous response induced with LPS will differ from that resulting from viral infection. The rapid transient upregulation of high levels of proinflammatory cytokines induced with LPS contrasts with the gradual cytokine response that characterizes viral infection. Despite the magnitude of the LPS response, nanogram quantities of CBP injected into the skin were able to effectively impair cellular trafficking.
The therapeutic potential of several poxvirus CBPs has been investigated in various rodent animal models. Cowpoxvirus vCCI was shown to suppress allergen-induced chronic pulmonary inflammation in BALB/cJ mice (Dabbagh et al., 2000), whilst myxomavirus MT1 suppressed the inflammatory response that develops immediately after allograft transplant vasculopathy surgery (Liu et al., 2004). The vaccinia virus 35 kDa protein has been shown to regulate leukocyte trafficking to the lungs during vaccinia virus infection (Reading et al., 2003). The murine models we have employed demonstrate unequivocally that CBP–FLAG is a potent inhibitor of leukocyte recruitment in acute inflammatory skin responses. The potency of this molecule is within the physiological range within which such cellular immune molecules operate (10–60 ng ml–1; Conti et al., 1997; Fahey et al., 2005). Furthermore, we have determined that approximately 60–120 ng CBP ml–1 is produced from recombinant ORFV infected cells in culture over a 2–5 day period (unpublished data). Our studies are the first to report the effects of a viral-encoded CBP on inflammatory cell trafficking to the skin and may have relevance to the treatment of inflammatory skin disorders.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Baggiolini, M. (1998). Chemokines and leukocyte traffic. Nature 392, 565–568.[CrossRef][Medline]
Charo, I. F. & Ransohoff, R. M. (2006). The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med 354, 610–621.
Conti, P., Pang, X., Boucher, W., Letourneau, R., Reale, M., Barbacane, R. C., Thibault, J. & Theoharides, T. C. (1997). Impact of Rantes and MCP-1 chemokines on in vivo basophilic cell recruitment in rat skin injection model and their role in modifying the protein and mRNA levels for histidine decarboxylase. Blood 89, 4120–4127.
Cyster, J. G. (1999). Chemokines and cell migration in secondary lymphoid organs. Science 286, 2098–2102.
Dabbagh, K., Xiao, Y., Smith, C., Stepick-Biek, P., Kim, S. G., Lamm, W. J. E., Liggitt, D. H. & Lewis, D. B. (2000). Local blockade of allergic airway hyperreactivity and inflammation by the poxvirus-derived pan-CC-chemokine inhibitor vCCI. J Immunol 165, 3418–3422.
Deane, D., McInnes, C. J., Percival, A., Wood, A., Thomson, J., Lear, A., Gilray, J., Fleming, S., Mercer, A. & Haig, D. (2000). Orf virus encodes a novel secreted protein inhibitor of granulocyte-macrophage colony-stimulating factor and interleukin-2. J Virol 74, 1313–1320.
Dieu-Nosjean, M. C., Vicari, A., Lebecque, S. & Caux, C. (1999). Regulation of dendritic cell trafficking: a process that involves the participation of selective chemokines. J Leukoc Biol 66, 252–262.[Abstract]
Fahey, J. V., Schaefer, T. M., Channon, J. Y. & Wira, C. R. (2005). Secretion of cytokines and chemokines by polarized human epithelial cells from the female reproductive tract. Hum Reprod 20, 1439–1446.
Fleming, S. B. & Mercer, A. A. (2007). Genus Parapoxvirus. In Poxviruses (in the series Birkhäuser Advances in Infectious Diseases, series Editors A. Schmidt, M. H. Wolff & S. H. E. Kaufmann), pp. 127–165. Edited by A. A. Mercer, A. Schmidt & O. Weber. Basel: Birkhäuser.
Fleming, S. B., McCaughan, C. A., Andrews, A. E., Nash, A. D. & Mercer, A. A. (1997). A homologue of interleukin-10 is encoded by the poxvirus orf virus. J Virol 71, 4857–4861.[Abstract]
Geissmann, F., Jung, S. & Littman, D. R. (2003). Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19, 71–82.[CrossRef][Medline]
González, L., Anderson, I., Deane, D., Summers, C. & Buxton, D. (2001). Detection of immune system cells in paraffin wax-embedded ovine tissues. J Comp Pathol 125, 41–47.[CrossRef][Medline]
Haberstroh, U., Pocock, J., Gómez-Guerrero, C., Helmchen, U., Hamann, A., Gutierrez-Ramos, J. C., Stahl, R. A. & Thaiss, F. (2002). Expression of the chemokines MCP-1/CCL2 and RANTES/CCL5 is differentially regulated by infiltrating inflammatory cells. Kidney Int 62, 1264–1276.[CrossRef][Medline]
Haig, D. M. & Mercer, A. A. (1998). Ovine diseases. Orf. Vet Res 29, 311–326.[Medline]
Haig, D. M., Thomson, J., McInnes, C. J., Deane, D. L., Anderson, I. E., McCaughan, C. A., Imlach, W., Mercer, A. A., Howard, C. J. & Fleming, S. B. (2002). A comparison of the anti-inflammatory and immunostimulatory activities of orf virus and ovine interleukin-10. Virus Res 90, 303–316.[CrossRef][Medline]
Huang, H., Li, F., Cairns, C. M., Gordon, J. R. & Xiang, J. (2001). Neutrophils and B cells express XCR1 receptor and chemotactically respond to lymphotactin. Biochem Biophys Res Commun 281, 378–382.[CrossRef][Medline]
Imhof, B. A. & Aurrand-Lions, M. (2004). Adhesion mechanisms regulating the migration of monocytes. Nat Rev Immunol 4, 432–444.[CrossRef][Medline]
Ishikawa, F. & Miyazaki, S. (2005). New biodefense strategies by neutrophils. Arch Immunol Ther Exp (Warsz) 53, 226–233.[Medline]
Kopydlowski, K. M., Salkowski, C. A., Cody, M. J., van Rooijen, N., Major, J., Hamilton, T. A. & Vogel, S. N. (1999). Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo. J Immunol 163, 1537–1544.
