1 Department of Medical Pathology and Laboratory Medicine, University of California, Davis, CA 95616, USA
2 Center for Comparative Medicine, University of California, Davis, CA 95616, USA
3 California National Primate Research Center, University of California, Davis, CA 95616, USA
Correspondence
James R. Carlson
jcarlson{at}focustechnologies.com
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ABSTRACT |
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MAIN TEXT |
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HCMV infects multiple cell types during both clinical and subclinical infections (Plachter et al., 1996). One potential advantage of a wide cell tropism is that it offers the virus multiple sites in which to establish and maintain a lifelong infection. Both monocytic precursor cells and endothelial cells (EC), in particular, have been implicated as key cell types in HCMV natural history. HCMV infection of EC has been documented in a variety of clinical settings (Plachter et al., 1996
), and EC are thought to play an important role in the haematogenous spread of the virus from the primary site of infection to distal tissues (Jarvis & Nelson, 2002
; Myerson et al., 1984
). In vitro studies have demonstrated that EC function is profoundly altered following HCMV infection in ways that may recapitulate mechanisms of HCMV pathogenesis in vivo (Grundy et al., 1998
; Maidji et al., 2002
; Sedmak et al., 1994
; Waldman et al., 1991
; Yamamoto-Tabata et al., 2004
). However, in vitro studies have been complicated by strain-related differences in EC-tropism due to incompletely defined changes in the viral genome (Baldanti et al., 2003
; Bolovan-Fritts & Wiedeman, 2001
, 2002
; Gerna et al., 2002
, 2003
; Hahn et al., 2002
; MacCormac & Grundy, 1999
; Sinzger et al., 1999
).
The present study was initiated to determine whether EC propagated in vitro were permissive for infection by the pathogenic strain 68-1 of rhesus CMV (RhCMV) (Chang et al., 2002b; Sequar et al., 2002
; Tarantal et al., 1998
). The results demonstrated that microvascular EC isolated from the brain of fetal rhesus macaques fully supported RhCMV replication.
Rhesus macaque microvascular endothelial cells (BrMVEC) were isolated from the brain tissue as described previously for human cells (Lamszus et al., 1999; Unger et al., 2002
). By day 6 after primary cell isolation, a subconfluent monolayer formed. These primary cells were subsequently purified further by using biotinylated Ulex europaeus agglutinin 1 (UEA-1) (Vector Laboratories) and anti-biotin microbeads (Miltenyi Biotec), i.e. by positive selection using UEA-I binding to EC.
This method resulted in a highly enriched (99 %) population of cells that were phenotypically and functionally consistent with EC. One percent or fewer cells were positive for either -smooth muscle actin or glial fibrillary acidic protein staining (data not shown), characteristic markers for smooth muscle cells or astrocytes, respectively. Confluent BrMVEC demonstrated characteristic cobblestone morphology with no evident gaps between cells (data not shown). The endothelial origin of the isolated cells was confirmed by positive immunostaining for von Willebrand factor (Factor VIII) (Dako) (Fig. 1a
) and positive uptake for Dil-Ac-LDL (1,1'-dioctadecyl-1,3,3,3'-tetramethylindocarbocyanine perchlorate acetylated low-density lipoprotein) (Fig. 1b
) in a reaction that is considered the most reliable parameter for the identification of EC (Craig et al., 1998
). Dil-AC-LDL uptake has been detected in BrMVEC derived from humans, as well as from other animal species (Carson et al., 1989
; de Vries et al., 1995
; Dorovini-Zis et al., 1991
; Gordon et al., 1991
; Greenwood, 1992
; Stanness et al., 1996
; Steffan et al., 1994
; Unger et al., 2002
). Positive immunostaining was also shown by using FITC-labelled UEA-1 (Vector Laboratories) (data not shown), and immunostaining for anti-CD105 antibody (data not shown) (clone SN6h, mouse IgG1; DAKO) (Lamszus et al., 1999
; MacLean et al., 2001
). EC organize into newly formed capillary structures during angiogenesis (Lamszus et al., 1999
). When rhesus BrMVEC suspensions were seeded onto Matrigel (Kubota et al., 1988
) thin extensions between cells or small cell clumps could be observed within 4 h. At 1216 h, a network of tubules was formed that organized in a way that left few solitary cells (Fig. 1c
). This phenotype was recently demonstrated for human BrMVEC (Lamszus et al., 1999
).
