Journal of Virological Methods, 39 (1992) 83-90 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00
83
VIRMET 01365
Viral susceptibility of an immortalized human microvascular endothelial cell line Edwin W. Ades”, John C. Hierholzer”, Velma Georgea, and Francisco Candal”
Jodi Blackb
“Biological Products Branch, bViral Exanthems and Herpesvirus Branch, and ‘Respiratory and Enteric Viruses Branch, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, GA (USA) (Accepted 28 February
1992)
Summary CDC/EU.HMEC1 is the first immortalized human microvascular endothelial cell line that retains morphologic, phenotypic, and functional characteristics of a normal human microvascular endothelial cell. This study evaluates a variety of viruses and their effects on this human endothelial cell line. The data indicate that adenoviruses, some herpesviruses, reoviruses and most picornaviruses grow well in HMEC-1, with distinctive cytopathic effects. The paramyxoviruses, however, do not appear to propagate, nor does HIV. The findings indicate that microvascular endothelial cells may act as a reservoir of these viruses; it also suggests the possibility that microvascular endothelium could be involved in the processing and presentation of antigen to immune cells. Human microvascular
endothelium; Viral susceptibility
Introduction Endothelial cells play a critical role in such important physiologic and pathophysiologic events as inflammation, wound healing, angiogenesis, and hemostasis (Cotran, 1987; Pober, 1988). Endothelium mediates the binding of peripheral blood leukocytes (Mantonvani et al., 1989; Gamble et al., 1991; Sica Correspondence to: E.W. Ades, Biological Products Branch, Center for Infectious Diseases, Centers for Disease Control, 1600 Clifton Road, l-3205 (D34). Atlanta, Georgia 30333, USA.
84
et al., 1990; Bevilacqua et al., 1987) and metastasizing tumor cells (Kramer et al., 1979; Rice et al., 1988; Nicolson, 1989) prior to their passage from the intravascular compartment into tissue, and endothelial cells also secrete proinflammatory cytokines (Rossi et al., 1985). Under certain circumstances, endothelial cells may act as antigen-presenting cells (Burger et al., 1981; Pober et al., 1983; Wagner et al., 1984; Wagner et al., 1985; Pober et al., 1986; Burger et al., 1987; Lawley et al., 1987). Studies utilizing microvascular endothelial cells are relatively few because of substantial difficulties in the isolation and growth of these cells (Folkman et al., 1979). We developed one such line of immortalized human microvascular endothelial cells, called CDC/EU.HMEC-1 (Ades et al., 1992). Briefly, human dermal microvascular endothelial cells from foreskin were transfected with a PBR-322-based plasmid, containing the coding region for the simian virus 40A gene product, large T antigen, and then the cells were immortalized. These cells have been passaged 60 times to date and show no signs of senescence. The cells exhibit typical ‘cobblestone’ morphology when grown in monolayer culture, express and secrete von Willebrand’s factor, take up acetylated low-density lipoprotein, and rapidly form tubes when cultured on matrigel. The viral spectra of these immortalized human microvascular endothelial cells were examined and their ability to support the growth of common viruses from several viral genera is described.
Materials and Methods Cell culture
CDC/EU.HMEC-1 cells were grown at 37°C in 5% CO2 in a growth medium consisting of the following components (Knedler et al., 1987): 200 ml of endothelial basal medium MCDB 131 (Clonetics, San Diego, CA)‘, 75 ml of normal heat-inactivated (HI) human serum (Irvine Scientific, Santa Ana, CA) or HI fetal bovine serum (Gibco, Grand Island, NY), 2.5 ml of 200 mM glutamine (Gibco), 2.8 ml of 100 x (final concentration) antibiotic-antimycotic solution (Gibco), hydrocortisone (HC) (2 PM, final concentration), and epidermal cell growth factor (EGF) (5 ng/ml, final concentration). Virus source, inoculation and quantitation Experiments six consecutive sterile gelatin HMEC-1 cells
were carried out, beginning with cell passage 19, for a total of passages of 7 days each. Glass tubes were pre-rinsed with 0.1% in 0.01 M phosphate-buffered saline (PBS), pH 7.2 diluent. in growth medium were added and incubated under CO* until
‘Use of trade names is for identification purposes only and does not imply endorsement Health Service or the U.S. Department of Health and Human Services.
