| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
Howard Hughes Medical Institute, G. W. Hooper Foundation and Departments of Microbiology and Medicine, University of California, San Francisco, CA 94143-0552, USA
Correspondence
Don Ganem
ganem{at}cgl.ucsf.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
B. CXCL16 induces the chemotaxis of activated T cells, whose products have been proposed to positively regulate KS tumour-cell survival and growth. Whilst CXCL16 has also been proposed as a direct endothelial chemoattractant and mitogen, neither proliferation nor chemotaxis of HUVECs was observed following CXCL16 exposure. These results suggest that CXCL16 induction by KSHV contributes to the inflammatory phenotype of KS, but plays little role in the recruitment of endothelial spindle cells. Supplementary figures are available in JGV Online.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). KS is an unusual neoplasm that displays a very strong relationship to chronic inflammation, which it both induces and appears to depend upon (Ensoli & Stürzl, 1998; Ensoli et al., 2001). Clinically, KS lesions often progress during or following states of systemic inflammation, and KS tumours sometimes arise precisely at sites of antecedent local inflammation, such as surgical wounds (a property known to clinicians as the Koebner phenomenon) (Maral, 2000). Histologically, KS lesions display proliferation of spindle-shaped endothelial cells (which ultimately comprise the bulk of the tumour mass), but also dramatic neoangiogenesis and inflammatory infiltration. Considerable importance has been attached to the inflammatory component of KS, as: (i) in early KS, inflammatory changes predate the development of a detectable mass; (ii) KS lesions display abundant expression of proinflammatory cytokines (Ensoli & Stürzl, 1998); and (iii) KS cells in culture require conditioned medium from activated T cells for their survival and proliferation in vitro (Ensoli & Stürzl, 1998; Nakamura et al., 1988).
In KS tumours, KSHV infection is detected primarily in the endothelial-cell compartment of the lesion; most of these cells display latent KSHV infection, with only a small subset entering the lytic cycle (Boshoff et al., 1995; Staskus et al., 1999). KSHV latency has been studied most extensively in B cells, which are thought to be the primary reservoir of infection. Latently infected B cells are known to produce a variety of proinflammatory cytokines. Moreover, lytic induction of such cells is associated with the induction of several viral proteins with known proinflammatory properties, including a viral G protein-coupled receptor, a viral interleukin-6 (IL-6) homologue and several virally encoded chemokine homologues (Arvanitakis et al., 1997; Moore et al., 1996; Nicholas, 2005; Nicholas et al., 1997) (host IL-6 production is also induced strongly; Glaunsinger & Ganem, 2004). These molecules are likely to play important roles in several KSHV-related B-cell disorders, most notably multicentric Castleman's disease, which is associated with pronounced lytic KSHV replication and striking IL-6 production (Oksenhendler et al., 2000; Parravicini et al., 2000; Staskus et al., 1999). Relatively less attention has been paid to the proinflammatory consequences of experimental endothelial infection by KSHV (although much has been written about the impact of KSHV infection on endothelial differentiation, proliferation and angiogenesis; Ciufo et al., 2001; Wang et al., 2004). Here, we have examined the impact of latent KSHV infection of primary vascular endothelial cells on the production and release of inflammatory mediators.