Larsen, C. G., Anderson, A. O., Appella, E., Oppenheim, J. J. & Matsushima, K. (1989). The neutrophil-activating protein (NAP-1) is also chemotactic for T lymphocytes. Science 243, 1464–1466.
Liu, L., Dai, E., Miller, L., Seet, B., Alshad, L., Macauley, C., Li, X., Virgin, H. W., Bunce, C. & other authors (2004). Viral chemokine-binding proteins inhibit inflammatory responses and aortic allograft transplant vasculopathy in rat models. Transplantation 77, 1652–1660.[CrossRef][Medline]
Lyttle, D. J., Fraser, K. M., Fleming, S. B., Mercer, A. A. & Robinson, A. J. (1994). Homologs of vascular endothelial growth factor are encoded by the poxvirus orf virus. J Virol 68, 84–92.
Reading, P. C., Symons, J. A. & Smith, G. L. (2003). A soluble chemokine-binding protein from vaccinia virus reduces virulence and the inflammatory response to infection. J Immunol 170, 1435–1442.
Reiner, S. L. & Locksley, R. M. (1993). Cytokines in the differentiation of Th1/Th2 CD4+ subsets in leishmaniasis. J Cell Biochem 53, 323–328.[Medline]
Rollins, B. J. (1997). Chemokines. Blood 90, 909–928.
Rossi, D. & Zlotnik, A. (2000). The biology of chemokines and their receptors. Annu Rev Immunol 18, 217–242.[CrossRef][Medline]
Schuetze, N., Schoeneberger, S., Mueller, U., Freudenberg, M. A., Alber, G. & Straubinger, R. K. (2005). IL-12 family members: differential kinetics of their TLR4-mediated induction by Salmonella Enteritidis and the impact of IL-10 in bone marrow-derived macrophages. Int Immunol 17, 649–659.
Seet, B. T., McCaughan, C. A., Handel, T. M., Mercer, A. A., Brunetti, C., McFadden, G. & Fleming, S. B. (2003). Analysis of an orf virus chemokine-binding protein: shifting ligand specificities among a family of poxvirus viroceptors. Proc Natl Acad Sci U S A 100, 15137–15142.
Sozzani, S., Luini, W., Borsatti, A., Polentarutti, N., Zhou, D., Piemonti, L., D'Amico, G., Power, C. A., Wells, T. N. & other authors (1997). Receptor expression and responsiveness of human dendritic cells to a defined set of CC and CXC chemokines. J Immunol 159, 1993–2000.[Abstract]
Takeda, K., Kaisho, T. & Akira, S. (2003). Toll-like receptors. Annu Rev Immunol 21, 355–376.
Uguccioni, M., D'Apuzzo, M., Loetscher, M., Dewald, B. & Baggiolini, M. (1995). Actions of the chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES, MIP-1 and MIP-1β on human monocytes. Eur J Immunol 25, 64–68.[Medline]
Wang, J. M., McVicar, D. W., Oppenheim, J. J. & Kelvin, D. J. (1993). Identification of RANTES receptors on human monocytic cells: competition for binding and desensitization by homologous chemotactic cytokines. J Exp Med 177, 699–705.
Wetzler, C., Kampfer, H., Pfeilschifter, J. & Frank, S. (2000). Keratinocyte-derived chemotactic cytokines: expressional modulation by nitric oxide in vitro and during cutaneous wound repair in vivo. Biochem Biophys Res Commun 274, 689–696.[CrossRef][Medline]
Yu, Y., Sweeney, M. D., Saad, O. M., Crown, S. E., Hsu, A. R., Handel, T. M. & Leary, J. A. (2005). Chemokine-glycosaminoglycan binding: specificity for CCR2 ligand binding to highly sulfated oligosaccharides using FTICR mass spectrometry. J Biol Chem 280, 32200–32208.
Received 11 December 2008;
accepted 16 February 2009.
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