by the Public
85
confluent (5-6 days). The viruses used were ‘wild’ strains representing the viruses often found in respiratory specimens, namely, those of the adenovirus, herpesvirus, paramyxovirus, and picornavirus genera. They were clinical isolates obtained from the past 10 years that had been stored at - 100°C. The inoculum size was 0.2 ml throughout. Inoculation of virus was performed as previously reported (Castells et al., 1990; Hierholzer et al., 1987, 1980, 1990). Briefly, the growth medium was decanted by inversion, and the cells were ready for inoculation at this point for most viruses; for paramyxovirus cultures, the tubes were decanted and then rinsed once with maintenance medium containing 1.5 pg/ml of trypsin, as described for NCI-H292 cells (Castells et al., 1990). The inoculum was adsorbed ‘dry’ to the cell monolayer for 1 h at ambient temperature, 1.O ml of maintenance medium was added, the tubes were tightly capped, and the cultures were incubated at 36.3”C for 7 days and read for cytopathologic effects (CPE) every other day. At the end of the incubation period, the monolayers were scraped, and 0.3 ml was passed into fresh monolayers in the usual fashion, without an intervening freezing step. For parainfluenza and mumps viruses, RPMI-1640 plus 1.5 pg/ml of trypsin was used as the maintenance medium rather than EMEMgsFCz (Castells et al., 1990). Further, respiratory syncytial virus (RSV), parainfluenza and mumps viruses were grown as roller cultures rather than stationary cultures, because these viruses grow best under roller conditions. All cultures were stored at -70°C after being subpassed until all six consecutive passages were complete; they were then tested for quantitative evidence of virus growth. Quantitation was done by TR-FIA for adenovirus (Hierholzer et al., 1987), RSV, and the paramyxoviruses (Hierholzer et al., 1989), and enterovirus 70 (24); by electron microscopy for herpes viruses; and by infectivity titration in HLF cells for the reoviruses and picornaviruses. Infection with human immunodeficiency virus (HIV) was done with two different strains (LAV, a T-lymphocyte tropic strain, and BAL, a monocytetropic strain) at multiplicities of infection from 1 to 100. We monitored reverse transcriptase activity and levels of gp24 antigen as indicators of infectivity. For infection with human herpesvirus(HHV-6), Epstein-Barr virus (EBV) and cytomegalovirus (CMV), media were removed from the confluent HMEC1 monolayers, replaced with 2 ml of virus inoculum and adsorbed for 2 h. The monolayers were then washed and fresh media were added. Monolayers were infected with HHV-6 strain 229 using cell-free virus obtained from lightly sonicated infected human cord blood lymphocytes (CBL) or intact infected cells. EBV was prepared from the cell culture supernatant of the B95.8 cell line. The AD169 strain of CMV was prepared by sonicating infected HLF cells to release virus, pelleting the debris, and using the supernatant as inoculum. Cultures were monitored for CPE for 1 week. The HHV-6-infected HMEC cells were passaged 4-times and CMV-infected HMEC cells were passaged twice. After the final passage, all cultures were analyzed for viral DNA by slot blot, as previously described (Black et al., 1989). Hybridizations were performed using a cocktail of HHV-6 plasmid clones (Linquester et al., unpublished results), a
86
plasmid containing the BumA fragment of the EBV genome (Dambaugh et al., 1980) and a cosmid containing the 1049 fragment of the CMV genome (Fleckenstein et al., 1982). Human placental DNA was used as a control. HHVB-infected cultures were also analyzed by an anti-complement immunofluorescence (ACIF) assay previously described (Black et al., 1989).
Results
Table 1 shows the results of replication of 20 representative respiratory viruses. Adenoviruses representing the predominant pathogenic subgroups (B, TABLE 1 Replication
of 20 representative
Genus
Virus
respiratory
viruses in CDC/EU.HMEC-1
CPE
Virus quantitation
Avg. score
1
endothelial
cells
after passage no.a 2
4
6
st.
rl.
st.
rl.
st.
rl.
st.
rl. 450 266 463
Adenoviridae Adeno. 3 (B) Adeno. 2 (C) Adeno. 8 (D)
4 + , rounding 4 + , rounding 4 + , rounding
353 429 369
350 421 366
342 452 761
359 419 499
471 473 515
391 203 636
458 416 556
Herpesviridae Herpes 1 Herpes 2
4 + , rounding 4 + , rounding
8 7
8 7
8 7
8 7
8 7
8 7
8 7
3
8
6
9
3
: 4 8 11
: 4 8 11
8 0 1 1
8 0 1 1
8 0 0 0
9 7 8
9 6 8
9 7 8
zl 7
8 6
: 7
t
:
:
8
8
5
Paramyxoviridae Mumps 2-3 + , degen. Para. 1 none Para. 4A none RSV, subgr. 1 none subgr. 1 none subgr. 2 none Picornaviridae Coxsackie Coxsackie Echovirus Poliovirus Enterovirus Enterovirus Rhinovirus Rhinovirus
A9 B2 15 1 70 71
Reoviridae Reovirus 3
1 + , degen. 2 + , degen. 3 + , degen. 2 + , degen. none 2 + , degen. 1 + , degen. 1 + , degen.
