Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, California 95616, USA1
Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717, USA2
Author for correspondence: N. James MacLachlan. Fax +1 530 754 8124. e-mail njmaclachlan{at}ucdavis.edu
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Abstract |
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Introduction |
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Despite the similarities in the pathogenesis of BTV infection of sheep and cattle, there are pronounced differences in the clinical manifestations of BTV infection in the two species. Cattle are considered reservoir hosts of BTV because most infections are asymptomatic, and the very sporadic occurrence of disease in BTV-infected cattle likely reflects a type-1 hypersensitivity reaction (Anderson et al., 1987 ). In contrast, bluetongue disease of sheep and deer is characterized by virus-induced injury to vascular endothelium (Mahrt & Osburn, 1986
; Howerth & Tyler, 1988
) and, in fulminant cases, by disseminated intravascular coagulation that leads to ischaemic necrosis in a variety of tissues, haemorrhagic diathesis and shock (reviewed in Parsonson, 1990
; MacLachlan, 1994
). The lungs are the shock organ of ruminants and the organ most susceptible to permeability disorders of the vasculature (Meyrick et al., 1989
, 1991
); thus severe pulmonary oedema is characteristic of bluetongue disease in sheep (Spruell, 1905
; Moulton, 1961
). Although high titres of BTV are present in the lungs of infected sheep (Pini, 1976
), it is uncertain whether BTV-mediated injury to the pulmonary vascular endothelium is entirely the result of direct virus-induced cytopathology or also from the activity of inflammatory mediators.
Endothelial cells (ECs) from different species or tissues have diverse properties, and may respond differently to the same stimulus (Gerritsen, 1987 ; Kumar et al., 1987
; Meyrick et al., 1989
; Page et al., 1992
; Craig et al., 1998
). Significantly, ECs cultured from different levels of the pulmonary circulation of sheep and cattle are heterogeneous and retain their phenotype in culture (Meyrick et al., 1991
). EC phenotype is central to the pathogenesis of endotheliotropic virus infections. For example, replication of American isolates of ovine lentivirus, as assessed by reverse transcriptase activity, was greater in cultures of sheep brain microvascular ECs than in cultures of ovine aortic ECs (Craig et al., 1997
). Similarly, EC tropism of African horse sickness virus varied between the organs of infected horses (Gomez-Villamondos et al., 1999
). Cytolysis and interferon production differed between BTV-infected ovine and bovine umbilical vein ECs (Russell et al., 1996
), and Coen et al. (1991)
proposed that species-specific differences in EC infection and cytokine production were responsible for the different clinical outcomes of BTV infection of sheep and cattle.
To further investigate the central role of pulmonary ECs in the expression of bluetongue disease in sheep but not cattle, we characterized the interaction of BTV with ovine and bovine ECs cultured from different regions of the pulmonary vasculature. Specific goals were to identify differences in virus production, the mechanism of cell death, and antithrombotic activity as assessed by prostacyclin production after BTV infection of the various EC cultures both in the presence and absence of inflammatory mediators.
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Methods |
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Lung microvascular ECs were obtained essentially as previously described (Meyrick et al., 1989 ; Carley et al., 1992
; Craig et al., 1998
). Briefly, the visceral pleura was removed, the lung parenchyma was finely minced, washed repeatedly with HBSS, and then digested for 30 min with 2 mg/ml collagenase IA/dispase (Sigma) in HBSS. The digested tissue was filtered through a screen, washed with MEM/D-Val (Life Technologies) that contained 20% foetal bovine serum (FBS; Hyclone) and the isolated cells were stimulated with vascular endothelial cell growth factor (VEGF) and EC mitogen (Biomedical Technologies). The procedure was repeated three times on any remaining tissue too large to pass through the screen. The cells from each digest were pooled in human fibronectin-coated dishes (Becton Dickinson) and grown in isolation medium [MEM/D-Val, 10% FBS, penicillinstreptomycin, MEM vitamins, non-essential amino acids (MediaTech); 16 U/ml heparin, 1 µg/ml hydrocortisone, sodium pyruvate, L-glutamine and 5% human serum from platelet-depleted plasma (Sigma)].
