Modulation of HLA class I antigen and ICAM-2 on endothelial cells after in vitro infection with human cytomegalovirus.

The aim of the present work is to investigate the expression of HLA class I antigen and intercellular adhesion molecule-2 (ICAM-2) on the surface of human umbilical cord endothelial cells (HUVEC) after infection with human cytomegalovirus (HCMV). The expression of HLA class I antigen and ICAM-2 were determined (using antibodies against HLA class I antigen and ICAM-2) by attachment inhibition assay and flow cytometric analysis. Attachment inhibition assay demonstrated that HCMV increased the expression of HLA class I antigen. This was confirmed by flow cytometric analysis, which showed an increase in HLA class I antigen expression on HCMV-infected HUVEC. The results of the expression of ICAM-2 using attachment inhibition assay revealed that ICAM-2 is involved significantly in the increased adhesion of T lymphocytes to HCMV-infected HUVEC. However, flow cytometric analysis revealed that there were no changes in the expression of ICAM-2 on HCMV-infected HUVEC. One possible explanation for this is that HCMV induces the activation of ICAM-2 on the surface of HCMV-infected endothelial cells without affecting its expression.

Human cytomegalovirus infection induces mRNA expression and secretion of plasminogen inhibitor type-1 in endothelial cells.

The aim of the study was to investigate whether infection of endothelial cells with human cytomegalovirus (HCMV) perturbs expression and production of plasminogen activator inhibitor type-1 (PAI-1). mRNA expression of PAI-1 was investigated by isolating total RNA from HCMV-infected and control cells, followed by Northern blotting and probing with 32P-labelled PAI-1 probe. Sandwich ELISA was used to investigate PAI-1 production. HCMV induced the expression of PAI-1-mRNA at 2-5 days postinfection (maximum expression was at 3 days postinfection which was 40% higher than control). HCMV also induced secretion of PAI-1 at 2-5 days postinfection. These results indicate that infection of endothelial cells with HCMV disturbs PAI-1 expression and production in these cells.

Alteration in the expression of endothelial cell integrin receptors alpha 5 beta 1 and alpha 2 beta 1 and alpha 6 beta 1 after in vitro infection with a clinical isolate of human cytomegalovirus.

Human cytomegalovirus infection of human umbilical vein endothelial cells reduces the ability of these cells to bind to fibronectin, collagen type IV and laminin. This suppression requires active virus, since UV-inactivated virus did not alter the binding ability of these cells to adhere to fibronectin, collagen type IV, and laminin. In an attempt to elucidate the molecular mechanism of this altered interaction, the surface expression of alpha 5 beta 1, alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 1 integrins on cytomegalovirus-infected endothelial cells was examined using attachment inhibition assay and flow cytometric analysis. The results presented here show that infection with human cytomegalovirus selectively alters the expression of integrin on human endothelial cells, with the ability to induce downregulation of alpha 5 beta 1 and alpha 2 beta 1 (p = 0.001) and p = 0.03, respectively), while significantly upregulating alpha 6 beta 1 (p = 0.03), and marginally upregulating alpha 3 beta 1 (p = 0.05).

Comparison of the infectivity of the laboratory strain AD169 and a clinical isolate of human cytomegalovirus to human smooth muscle cells.

The results displayed by human cytomegalovirus (HCMV) IE/E antigen expression and virus release into the supernatant at various times post infection with a clinical isolate (C3/p5) and AD169 laboratory strain of HCMV, illustrated differences in the biology of these viruses on the various cell lines. While AD169 strain was shown to infect fibroblasts efficiently, it showed a low infectivity profile to smooth muscle cells, whereas C3/p5 displayed comparable infectivity characteristics on both cell lines. Neither virus demonstrated propensity for infecting endothelial cells, although passaging of the C3/p5 for additional 11 passages in endothelial cells provided virus capable of infecting endothelial cells efficiently. These results show that HCMV is capable of infecting smooth muscle cells which could be of relevance to the proposed role of HCMV in atherogenesis and provides further evidence on the adaptation of AD169 to fibroblasts.

Alterations in the expression of ELAM-1, ICAM-1 and VCAM-1 after in vitro infection of endothelial cells with a clinical isolate of human cytomegalovirus.

Human cytomegalovirus (HCMV) infection of endothelial cells resulted in increased adhesion of the cells to peripheral blood leukocytes. It was demonstrated by flow cytometry that increased adhesiveness parallels the increased expression of cell surface adhesion molecules (ELAM-1, ICAM-1, VCAM-1). The increased adhesion of PMN and T-lymphocytes was due to upregulation in the expression of ELAM-1 and ICAM-1. The upregulation of VCAM-1 resulted in the increased adhesiveness of monocytes and T-lymphocytes to HCMV-infected HUVEC. The increased adhesiveness to leukocytes was caused by HCMV replication since endothelial cells exposed to HCMV-free supernatants and UV-inactivated HCMV did not show any increase in adhesiveness to any of the leukocytes tested.

Cytomegalovirus infection of vascular endothelial cells alters production of GM-CSF and G-CSF.

Infection of human umbilical vein endothelial cells with the clinical isolate of human cytomegalovirus (HCMV; at a multiplicity of infection of 2) severely suppresses the production of granulocyte-CSF and granulocyte-macrophage-CSF at late stages of infection (6 days post infection onwards). The effect was produced by actively multiplying virus which indicates that HCMV antigen expression is important for this suppression. The suppression in the production of these two cytokines was not due to their accumulation inside the cell nor to cell damage or lysis after infection.

Interleukin-1 production and cell-activation response to cytomegalovirus infection of vascular endothelial cells.

Human cytomegalovirus (HCMV) is a source of major complications in immunosuppressed individuals, and endothelial involvement in HCMV infection is well documented. Traditionally laboratory strains of HCMV have been used in experimental investigations in vitro; however the continuous propagation of these strains in fibroblasts have attenuated the virus making it unsuitable for infecting other cell systems such as endothelial cells. In this study a recent clinical isolate of HCMV was propagated through several passages in endothelial cells and was used to investigate the effect of HCMV infection of human umbilical vein endothelial cells (HUVEC) on IL-1 production and cell proliferation. Infection of HUVEC with the clinical isolate of HCMV (at multiplicity of infection 5:1) suppressed the production of IL-1 alpha (82%) and IL-1 beta (99%) at 5 h post infection; the levels returned to that of the control within 24h post infection. Ultraviolet inactivated (but not heat killed) virus produced similar suppression confirming that a thermolabile viral structural protein or intact virion were responsible for this suppression. Infection of HUVEC with the clinical isolate increased the number of these cells and the rate of their proliferation. An increase of infected HUVEC number under quiescent growth conditions continued as the infection progressed (6-10 days post infection), exhibiting, at 3 days post infection, 5 times the number of uninfected HUVEC (control) which did not tolerate the quiescent culture conditions for more than 4 days. Live virus is responsible for this increase because UV-inactivated virus did not maintain the proliferation of HUVEC. These studies suggest that while infection of HUVEC with a recent clinical isolate of HCMV suppressed the production of IL-1 at early hours after infection, it increased the proliferation of these cells at later stages of infection.