Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print May 12, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028118
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/3/797    most recent
biolreprod.104.028118v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chan, G.
Right arrow Articles by Guilbert, L.J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chan, G.
Right arrow Articles by Guilbert, L.J.
Agricola
Right arrow Articles by Chan, G.
Right arrow Articles by Guilbert, L.J.
BIOLOGY OF REPRODUCTION 71, 797–803 (2004)
DOI: 10.1095/biolreprod.104.028118
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Human Cytomegalovirus-Induced Upregulation of Intercellular Cell Adhesion Molecule-1 on Villous Syncytiotrophoblasts

G. Chan2,3, M.F. Stinski4, and L.J. Guilbert1,2,3

The Department of Medical Microbiology and Immunology2 the Perinatal Research Centre,3 University of Alberta, Edmonton, Alberta, Canada T6G 2S2 The Department of Microbiology,4 College of Medicine, University of Iowa, Iowa City, Iowa


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human cytomegalovirus (HCMV) is secreted apically from villous trophoblasts, thus congenital infection is not likely to occur by basal release across the basement membrane. As an alternative route, we hypothesize that an HCMV-infected villous syncytiotrophoblast (ST) upregulates intercellular adhesion molecule (ICAM)-1, causing blood monocytes to bind to the ST and induce apoptosis. Purified (>99.99%) populations of human villous trophoblasts were differentiated into an ST-like culture, infected with HCMV strain AD169, and assessed for ICAM-1 expression by immunofluorescence. Infection strongly upregulated ICAM-1 24 h after challenge. ICAM-1 was also stimulated by transfection with viral genes IE2-55, IE1-72, and IE2-86, but not by UV-inactivated virus. Infection with a green fluorescent protein recombinant virus allowed infection and ICAM-1 expression to be topographically located. We found that ICAM-1 was expressed on both infected and noninfected cells. Furthermore, antibody to tumor necrosis factor (TNF){alpha} and, to a lesser extent, interleukin (IL)1ß inhibited ICAM-1 upregulation on noninfected cells but not on infected cells. We conclude that HCMV IE proteins stimulate ICAM-1 expression on villous trophoblasts by paracrine release of TNF{alpha} and IL1ß, as well as by a direct effect on infected cells.

cytokines, immunology, placenta, syncytiotrophoblast, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human cytomegalovirus (HCMV), a member of the Herpesviridae family, is found in 50%–90% of the adult population. Infection with HCMV can have serious consequences in individuals with acquired immune deficiency syndrome or in those who have been immunocompromised, and to the fetus during pregnancy [1, 2]. Of mothers infected for the first time during pregnancy, 40% transmit HCMV to their fetuses, whereas only 0.5% of mothers who are seropositive prior to pregnancy transmit the virus [1– 3]. It is thus among one of the most commonly vertically transmitted diseases with a frequency of 0.5% to 2.0%.

During pregnancy, the placenta is the interface between the mother and the fetus. At term, maternal blood flows through the ~10 m2 of intervillous space to exchange oxygen and nutrients with the fetal circulation by way of the villous trophoblast and the fetal capillary circulation [4]. The villous trophoblast comprises a continuous nonreplicative outer layer, the syncytiotrophoblast (ST), which is in contact with maternal blood, and a discontinuous proliferative layer, the cytotrophoblast (CT), which fuses with and replenish the ST layer as it thins. Beneath the CT, and much of the ST, run the basement membrane and the villous stroma, which contain fibroblasts, macrophages, and the fetal endothelium. Villous trophoblast involvement in the vertical transmission of HCMV is indicated by its infection in vivo [5–7] and the productive infection of primary trophoblasts in vitro [8, 9].

HCMV gene expression occurs in three temporal classes: immediate early (IE), early, and late. The most abundantly transcribed IE region is the ie1/ie2 locus, which encodes proteins of 72 kDa (IE1-72), 86 kDa (IE2-86), and 55 kDa (IE2-55, a splice variant of IE2-86), [10, 11]. These viral proteins are potent transactivators that are necessary for productive infection and are involved in the regulation of cellular gene expression [10, 11].

