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Journal of Virology, December 2002, p. 12803-12812, Vol. 76, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.24.12803-12812.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Upregulation of CD40 Expression on Endothelial Cells Infected with Human Cytomegalovirus

Tim Maisch,1 Barbara Kropff,1 Christian Sinzger,2 and Michael Mach1*

Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, 91054 Erlangen,1 Institut für Medizinische Virologie und Epidemiologie der Viruskrankheiten, Universität Tübingen, 72076 Tübingen, Germany2

Received 28 May 2002/ Accepted 4 September 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD40 has been identified as an important molecule for a number of processes, such as immune responses, inflammation, and the activation of endothelia. We investigated CD40 in endothelial cells (EC) following infection with an endotheliotropic strain of human cytomegalovirus (HCMV). Between 8 and 72 h postinfection, we observed a significant increase in CD40 levels on the surface of infected EC, as measured by fluorescence-activated cell sorting analysis. As a consequence of CD40 upregulation, increased levels of E-selectin were found on infected EC after stimulation with CD154-expressing T cells. Enhanced expression of CD40 was specific for EC, since infection of fibroblasts did not result in the upregulation of CD40. The addition of neutralizing antibodies as well as UV inactivation of virus completely prevented the upregulation of CD40 on EC. Also, laboratory-adapted HCMV strain AD169 was not able to induce CD40 on EC. De novo protein synthesis was necessary for the increased surface expression. At early times (4 to 24 h) postinfection, this change was not accompanied by increased levels of CD40 protein or mRNA. At late times (48 to 96 h) postinfection, increased amounts of CD40 protein and mRNA were detected. Immunohistochemical analysis of infected tissues demonstrated elevated levels of CD40 on HCMV-infected EC in vivo. Thus, infection of EC by HCMV may result in the activation of endothelia and in the augmentation of inflammatory processes.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human cytomegalovirus (HCMV) infects the majority of the population worldwide. As a member of the herpesvirus family, HCMV establishes in the infected individual a lifelong latent or persistent infection with the potential for reactivation. Reinfections with genetically different HCMV strains are also possible (8). Fortunately, this infection usually remains clinically asymptomatic in immunocompetent individuals. However, in immunologically impaired hosts, such as transplant recipients, newborn infants, or patients with AIDS, HCMV can cause a wide spectrum of clinical syndromes (2). In recent years, evidence has accumulated to suggest a role for HCMV in vascular alterations, such as rapidly progressive coronary artery disease, and endothelial alterations in cardiac transplant patients (14, 33); coronary artery restenosis after angioplasty (47, 57); and inflammatory aortic diseases (55). Although these studies have suggested a role for HCMV in human vascular disease, there is still controversy (24, 35).

Substantial evidence has implicated endothelial cells (EC) as natural hosts during HCMV infection (42). HCMV immediate-early (IE) gene products have been identified during the latent state in EC, suggesting that these cells may be reservoirs for the maintenance of infection (12). In addition, HCMV is capable of efficiently replicating in EC in vitro, provided the natural tropism of virus strains is conserved by serial passages in EC. The transmission of virus from EC to blood cells in cell culture models has underscored the emerging role of infected EC in the dissemination of virus (52).

CD40 is a cell surface receptor that belongs to the tumor necrosis factor (TNF) receptor family and that was first identified and functionally characterized on B lymphocytes (5). Its critical role in T-cell-dependent humoral immune responses was demonstrated in patients with the X-linked immunodeficiency hyperimmunoglobulin M (IgM) syndrome as well as by gene targeting in mice (15). However, in recent years, it has become clear that the tissue distribution of CD40 is much broader and includes expression on monocytes, dendritic cells, epithelial cells, and EC (15, 49). Thus, CD40 may play an important functional role in a wide spectrum of cells and tissues. The ligand for CD40, CD154, is a membrane-bound member of the TNF receptor family that is prominently expressed by activated CD4+ T cells. In addition, CD154 has been found on mast cells, professional antigen-presenting cells, blood dendritic cells, NK cells, activated platelets, and EC at sites of inflammation (32, 50). The critical role played by this ligand-receptor pair is underscored by the fact that its blockade globally inhibits humoral immunity and many aspects of cellular immunity (15). Furthermore, the development and progression of a wide spectrum of autoimmune diseases as well as the response to transplantation antigens are halted upon therapeutic intervention with anti-CD154 antibodies (10, 26). Increasing evidence supports the importance of CD40-CD154 interactions among EC, activated T cells, and circulating monocytes in atherosclerosis. CD40 is present on atheroma-derived cells in vitro and in human atheromata in situ (32). Ligation of CD40 on atheroma-associated cells in vitro activates a number of processes responsible for lesion progression and plaque destabilization, such as the production of chemokines, cytokines, adhesion molecules, and tissue factors (31). Inhibition of CD40-CD154 interactions by the administration of antibodies has been shown to reduce atherosclerosis in mice (30, 38).

We examined the impact of HCMV infection on CD40 expression by EC in vivo and in vitro. Our data reveal a considerable increase in CD40 levels on the surface of infected cultured EC between 8 and 96 h postinfection (p.i.). The effect was cell type specific, since fibroblasts infected in vitro did not show increased CD40 levels. At early times (4 to 24 h) p.i., this change was not accompanied by increased de novo synthesis of CD40 protein or mRNA. At late times (48 to 96 h) p.i., increased levels of CD40 protein and mRNA were detected. The upregulation of CD40 on EC early after infection was not inhibited by conventional antiviral drugs. Immunohistochemical analysis of HCMV-infected tissues demonstrated elevated levels of CD40 on infected EC in vivo.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures. Primary human umbilical vein EC (HUVEC) were obtained by chymotrypsin treatment of single umbilical vein cords and cultured in RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 10% heat-inactivated (56°C, 45 min) HCMV-negative human serum, 5 U of heparin (Sigma, Deisenhofen, Germany)/ml, 50 µg of EC growth factor (Becton Dickinson, Bedford, Mass.)/ml, and 100 mg of gentamicin/liter (complete RPMI). Human pulmonary artery EC (HPAEC) (Promocell, Heidelberg, Germany) were propagated in EBM-2 bullet kit medium (Clonetics, BioWhittaker Inc., Walkersville, Md.) containing 10% HCMV-negative human serum. HUVEC and HPAEC were grown in tissue culture material that had been precoated with 0.1% gelatin (Sigma) and were used between passages 2 and 6. The purity of HUVEC was confirmed by the presence of von Willebrand factor in >97% of the cells. Human foreskin fibroblasts (HFF) were grown as described previously (23).

