Department of Medical Virology, University of Tübingen, Calwerstrasse 7/6, D-72076 Tübingen, Germany1
Department of Medicine, University of Tübingen, Tübingen, Germany2
Author for correspondence: Christian Sinzger. Fax +49 7071 295790.e-mail christian.sinzger{at}med.uni-tuebingen.de
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Abstract |
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Immature DC were generated from adherent peripheral blood mononuclear cells (PBMC) as recently described (Brossart & Bevan, 1997 ; Sallusto & Lanzavecchia, 1994
). Briefly, peripheral blood was obtained from HCMV-seronegative donors. PBMC were prepared by centrifugation on a FicollHypaque (Lymphoprep; Nycomed) density gradient, resuspended in RPMI containing 10% FCS, 2·4 mmol/l glutamine and 100 µg/ml gentamicin, and allowed to adhere to 6-well tissue culture dishes. After 2 h at 37 °C in 5% CO2 atmosphere, the non-adherent cells were removed. The adherent fractions were cultured in RPMI10% FCS (Gibco), supplemented with 1000 U/ml interleukin-4 (IL-4; Genzyme) and 100 ng/ml granulocytemacrophage colony-stimulating factor (GM-CSF; Leukomax, Sandoz) on the initial day of culture. Cytokines were replenished on days 2, 4 and 6. During this incubation period a homogeneous non-adherent cell population with typical immature DC morphology developed (Fig. 1b
). On day 7, non-adherent cells were collected by moderate aspiration and transferred to fresh 6-well plates. These cells consisted of 90% immature DC as determined by the CD1a+/CD40+/CD80+/CD86+/HLA-DR+/CD14- phenotype (Fig. 1a
) and were used for all experiments described. The phenotype of these immature DC was further confirmed by their ability to differentiate into mature DC when TNF
was added after 7 days incubation with IL4+GM-CSF (Brossart et al., 1998
).
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We next examined whether immature DC infected by HCMV strains TB40/E and VHL/E were permissive for the complete virus replication cycle. We analysed IE, early and late antigen expression in immature DC at various intervals after infection. In particular, we detected the IE1 and IE2 antigens (UL122/123; Biosoft), the early antigen p52 (UL44, MAb BS510; Biotest), and the late viral proteins pp150 (UL32, MAb XP1; Behringwerke) and major capsid antigen (MCP, UL86, MAb 28-4; kindly provided by W. Britt) in cytospin preparations by an indirect immunoperoxidase technique (Fig. 2). Antigens of all phases of HCMV replication were detectable in up to 93% of cells. All antigens occurred with typical localization but with slightly delayed kinetics as compared to standard fibroblast cultures. IE antigens were detectable from day 1 after infection, early antigen was detectable from day 2 after infection, and late antigen was detectable from day 3 after infection. At that time-point, CPE occurred in infected cultures, namely re-adherence of the cells, formation of syncytia, and formation of nuclear inclusions. Starting on day 68 after infection, the number of viable cells decreased significantly and cell detritus appeared. Lysis of the infected cultures was complete on day 12 after infection. Cell lysis appeared to be caused by HCMV infection, since lysis of DC cultures was not detected during 12 days after mock-infection. In summary, these experiments demonstrated that immature DC were permissive to the complete virus replication cycle and that infection was cytopathic and lytic.
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If interstrain differences in the infection of DC also applied to the in vivo situation, this would provide one explanation for the highly variable course of HCMV infections in the host. In terms of virus pathogenesis, this may include a variable degree of primary replication in DC at the sites of virus entry to mucosal tissues, a variable degree of virus dissemination by infected DC via the lymph, and a variable degree of interaction with immune functions of DC. HCMV infection of DC could have different effects on the immune function of DC. Whereas efficient presentation of viral antigens has been reported for DC infected by influenza virus (Bender et al., 1998 ; Bhardwaj et al., 1994
) or replication-deficient adenovirus (Brossart et al., 1997
), measles virus caused suppression of immune functions in infected DC (Fugier Vivier et al., 1997
; Grosjean et al., 1997
; Kaiserlian et al., 1997
; Schnorr et al., 1997
). As down-regulation of the MHC I pathway has been demonstrated in HCMV-infected fibroblasts (Hengel et al., 1995
; Steinmassl & Hamprecht, 1994
), it will be interesting to analyse whether the same effect occurs in infected DC. In this context, the dramatic interstrain differences that we found regarding efficiency of immature DC infection may be relevant. While the expression of gene products down-regulating MHC I or II (Ahn et al., 1996
; Fruh et al., 1997
; Hengel et al., 1996
; Jones et al., 1996
; Lehner et al., 1997
; Machold et al., 1997
; Miller et al., 1998
; Wiertz et al., 1996
) is likely to occur during lytic infection of DC, such HCMV variants that cannot initiate viral gene expression in DC will most likely not interfere with the immune functions of these cells. These considerations are hypothetical, but the cell culture model presented here will enable future analyses of the functional effects of HCMV variants on infected immature DC.