4 + , degen.
8
8
8
8
8
‘st. = stationary culture 7 days, 36.3”C; rl. = roller culture, same conditions. Numbers in body of table are: for Adenoviridae, Paramyxoviridae, and enterovirus 70 x lo3 counts/s in TR-FIA; for Herpesviridae, virus titer as log,e/ml, as estimated by pseudoreplica electron microscopy; for the remaining Picornaviridae and Reoviridae, virus titer as logrs/ml as determined by 7-day infectivity titrations in HLF cells. All TR-FIA counts and virus titers are rounded off for ease in presentation.
87
C, and D) grew very well in the HMEC-1, with significant CPE and no loss of titer between the first and sixth passage under either roller or stationary conditions. Results were similar for herpes simplex types 1 and 2. Mumps virus replicated well in the endothelial cells, and from this we can infer that parainfluenzavirus 2 and 3 would also grow well, because these three viruses usually have the same cell spectrum. Parainfluenza types 1 and 4, on the other hand, have a very restricted spectrum (Castells et al., 1990) and neither virus replicated in the endothelial cells. Two strains of subgroup 1 RSV and one strain of subgroup 2 RSV also failed to replicate in these cells. Most of the picornaviruses examined replicated to high infectivity titers in the new cell line. Coxsackievirus A9, which grows well in many epithelial and fibroblast cells, grew well here also; however, this result cannot be extrapolated to other coxsackie A serotypes because most of them do not grow well in cell culture (their growth in suckling mouse brain is a principal marker in picornavirus classification). The more homogenous coxsackie B, echo and polio viruses would probably all do well, as evidenced by the representative coxsackievirus B2, echovirus type 15, and polio type 1 strains used here. Among the newer enteroviruses, EV 70, the principal cause of acute hemorrhagic conjunctivitis, failed to grow, while the less fastidious EV 71, which causes hand-foot-mouth disease, did grow. Two rhinovirus isolates, one from Georgia in 1982 and one from Texas in 1985, grew to increasingly higher titers from the first through the sixth passage. In general, all the Picornaviridae replicated to higher titers when rolled rather than in stationary culture, the same as they do in other cell lines. Finally, reovirus type 3, a common throat and stool isolate, with no proven disease association, grew well in the HMEC-1 cells and was identifiable by a hemagglutination-inhibition test with human ‘0’ cells. The remaining Herpesviridae (CMV, EBV, HHV-6) and the Retroviridae (LAV, BAL) we inoculated did not replicate in HMEC-1 cells. Very low levels of HHV-6 DNA were detected in cultures infected with both infected intact and sonicated CBL; however, we observed no CPE or positive reaction in the ACIF analysis. Therefore, HMEC may be susceptible to HHV-6 but unable to support virus growth. Neither CPE nor viral DNA was observed in EBV- or CMV-infected HMEC cells or in uninfected controls. In four separate experiments using different passages of the HMEC-1 cells, we found no indication of HIV infection by either reverse transcriptase activity or levels of gp24 antigen.
Discussion This study was designed to assess whether the unique immortalized human microvascular endothelial cell line, CDC/EU.HMEC1, was susceptible to common viruses and had potential uses in the virology laboratory. The cell line was tested for its sensitivity to 25 laboratory strains of Adenoviridae,
88
Herpesviridae, Paramyxoviridae, Picornaviridae, Reoviridae, and Retroviridae. A wide range of virus susceptibility was demonstrated for this cell line. Clearly, adenovirus and herpes simplex virus grew the best, with significant CPE. Of the paramyxoviruses studied, only mumps virus grew well and generated CPE. This is unfortunate because we had hoped that these cells could serve as a substitute for primary rhesus monkey kidney cells in the primary isolation of paramyxoviruses from clinical specimens. Most of the enteroviruses, rhinoviruses, and reoviruses studied grew well, with typical CPE and production of infectious virus. Notably, however, EV 70, a fastidious virus causing epidemics of eye disease (Hierholzer and Pallanshr, 1989), did not replicate in these cells. We examined and found these cells to be susceptible to HHV-6 virus; however, CPE was not observed. The cells were not susceptible to HIV infection, even in the presence of enriched cell growth factors. Numerous reports have previously found by histological detection that vascular endothelial cells were susceptible to viral infection including the Picornaviridae in pigs (Tsangaris et al., 1989), Theilers murine encephalomyelitis virus in nu/nu mice (Zurbriggen and Fujinamir, 1988) and coxsackievirus in humans (Iwasaki et al., 1985a; Iwasaki et al., 1985b). This latter report goes on to suggest that blood flow disturbance due to endothelial cell damage is a cause of myocardial lesions. These cells are susceptible to infection with some coxsackieviruses and may thus be a very useful tool to extrapolate the interplay of vascular endothelial cells in myocardium. There is considerable evidence indicating that certain viruses attack distinct cell types with resultant pathogenesis. Since viruses of different viral genera infected these endothelial cells, an element, such as a common integrin, could explain the growth of these viruses. Alternatively, several reports have indicated that specially functioning cells play an essential role in the presentation of antigens to effector lymphocytes in many immune responses (Katz et al., 1985). Our current research involves investigation of whether these cells possess the ability to process and present viral antigen, as well as whether this immunologic response is major histocompatibility complex-dependent.