Viruses. The HCMV laboratory strain AD169 and the HCMV isolate TB40E (43) were harvested from the supernatants of infected HFF at 10 days p.i. by clarification at 500 x g for 10 min, and aliquots were stored at -80°C. Virus stocks were titrated by using a plaque assay and were used at a multiplicity of infection (MOI) of 1 or 2. Virions were isolated by glycerol-tartrate gradient centrifugation (3). For infection of HUVEC and HPAEC with HCMV, the medium was replaced with incomplete RPMI 1640 (without heparin and EC growth factor) for 1 h before virus inoculation. At 90 min after virus inoculation, the cells were washed twice with incomplete RPMI 1640. Complete RPMI or EBM-2 bullet kit medium was then added, and the cells were cultured for various time periods. Ganciclovir (Synthex Arzneimittel GmbH, Aachen, Germany) was present at 20 µM (final concentration) throughout the entire culture period after infection. Cycloheximide (Sigma) at a final concentration of 150 µg/ml was added to cell monolayers 30 min before viral or mock infection and left on the cells for 8 h after infection at 100 µg/ml. The medium then was replaced with complete RPMI. In some experiments, 50 U of human TNF alpha (TNF-{alpha}) (a gift from I. Strobel, Institute of Dermatology, University of Erlangen, Erlangen, Germany)/ml was added to the cells. The percentage of infected cells was determined by indirect immunofluorescence as described previously (23). Infectious virus was neutralized with human monoclonal antibody C23 exactly as described previously (23). To inactivate virus, stocks were placed in 1.5-ml Eppendorf reaction tubes and exposed to 254-nm UV light (40 W) for 1 h.

Antibodies. The following monoclonal antibodies or polyclonal sera were used: mouse IgG1-fluorescein isothiocyanate (FITC)-conjugated anti-human CD54 (Calbiochem, La Jolla, Calif.), anti-human major histocompatibility complex class I (MHC-I) (BD PharMingen, San Diego, Calif.), mouse IgG1-FITC anti-human CD40 (Dianova, Hamburg, Germany), mouse IgG1-FITC anti-human CD62E (Calbiochem), antibodies G28.5 and Ro1 (anti-human CD40) (gifts from H. Engelmann, Institute for Immunology, University of Munich, Munich, Germany) (39), polyclonal rabbit anti-CD40 serum (Santa Cruz Biotechnology), rabbit antiserum against human calreticulin (Dianova), biotin-conjugated rabbit anti-mouse antibodies, and biotinylated swine anti-rabbit antibodies (Dako, Hamburg, Germany).

Flow cytometry. Mock- or HCMV-infected cell monolayers were treated with 0.5 mM EDTA in phosphate-buffered saline (PBS) for detachment from the flasks and were incubated with an FITC-phycoerythrin-conjugated specific antibody (4°C, 45 min). All antibody incubations were performed with PBS containing 5% fetal calf serum and 0.1% NaN3 (FNCS/PBS). After incubation with the antibody, the cells were washed three times in FNCS/PBS and subsequently analyzed with a FACScalibur flow cytometer (Becton Dickinson, Heidelberg, Germany). Nonspecific fluorescence was assessed by staining the cells with irrelevant isotype-matched FITC-labeled monoclonal antibodies (BD PharMingen). A total of 5,000 gated events were collected and analyzed by using CellQuest software (BD PharMingen). For each treatment, the mean fluorescence intensity (MFI) of the control stained population was subtracted from the MFI of the positive stained sample. MFI values are geometric mean intensities and represent values normalized to the log scale, as calculated with CellQuest software.

Immunoblot analysis. Polyacrylamide gel electrophoresis and immunoblotting were performed by standard methods. Lysates from 1.5 x 105 cells were analyzed per time point. Blots were developed by enhanced chemiluminescence according to the manufacturer's protocol (ECL Western detection kit; Amersham Pharmacia Biotech Europe, Freiburg, Germany). The levels of CD40 protein expression were normalized to that of a stable protein (calreticulin) by densitometric analysis with a scanner (UMAX PowerlookIII) and Aida 2.0 software (Raytest, Straubenhardt, Germany). For N-glycosidase F treatment, 7.5 x 105 infected or mock-infected HUVEC were harvested per time point and resuspended in denaturing buffer (5% sodium dodecyl sulfate, 10% ß-mercaptoethanol) at 100°C for 10 min. After the addition of 0.5 M sodium phosphate (pH 7.5), the solutions were divided into aliquots; one received 500 U of PNGase F (New England BioLabs, Schwalbach, Germany), and the other served as a control. Digestion was carried out for 3 h at 37°C and was terminated by the addition of 2x sodium dodecyl sulfate sample buffer containing 10% ß-mercaptoethanol.

RNA isolation and RT-PCR. Total RNA was prepared from infected and mock-infected HUVEC by using a High Pure RNA isolation kit (Roche, Mannheim, Germany). RNA preparations were digested with 10 U of DNase I (Amersham Pharmacia Biotech Europe) for 2 h at 37°C. The enzyme was inactivated by heating the samples to 60°C for 20 min. By use of a Titan One Tube reverse transcription (RT)-PCR system (Roche), cDNA was synthesized with 0.5 µg of total RNA per reaction. RT was carried out at 58°C for 30 min. PCR was performed for 35 cycles at 94°C (30 s), primer annealing was performed at 50°C (30 s), and elongation was performed at 68°C (45 s; 5 s of extension per cycle) after a hot start.

The nucleotide sequences of primers were as follows: for human CD40, antisense, 5'-GGGACCACAGACAACATCAG-3' (nucleotides [nt] 611 to 592) (32), and sense, 5'-TGCCAGCCAGGGACAGAAACT-3' (nt 168 to 187); for ICAM-1, sense, 5'-ACATGCAGCACCTCCTGTG-3' (nt 196 to 214) and antisense, 5'-CACCGTGGTCGTGACCTCAG-3' (nt 576 to 557); and for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), antisense, 5'-TCCACCACCCTGTTGCTGTA-3' (nt 1037 to 1019) (48), and sense, 5'-GTACGTCGTGGAGTCCACTG-3' (nt 339 to 358). The primers were obtained from ARK Scientific Biosystems (Darmstadt, Germany).

Controls in the absence of RT were included. Southern blot analysis of cDNA was carried out according to standard procedures. Probes consisting of the respective RT-PCR products were radioactively labeled with 32P by using an NEBlot-kit (New England Biolabs, Beverly, Mass.). Signals were quantified by using a phosphorimager (bioimaging analyzer; Fuji, Tokyo, Japan) and appropriate software (Aida 2.0).