Primary human umbilical vein endothelial cells (HUVECs) were chosen for this study because of their ready availability and their susceptibility to KSHV infection. In fact, KSHV infection of such cells results in morphological changes typical of KS spindle cells in vivo – rearrangement of the actin cytoskeleton and cell elongation (Grossmann et al., 2006; Zhou et al., 2002). HUVECs were divided into two aliquots: one was mock-infected, whilst the other was infected with KSHV under conditions resulting in latent infection of virtually 100 % of cells (as judged by expression of LANA protein visualized by indirect immunofluorescence). At 6 days post-infection, by which time the infected cells had undergone obvious morphological changes, culture supernatants were removed and assayed for chemokines by using a solid-phase immunoblotting procedure (RayBio Human Chemokine Antibody Array 1.1; RayBiotech) (Fig. 1a). The supernatants were applied to membranes containing antibodies to 38 human chemokines, each spotted in duplicate. Following binding, the membranes were washed and incubated with biotin-conjugated anti-chemokine antibodies, followed by incubation with horseradish peroxidase-conjugated streptavidin and development according to the instructions of the manufacturer. As shown in Fig. 1(b), the medium of uninfected HUVECS has considerable basal levels of several chemokines (including GRO, IL-8, MCP-1, MIP-1
, MIP-1
, eotaxin-3 and NAP 2). Following infection, several of these undergo significant upregulation – most notably MCP-1, NAP 2 and RANTES. Notably, one chemokine, CXCL16, was nearly undetectable in the basal medium, but induced strongly by infection. This induction was confirmed by quantitative ELISA (Fig. 1c).
|
(a), vFLIP expression upregulated CXCL16 expression strongly, whereas the other loci induced little (kaposin A) or no CXCL16 induction above the basal level. Examination of the vFLIP transductants for the levels of CXCL16 mRNA by quantitative RT-PCR revealed that the induction was at the level of mRNA accumulation (Fig. 2b); the same is true of HUVECs infected authentically by KSHV (Fig. 2c), although the induction ratio is of lesser magnitude.
|
) and cleavage of this form by the protease ADAM10 in its ectodomain leads to release of the extracellular ligand (Ludwig et al., 2005). To see whether upregulation of ADAM10 might contribute to enhanced expression of soluble CXCL16, we assayed levels of ADAM10 mRNA by quantitative RT-PCR in both KSHV-infected and vFLIP-transduced HUVECs. In neither case was ADAM10 mRNA induced (see Supplementary Fig. S1, available in JGV Online); although we cannot exclude a component of post-transcriptional regulation of ADAM10 activity, the induction of CXCL16 mRNA by vFLIP would appear to be sufficient to explain the upregulation of the secreted chemokine.
vFLIP's primary biochemical activity is the induction of NF-B activation via stimulation of IKK activity (Chaudhary et al., 1999; Field et al., 2003; Liu et al., 2002; Matta & Chaudhary, 2004), although other signalling activities of vFLIP have been reported (An et al., 2003). To validate that the upregulation of CXCL16 was dependent upon NF-B activation, we tested the ability of a clone expressing an IB super-repressor (IB-SR) to block induction of the chemokine (IB-SR lacks sites for phosphorylation by IKK, and hence cannot release active NF-B from IB after IKK stimulation). HUVECs were transduced with a murine retroviral vector expressing IB-SR (or with the empty vector only). After 48 h, the cells were then transduced with the vFLIP-expressing retrovirus; 4 days later, conditioned medium was harvested and assayed for CXCL16 production by ELISA. Fig. 3(a) shows that expression of IB-SR reduces induction of CXCL16 substantially, whereas infection with the empty vector had no effect.
|
) or vFLIP transduction by MuLV-based vectors (Fig. 3c). CXCL16 induction was not observed in any of the established cell lines that we tested, following either viral infection or vFLIP overexpression; this survey included immortalized endothelial lines (SLK and TIME), as well as transformed cells of epithelial (RPE) and fibroblastic (MCF10A) lineage. To date, we have observed vFLIP induction of CXCL16 only in differentiated primary endothelial cells. Consistent with this, vFLIP transduction into primary microvascular endothelial cells of blood (BEC) or lymphatic (LEC) lineage also displayed CXCL16 induction (data not shown).
As most cells expressing vFLIP display NF-B induction (An et al., 2003; Chaudhary et al., 1999; Matta & Chaudhary, 2004; Sun et al., 2003a, b, 2005, 2006), it seems clear that the ability to upregulate CXCL16 mRNA must be controlled by additional factors. Most likely, HUVECs are permissive for a post-NF-B regulatory step that most lines do not support. Possibilities for such steps include transcript elongation, termination, stabilization or export. Alternatively, non-responsive cells might harbour repressive chromatin structures at this locus that prevent transcription induction by NF-B.