HCMV is often found in placentas with villitis, inflammation of the placenta [4, 5, 12, 13], which is accompanied by a focal loss of the trophoblast [4]. The major infiltrating leukocytes in villitis associated with HCMV or with villitis of unknown etiology are mononuclear phagocytes [14, 15], which suggests these are involved in the disease. Monocyte adhesion to ST-like cultures in vitro is mediated by intercellular cell adhesion molecule (ICAM)-1, which can be stimulated by the inflammatory cytokines interleukin (IL)1ß, tumor necrosis factor (TNF){alpha}, and interferon (IFN){gamma} [16]. Such adhesion leads to TNF{alpha}-mediated apoptosis [17], suggesting that villous ST upregulation of ICAM-1 is an early step in villitis.

ICAM-1 is a ~100-kDa membrane glycoprotein, the expression of which is associated with tight immune-cell binding to the vasculature [18]. It mediates cellular interactions by binding to its receptors on leukocytes, leukocyte function-associated molecule (LFA)-1, and the C3 complement receptor MAC-1 [19, 20], although on trophoblasts, ICAM-1 appears to mediate monocyte binding solely through LFA-1 [16]. ICAM-1 is expressed at relatively low levels on the villous trophoblast unless stimulated by an infection such as Plasmodium falciparum [16, 21, 22]. In a number of endothelial and epithelial cell lines, HCMV infection results in the increased expression of ICAM-1 [23–28]. However, nothing is known of the effect of HCMV infection on villous trophoblast expression of ICAM-1.

Using highly purified (>99.99%) CT differentiated into ST-like cultures, we show here that HCMV-1 infection (or expression of IE1-72, IE2-86, and IE2-55 genes) upregulates ICAM-1 expression. It is interesting that ICAM-1 expression is stimulated by HCMV in a paracrine fashion (on noninfected cells) by TNF{alpha} and to a lesser extent by IL1ß, but its expression in infected cells is induced in a manner independent of these two cytokines.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells

Human term villous CTs were isolated from placentas obtained after normal term delivery or elective cesarean delivery from uncomplicated pregnancies (with patient consent and ethics approval from the Capital Health Region, Edmonton, Alberta, Canada) and purified by immunoelimination of CD9-, major histocompatibility complex (MHC) class I-, and MHC class II-expressing cells as previously described [29]. The purified cells were routinely cryopreserved [29, 30] and, after thawing, were washed twice in Iscove modified Dulbecco medium (IMDM; Gibco, Grand Island, NY) supplemented with 2% fetal bovine serum (FBS; Gibco). The cells were seeded at 105 per microwell per 100 µl of 10% FBS-IMDM (Gibco) and incubated for 4 h at 37°C in a 5% CO2 humidified atmosphere. Nonadherent cells and debris were removed with prewarmed IMDM and the cells were replenished with 10% FBS-IMDM. All preparations contained fewer than 10 vimentin-positive cells (fibroblasts) per microwell after the 4-h wash. Syncytialization of cultured CTs was induced by treatment with 10 ng/ml of recombinant human epidermal growth factor (EGF) (Pepro-Tech, Rocky Hill, NJ) for 5 days as previously described (operationally termed ST-like cultures) [31].

Human embryonic lung fibroblasts (HELs) were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and propagated in Eagle minimum essential medium (MEM) supplemented with 10% FBS and 50 µg of gentamicin per milliliter as previously described [8, 29].

Virus Preparation, Culture Challenge, and Assessment of Infection

Cytomegalovirus (CMV) laboratory strain AD169 and a green fluorescent protein (GFP)-expressing CMV recombinant strain, RVdlMwt-GFP, henceforth termed gfp-CMV [32], were passaged on confluent HEL cells in 2% FBS-MEM as previously described [8]. Infectious virus was recovered by freezing and thawing the cultures three times. The lysate was passed through 0.45-µm pore-size filters (MILLEX-HV; Millipore Products Division, Bedford, MA) and stored in liquid nitrogen until use. Virus titers were determined by inoculating confluent HEL cultures in 96-well plates with dilutions of each virus in serum-free MEM. The plates were then centrifuged for 45 min at 2500 x g in a GCL-2 Sorvall centrifuge, the wells were washed five times with warm MEM, and the plates were incubated for a further 18 to 20 h in fresh 2% FBS-MEM. The cultures were fixed in ice-cold methanol and immunohistochemically stained for CMV IE antigen as described below. Each IE-positive nucleus is equated to an infection focus (IF) of virus, and the titer of virus was determined within a linear dose-response concentration range as IF/milliliter.