Immunohistochemical staining. For the analysis of CD40 expression in HCMV-infected tissues, simultaneous immunodetection of viral antigen and CD40 was performed with three-step immunoperoxidase staining and three-step immunoalkaline phosphatase staining, respectively. Formalin-fixed, paraffin-embedded tissue sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked by incubation with 0.6% H2O2. Epitopes were unmasked by using Dako antigen retrieval solution according to the supplier's recommendations. Sections were then incubated with monoclonal antibody E13 directed against the IE proteins of HCMV (UL122/123; Biosoft, Paris, France), with biotinylated rabbit anti-mouse antibodies (Dako), and with streptavidin-biotin-peroxidase complexes. Subsequent incubation with the chromogen diaminobenzidine (Sigma) yielded brown nuclear staining of HCMV-infected cells. Sections were then incubated with polyclonal rabbit serum directed against CD40, with biotinylated swine anti-rabbit antibodies (Dako), and with streptavidin-biotin-alkaline phosphatase complexes. Subsequent incubation with the chromogen fast red (Sigma) resulted in red cytoplasmic staining. Sections were then counterstained with hematoxylin and mounted with glycerol-gelatin. Stained slides were analyzed under a Polyvar microscope. Specimens included samples from a congenitally infected child (one slide), a liver transplant patient (one slide), a patient with Crohn's disease (one slide), and human immunodeficiency virus-infected persons (three slides). Informed consent was obtained from the patient or a responsible family member.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD40 expression on HUVEC following HCMV infection. The laboratory-adapted strains of HCMV that have been passaged for decades on human fibroblasts differ in at least two important aspects from recent clinical isolates: (i) they have lost the capacity to replicate to a significant extent in a number of cell types shown to be infected in vivo, including EC, and (ii) they lack approximately 15 kb of coding sequence (6). To investigate the effect of HCMV infection in HUVEC, we therefore used the recently isolated HCMV strain TB40E. This virus strain replicates efficiently in a number of different human cell types, including fibroblasts and EC (43).

HUVEC were infected at a multiplicity of infection of 1 or mock infected, and CD40 expression was determined at various times p.i. by indirect immunofluorescence flow cytometric (fluorescence-activated cell sorting [FACS]) analysis. In general, >80% of cells were found to be infected at 24 h p.i. (data not shown). In accordance with published data, we observed a constitutive low level of expression of CD40 on uninfected HUVEC (21) (Fig. 1). This expression was maintained at similar levels over several early passages (3 to 6) (data not shown). Infection with TB40E drastically increased the expression of CD40 on the surface of HUVEC (Fig. 1A). CD40 levels started to increase at 4 h p.i. and reached peak levels at 48 to 72 h p.i. Histograms for the FACS analysis at 2 and 48 h p.i. are shown in Fig. 1B. In repeated experiments, we observed an increase in CD40-specific MFI of between three- and fivefold in infected HUVEC. Increased expression of CD40 on the surface of infected cells was confirmed with a number of independent CD40-specific antibodies, including CD40-activating antibodies (data not shown) (39). In contrast to the results obtained after infection of HUVEC with TB40E, no upregulation of CD40 was observed in fibroblasts (Fig. 1). The addition of TNF-{alpha} to the culture medium resulted in the expected CD40 expression pattern, i.e., a slight increase on uninfected HUVEC but not on fibroblasts (Fig. 1A). These results are consistent with previous reports (21, 54). Control experiments analyzing the surface expression of MHC-I and ICAM-1 on HUVEC as well as fibroblasts gave results which were in agreement with published data (9, 18, 41, 53). The expression of MHC-I was downregulated, whereas that of ICAM-1 was upregulated (Fig. 2). Note that ICAM-1 surface expression on HUVEC differed from that of CD40 in that it started to increase between 24 and 48 h p.i. (Fig. 2, left panel).




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  		FIG. 1. CD40 expression on the surface of HUVEC and HFF after infection with HCMV. HUVEC and HFF were infected with HCMV TB40E at an MOI of 1, mock infected, or cultured in the presence of TNF-{alpha} (50 U/ml). CD40 expression was determined at the indicated times p.i. by FACS analysis. (A) MFIs determined by FACS analysis plotted against the time course of infection. inf., infected; n.i., noninfected. (B) Histograms representing cell number versus fluorescence intensity for 5,000 cells. Numbers in the panels indicate MFIs. Symbols: dark grey, infected cells; black, noninfected cells; light grey, isotype control.

 


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  		FIG. 2. Effect of HCMV TB40E infection on the expression of ICAM-1 and MHC-I. HUVEC or HFF were infected (inf.) at an MOI of 1 or mock infected (noninfected [n.i.]). The expression of ICAM-1 and MHC-I was determined at the indicated times p.i. by FACS analysis.

 
To test whether the observed upregulation of CD40 was also seen in EC from other sources, HPAEC were infected under conditions identical to those used for HUVEC, and CD40 expression levels were determined by FACS analysis. In repeated experiments, we detected an increase in CD40 expression of between 1.5- and 2.5-fold after infection with TB40E, indicating that the upregulation of CD40 was a more general phenomenon associated with HCMV infection of EC (Table 1).


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  		TABLE 1. Effect of HCMV infection on CD40 expression in HPAEC

 
Binding of the CD40 ligand results in increased expression of E-selectin on infected HUVEC. The stimulation of CD40 by its ligand, CD154, results in the increased expression of adhesion molecules in EC (22). To test whether HCMV infection can augment this process, we analyzed the expression of E-selectin (CD62E) following coculturing of HUVEC with Jurkat T-cell clone D1.1, expressing CD154, or Jurkat T-cell clone B2.7, which is negative for CD154 (28). HUVEC were infected for 60 h with HCMV, cocultured with the respective Jurkat cells for 6 h, and analyzed for the expression of E-selectin by FACS analysis. As shown in Table 2, E-selectin levels were comparable between infected and noninfected HUVEC, and coculturing with Jurkat T-cell clone B2.7 had no effect. Unchanged levels of E-selectin following HCMV infection of HUVEC have also been seen by others (41). In contrast, a clear difference in E-selectin levels was seen in infected and noninfected HUVEC cocultured with CD154-expressing Jurkat cells. Infected HUVEC showed MFIs almost threefold higher than those of HUVEC cocultured with CD154-negative Jurkat cells. The elevated levels of E-selectin seen in noninfected HUVEC cocultured with Jurkat T-cell clone D1.1 were most probably due to the stimulation of CD40 constitutively expressed by these cells (as demonstrated in Fig. 1). As expected, the addition of TNF-{alpha} to HUVEC resulted in a drastic increase in E-selectin expression in both infected and noninfected HUVEC (Table 2).