Pulmonary artery ECs were obtained essentially as previously described (Meyrick et al., 1989 ; Visner et al., 1994
) and grown in isolation medium. Briefly, the pulmonary artery was filled with collagenase/dispase and the ECs were removed after a 5 min digestion by gently rinsing the lumen with HBSS. The isolated cells were then washed and stimulated as described. The enzyme digestion was repeated twice and cells from each digest were plated separately on fibronectin-coated dishes. Cultures derived from the pulmonary artery were incubated until colonies exhibiting the cobblestone morphology characteristic of ECs were identified. Spindle-shaped cells were considered to be contaminants and those in the vicinity of EC colonies were removed by aspiration. Multiple EC colonies were then selected with trypsin-soaked cloning discs (Scienceware), pooled and expanded.
Purification and characterization of ECs.
Cultures of ECs were purified by fluorescence-activated cell sorting (FACS) with a MoFlo instrument (Cytomation). ECs have a scavenger pathway for low-density lipoproteins and rapidly uptake the acetylated forms (Voyta et al., 1984 ); thus FACS selection of ECs was based on the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate conjugated to acetylated low-density lipoproteins (DiI-Ac-LDL; Biomedical Technologies;). This method has been used previously to obtain pure cultures of ECs from a variety of species and tissues (Voyta et al., 1984
; Carley et al., 1992
; Visner et al., 1994
; Magee et al., 1994
; Craig et al., 1998
).
Cultures of lung microvascular ECs were enriched by FACS at approximately 2 days after isolation and transferred to dishes coated with 1 µg/cm2 natural mouse laminin (Life Technologies). Multiple colonies of microvascular ECs were then selected with cloning discs, pooled and expanded. The cultures of pulmonary artery and lung microvascular ECs were labelled with DiI-Ac-LDL and purified by FACS three additional times to remove any contaminating cells that remained after the primary isolation. Purified EC cultures were screened for contaminating cells with monoclonal antibodies (MAbs; Dako) that recognize epithelial and mesothelial cells (MAbs AE1/AE3, cytokeratin-specific), pericytes (MAb D33, desmin-specific) and smooth muscle cells (MAb 1A4, actin-specific; Hewett & Murray, 1993 ; McDouall et al., 1996
). The phenotype of the purified ECs was confirmed with rabbit antiserum to von Willebrand factor (Dako), as von Willebrand factor contained in WeibelPalade bodies is highly characteristic of ECs (Jaffe et al., 1973
; Gerritsen et al., 1988
). Ovine and bovine ECs were further characterized for expression of a recently identified EC-specific adhesion molecule (GR antigen; Jutila et al., 1997
). Bovine ECs also were characterized for E-selectin expression, an EC-specific surface receptor (CD62E) that interacts with leukocytes (reviewed in Vestweber & Blanks, 1999
). Briefly, selected cultures were activated with 10 ng/ml endotoxin from Serratia marcescens (Sigma) for 4 h and labelled with MAb GR113 (GR antigen) or MAb EL-246 (E-selectin; Jutila et al., 1992
, 1997
). Antibody binding was detected with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated secondary antibodies (Sigma), fluorescence microscopy and FACS. The purified ECs were then expanded in laminin-coated flasks containing maintenance medium [ECMM; Dulbeccos MEM (MediaTech), FBS, penicillinstreptomycin, non-essential amino acids, L-glutamine and heparin] and low-passage stocks were cryopreserved. All experiments were performed on EC cultures between passages 7 and 15.
Virus.
BTV serotype 17 in the blood of a sheep that died of bluetongue disease after natural infection was passaged twice in seronegative cattle and isolated from blood essentially as previously described (MacLachlan & Fuller, 1986 ; Richards et al., 1988
). The virus was isolated in bovine lung microvascular ECs, amplified by two additional passages, and a stock of BTV-infected EC lysate was prepared. Briefly, the cells and medium were frozen at -70 °C and then thawed, homogenized, sonicated and filtered (0·2 µm). A similar lysate of uninfected lung microvascular ECs was produced for mock infection of ECs. Partially purified BTV17 was prepared from BTV-infected bovine microvascular ECs that were pelleted from the medium, washed with PBS, resuspended in ECMM, and then disrupted and filtered as above. Virus was pelleted by centrifugation at 69000 g for 3 h through a 20% sucrose cushion. The virus-containing pellets were resuspended in ECMM and the supernatant, containing cellular components of infected ECs, also was harvested. Virus titres (TCID50) were determined in BHK-21 cells as described previously (MacLachlan et al., 1984
; Barratt-Boyes et al., 1992
).
BTV infection of ECs.