References Ades, E.W., Candal, F.J., Swerlick, R.A., George, V.G., Summers, S., Bosse, D. and Lawley, T.J. (1992) HMEC-I : Establishment of an Immortalized Human Microvascular Endothelial Cell Line. J. Invest. Derm., in press. Bevilacqua, M.P., Pober, J.S., Mendrick, D.L., Cotran, R.S. and Gimbrone, Jr., M.A. (1987) Identification of an inducible endothelial-leukocyte adhesion molecule. Proc. Natl. Acad. Sci. USA 84, 9238-9242. Black, J.B., Sanderlin, K.C., Goldsmith, C.S., Gary, H.E., Lopez, C. and Pellett, P.E. (1989) Growth properties of human herpesvirusstrain 229. J. Virol. Methods 26, 133-146. Burger, D.R., Ford, D., Vetto, R.M., Hamblin, A., Goldstein, A., Hubbard, M. and Dumonde, D.C. (1981) Endothelial cell presentation of antigen to human T cells. Human Immunol. 3, 209230.
89 Burger, D.R., Jones, R.E. and Vetto, R.M. (1987) Accessory cell function of the endothelial cell: its role in the cellular immune response. Int. J. Tiss. Reac. IX, 365-370. Castells, E., George, V.G. and Hierholzer, J.C. (1990) NCI-H292 as an alternative cell line for the isolation and propagation of the human paramyxoviruses. Arch. Viral. 115, 2777288. Cotran, R.S. (1987) New roles for the endothelium in inflammation and immunity. Am. J. Pathol. 129, 407413. Dambaugh, T., Beisel, C., Hummel, M., King, W., Fennewald, S., Cheung, A., Heller, M., RaabTraub, N. and Kieff. E. (1980) Epstein-Barr virus (B95-8) DNA VII: Molecular cloning and detailed mapping. Proc. Nat]. Acad. Sci. USA 77, 299993003. Fleckenstein, B., Muller, I. and Collins, J. (1982) Cloning of the complete human cytomegalovirus genus in cosmids. Gene 18, 3946. Folkman, J.. Haudenschild, C.C. and Zetter, B.R. (1979) Long-term culture of capillary endothelial cells. Proc. Natl. Acad. Sci. USA 76, 5217-5221. Gamble, J.R. and Vadas, M.A. (1991) Endothelial cell adhesiveness for human T-lymphocytes is inhibited by transforming growth factor-beta. J. Immunol. 146, 1149-1154. Hierholzer, J.C., Anderson, L.J. and Halonen, P.E. (1990) Monoclonal time-resolved fluoroimmunoassay: sensitive systems for the rapid diagnosis of respiratory virus infections. Med. Virol. 9, 1745. Hierholzer, J.C., Bingham, P.G., Coombs, R.A., Johansson, K.H.. Anderson, L.J. and Halonen, P.E. (1989) Comparison of monoclonal antibody time-resolved fluoroimmunoassay with monoclonal antibody capture-biotinylated detector enzyme immunoassay for respiratory syncytial virus and parainfluenza virus antigen detection. J. Clin. Microbial. 27, 1243-1249. Hierholzer, J.C., Johansson, K.H., Anderson, L.J., Tsou, C.J. and Halonen, P.E. (1987) Comparison of monoclonal time-resolved fluoroimmunoassay with monoclonal capturebiotinylated detector enzyme immunoassay for adenovirus antigen detection. J. Clin. Microbial. 25, 166221667. Hierholzer, J.C. and Pallansch. M.A. (1989) Acute Hemorrhagic Conjunctivitis in the Western Hemisphere. In: K. Ishii, Y. Uchida, K. Miyamura and S. Yamazaki (Eds), Acute Hemorrhagic Conjunctivitis, University of Tokyo Press. Iwasaki. T., Monma. N., Satodate, R., et al. (1985) An immunofluorescent study of generalized Coxsackie virus B3 infection in a newborn infant. Acta Pathol. Jpn. 35, 741-748. Iwasaki, T., Monma, N., Satodate, R., et al. (1985) Myocardial lesions by Coxsackie virus B-3 and cytomegalovirus infection in infants. Heart Vessels Suppl. 167-172. Katz, S.I., Cooper, K.D., Iijima, M. and Tsuchida. T. (1985) The role of Langerhans cells in antigen presentation. J. Invest. Derm. 85, 9698. Knedler, A. and Ham, K.G. (1987) Optimized medium for clonal growth of human microvascular endothelial cells with minimal serum. In Vitro 23, 481491. Kramer, R.H. and Nicholson, G.L. (1979) Interactions of tumor cells with vascular endothelial cell monolayers: a model of metastatic invasion. Proc. Natl. Acad. Sci. USA 76, 57045708. Lawley, T.J. and Kubota, Y. (1987) Cutaneous microvascular endothehal cells. In: Una S. Ryan (ed), Endothehal Cells, Vol. III, CRC Press, Boca Raton, pp. 2299240. Mantonvani, A. and Dejana, E. (1989) Cytokines as communication b753 signals between leukocytes and endothelial cells. Immunol. Today 10, 37&375. Nicolson, G.L. (1989) Metastatic tumor cell interactions with endothehum, basement membrane and tissue. Curr. Opinion Cell Biol. 1, 1009-1019. Pober, J.S. (1988) Cytokine-mediated activation of vascular endothehum. Am. J. Pathol. 