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Acknowledgments |
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References |
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Bender, A., Albert, M., Reddy, A., Feldman, M., Sauter, B., Kaplan, G., Hellman, W. & Bhardwaj, N. (1998). The distinctive features of influenza virus infection of dendritic cells. Immunobiology 198, 552-567.[Medline]
Bhardwaj, N., Bender, A., Gonzalez, N., Bui, L. K., Garrett, M. C. & Steinman, R. M. (1994). Influenza virus-infected dendritic cells stimulate strong proliferative and cytolytic responses from human CD8+ T cells. Journal of Clinical Investigation 94, 797-807.[Medline]
Brossart, P. & Bevan, M. J. (1997). Presentation of exogenous protein antigens on major histocompatibility complex class I molecules by dendritic cells: pathway of presentation and regulation by cytokines. Blood 90, 1594-1599.
Brossart, P., Goldrath, A. W., Butz, E. A., Martin, S. & Bevan, M. J. (1997). Virus-mediated delivery of antigenic epitopes into dendritic cells as a means to induce CTL. Journal of Immunology 158, 3270-3276.[Abstract]
Brossart, P., Grunebach, F., Stuhler, G., Reichardt, V. L., Mohle, R., Kanz, L. & Brugger, W. (1998). Generation of functional human dendritic cells from adherent peripheral blood monocytes by CD40 ligation in the absence of granulocyte-macrophage colony-stimulating factor. Blood 92, 4238-4247.
Canque, B., Rosenzwajg, M., Camus, S., Yagello, M., Bonnet, M. L., Guigon, M. & Gluckman, J. C. (1996). The effect of in vitro human immunodeficiency virus infection on dendritic-cell differentiation and function. Blood 88, 4215-4228.
Cella, M., Sallusto, F. & Lanzavecchia, A. (1997). Origin, maturation and antigen presenting function of dendritic cells. Current Opinion in Immunology 9, 10-16.[Medline]
Cha, T. A., Tom, E., Kemble, G. W., Duke, G. M., Mocarski, E. S. & Spaete, R. R. (1996). Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. Journal of Virology 70, 78-83.[Abstract]
Einsele, H., Ehninger, G., Steidle, M., Fischer, I., Bihler, S., Gerneth, F., Vallbracht, A., Schmidt, H., Waller, H. D. & Muller, C. A. (1993). Lymphocytopenia as an unfavorable prognostic factor in patients with cytomegalovirus infection after bone marrow transplantation. Blood 82, 1672-1678.[Abstract]
Fruh, K., Ahn, K. & Peterson, P. A. (1997). Inhibition of MHC class I antigen presentation by viral proteins. Journal of Molecular Medicine 75, 18-27.[Medline]
Fugier Vivier, I., Servet Delprat, C., Rivailler, P., Rissoan, M. C., Liu, Y. J. & Rabourdin Combe, C. (1997). Measles virus suppresses cell-mediated immunity by interfering with the survival and functions of dendritic and T cells. Journal of Experimental Medicine 186, 813-823.
Grosjean, I., Caux, C., Bella, C., Berger, I., Wild, F., Banchereau, J. & Kaiserlian, D. (1997). Measles virus infects human dendritic cells and blocks their allostimulatory properties for CD4+ T cells. Journal of Experimental Medicine 186, 801-812.
Hahn, G., Jores, R. & Mocarski, E. S. (1998). Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells. Proceedings of the National Academy of Sciences, USA 95, 3937-3942.
Hart, D. N. (1997). Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 90, 3245-3287.
Hengel, H., Esslinger, C., Pool, J., Goulmy, E. & Koszinowski, U. H. (1995). Cytokines restore MHC class I complex formation and control antigen presentation in human cytomegalovirus-infected cells. Journal of General Virology 76, 2987-2997.[Abstract]
Hengel, H., Flohr, T., Hammerling, G. J., Koszinowski, U. H. & Momburg, F. (1996). Human cytomegalovirus inhibits peptide translocation into the endoplasmic reticulum for MHC class I assembly. Journal of General Virology 77, 2287-2296.[Abstract]
Ibanez, C. E., Schrier, R., Ghazal, P., Wiley, C. & Nelson, J. A. (1991). Human cytomegalovirus productively infects primary differentiated macrophages. Journal of Virology 65, 6581-6588.[Medline]
Jones, T. R., Wiertz, E. J., Sun, L., Fish, K. N., Nelson, J. A. & Ploegh, H. L. (1996). Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains. Proceedings of the National Academy of Sciences, USA 93, 11327-11333.