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  		TABLE 2. E-selectin expression on HUVEC following coculturing with Jurkat T cells

 
Adsorption and penetration of virions are insufficient events for the upregulation of CD40. The binding of HCMV virion envelope molecules to the plasma membrane of target cells has been reported to rapidly upregulate cellular transcription factors, such as Sp-1 and NF-{kappa}B which, in turn, could potentially activate CD40 transcription (56). We therefore next investigated whether the observed CD40 upregulation was related to the initial physical interaction between the virus and the target cell. First, we used UV-inactivated virus to determine the effect on CD40 expression. No upregulation of CD40 was seen when UV-inactivated virions were used for infection (data not shown). Next, the human anti-HCMV monoclonal antibody C23 was used to neutralize the infectivity of TB40E. This antibody allows the attachment of virions to cells but inhibits the penetration of viral particles into target cells (34). A total of 106 PFU of TB40E virions were incubated with C23 for 4 h, and the mixture was used to infect HUVEC. This treatment resulted in 100% neutralization of viral infectivity (data not shown). The upregulation of CD40 was completely inhibited following the neutralization of virus (Fig. 3, left panel). Whether the penetration of virions into HUVEC contributed to the CD40 upregulation was tested with HCMV strain AD169. Although laboratory-adapted HCMV strain AD169 replicates inefficiently in EC, it can attach to and penetrate these cells at rates comparable to those for fibroblasts (44). We therefore used this experimental tool to test whether events following adsorption, such as fusion between viral and cellular membranes and/or penetration of target cells, were involved in CD40 upregulation. As shown in Fig. 3, CD40 expression levels remained unchanged after HUVEC infection with strain AD169. Taken together, these data indicate that adsorption and penetration of HCMV virions are insufficient for the upregulation of CD40 expression on EC.



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  		FIG. 3. Effect of adsorption or penetration of HCMV virions on CD40 expression in HUVEC. (Left) A total of 106 PFU of HCMV TB40E were incubated with human monoclonal antibody C23 (4 µg/ml) for 4 h, and the mixture was used to infect HUVEC. At the indicated times, CD40 expression was analyzed by FACS analysis, and MFIs were plotted. inf., infected; n.i., noninfected. (Right) HUVEC were infected with HCMV strain AD169 at an MOI of 2 or mock infected, and CD40 expression was determined at the indicated times p.i. by FACS analysis.

 
De novo protein synthesis is required for the upregulation of CD40 on infected HUVEC. The above results suggested that events following viral penetration are required for the upregulation of CD40. To analyze whether de novo protein synthesis was necessary for the observed effect, we infected HUVEC in the presence of cycloheximide. Cells were treated with the drug during the first 8 h of infection. No increase in CD40 levels was seen in the cultures during that period, whereas control cultures showed the expected upregulation of CD40 (Fig. 4). Removal of the cycloheximide block at 8 h p.i. resulted in an increase in CD40 levels to values comparable to those in control cultures at 48 and 72 h p.i. In contrast, treatment of cells with ganciclovir, an inhibitor of viral DNA synthesis, allowed CD40 upregulation with kinetics identical to those in infected cells not treated with the drug (Fig. 4). Thus, de novo protein synthesis is essential for the enhanced expression of CD40 in infected EC.



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  		FIG. 4. Effect of cycloheximide or ganciclovir on EC CD40 expression following HCMV infection. HUVEC were infected (inf.) at an MOI of 1 or mock infected (noninfected [n.i.]) in the presence of cycloheximide (Cyc.) or ganciclovir (GCV) as described in Materials and Methods. Cycloheximide was removed from the cultures at 8 h p.i. Treatment with ganciclovir was continued throughout the entire infection period. CD40 expression was determined at the indicated times by FACS analysis.

 
CD40 mRNA and protein levels remain constant during early times after HCMV infection. Next, we investigated whether the cell surface expression of CD40 molecules on infected HUVEC was paralleled by an increase in the total CD40 protein level. Cells were infected for 0 to 96 h with TB40E, and lysates were prepared and subjected to immunoblot analysis with a polyclonal rabbit serum against CD40. Signals were obtained at 45 kDa, which is in agreement with the reported molecular mass of CD40 (Fig. 5A). Following infection, the signal intensity remained constant at 0 to 24 h p.i. and was clearly enhanced at 48 to 96 h p.i. The increase at later times p.i. was accompanied by the presence of slower-migrating forms of CD40 beginning at 72 h p.i. (Fig. 5A). A slight increase in the CD40 protein level was also seen in mock-infected HUVEC at 72 and 96 h. Levels of calreticulin, an endoplasmic reticulum-resident protein, remained constant over the entire observation period in both infected and mock-infected cells (Fig. 5B). Densitometric evaluation of the signals obtained on immunoblots confirmed the constant level of CD40 between 0 and 48 h p.i. as well as an approximately threefold increase in the level in infected cells at later times (Fig. 5C). To test whether the slower-migrating proteins which were synthesized late after infection represented CD40, cell lysates were treated with endoglycosidase F prior to immunoblot analysis to remove N-linked carbohydrates. As shown in Fig. 5D, all forms of the antibody-reactive protein could be converted to the 40-kDa nonglycosylated form of CD40, indicating that the slower-migrating proteins represented CD40.



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  		FIG. 5. Immunoblot analysis of CD40 in whole-cell lysates. Cell monolayers were infected with HCMV TB40E at an MOI of 1 (inf.) or mock infected (noninfected [n.i.]). Cell lysates were prepared at the indicated times and analyzed by immunoblotting. (A) CD40 protein levels were detected with a polyclonal rabbit serum directed against human CD40. (B) Blots from panel A were stripped and reprobed with a rabbit serum directed against calreticulin. (C) Signal intensities from panels A and B were densitometrically quantified; data represent the ratio of CD40 to calreticulin (y axis) versus time p.i. (x axis). (D) Protein lysates from infected or noninfected cells were treated with N-glycosidase F (+) or left untreated (-) prior to immunoblot analysis with anti-CD40 antibodies. Molecular masses are given in kilodaltons.

 
To investigate whether mRNA levels for CD40 paralleled increased protein levels, we analyzed the mRNA concentrations for CD40 in infected and mock-infected HUVEC by using RT-PCR. The amplification of total mRNA with CD40-specific primers revealed products of the expected size (443 bp) (Fig. 6A). Genomic contamination was excluded by the absence of a product following PCR of non-reverse-transcribed mRNA preparations (data not shown). The RT-PCR products were subjected to Southern blot analysis with 32P-labeled CD40 cDNA as a probe. Signals were collected by phosphorimaging. As an internal control, mRNA specific for GAPDH was amplified together with CD40 and probed with the respective radiolabeled cDNA fragment (Fig. 6B). In general, CD40 mRNA levels showed kinetics identical to those found for CD40 protein levels, i.e., a constant level at early times p.i. (0 to 8 h) followed by a slight increase at later times (24 to 72 h) (Fig. 6). Signal quantification after phosphorimaging revealed that at 72 h p.i., relative to GAPDH expression, CD40 mRNA levels were 1.3- to 1.5-fold higher in infected HUVEC than in noninfected HUVEC (data not shown). In agreement with previously published data, we observed a 4- to 5.8-fold increase in ICAM-1 mRNA levels at 24 and 48 h p.i. (Fig. 6) (4).