CXCL16 is a recently identified chemokine first identified in mice (Matloubian et al., 2000). There, it is associated with chemotaxis of activated (but not resting) T cells in vivo and in vitro. More recent in vitro studies have suggested that the protein can trigger chemotaxis of endothelial cells (HUVECs) in vitro (Zhuge et al., 2005). As such activities could be very germane to KS pathogenesis in vivo, we deemed it important to verify them independently. Accordingly, HUVECs were placed in the upper well of a Transwell chamber (Corning) and recombinant CXCL16 was added to the lower well of the chamber; cells that migrated across the chamber filter toward the lower surface were counted after 3 h incubation at 37 °C. However, no significant chemotaxis was observed, even though chemotaxis to a known endothelial attractant, bFGF, was unimpaired (see Supplementary Fig. S2, available in JGV Online). We also examined whether HUVECs can be stimulated to proliferate following exposure to the chemokine, as judged by BrdU incorporation. As shown in Supplementary Fig. S3 (available in JGV Online), CXCL16 did not stimulate BrdU incorporation into HUVECs, whereas known endothelial mitogens such as bFGF were highly active in this assay.
These data indicate that viral infection of differentiated primary endothelial cells can trigger a programme of proinflammatory gene expression that differs somewhat from that observed in non-endothelial cells and includes the induction of the unusual chemokine CXCL16. The principal KSHV determinant of CXCL16 production appears to be expression of vFLIP, although lesser contributions by other viral latency proteins may occur. Upregulation by vFLIP appears to be primarily via NF-B-mediated induction of CXCL16 mRNA production. The fact that CXCL16 induces migration of activated T cells is of interest as, in cell-culture systems, KS cells appear to require conditioned medium from activated T cells for survival and proliferation. It is therefore possible that CXCL16 induction may play an indirect, paracrine role in promoting survival and expansion of the tumour. A direct role for CXCL16 in endothelial chemotaxis and growth has also been postulated by others (Zhuge et al., 2005), but we cannot confirm their observations and think it likely that the protein does not itself stimulate endothelial migration or proliferation directly. Finally, we note that the induction of CXCL16 occurs only in primary endothelial cells and not in established lines of other lineages (even immortalized endothelial cells do not display this behaviour). This re-emphasizes the need to use cells as close as possible to those targeted naturally for infection when examining host–viral interactions designed to model one or more aspects of virus-induced disease.
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
B and JNK/AP1 pathways. Oncogene 22, 3371–3385.[CrossRef][Medline]Arvanitakis, L., Geras-Raaka, E., Varma, A., Gershengorn, M. C. & Cesarman, E. (1997). Human herpesvirus KSHV encodes a constitutively active G-protein-coupled receptor linked to cell proliferation. Nature 385, 347–350.[CrossRef][Medline]
Boshoff, C., Schulz, T. F., Kennedy, M. M., Graham, A. K., Fisher, C., Thomas, A., McGee, J. O., Weiss, R. A. & O'Leary, J. J. (1995). Kaposi's sarcoma-associated herpesvirus infects endothelial and spindle cells. Nat Med 1, 1274–1278.[CrossRef][Medline]
Chaudhary, P. M., Jasmin, A., Eby, M. T. & Hood, L. (1999). Modulation of the NF-B pathway by virally encoded death effector domains-containing proteins. Oncogene 18, 5738–5746.[CrossRef][Medline]
Ciufo, D. M., Cannon, J. S., Poole, L. J., Wu, F. Y., Murray, P., Ambinder, R. F. & Hayward, G. S. (2001). Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures. J Virol 75, 5614–5626.