UV inactivation of HCMV occurred by exposure to UV light (30 W germicidal; samples were exposed at a distance of 20 cm from the UV source) on ice for 20 min. Virus-free supernatant was obtained by filtering HCMV batches through a 0.1-µm pore-size syringe top filter (Millipore). UV inactivation and complete filtration was assured by the absence of IE-positive nuclei in trophoblast cultures.

Infections were carried out at a multiplicity of infection (MOI) (the ratio of inoculating virus IF to the number of nuclei in the culture to be infected) of 10 in serum-free IMDM for 2 h at 37°C in 5% CO2. The number of trophoblasts present in microwells was determined from parallel cultures by enumerating the number of nuclei in CT and ST-like cultures by 4,6-diamidino-2-phenylindole (DAPI) staining (see below). A 10-fold higher IF of virus (or an equal volume of UV-inactivated or virus-free supernatant from the same preparation) was then added. Cultures were infected with AD169, washed twice with PBS, and fixed with 4% paraformaldehyde for 10 min at room temperature (RT). Trophoblasts were then washed three times with PBS in preparation for immunofluorescence (see below). Cultures infected with gfp-CMV were washed five times on Day 1 with warm 2% FBS IMDM. Next, 10% FBS IMDM containing EGF was added and changed at Day 3. The culture was continued to Day 4 (GFP expression can be seen at this time) and then fixed with 4% paraformaldehyde.

Plasmids and Transient Transfections

All plasmids were propagated in Escherichia coli DH5{alpha} isolated by standard procedures, and the plasmid DNA was purified with a Qiagen Plasmid Maxiprep kit (Qiagen, Mississauga, ON, Canada). Plasmids pcDNA3-IE1-72, pcDNA3-IE2-55, and pcDNA-IE2-86 (a kind gift of A. Yurochko, Louisiana State University, Baton Rouge, LA) express the HCMV IE proteins IE72, IE55, and IE86, respectively [11]. The backbone vector pcDNA3 (Invitrogen, San Diego, CA) was used as a negative control. Trophoblasts were cultured in microwells as described above and transfected with Lipofectamine 2000 (Invitrogen)/plasmid DNA complexes as follows: 700 µg of DNA in 25 µl of Opti-MEM (Invitrogen) was mixed with 0.5 µl of 1 mg/ml Lipofectamine 2000 diluted with 25 µl of Opti-MEM, then added to a microwell containing 100 µl of 2% FBS/ IMDM. The transfection efficiency was 15% to 25%, as determined IE by immunofluorescence as described below.

Immunofluorescence Staining

ST-like cultures were challenged with virus-free supernatant, UV-inactivated virus, AD169, or gfp-CMV as described above. Following incubation, cells were washed twice with PBS, fixed with 4% phosphate-buffered paraformaldehyde for 10 min at RT, and washed three times with PBS again. Trophoblasts were then incubated with 10% goat serum (Zymed/Intermedico, Markham, CA) to block nonspecific binding. Primary antibody against ICAM-1 (1 µg/ml) (ICOS Corporation, Bothell, WA) or its isotype control, immunoglobulin G1 (IgG1; DAKO Corporation, Carpinteria, CA), was then added and allowed to incubate for 60 min at RT. After thorough washing with PBS, Alexa Fluor 546 goat anti-mouse IgG conjugate (Molecular Probes, Eugene, OR) was diluted in PBS to 1 µg/ml, and 50 µl was added to each well for 60 min at RT, after which cells were washed five times with PBS. Visualization and analysis of immunofluorescence is described below.