Kacani, L., Frank, I., Spruth, M., Schwendinger, M. G., Mullauer, B., Sprinzl, G. M., Steindl, F. & Dierich, M. P. (1998). Dendritic cells transmit human immunodeficiency virus type 1 to monocytes and monocyte-derived macrophages. Journal of Virology 72, 6671-6677.
Kaiserlian, D., Grosjean, I. & Caux, C. (1997). Infection of human dendritic cells by measles virus induces immune suppression. Advances in Experimental Medicine and Biology 417, 421-423.[Medline]
Kemble, G., Duke, G., Winter, R. & Spaete, R. (1996). Defined large-scale alterations of the human cytomegalovirus genome constructed by cotransfection of overlapping cosmids. Journal of Virology 70, 2044-2048.[Abstract]
Klagge, I. M. & Schneider-Schaulies, S. (1999). Virus interactions with dendritic cells. Journal of General Virology 80, 823-833.
Lathey, J. L. & Spector, S. A. (1991). Unrestricted replication of human cytomegalovirus in hydrocortisone-treated macrophages. Journal of Virology 65, 6371-6375.[Medline]
Lehner, P. J., Karttunen, J. T., Wilkinson, G. W. & Cresswell, P. (1997). The human cytomegalovirus US6 glycoprotein inhibits transporter associated with antigen processing-dependent peptide translocation. Proceedings of the National Academy of Sciences, USA 94, 6904-6909.
Machold, R. P., Wiertz, E. J., Jones, T. R. & Ploegh, H. L. (1997). The HCMV gene products US11 and US2 differ in their ability to attack allelic forms of murine major histocompatibility complex (MHC) class I heavy chains. Journal of Experimental Medicine 185, 363-366.
Miller, D. M., Rahill, B. M., Boss, J. M., Lairmore, M. D., Durbin, J. E., Waldman, J. W. & Sedmak, D. D. (1998). Human cytomegalovirus inhibits major histocompatibility complex class II expression by disruption of the Jak/Stat pathway. Journal of Experimental Medicine 187, 675-683.
Minton, E. J., Tysoe, C., Sinclair, J. H. & Sissons, J. G. (1994). Human cytomegalovirus infection of the monocyte/macrophage lineage in bone marrow. Journal of Virology 68, 4017-4021.[Abstract]
Sallusto, F. & Lanzavecchia, A. (1994). Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. Journal of Experimental Medicine 179, 1109-1118.[Abstract]
Schnorr, J. J., Xanthakos, S., Keikavoussi, P., Kampgen, E., ter Meulen, V. & Schneider Schaulies, S. (1997). Induction of maturation of human blood dendritic cell precursors by measles virus is associated with immunosuppression. Proceedings of the National Academy of Sciences, USA 94, 5326-5331.
Sinzger, C., Grefte, A., Plachter, B., Gouw, A. S., The, T. H. & Jahn, G. (1995). Fibroblasts, epithelial cells, endothelial cells and smooth muscle cells are major targets of human cytomegalovirus infection in lung and gastrointestinal tissues. Journal of General Virology 76, 741-750.[Abstract]
Sinzger, C., Plachter, B., Grefte, A., The, T. H. & Jahn, G. (1996). Tissue macrophages are infected by human cytomegalovirus in vivo. Journal of Infectious Diseases 173, 240-245.[Medline]
Sinzger, C., Knapp, J., Plachter, B., Schmidt, K. & Jahn, G. (1997). Quantification of replication of clinical cytomegalovirus isolates in cultured endothelial cells and fibroblasts by a focus expansion assay. Journal of Virological Methods 63, 103-112.[Medline]
Soderberg-Naucler, C., Fish, K. N. & Nelson, J. A. (1998). Growth of human cytomegalovirus in primary macrophages. Methods 16, 126-138.[Medline]
Steinmassl, M. & Hamprecht, K. (1994). Double fluorescence analysis of human cytomegalovirus (HCMV) infected human fibroblast cultures by flow cytometry: increase of class I MHC expression on uninfected cells and decrease on infected cells. Archives of Virology 135, 75-87.[Medline]
Torok Storb, B., Fries, B., Stachel, D. & Khaira, D. (1993). Cytomegalovirus: variations in tropism and disease. Leukemia 7, S83-85.[Medline]
Waldman, W. J., Roberts, W. H., Davis, D. H., Williams, M. V., Sedmak, D. D. & Stephens, R. E. (1991). Preservation of natural endothelial cytopathogenicity of cytomegalovirus by propagation in endothelial cells. Archives of Virology 117, 143-164.[Medline]
Wiertz, E. J., Jones, T. R., Sun, L., Bogyo, M., Geuze, H. J. & Ploegh, H. L. (1996). The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell 84, 769-779.[Medline]
Received 26 July 1999;
accepted 22 October 1999.