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  		FIG. 6. RT-PCR analysis of CD40- and ICAM-1-specific mRNA levels in HCMV-infected HUVEC. RNA was extracted from infected (inf.) and mock-infected (noninfected [n.i.]) cells at the indicated times, and RT-PCR was performed. (A) PCR products were subjected to Southern blot analysis and probed with the indicated probes. (B) Blots from panel A were stripped and rehybridized with a GAPDH-specific probe. Numbers at right are base pairs.

 
HCMV-infected EC express elevated levels of CD40 in vivo. To analyze the expression of CD40 in HCMV-infected EC in vivo, immunohistochemical staining of tissue sections from patients with acute HCMV pneumonitis and gastroenteritis was performed. Viral IE antigen and CD40 were simultaneously detected by indirect immunoperoxidase staining and indirect alkaline phosphatase staining, respectively. With this approach, three patterns of antigen expression were revealed. (i) In general, foci of HCMV-infected cells were surrounded by an inflammatory reaction, and within such foci of inflammation, enhanced CD40 expression was detectable (Fig. 7A and C). This finding was mainly due to infiltration of CD40-positive monocytes. In addition, moderate CD40 expression was detectable on endothelial layers within such foci of infection. CD3+ T cells were also present in the surrounding infected vessels, with CD40 upregulation (data not shown). (ii) With regard to CD40 expression on HCMV-infected EC, the majority of infected EC showed moderate CD40 signals comparable to those shown by adjacent uninfected cells (Fig. 7B). (iii) However, on a fraction of infected cells, overexpression of CD40 relative to that seen on adjacent uninfected cells occurred (Fig. 7D). In total, 42 infected EC were detected on six slides from different patients. Eight (19%) were negative for CD40, 17 (40%) expressed moderate CD40 levels, and 17 (40%) showed markedly enhanced CD40 levels. Various other surface molecules were expressed differently on infected EC. In contrast to the upregulation observed with CD40, the expression of VCAM-1 was unaltered, and MHC-I molecules were downregulated (data not shown). Enhanced CD40 signals were found on HCMV-infected EC with or without nuclear inclusions, indicating that this effect may occur early in the infectious cycle of HCMV. This effect seemed to be cell type specific, as CD40 induction was never observed in HCMV-infected epithelial cells (Fig. 7E).



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  		FIG. 7. CD40 expression in HCMV-infected EC in vivo. Immunohistochemical staining of tissue sections from a patient with acute HCMV gastroenteritis was performed. Viral IE antigen and CD40 were simultaneously detected by indirect immunoperoxidase staining and indirect alkaline phosphatase staining, respectively, as described in Materials and Methods. Nuclear staining of HCMV-infected cells is brown, and cytoplasmic staining of CD40 antigen is red. (A to D) Tissue sections from a colon resection. Panels B and D are magnifications of the boxed areas in panels A and C, respectively. (E) Tissue section from an esophageal biopsy. Arrowheads indicate infected cells.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
EC represent a major target for HCMV. In vivo studies showed that EC are frequently infected in a variety of organs (42). Moreover, the appearance of HCMV-infected EC in peripheral blood of patients demonstrates vascular injury following HCMV infection (36). In vitro experiments with HCMV-infected EC have revealed a number of different effects which could negatively affect EC function and/or induce inflammatory reactions (40, 41). Given the essential role of the endothelium for many physiological processes, it can be expected that infection of EC with HCMV in vivo will have profound effects on EC-associated pathology.

Our data clearly show that the expression of CD40 on the surface of EC is enhanced following infection with HCMV and that subsequent stimulation by CD154 activates EC to express adhesion molecules. The increase in CD40 expression was specific for EC infected with an HCMV isolate which can efficiently replicate in these cells. Infection of fibroblasts had no effect on the expression of CD40. Likewise, infection of EC with HCMV isolates that penetrate well but replicate inefficiently in these cells did not alter CD40 expression. Interestingly, the data suggested that the increased expression of CD40 on the surface of EC was mediated by more than one mechanism. During the early phase p.i. (8 to 24 h), increased expression of CD40 on the surface of EC was observed in FACS analyses. However, this increase was not accompanied by elevated concentrations of CD40 protein in whole-cell lysates. Likewise, CD40 mRNA levels remained constant during this period. In contrast, elevated protein and mRNA levels were seen at late times p.i. (48 to 96 h).

The in vitro findings were confirmed by immunohistochemical analyses of HCMV-infected tissue sections. In a significant fraction of infected EC, increased levels of CD40 were observed. The fact that not all infected EC showed enhanced CD40 staining was most probably related to the state of infection in individual cells. Since at an early stage of infection the increase in CD40 levels on the surface of EC is not accompanied by an increase in overall protein levels, such cells will not stain more intensely than noninfected EC. Only the fraction of cells at a late stage of infection will contain elevated levels of CD40 and stain more prominently. The immunohistochemical analyses also confirmed the cell type specificity of the upregulation of CD40. In no case did we detect infected epithelial cells that contained enhanced CD40 levels, although these cells are capable of expressing CD40 (49). Moreover, the in vivo detection of elevated CD40 levels in tissues from different donors suggested that the upregulation of this molecule was not secondary to tissue culture adaptation of our HCMV isolate to EC.

HCMV has the potential to alter cellular gene expression through multiple mechanisms. Physical interactions of viral surface glycoproteins with target cells can result in changes in cellular transcription factors and immunoregulatory molecule expression (56). Also, constituents of virions, such as tegument protein pp71, migrate to the nucleus and activate transcription after infection (29). These mechanisms were not operative in the upregulation of CD40 at early times after infection. This conclusion can be drawn from experiments in which (i) adsorption of virus was allowed but penetration was inhibited by antibodies; (ii) UV-inactivated virus was used for infection; and (iii) HCMV strain AD169, which efficiently penetrates EC but replicates poorly, was used. In no case were elevated levels of CD40 seen on infected EC. Instead, de novo protein synthesis was shown to be essential for the early upregulation of CD40.

Although we have no formal proof at the moment, the upregulation of CD40 as early as 6 to 8 h p.i. strongly suggests the participation of viral IE proteins. Transcription of IE genes occurs immediately after entry of the virus into the host cell. We were able to detect IE proteins between 2 and 4 h after infection of EC (data not shown). HCMV encodes a number of IE proteins which could be involved in the upregulation of CD40 expression. Only a fraction of these proteins have been functionally analyzed. The best-characterized proteins are IE1 and IE2. They represent nuclear proteins which modulate the transcription of a number of viral and cellular promoters (13). Particularly relevant for the interpretation of our data is the fact that the IE1 and IE2 proteins synergistically activate ICAM-1 gene expression, which results in an increase in ICAM-1 mRNA levels following infection (4). The increase in ICAM-1 protein levels in our experiments was also accompanied by a parallel increase in ICAM-1 mRNA levels (data not shown). However, the kinetics of expression of ICAM-1 on the surface of infected cells differed significantly from those of CD40. Whereas increased CD40 concentrations on the surface of EC were detected between 6 and 8 h p.i., ICAM-1 levels started to increase between 24 and 48 h p.i. Moreover, characteristically for transactivation through IE1 and IE2 proteins, the upregulation of ICAM-1 gene expression is independent of the cell type and can also be seen in fibroblasts (16; this work). Thus, the available data suggest that the upregulation of CD40 at early times after infection may be mediated by mechanisms other than direct transactivation via the IE1 and IE2 proteins.