Two-color fluorescence analysis was carried out in parallel cultures to determine total nuclei number and the fraction of nuclei expressing HCMV IE proteins. After methanol fixation and PBS washing, nonspecific binding sites were blocked with 3% skim milk/0.5% Tween 20/PBS at RT for 30 min. Primary antibodies detecting CMV IE (detecting p72, p55, and p86; Specialty Diagnostics, Dupont, Billerica, MA) or its isotype control, IgG1 (DAKO Corporation), were added and incubated at RT for 1 h. The primary antibody was then removed and the cells were washed five times with PBS. Next, Alexa Fluor 546 goat anti-mouse IgG conjugate (Molecular Probes) was diluted in 3% skim milk/0.5% Tween 20/PBS to 1µg/ml each, and 50 µl was added to each well for 60 min at RT. The cells were washed with PBS five times and nuclei were visualized with 100 µl of 1.4 µg/ml DAPI (Molecular Probes; 10 min, RT). The frequency of infection or transfection was determined by the number of IE-positive nuclei relative to the total number of nuclei.

Digital Photography and Analysis

Immunofluorescence was examined using an inverted phase contrast microscope (model Ds-IRB; Leica, Heerbrugg, Switzerland) equipped for epifluorescence with a 100 W high-pressure mercury lamp driven by a Ludl power source (Ludl Electronic Products, Hawthorne, NY). Nuclei and IE-positive nuclei were visualized under a DAPI filter (blue) and a rhodamine filter (red), respectively. ICAM-1 staining was visualized with a rhodamine filter, and digital images from each well were taken with a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI). The mean fluorescent intensity was determined by a digital analysis program, Image-Pro Plus (Media Cybernetics, Del Mar, CA) and normalized to the total number of nuclei per image. It should be noted that background fluorescence was corrected for each image by subtracting mean fluorescent intensity from IgG1-stained cultures. GFP expression from the gfp-CMV infected cultures was visualized using a fluorescein isothiocyanate filter (green). The ICAM-1 (red) and GFP (green) images were superimposed, and the percent areas of ICAM-1, GFP, and overlap (yellow) were determined using Image-Pro Plus. To determine the percent area of high ICAM-1 expression, a baseline fluorescence was first selected based on the average fluorescence intensity of control cultures. The area of fluorescence that was above the baseline fluorescence in infected or antibody-treated ST cultures was then measured using Image-Pro Plus. The following formula was used to determine the percent area of high ICAM-1 expression: Percent area = (area of high ICAM-1 expression/total cell area) x 100.

TNF{alpha} and IL1ß Neutralization

To neutralize TNF{alpha} and IL1ß released in trophoblast cultures, 20 µg/ ml of polyclonal anti-human TNF{alpha} (Upstate Biotechnology, Lake Placid, NY), 20 µg/ml of polyclonal anti-human IL1ß (R&D Systems, Minneapolis, MN), or both was added to the culture 2 h before virus challenge. After incubations, cells were washed with PBS three times, fixed with 4% paraformaldehyde, and ICAM-1 expression was analyzed as described above. The anti-IL1ß antibody inhibited IL1ß, but not IL1{alpha}, and stimulated ICAM-1 expression in trophoblasts (data not shown).

Statistical Analysis

Experiments for each figure were performed at least three times on trophoblasts isolated from two different placentas. Differences between experimental groups were evaluated by one-way analysis of variance with pair-wise multiple comparison procedures (Tukey test) using the SigmaStat program (Jandel Scientific, San Rafael, CA). Results were considered to be significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HCMV Induces ICAM-1 Expression on the Surface of ST-Like Cultures

In order to determine whether HCMV infection of ST-like cultures upregulated cell surface expression of ICAM-1, we carried out immunofluorescence analysis on paraformaldehyde-fixed cells treated with IFN{gamma}, which is known to upregulate trophoblast ICAM-1 expression [16], or challenged them with HCMV for 24 h (Fig. 1). As expected, there was much greater cell surface immunofluorescence on IFN{gamma}-treated cells than on untreated cells (Fig. 1, A and B). HCMV-infected cells had, visually, increased surface expression (Fig. 1A) by digital analysis of fluorescence emissions (Fig. 1B) and by Western blot analysis (data not shown) from three independent experiments. In contrast, treatment with UV-inactivated virus and virus-free supernatant did not induce ICAM-1 expression, suggesting that viral replication is required for upregulation.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 1. Immunofluorescence of cell surface ICAM-1 from ST-like cultures after treatment with IFN{gamma} and HCMV. A) Photomicrographs of a single experiment in which ST-like cultures were either untreated (control) or treated for 24 h with IFN (100 U/ml) or challenged at an MOI of 10 with an HCMV preparation that had been filtered through a 0.1-µm filter to remove virus particles (filtered), UV-inactivated, or not treated (HCMV). B) Digital analysis of fluorescence emissions shown in (A). Depicted are the average ± SD of emissions (expressed as arbitrary units from three independent experiments carried out on two different placental preparations). Emission units in the different cultures have been normalized to the number of nuclei (determined by DAPI staining) per image. Experimental groups within each panel labeled with different letters (a, b, or c) are significantly different (P < 0.05)