Ensoli, B. & Stürzl, M. (1998). Kaposi's sarcoma: a result of the interplay among inflammatory cytokines, angiogenic factors and viral agents. Cytokine Growth Factor Rev 9, 63–83.[CrossRef][Medline]
Ensoli, B., Sgadari, C., Barillari, G., Sirianni, M. C., Stürzl, M. & Monini, P. (2001). Biology of Kaposi's sarcoma. Eur J Cancer 37, 1251–1269.[CrossRef][Medline]
Field, N., Low, W., Daniels, M., Howell, S., Daviet, L., Boshoff, C. & Collins, M. (2003). KSHV vFLIP binds to IKK-
to activate IKK. J Cell Sci 116, 3721–3728.
Glaunsinger, B. & Ganem, D. (2004). Highly selective escape from KSHV-mediated host mRNA shutoff and its implications for viral pathogenesis. J Exp Med 200, 391–398.
Grossmann, C., Podgrabinska, S., Skobe, M. & Ganem, D. (2006). Activation of NF-B by the latent vFLIP gene of Kaposi's sarcoma-associated herpesvirus is required for the spindle shape of virus-infected endothelial cells and contributes to their proinflammatory phenotype. J Virol 80, 7179–7185.
Liu, L., Eby, M. T., Rathore, N., Sinha, S. K., Kumar, A. & Chaudhary, P. M. (2002). The human herpes virus 8-encoded viral FLICE inhibitory protein physically associates with and persistently activates the IB kinase complex. J Biol Chem 277, 13745–13751.
Ludwig, A., Hundhausen, C., Lambert, M. H., Broadway, N., Andrews, R. C., Bickett, D. M., Leesnitzer, M. A. & Becherer, J. D. (2005). Metalloproteinase inhibitors for the disintegrin-like metalloproteinases ADAM10 and ADAM17 that differentially block constitutive and phorbol ester-inducible shedding of cell surface molecules. Comb Chem High Throughput Screen 8, 161–171.[CrossRef][Medline]
Maral, T. (2000). The Koebner phenomenon in immunosuppression-related Kaposi's sarcoma. Ann Plast Surg 44, 646–648.[Medline]
Matloubian, M., David, A., Engel, S., Ryan, J. E. & Cyster, J. G. (2000). A transmembrane CXC chemokine is a ligand for HIV-coreceptor Bonzo. Nat Immunol 1, 298–304.[CrossRef][Medline]
Matta, H. & Chaudhary, P. M. (2004). Activation of alternative NF-B pathway by human herpes virus 8-encoded Fas-associated death domain-like IL-1-converting enzyme inhibitory protein (vFLIP). Proc Natl Acad Sci U S A 101, 9399–9404.
Moore, P. S., Boshoff, C., Weiss, R. A. & Chang, Y. (1996). Molecular mimicry of human cytokine and cytokine response pathway genes by KSHV. Science 274, 1739–1744.
Nakamura, S., Salahuddin, S. Z., Biberfeld, P., Ensoli, B., Markham, P. D., Wong-Staal, F. & Gallo, R. C. (1988). Kaposi's sarcoma cells: long-term culture with growth factor from retrovirus-infected CD4+ T cells. Science 242, 426–430.
Nicholas, J. (2005). Human gammaherpesvirus cytokines and chemokine receptors. J Interferon Cytokine Res 25, 373–383.[CrossRef][Medline]
Nicholas, J., Ruvolo, V. R., Burns, W. H., Sandford, G., Wan, X., Ciufo, D., Hendrickson, S. B., Guo, H.-G., Hayward, G. S. & Reitz, M. S. (1997). Kaposi's sarcoma-associated human herpesvirus-8 encodes homologues of macrophage inflammatory protein-1 and interleukin-6. Nat Med 3, 287–292.[CrossRef][Medline]
Oksenhendler, E., Carcelain, G., Aoki, Y., Boulanger, E., Maillard, A., Clauvel, J.-P. & Agbalika, F. (2000). High levels of human herpesvirus 8 viral load, human interleukin-6, interleukin-10, and C reactive protein correlate with exacerbation of multicentric Castleman disease in HIV-infected patients. Blood 96, 2069–2073.