Apart from the IE1 and IE2 proteins, HCMV strain AD169 codes for a number of additional IE proteins (7). Uncharacteristically for human herpesviruses, several of these IE proteins are type I integral membrane glycoproteins, suggesting a potential role in cellular transport mechanisms. Moreover, recent clinical isolates that effectively infect EC carry a number of additional glycoprotein genes whose temporal expression patterns have not yet been explored (6). With respect to already characterized IE proteins from strain AD169, it is interesting that IE protein US3 is known to bind and retain MHC-I heavy chains in the endoplasmic reticulum, thereby inhibiting antigen presentation (19). Another example of an IE glycoprotein is US37, a glycoprotein which travels through the secretory pathway to the plasma membrane (1). Thus, it is tempting to speculate that IE glycoproteins may be involved in the early appearance of enhanced CD40 levels on the surface of infected EC. No matter what the underlying mechanism is, currently available antiviral drugs, such as ganciclovir, were unable to inhibit the effect. The mechanism for the increase in CD40-specific mRNA and protein levels at late times after infection is also unknown at present. However, it is well documented that HCMV, unlike other herpesviruses, is capable of stimulating host cell macromolecule synthesis throughout the entire infection cycle.

What might be the relevance of our findings for the in vivo situation? HCMV infection has been linked to a number of pathological situations resulting from alterations of the endothelium, in particular, transplantation-associated vasculopathy, restenosis after angioplasty, and atherosclerosis (11, 25, 45, 46). Transplantation-associated vasculopathy has emerged as a major limitation to long-term graft survival in transplantation (20). A number of reports have implicated HCMV as an aggravating agent in the development of transplantation-associated vasculopathy (17, 25). The findings were also supported by animal studies (27). Our data suggest a potential pathogenic role for HCMV infection in transplantation-associated vasculopathy. As we have shown here, HCMV infection will lead to increased CD40 levels on EC. Chronically activated alloreactive T cells carrying CD154 can bind to EC and lead to further activation of EC, manifested by the expression of elevated levels of adhesion molecules, such as E-selectin and ICAM-1. The presence of E-selectin and ICAM-1, both critical mediators of leukocyte recruitment, will result in enhanced adhesion of leukocytes. On the other hand, T cells will also be stimulated to produce proinflammatory cytokines, such as gamma interferon and TNF-{alpha}. In fact, it was shown that HCMV-infected EC are capable of directly eliciting vigorous activation responses by T cells and that these T cells produce interleukin 2, TNF-{alpha}, and gamma interferon (51). Thus, the total effects mediated by infection of EC by HCMV clearly have the potential to exacerbate transplantation-associated vasculopathy. Interestingly, HCMV also seems to be capable of upregulating CD40 on dentritic cells, a cell population that also interacts with T cells (37).

In conclusion, we have shown that HCMV infection results in rapid and sustained upregulation of CD40 on the surface of EC. The effect is specific, since neither fibroblasts (infected in vitro) nor epithelial cells (infected in vivo) showed increased levels of CD40. Considering the widespread distribution of HCMV in humans, CD40 upregulation could represent an important factor contributing to the development of vascular alterations, such as transplantation-associated vasculopathy or atherosclerosis. Further efforts will be directed toward studying the mechanism of CD40 upregulation by HCMV.


   ACKNOWLEDGMENTS
 
We thank Hartmut Engelman (Institut für Immunologie, Munich, Germany) for monoclonal antibodies.

This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Bundesministerium für Forschung und Technologie (IZKF Erlangen).


   FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Klinische und Molekulare Virologie, Schlossgarten 4, 91054 Erlangen, Germany. Phone: 49 9131 8522107. Fax: 49 9131 8522101. E-mail: mlmach{at}viro.med.uni-erlangen.de. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Al Barazi, H. O., and A. M. Colberg-Poley. 1996. The human cytomegalovirus UL37 immediate-early regulatory protein is an integral membrane N-glycoprotein which traffics through the endoplasmic reticulum and Golgi apparatus. J. Virol. 70:7198-7208.[Abstract/Free Full Text]
  2. Alford, C. A., and W. J. Britt. 1990. Cytomegalovirus, p. 1981-2010. In B. N. Fields and D. M. Knipe (ed.), Virology. Raven Press, Ltd., New York, N.Y.
  3. Almeida, J., D. Lang, and P. Talbot. 1978. Herpesvirus morphology: visualization of a structural subunit. Intervirology 10:318-320.[Medline]
  4. Burns, L. J., J. C. Pooley, D. J. Walsh, G. M. Vercellotti, M. L. Weber, and A. Kovacs. 1999. Intercellular adhesion molecule-1 expression in endothelial cells is activated by cytomegalovirus immediate early proteins. Transplantation 67:137-144.[CrossRef][Medline]
  5. Calderhead, D. M., Y. Kosaka, E. M. Manning, and R. J. Noelle. 2000. CD40-CD154 interactions in B-cell signaling. Curr. Top. Microbiol. Immunol. 245:73-99.[Medline]
  6. Cha, T. A., E. Tom, G. W. Kemble, G. M. Duke, E. S. Mocarski, and R. R. Spaete. 1996. Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J. Virol. 70:78-83.[Abstract]
  7. Colberg-Poley, A. M. 1996. Functional roles of immediate early proteins encoded by the human cytomegalovirus UL36-38, UL115-119, TRS1/IRS1 and US3 loci. Intervirology 39:350-360.[Medline]
  8. Collier, A. C., S. H. Chandler, H. H. Handsfield, L. Corey, and J. K. McDougall. 1989. Identification of multiple strains of cytomegalovirus in homosexual men. J. Infect. Dis. 159:123-126.[Medline]
  9. Craigen, J. L., and J. E. Grundy. 1996. Cytomegalovirus induced up-regulation of LFA-3 (CD58) and ICAM-1 (CD54) is a direct viral effect that is not prevented by ganciclovir or foscarnet treatment. Transplantation 62:1102-1108.[CrossRef][Medline]
  10. Durie, F. H., R. A. Fava, T. M. Foy, A. Aruffo, J. A. Ledbetter, and R. J. Noelle. 1993. Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40. Science 261:1328-1330.[Abstract/Free Full Text]
  11. Epstein, S. E., Y. F. Zhou, and J. Zhu. 1999. Infection and atherosclerosis: emerging mechanistic paradigms. Circulation 100:e20-e28.[Abstract/Free Full Text]
  12. Fish, K. N., C. Soderberg-Naucler, L. K. Mills, S. Stenglein, and J. A. Nelson. 1998. Human cytomegalovirus persistently infects aortic endothelial cells. J. Virol. 72:5661-5668.[Abstract/Free Full Text]
  13. Fortunato, E. A., and D. H. Spector. 1999. Regulation of human cytomegalovirus gene expression. Adv. Virus Res. 54:61-128.[Medline]
  14. Grattan, M. T., C. E. Moreno-Cabral, V. A. Starnes, P. E. Oyer, E. B. Stinson, and N. E. Shumway. 1989. Cytomegalovirus infection is associated with cardiac allograft rejection and atherosclerosis. JAMA 261:3561-3566.[Abstract]
  15. Grewal, I. S., and R. A. Flavell. 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16:111-135.[CrossRef][Medline]
  16. Grundy, J. E., and K. L. Downes. 1993. Up-regulation of LFA-3 and ICAM-1 on the surface of fibroblasts infected with cytomegalovirus. Immunology 78:405-412.[Medline]
  17. Hosenpud, J. D. 1999. Coronary artery disease after heart transplantation and its relation to cytomegalovirus. Am. Heart J. 138:S469-S472.[CrossRef][Medline]
  18. Ito, M., M. Watanabe, T. Ihara, H. Kamiya, and M. Sakurai. 1995. Increased expression of adhesion molecules (CD54, CD29 and CD44) on fibroblasts infected with cytomegalovirus. Microbiol. Immunol. 39:129-133.[Medline]
  19. Jones, T. R., E. J. Wiertz, L. Sun, K. N. Fish, J. A. Nelson, and H. L. Ploegh. 1996. Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains. Proc. Natl. Acad. Sci. USA 93:11327-11333.[Abstract/Free Full Text]
  20. Julius, B. K., C. H. Attenhofer Jost, G. Sutsch, H. P. Brunner, A. Kuenzli, P. R. Vogt, M. Turina, O. M. Hess, and W. Kiowski. 2000. Incidence, progression and functional significance of cardiac allograft vasculopathy after heart transplantation. Transplantation 69:847-853.[CrossRef][Medline]
  21. Karmann, K., C. C. Hughes, J. Schechner, W. C. Fanslow, and J. S. Pober. 1995. CD40 on human endothelial cells: inducibility by cytokines and functional regulation of adhesion molecule expression. Proc. Natl. Acad. Sci. USA 92:4342-4346.[Abstract/Free Full Text]
  22. Karmann, K., W. Min, W. C. Fanslow, and J. S. Pober. 1996. Activation and homologous desensitization of human endothelial cells by CD40 ligand, tumor necrosis factor, and interleukin 1. J. Exp. Med. 184:173-182.[Abstract/Free Full Text]
  23. Klein, M., K. Schoppel, N. Amvrossiadis, and M. Mach. 1999. Strain-specific neutralization of human cytomegalovirus isolates by human sera. J. Virol. 73:878-886.[Abstract/Free Full Text]
  24. Kol, A., G. Sperti, J. Shani, N. Schulhoff, W. van-de-Greef, M. P. Landini, M. La-Placa, A. Maseri, and F. Crea. 1995. Cytomegalovirus replication is not a cause of instability in unstable angina. Circulation 91:1910-1913.[Abstract/Free Full Text]
  25. Koskinen, P. K., M. S. Nieminen, L. A. Krogerus, K. B. Lemstrom, S. P. Mattila, P. J. Hayry, and I. T. Lautenschlager. 1993. Cytomegalovirus infection and accelerated cardiac allograft vasculopathy in human cardiac allografts. J. Heart Lung Transplant. 12:724-729.[Medline]
  26. Larsen, C. P., E. T. Elwood, D. Z. Alexander, S. C. Ritchie, R. Hendrix, C. Tucker-Burden, H. R. Cho, A. Aruffo, D. Hollenbaugh, P. S. Linsley, K. J. Winn, and T. C. Pearson. 1996. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381:434-438.[CrossRef][Medline]
  27. Lautenschlager, I., A. Soots, L. Krogerus, H. Kauppinen, O. Saarinen, C. Bruggeman, and J. Ahonen. 1997. Effect of cytomegalovirus on an experimental model of chronic renal allograft rejection under triple-drug treatment in the rat. Transplantation 64:391-398.[CrossRef][Medline]
  28. Lederman, S., M. J. Yellin, A. Krichevsky, J. Belko, J. J. Lee, and L. Chess. 1992. Identification of a novel surface protein on activated CD4+ T cells that induces contact-dependent B cell differentiation (help). J. Exp. Med. 175:1091-1101.[Abstract/Free Full Text]
  29. Liu, B., and M. F. Stinski. 1992. Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements. J. Virol. 66:4434-4444.[Abstract/Free Full Text]
  30. Lutgens, E., L. Gorelik, M. J. Daemen, E. D. de Muinck, I. S. Grewal, V. E. Koteliansky, and R. A. Flavell. 1999. Requirement for CD154 in the progression of atherosclerosis. Nat. Med. 5:1313-1316.[CrossRef][Medline]