133, 426 433. Pober, J.S., Collins, T.. Gimbrone, M.A., Libby, P. and Reiss, C.S. (1986) Inducible expression of class II major histocompatibility complex antigens and the immunogenicity of vascular endothelium. Transplantation 41, 141-146. Pober, J.S., Gimbrone, Jr., M.A., Cotran, R.S., Reiss, C.S., Burakoff, S.J., Fiers, W. and Ault, K.A. (1983) Ia expression by vascular endothehum is inducible by activated T cells and by human gamma interferon. J. Exp. Med. 157, 1339-1353. Rice, G.E.. Gimbrone, Jr., M.A. and Bevilacqua M.P. (1988) Tumor cell-endothelial interactions.
90 Increased adhesion of human melanoma cells to activated vascular endothelium. Am. J. Pathol. 133, 204210. Rossi, V., Breviario, F., Ghezzi, P., Dejana, E. and Mantovani, A. (1985) Prostacyclin synthesis induced in vascular cells by interleukin- 1. Science 229, 174-l 76. Sica, A., Wang, J.M., Colotta, F., Dejana, E., Mantovani, A., Oppenheim, J.J., Larsen, C.G., Zachariae, C.O.C. and Matsushima, K. (1990) Monocyte chemotactic and activating factor gene expression induced in endothelial cells by IL-l and tumor necrosis factor. J. Immunol. 144, 30343038. Tsangaris, T., Lekkas, S. and Kanakoudis, G. (1989) Changes in the myocardium in encephalomyocarditis of pigs. DTW Dtsch TierarzH Wochenschr (Germany) 96, 301-330. Wagner, C.R., Vetto, R.M. and Burger, D.R. (1985) Subcultured human endothelial cells function independehtly as fully competent antigen presenting cells. Human Immunol. 13, 3347. Wagner, C.R., Vetto, R.M. and Burger, D.R. (1984) The mechanisms of antigen presentation by endothelial cells. Immunobiol. 168, 453-469. Zurbriggen, A. and Fujinami, R.S. (1988) Theilers virus infection in nude mice: Viral RNA in vascular endothelial cells. J. Virol. 62, 3589-3596.
83
VIRMET 01365
Viral susceptibility of an immortalized human microvascular endothelial cell line Edwin W. Ades”, John C. Hierholzer”, Velma Georgea, and Francisco Candal”
Jodi Blackb
“Biological Products Branch, bViral Exanthems and Herpesvirus Branch, and ‘Respiratory and Enteric Viruses Branch, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, GA (USA) (Accepted 28 February
1992)
Summary CDC/EU.HMEC1 is the first immortalized human microvascular endothelial cell line that retains morphologic, phenotypic, and functional characteristics of a normal human microvascular endothelial cell. This study evaluates a variety of viruses and their effects on this human endothelial cell line. The data indicate that adenoviruses, some herpesviruses, reoviruses and most picornaviruses grow well in HMEC-1, with distinctive cytopathic effects. The paramyxoviruses, however, do not appear to propagate, nor does HIV. The findings indicate that microvascular endothelial cells may act as a reservoir of these viruses; it also suggests the possibility that microvascular endothelium could be involved in the processing and presentation of antigen to immune cells. Human microvascular
endothelium; Viral susceptibility
Introduction Endothelial cells play a critical role in such important physiologic and pathophysiologic events as inflammation, wound healing, angiogenesis, and hemostasis (Cotran, 1987; Pober, 1988). Endothelium mediates the binding of peripheral blood leukocytes (Mantonvani et al., 1989; Gamble et al., 1991; Sica Correspondence to: E.W. Ades, Biological Products Branch, Center for Infectious Diseases, Centers for Disease Control, 1600 Clifton Road, l-3205 (D34). Atlanta, Georgia 30333, USA.