All ovine and bovine pulmonary artery and lung microvascular EC cultures were infected with BTV that was derived from bovine lung microvascular ECs. Cultures of similar passage and cell density were infected at high multiplicity (m.o.i.=3) for a one-step analysis of virus growth. Briefly, cultures were washed with DMEM and adsorbed with partially purified BTV for 1 h at 37 °C. The inoculum was then removed and the cultures were washed with DMEM. The infected cultures were maintained in ECMM and replicate cultures were harvested at 4 h intervals by scraping. Samples of each culture were frozen and thawed, sonicated, and virus titres (TCID50) were determined.
EC cultures of similar passage and cell density also were infected at low m.o.i. (0·05) with either partially purified BTV or the BTV-infected EC lysate. Individual EC cultures were infected at 48 h intervals, and replicate cultures were included at 24, 48, 72 and 96 h intervals. Control flasks were inoculated (mock infected) with the uninfected EC lysate at 24 h intervals. Cytopathic effect was estimated as a percentage of the monolayer at each interval, and cultures were then harvested and simultaneously prepared for virus titration and FACS analyses. Culture medium from each flask was clarified by centrifugation at 400 g prior to virus titration. Cells remaining in each flask were removed by trypsinization, pooled with those previously pelleted from the medium, and washed once with PBS. Titres (TCID50) of BTV in the supernatant and cell fraction of individual EC cultures were determined by microtitre assay. Infection of ECs was detected by labelling virus proteins with BTV-specific rabbit antiserum (Heidner et al., 1988 , 1990
), FITC- or PE-conjugated goat anti-rabbit immunoglobulin (Sigma), and FACS analyses as previously described (Barratt-Boyes et al., 1992
).
Quantification of cell death in BTV-infected EC cultures.
The incidence of cell death in BTV-infected EC cultures was quantified by determining the percentage of apoptotic and necrotic cells in each culture at 48 h intervals after BTV infection. Apoptosis and necrosis were quantified in EC cultures by double-label FACS analysis based on the exclusion of propidium iodide (Sigma) and the binding of annexin V (R&D Systems), as previously described (Vermes et al., 1995 ).
Influence of inflammatory mediators on BTV infection of ECs.
The levels of prostacyclin released following BTV infection of ECs were determined at 24 h intervals by measuring its stable metabolite, 6-ketoprostaglandin F1, in clarified culture medium with a competitive ELISA (R&D Systems). The potential role of cytokine mediators in BTV-induced EC injury was investigated also. EC cultures were treated with 10 ng/ml mouse recombinant interleukin-1
(IL-1
; Sigma) and infected with partially purified BTV. Uninfected EC cultures were treated with IL-1
alone, the EC supernatant collected during the purification of BTV, or a cocktail of cytokines [10 ng/ml IL-1
, 1·64 ng/ml mouse recombinant tumour necrosis factor-
(TNF-
) and 1 µM platelet-activating factor (PAF); Sigma]. These cytokines previously have been used to activate ECs isolated from humans and ruminants (Meyrick et al., 1991
; Jutila et al., 1994
; Bargatze et al., 1994
; Sterner-Kock et al., 1996
). The percentage of apoptotic and necrotic cells in cultures treated with cytokines then was determined at 48 h intervals.
Data analysis.
Data were analysed with CELLQuest version 3.1 (Becton Dickinson), Excel 97 (Microsoft) and MINITAB (Minitab Inc.) software packages. Polynomial regression analyses were performed on FACS and ELISA data, and moving average regression analyses were done on virus titration data obtained at different times from the various EC cultures. All experiments were repeated at least once and the data from replicate experiments were compared at 0, 24, 48, 72 and 96 h post-infection (p.i.). The mean values and standard deviations were calculated for each parameter and a Students t-test was applied to determine differences between mean values of the data at each time-point, with P0·05 considered significant. The comparison yielding the highest P value
0·05 is reported in cases where multiple t-tests were used to identify statistical differences at various time-points between the four types of ECs.
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Results |
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Infection of ECs with partially purified BTV
The various EC cultures were inoculated at both high and low m.o.i. to identify any differences in the kinetics of BTV infection of ovine and bovine microvascular and pulmonary artery ECs. The kinetics of BTV infection were similar in the four types of EC cultures inoculated at high m.o.i. with partially purified BTV, and there were no significant differences in the maximum titres of BTV produced in each type of EC (Fig. 1). In contrast, the species and site of origin of the ECs clearly influenced the kinetics of BTV infection in cultures inoculated at low m.o.i. (Fig. 2
). Specifically, bovine microvascular EC cultures produced significantly higher titres of virus than did the ovine microvascular EC cultures (108·9 versus 106·0 TCID50 respectively; P=0·0014), whereas the maximum titres of BTV in ovine and bovine pulmonary artery EC cultures were not significantly different. Furthermore, the percentage of BTV-infected cells, as determined by FACS, was significantly lower in ovine lung microvascular ECs at 24 h p.i. than in any of the other EC types (P
0·011), and was significantly higher in both types of bovine ECs at 72 and 96 h p.i. than in either of the ovine EC types (P
0·033). Similar results were consistently obtained in replicate experiments, confirming that both the m.o.i. of the cultures, and the species and site of origin of the ECs can significantly influence the kinetics of BTV infection of cultured ovine and bovine pulmonary ECs.