  31. Mach, F., U. Schonbeck, and P. Libby. 1998. CD40 signaling in vascular cells: a key role in atherosclerosis? Atherosclerosis 137(Suppl.):S89-S95.
  32. Mach, F., U. Schonbeck, G. K. Sukhova, T. Bourcier, J. Y. Bonnefoy, J. S. Pober, and P. Libby. 1997. Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40-CD40 ligand signaling in atherosclerosis. Proc. Natl. Acad. Sci. USA 94:1931-1936.[Abstract/Free Full Text]
  33. Muhlestein, J. B., B. D. Horne, J. F. Carlquist, T. E. Madsen, T. L. Bair, R. R. Pearson, and J. L. Anderson. 2000. Cytomegalovirus seropositivity and C-reactive protein have independent and combined predictive value for mortality in patients with angiographically demonstrated coronary artery disease. Circulation 102:1917-1923.[Abstract/Free Full Text]
  34. Ohizumi, Y., H. Suzuki, Y. Matsumoto, Y. Masuho, and Y. Numazaki. 1992. Neutralizing mechanisms of two human monoclonal antibodies against human cytomegalovirus glycoprotein 130/55. J. Gen. Virol. 73:2705-2707.[Abstract/Free Full Text]
  35. Pauletto, P., G. Pisoni, R. Boschetto, M. Zoleo, A. C. Pessina, and G. Palu. 1996. Human cytomegalovirus and restenosis of the internal carotid artery. Stroke 27:1669-1671.[Abstract/Free Full Text]
  36. Percivalle, E., M. G. Revello, L. Vago, F. Morini, and G. Gerna. 1993. Circulating endothelial giant cells permissive for human cytomegalovirus (HCMV) are detected in disseminated HCMV infections with organ involvement. J. Clin. Investig. 92:663-670.
  37. Raftery, M. J., M. Schwab, S. M. Eibert, Y. Samstag, H. Walczak, and G. Schonrich. 2001. Targeting the function of mature dendritic cells by human cytomegalovirus: a multilayered viral defense strategy. Immunity 15:997-1009.[CrossRef][Medline]
  38. Schonbeck, U., G. K. Sukhova, K. Shimizu, F. Mach, and P. Libby. 2000. Inhibition of CD40 signaling limits evolution of established atherosclerosis in mice. Proc. Natl. Acad. Sci. USA 97:7458-7463.[Abstract/Free Full Text]
  39. Schwabe, R. F., S. Hess, J. P. Johnson, and H. Engelmann. 1997. Modulation of soluble CD40 ligand bioactivity with anti-CD40 antibodies. Hybridoma 16:217-226.[Medline]
  40. Sedmak, D. D., A. M. Guglielmo, D. A. Knight, D. J. Birmingham, E. H. Huang, and W. J. Waldman. 1994. Cytomegalovirus inhibits major histocompatibility class II expression on infected endothelial cells. Am. J. Pathol. 144:683-692.[Abstract]
  41. Sedmak, D. D., D. A. Knight, N. C. Vook, and J. W. Waldman. 1994. Divergent patterns of ELAM-1, ICAM-1, and VCAM-1 expression on cytomegalovirus-infected endothelial cells. Transplantation 58:1379-1385.[Medline]
  42. Sinzger, C., A. Grefte, B. Plachter, A. S. Gouw, T. H. The, and G. Jahn. 1995. Fibroblasts, epithelial cells, endothelial cells and smooth muscle cells are major targets of human cytomegalovirus infection in lung and gastrointestinal tissues. J. Gen. Virol. 76:741-750.[Abstract/Free Full Text]
  43. Sinzger, C., K. Schmidt, J. Knapp, M. Kahl, R. Beck, J. Waldman, H. Hebart, H. Einsele, and G. Jahn. 1999. Modification of human cytomegalovirus tropism through propagation in vitro is associated with changes in the viral genome. J. Gen. Virol. 80:2867-2877.[Abstract/Free Full Text]
  44. Slobbe-van Drunen, M. E., A. T. Hendrickx, R. C. Vossen, E. J. Speel, M. C. Dam-Mieras, and C. A. Bruggeman. 1998. Nuclear import as a barrier to infection of human umbilical vein endothelial cells by human cytomegalovirus strain AD169. Virus Res. 56:149-156.[CrossRef][Medline]
  45. Sorlie, P. D., E. Adam, S. L. Melnick, A. Folsom, T. Skelton, L. E. Chambless, R. Barnes, and J. L. Melnick. 1994. Cytomegalovirus/herpesvirus and carotid atherosclerosis: the ARIC study. J. Med. Virol. 42:33-37.[Medline]
  46. Speir, E., E. S. Huang, R. Modali, M. B. Leon, F. Shawl, T. Finkel, and S. E. Epstein. 1995. Interaction of human cytomegalovirus with p53: possible role in coronary restenosis. Scand. J. Infect. Dis. Suppl. 99:78-81.[Medline]
  47. Speir, E., R. Modali, E. S. Huang, M. B. Leon, F. Shawl, T. Finkel, and S. E. Epstein. 1994. Potential role of human cytomegalovirus and p53 interaction in coronary restenosis. Science 265:391-394.[Abstract/Free Full Text]
  48. Tokunaga, K., Y. Nakamura, K. Sakata, K. Fujimori, M. Ohkubo, K. Sawada, and S. Sakiyama. 1987. Enhanced expression of a glyceraldehyde-3-phosphate dehydrogenase gene in human lung cancers. Cancer Res. 47:5616-5619.[Abstract/Free Full Text]
  49. van Kooten, C., and J. Banchereau. 1997. Functions of CD40 on B cells, dendritic cells and other cells. Curr. Opin. Immunol. 9:330-337.[CrossRef][Medline]
  50. van Kooten, C., and J. Banchereau. 2000. CD40-CD40 ligand. J. Leukoc. Biol. 67:2-17.[Abstract]
  51. Waldman, W. J., P. W. Adams, C. G. Orosz, and D. D. Sedmak. 1992. T lymphocyte activation by cytomegalovirus-infected, allogeneic cultured human endothelial cells. Transplantation 54:887-896.[Medline]
  52. Waldman, W. J., D. A. Knight, E. H. Huang, and D. D. Sedmak. 1995. Bidirectional transmission of infectious cytomegalovirus between monocytes and vascular endothelial cells: an in vitro model. J. Infect. Dis. 171:263-272.[Medline]
  53. Yamashita, Y., K. Shimokata, S. Mizuno, H. Yamaguchi, and Y. Nishiyama. 1993. Down-regulation of the surface expression of class I MHC antigens by human cytomegalovirus. Virology 193:727-736.[CrossRef][Medline]
  54. Yellin, M. J., S. Winikoff, S. M. Fortune, D. Baum, M. K. Crow, S. Lederman, and L. Chess. 1995. Ligation of CD40 on fibroblasts induces CD54 (ICAM-1) and CD106 (VCAM-1) up-regulation and IL-6 production and proliferation. J. Leukoc. Biol. 58:209-216.[Abstract]
  55. Yonemitsu, Y., K. Nakagawa, S. Tanaka, R. Mori, K. Sugimachi, and K. Sueishi. 1996. In situ detection of frequent and active infection of human cytomegalovirus in inflammatory abdominal aortic aneurysms: possible pathogenic role in sustained chronic inflammatory reaction. Lab. Investig. 74:723-736.[Medline]
  56. Yurochko, A. D., E. S. Hwang, L. Rasmussen, S. Keay, L. Pereira, and E. S. Huang. 1997. The human cytomegalovirus UL55 (gB) and UL75 (gH) glycoprotein ligands initiate the rapid activation of Sp1 and NF-{kappa}B during infection. J. Virol. 71:5051-5059.[Abstract]
  57. Zhou, Y. F., M. B. Leon, M. A. Waclawiw, J. J. Popma, Z. X. Yu, T. Finkel, and S. E. Epstein. 1996. Association between prior cytomegalovirus infection and the risk of restenosis after coronary atherectomy. N. Engl. J. Med. 335:624-630.[Abstract/Free Full Text]

Journal of Virology, December 2002, p. 12803-12812, Vol. 76, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.24.12803-12812.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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