84
et al., 1990; Bevilacqua et al., 1987) and metastasizing tumor cells (Kramer et al., 1979; Rice et al., 1988; Nicolson, 1989) prior to their passage from the intravascular compartment into tissue, and endothelial cells also secrete proinflammatory cytokines (Rossi et al., 1985). Under certain circumstances, endothelial cells may act as antigen-presenting cells (Burger et al., 1981; Pober et al., 1983; Wagner et al., 1984; Wagner et al., 1985; Pober et al., 1986; Burger et al., 1987; Lawley et al., 1987). Studies utilizing microvascular endothelial cells are relatively few because of substantial difficulties in the isolation and growth of these cells (Folkman et al., 1979). We developed one such line of immortalized human microvascular endothelial cells, called CDC/EU.HMEC-1 (Ades et al., 1992). Briefly, human dermal microvascular endothelial cells from foreskin were transfected with a PBR-322-based plasmid, containing the coding region for the simian virus 40A gene product, large T antigen, and then the cells were immortalized. These cells have been passaged 60 times to date and show no signs of senescence. The cells exhibit typical ‘cobblestone’ morphology when grown in monolayer culture, express and secrete von Willebrand’s factor, take up acetylated low-density lipoprotein, and rapidly form tubes when cultured on matrigel. The viral spectra of these immortalized human microvascular endothelial cells were examined and their ability to support the growth of common viruses from several viral genera is described.
Materials and Methods Cell culture
CDC/EU.HMEC-1 cells were grown at 37°C in 5% CO2 in a growth medium consisting of the following components (Knedler et al., 1987): 200 ml of endothelial basal medium MCDB 131 (Clonetics, San Diego, CA)‘, 75 ml of normal heat-inactivated (HI) human serum (Irvine Scientific, Santa Ana, CA) or HI fetal bovine serum (Gibco, Grand Island, NY), 2.5 ml of 200 mM glutamine (Gibco), 2.8 ml of 100 x (final concentration) antibiotic-antimycotic solution (Gibco), hydrocortisone (HC) (2 PM, final concentration), and epidermal cell growth factor (EGF) (5 ng/ml, final concentration). Virus source, inoculation and quantitation Experiments six consecutive sterile gelatin HMEC-1 cells
were carried out, beginning with cell passage 19, for a total of passages of 7 days each. Glass tubes were pre-rinsed with 0.1% in 0.01 M phosphate-buffered saline (PBS), pH 7.2 diluent. in growth medium were added and incubated under CO* until
‘Use of trade names is for identification purposes only and does not imply endorsement Health Service or the U.S. Department of Health and Human Services.