Parravicini, C., Chandran, B., Corbellino, M., Berti, E., Paulli, M., Moore, P. S. & Chang, Y. (2000). Differential viral protein expression in Kaposi's sarcoma-associated herpesvirus-infected diseases: Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Am J Pathol 156, 743–749.
Schulz, T. F. (1999). Epidemiology of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8. Adv Cancer Res 76, 121–160.[Medline]
Staskus, K. A., Sun, R., Miller, G., Racz, P., Jaslowski, A., Metroka, C., Brett-Smith, H. & Haase, A. T. (1999). Cellular tropism and viral interleukin-6 expression distinguish human herpesvirus 8 involvement in Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. J Virol 73, 4181–4187.
Sun, Q., Matta, H. & Chaudhary, P. M. (2003a). The human herpes virus 8-encoded viral FLICE inhibitory protein protects against growth factor withdrawal-induced apoptosis via NF-B activation. Blood 101, 1956–1961.
Sun, Q., Zachariah, S. & Chaudhary, P. M. (2003b). The human herpes virus 8-encoded viral FLICE-inhibitory protein induces cellular transformation via NF-B activation. J Biol Chem 278, 52437–52445.
Sun, Q., Matta, H. & Chaudhary, P. M. (2005). Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 long terminal repeat via the classical NF-B pathway and functionally cooperates with Tat. Retrovirology 2, 9.[CrossRef][Medline]
Sun, Q., Matta, H., Lu, G. & Chaudhary, P. M. (2006). Induction of IL-8 expression by human herpesvirus 8 encoded vFLIP K13 via NF-B activation. Oncogene 25, 2717–2726.[CrossRef][Medline]
Wang, H.-W., Trotter, M. W. B., Lagos, D., Bourboulia, D., Henderson, S., Mäkinen, T., Elliman, S., Flanagan, A. M., Alitalo, K. & Boshoff, C. (2004). Kaposi sarcoma herpesvirus-induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma. Nat Genet 36, 687–693.[CrossRef][Medline]
Zhou, F.-C., Zhang, Y.-J., Deng, J.-H., Wang, X.-P., Pan, H.-Y., Hettler, E. & Gao, S.-J. (2002). Efficient infection by a recombinant Kaposi's sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: application for genetic analysis. J Virol 76, 6185–6196.
Zhuge, X., Murayama, T., Arai, H., Yamauchi, R., Tanaka, M., Shimaoka, T., Yonehara, S., Kume, N., Yokode, M. & Kita, T. (2005). CXCL16 is a novel angiogenic factor for human umbilical vein endothelial cells. Biochem Biophys Res Commun 331, 1295–1300.[CrossRef][Medline]
Received 11 July 2006;
accepted 23 August 2006.
This article has been cited by other articles:
|
|
 |
|
  V. Punj, H. Matta, S. Schamus, T. Yang, Y. Chang, and P. M. Chaudhary Induction of CCL20 production by Kaposi sarcoma-associated herpesvirus: role of viral FLICE inhibitory protein K13-induced NF-{kappa}B activation Blood, May 28, 2009; 113(22): 5660 - 5668. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L.-W. Qian, W. Greene, F. Ye, and S.-J. Gao Kaposi's Sarcoma-Associated Herpesvirus Disrupts Adherens Junctions and Increases Endothelial Permeability by Inducing Degradation of VE-Cadherin J. Virol., December 1, 2008; 82(23): 11902 - 11912. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Efklidou, R. Bailey, N. Field, M. Noursadeghi, and M. K. Collins vFLIP from KSHV inhibits anoikis of primary endothelial cells J. Cell Sci., February 15, 2008; 121(4): 450 - 457. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