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Virus replication kinetics demonstrated a large burst in virus production between 24 and 48 h post-infection that reached a plateau at 96 h (Fig. 2b). The kinetics of RhCMV replication in BrMVEC were comparable to the pattern of infection in telomerized rhesus fibroblasts (Chang et al., 2002a
). These results demonstrated that rhesus BrMVEC were fully permissive for RhCMV infection.
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Rhesus CMV infection of BrMVEC was detected by strongly positive staining for IE1 gene product at 24 h post-infection. The infection was rapidly lytic with visible plaque formation within 5 days (Fig. 2a). These results indicated efficient cell-to-cell spread of RhCMV in BrMVEC.
For cellular activation experiments, BrMVEC were pre-incubated for 2 h with plain MV-EGM-2 with FBS-1 % as the only supplement. IL1 (2 ng ml1; Roche Molecular Biochemicals), TNF-
(10 ng ml1; R&D Systems) or phorbol 12-myristate 13-acetate (PMA) (100 ng ml1; Calbiochem) (diluted in MV-EGM-2/FBS-1 %) were added at selected intervals before, during or after infection. Untreated cells served as controls for the IL1
and TNF-
treatments, whereas cells incubated with MV-EGM-2/FBS-1 %/0·1 % DMSO served as the controls for PMA treatment.
BrMVEC were treated with TNF-, IL1
or PMA for 1 h intervals either (i) 1 h prior to infection (Fig. 3a and b
), (ii) 1 h during infection (data not shown) or (iii) 2 h after infection (data not shown). Results of these experiments showed that treatment with all activators given prior to or during infection significantly reduced RhCMV infection of BrMVEC when assaying the number of infected cells 24 h post-infection (Fig. 3a
) or the number of plaques 5 days post-infection (Fig. 3b
). In contrast, when the cells were treated with each at an interval of 2 h immediately following infection, no significant differences were observed between treated and untreated cells. Since RhCMV infection of BrMVEC was not diminished by post-infection treatment with the activators, the results suggested that treatment of cells before or during infection altered the earliest steps in virus infection, such as attachment and/or entry.
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The BrMVEC that we isolated from fetal macaques demonstrated phenotypic characteristics similar to those described previously (MacLean et al., 2001; Strelow et al., 1998
). In addition, we report an efficient method for purifying primary cultures by using biotin-labelled UEA-1 that bound to the BrMVEC before subsequent capture by anti-biotin antibody-coated magnetic beads. This method eliminated the need for the use of cloning rings (MacLean et al., 2001
; Strelow et al., 1998
) with no diminution in cell numbers or viability that has been reported previously (Lamszus et al., 1999
).
Although all EC share the property of forming a single cell layer separating flowing blood from the vessel walls and underlying tissues (Manconi et al., 2000), EC isolated from different anatomical sites have demonstrated differences in physiological and biochemical characteristics (Page et al., 1992
; Turner et al., 1987
). It has been reported that HCMV infects human BrMVEC more efficiently than human umbilical vein EC (Fish et al., 1998
; Jarvis & Nelson, 2002
), although others have found no EC-specific differences in infectivity (Kahl et al., 2000b
).
In the present study, we found that RhCMV strain 68-1 efficiently infects and replicates to high levels in BrMVEC. The formation of plaques demonstrates that infection is cytolytic, similar to HCMV infection of human BrMVEC (Jarvis & Nelson, 2002). Comparative studies using rhesus macaque EC derived from large vessels is now warranted to determine whether RhCMV replication is affected by the anatomical origin of the EC similar to HCMV (Jarvis & Nelson, 2002
; Kahl et al., 2000a
). Treatment of rhesus BrMVEC with TNF-
, IL1
or PMA each caused a significant decrease in RhCMV infection. It should be noted, that however, this may be a reaction specific for BrMVEC, since our observation stands in contrast to the effects of PMA treatment on HCMV infectivity of human umbilical vein EC where CMV infection was enhanced by treatment (Slobbe-van Drunen et al., 1997
).
Our data show that BrMVEC from rhesus macaques can be easily isolated and cultured. These cells demonstrate phenotypic characteristics similar to those derived from human tissue and the replication kinetics of the lytic infection produced by RhCMV was similar to HCMV in human microvascular EC derived from brain tissue (Jarvis & Nelson, 2002; Kahl et al., 2000a
). These experiments indicate that in vitro assays using rhesus macaque-derived BrMVEC will enhance the RhCMV model for further neuropathogenesis research.
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ACKNOWLEDGEMENTS |
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Received 8 July 2004;
accepted 12 November 2004.
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