by the Public
85
confluent (5-6 days). The viruses used were ‘wild’ strains representing the viruses often found in respiratory specimens, namely, those of the adenovirus, herpesvirus, paramyxovirus, and picornavirus genera. They were clinical isolates obtained from the past 10 years that had been stored at - 100°C. The inoculum size was 0.2 ml throughout. Inoculation of virus was performed as previously reported (Castells et al., 1990; Hierholzer et al., 1987, 1980, 1990). Briefly, the growth medium was decanted by inversion, and the cells were ready for inoculation at this point for most viruses; for paramyxovirus cultures, the tubes were decanted and then rinsed once with maintenance medium containing 1.5 pg/ml of trypsin, as described for NCI-H292 cells (Castells et al., 1990). The inoculum was adsorbed ‘dry’ to the cell monolayer for 1 h at ambient temperature, 1.O ml of maintenance medium was added, the tubes were tightly capped, and the cultures were incubated at 36.3”C for 7 days and read for cytopathologic effects (CPE) every other day. At the end of the incubation period, the monolayers were scraped, and 0.3 ml was passed into fresh monolayers in the usual fashion, without an intervening freezing step. For parainfluenza and mumps viruses, RPMI-1640 plus 1.5 pg/ml of trypsin was used as the maintenance medium rather than EMEMgsFCz (Castells et al., 1990). Further, respiratory syncytial virus (RSV), parainfluenza and mumps viruses were grown as roller cultures rather than stationary cultures, because these viruses grow best under roller conditions. All cultures were stored at -70°C after being subpassed until all six consecutive passages were complete; they were then tested for quantitative evidence of virus growth. Quantitation was done by TR-FIA for adenovirus (Hierholzer et al., 1987), RSV, and the paramyxoviruses (Hierholzer et al., 1989), and enterovirus 70 (24); by electron microscopy for herpes viruses; and by infectivity titration in HLF cells for the reoviruses and picornaviruses. Infection with human immunodeficiency virus (HIV) was done with two different strains (LAV, a T-lymphocyte tropic strain, and BAL, a monocytetropic strain) at multiplicities of infection from 1 to 100. We monitored reverse transcriptase activity and levels of gp24 antigen as indicators of infectivity. For infection with human herpesvirus(HHV-6), Epstein-Barr virus (EBV) and cytomegalovirus (CMV), media were removed from the confluent HMEC1 monolayers, replaced with 2 ml of virus inoculum and adsorbed for 2 h. The monolayers were then washed and fresh media were added. Monolayers were infected with HHV-6 strain 229 using cell-free virus obtained from lightly sonicated infected human cord blood lymphocytes (CBL) or intact infected cells. EBV was prepared from the cell culture supernatant of the B95.8 cell line. The AD169 strain of CMV was prepared by sonicating infected HLF cells to release virus, pelleting the debris, and using the supernatant as inoculum. Cultures were monitored for CPE for 1 week. The HHV-6-infected HMEC cells were passaged 4-times and CMV-infected HMEC cells were passaged twice. After the final passage, all cultures were analyzed for viral DNA by slot blot, as previously described (Black et al., 1989). Hybridizations were performed using a cocktail of HHV-6 plasmid clones (Linquester et al., unpublished results), a
86
plasmid containing the BumA fragment of the EBV genome (Dambaugh et al., 1980) and a cosmid containing the 1049 fragment of the CMV genome (Fleckenstein et al., 1982). Human placental DNA was used as a control. HHVB-infected cultures were also analyzed by an anti-complement immunofluorescence (ACIF) assay previously described (Black et al., 1989).
Results
Table 1 shows the results of replication of 20 representative respiratory viruses. Adenoviruses representing the predominant pathogenic subgroups (B, TABLE 1 Replication
of 20 representative
Genus
Virus
respiratory
viruses in CDC/EU.HMEC-1
CPE
Virus quantitation
Avg. score
1
endothelial
cells
after passage no.a 2
4
6
st.
rl.
st.
rl.
st.
rl.
st.
rl. 450 266 463
Adenoviridae Adeno. 3 (B) Adeno. 2 (C) Adeno. 8 (D)
4 + , rounding 4 + , rounding 4 + , rounding
353 429 369
350 421 366
342 452 761
359 419 499
471 473 515
391 203 636
458 416 556
Herpesviridae Herpes 1 Herpes 2
4 + , rounding 4 + , rounding
8 7
8 7
8 7
8 7
8 7
8 7
8 7
3
8
6
9
3
: 4 8 11
: 4 8 11
8 0 1 1
8 0 1 1
8 0 0 0
9 7 8
9 6 8
9 7 8
zl 7
8 6
: 7
t
:
:
8
8
5
Paramyxoviridae Mumps 2-3 + , degen. Para. 1 none Para. 4A none RSV, subgr. 1 none subgr. 1 none subgr. 2 none Picornaviridae Coxsackie Coxsackie Echovirus Poliovirus Enterovirus Enterovirus Rhinovirus Rhinovirus
A9 B2 15 1 70 71
Reoviridae Reovirus 3
1 + , degen. 2 + , degen. 3 + , degen. 2 + , degen. none 2 + , degen. 1 + , degen. 1 + , degen.
4 + , degen.
8
8
8
8
8
‘st. = stationary culture 7 days, 36.3”C; rl. = roller culture, same conditions. Numbers in body of table are: for Adenoviridae, Paramyxoviridae, and enterovirus 70 x lo3 counts/s in TR-FIA; for Herpesviridae, virus titer as log,e/ml, as estimated by pseudoreplica electron microscopy; for the remaining Picornaviridae and Reoviridae, virus titer as logrs/ml as determined by 7-day infectivity titrations in HLF cells. All TR-FIA counts and virus titers are rounded off for ease in presentation.