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Prostacyclin production in BTV-infected ECs
Levels of prostacyclin were determined at regular intervals after inoculation of the various EC cultures to further investigate the role of inflammatory mediators in the in vitro pathogenesis of BTV infection of ECs (Fig. 4). Ovine ECs released significantly less prostacyclin than did bovine ECs at 48, 72 and 96 h p.i. with partially purified BTV (P
0·036). With the notable exception of ovine microvascular ECs, ECs inoculated with the BTV-infected EC lysate also released considerable amounts of prostacyclin (data not shown). Ovine lung microvascular ECs consistently released relatively little prostacyclin after infection with either BTV inoculum, indicating for the first time that their antithrombotic response to BTV infection is distinctly different from that of other types of ECs.
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Discussion |
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Mediators released by ECs clearly modulated the course of BTV infection in ECs, as previously described for BTV infection of ovine and bovine umbilical vein ECs and a bovine cardiopulmonary EC line (Coen et al., 1991 ; Russell et al., 1996
). EC-derived mediators that modulate thrombosis and inflammation include IL-1, IL-6, interferons, prostacyclin and chemokines such as IL-8 (Mantovani et al., 1992
), and likely were present in the BTV-infected EC lysate but not in the partially purified BTV inoculum used in our studies. Thus, virus yields consistently were lower in ECs infected with the BTV-infected EC lysate as compared to those infected with partially purified BTV. Furthermore, necrosis was the major cause of cell death in all ECs infected with partially purified BTV except ovine pulmonary artery ECs, whereas apoptosis predominated in all ECs infected with the BTV-infected cell lysate. Similarly, addition of IL-1
to the partially purified BTV inoculum caused apoptosis of virus-infected ovine and bovine microvascular ECs, whereas infection of these cultures with partially purified BTV alone resulted only in necrosis of virus-infected ECs. Apoptosis of ECs is linked to paracrine release of IL-1, and genes such as that encoding IL-1-converting enzyme (caspase-1) also regulate release of inflammatory cytokines (Steller, 1995
; Henkart, 1996
; Vaux & Strasser, 1996
; Hebert et al., 1998
). The expression of clinical bluetongue disease in sheep but not cattle might, therefore, reflect inherent differences in the susceptibility of ovine and bovine pulmonary microvascular ECs to induction of apoptosis by IL-1 or other virus-induced EC-derived mediators.
Prostacyclin is a potent antithrombotic agent that is produced primarily by ECs, and there were significant differences in production of prostacyclin following BTV infection of the four types of ECs. BTV-infected ovine lung microvascular ECs consistently and reproducibly produced significantly lower levels of prostacyclin than did the other types of ECs. Similarly, ovine and bovine pulmonary artery and microvascular ECs produced different amounts of prostacyclin after activation with endotoxin (Meyrick et al., 1989 ). The very low levels of prostacyclin produced in BTV-infected ovine microvascular ECs suggest that sheep might be less able than cattle to regulate platelet aggregation subsequent to BTV-induced endothelial injury, consistent with the consumptive coagulopathy, haemorrhagic diathesis and severe pulmonary oedema that are characteristic of bluetongue disease of sheep but which rarely, if ever, occur in BTV-infected cattle.
In summary, our studies indicate that the site and species of EC origin can markedly influence the outcome of BTV infection in vitro. Thus, endothelial injury in BTV-infected sheep but not cattle could reflect differences in either the direct pathogenic effects of BTV infection of ECs, or their response to inflammatory mediators released by virus-infected ECs and, perhaps, other cell types such as monocytes (Whetter et al., 1989 ). Although precise characterization clearly will require additional studies, data from these in vitro studies strongly suggest that inherent differences in the susceptibility of ruminant microvascular ECs to BTV infection, and their production and response to mediators such as prostacyclin and IL-1, contribute to endothelial injury and subsequent expression of bluetongue disease in sheep but not cattle.
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Acknowledgments |
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Received 27 October 2000;
accepted 18 December 2000.