87
C, and D) grew very well in the HMEC-1, with significant CPE and no loss of titer between the first and sixth passage under either roller or stationary conditions. Results were similar for herpes simplex types 1 and 2. Mumps virus replicated well in the endothelial cells, and from this we can infer that parainfluenzavirus 2 and 3 would also grow well, because these three viruses usually have the same cell spectrum. Parainfluenza types 1 and 4, on the other hand, have a very restricted spectrum (Castells et al., 1990) and neither virus replicated in the endothelial cells. Two strains of subgroup 1 RSV and one strain of subgroup 2 RSV also failed to replicate in these cells. Most of the picornaviruses examined replicated to high infectivity titers in the new cell line. Coxsackievirus A9, which grows well in many epithelial and fibroblast cells, grew well here also; however, this result cannot be extrapolated to other coxsackie A serotypes because most of them do not grow well in cell culture (their growth in suckling mouse brain is a principal marker in picornavirus classification). The more homogenous coxsackie B, echo and polio viruses would probably all do well, as evidenced by the representative coxsackievirus B2, echovirus type 15, and polio type 1 strains used here. Among the newer enteroviruses, EV 70, the principal cause of acute hemorrhagic conjunctivitis, failed to grow, while the less fastidious EV 71, which causes hand-foot-mouth disease, did grow. Two rhinovirus isolates, one from Georgia in 1982 and one from Texas in 1985, grew to increasingly higher titers from the first through the sixth passage. In general, all the Picornaviridae replicated to higher titers when rolled rather than in stationary culture, the same as they do in other cell lines. Finally, reovirus type 3, a common throat and stool isolate, with no proven disease association, grew well in the HMEC-1 cells and was identifiable by a hemagglutination-inhibition test with human ‘0’ cells. The remaining Herpesviridae (CMV, EBV, HHV-6) and the Retroviridae (LAV, BAL) we inoculated did not replicate in HMEC-1 cells. Very low levels of HHV-6 DNA were detected in cultures infected with both infected intact and sonicated CBL; however, we observed no CPE or positive reaction in the ACIF analysis. Therefore, HMEC may be susceptible to HHV-6 but unable to support virus growth. Neither CPE nor viral DNA was observed in EBV- or CMV-infected HMEC cells or in uninfected controls. In four separate experiments using different passages of the HMEC-1 cells, we found no indication of HIV infection by either reverse transcriptase activity or levels of gp24 antigen.
Discussion This study was designed to assess whether the unique immortalized human microvascular endothelial cell line, CDC/EU.HMEC1, was susceptible to common viruses and had potential uses in the virology laboratory. The cell line was tested for its sensitivity to 25 laboratory strains of Adenoviridae,
88
Herpesviridae, Paramyxoviridae, Picornaviridae, Reoviridae, and Retroviridae. A wide range of virus susceptibility was demonstrated for this cell line. Clearly, adenovirus and herpes simplex virus grew the best, with significant CPE. Of the paramyxoviruses studied, only mumps virus grew well and generated CPE. This is unfortunate because we had hoped that these cells could serve as a substitute for primary rhesus monkey kidney cells in the primary isolation of paramyxoviruses from clinical specimens. Most of the enteroviruses, rhinoviruses, and reoviruses studied grew well, with typical CPE and production of infectious virus. Notably, however, EV 70, a fastidious virus causing epidemics of eye disease (Hierholzer and Pallanshr, 1989), did not replicate in these cells. We examined and found these cells to be susceptible to HHV-6 virus; however, CPE was not observed. The cells were not susceptible to HIV infection, even in the presence of enriched cell growth factors. Numerous reports have previously found by histological detection that vascular endothelial cells were susceptible to viral infection including the Picornaviridae in pigs (Tsangaris et al., 1989), Theilers murine encephalomyelitis virus in nu/nu mice (Zurbriggen and Fujinamir, 1988) and coxsackievirus in humans (Iwasaki et al., 1985a; Iwasaki et al., 1985b). This latter report goes on to suggest that blood flow disturbance due to endothelial cell damage is a cause of myocardial lesions. These cells are susceptible to infection with some coxsackieviruses and may thus be a very useful tool to extrapolate the interplay of vascular endothelial cells in myocardium. There is considerable evidence indicating that certain viruses attack distinct cell types with resultant pathogenesis. Since viruses of different viral genera infected these endothelial cells, an element, such as a common integrin, could explain the growth of these viruses. Alternatively, several reports have indicated that specially functioning cells play an essential role in the presentation of antigens to effector lymphocytes in many immune responses (Katz et al., 1985). Our current research involves investigation of whether these cells possess the ability to process and present viral antigen, as well as whether this immunologic response is major histocompatibility complex-dependent.
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