HCMV-Induced ICAM-1 Expression Is Mediated by IE Proteins

The rapid nature of HCMV-induced ICAM-1 expression indicates that an early viral event is responsible. Trophoblast cell surface interactions with virus coat proteins are the earliest possible event; however, it is unlikely that virus entry would induce ICAM-1 expression, because UV-inactivated virus failed to do so. HCMV-induced ICAM-1 expression by human umbilical vein endothelial cells (HUVECs) is mediated by viral IE genes [23]. We therefore examined whether transcription of viral IE genes alone might induce trophoblast ICAM-1 levels.

HCMV IE promoter-driven IE expression plasmids carrying IE1-72, IE2-55, and IE2-86 [11] genes or the empty vector plasmid expressing only GFP were individually transfected into ST-like cultures, then cultured for 24 h. A transfection frequency of 15%–25% was achieved as previously described [31]. Following incubation, cell surface ICAM-1 expression was measured by immunofluorescence. All three IE proteins strongly induced ICAM-1 expression in the order of IE1-72>IE2-55>IE2-86, with IE1-72 stimulating expression almost as well as the positive control (IFN{gamma}-stimulated cultures) (Fig. 2).



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 2. Immunofluorescence of cell surface ICAM-1 from ST-like cultures after no treatment (control); treatment with IFN{gamma} or transfection with an empty expression plasmid (mock); or with expression plasmids containing CMV-IE2-55, IE1-72, or IE2-86 genes. A) Photomicrographs of trophoblast cultures after the indicated treatments. B) Digital analysis of fluorescence emission shown in (A). Depicted are the average ± SD of emissions (expressed as arbitrary units) from three independent experiments carried out on two different placental preparations. Emission units in the different cultures have been normalized to the number of nuclei (determined by DAPI staining) per image. Experimental groups within each panel labeled with different letters (a, b, or c) are significantly different (P < 0.05)

The photomicrographs (Figs. 1A and 2A) combined with data from earlier experiments that show the infection frequency is <10%, indicating that ICAM-1 expression is rather widespread, and thus suggesting that cells other than those infected or transfected express higher levels of ICAM-1. To test this idea the distribution of ICAM-1 (cell surface [red]) and virus replication (GFP expression [green]) was determined by two-color immunofluorescence (see Fig. 5A). GFP expression was driven from the early gene UL127, which is nonessential for replication in culture. The results indicate that ICAM-1 is expressed both in culture areas near and at sites of viral replication, and therefore suggest some form of paracrine induction. However, they do not exclude concomitant direct induction.



View larger version (133K):
[in this window]
[in a new window]
  		FIG. 5. Immunofluorescence of cell surface ICAM-1 (red) and GFP expression (green) from ST-like cultures infected with gfp-HCMV (MOI 10). A) Photomicrographs of trophoblast cultures infected with HCMV. B) Photomicrographs of trophoblast cultures infected with HCMV in the presence of 20 µg/ml of anti-IL1ß antibody. C) Photomicrographs of trophoblast cultures infected with HCMV in the presence of 20 µg/ml of anti-TNF{alpha} antibody. D) Photomicrographs of trophoblast cultures infected with HCMV in the presence of 20 µg/ml of anti-TNF{alpha} antibody and anti-IL1ß. Overlay images depicting ICAM-1 and GFP colocalization (yellow) were created using Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA)

TNF{alpha} Mediates Most of HCMV-Induced ICAM-1 Expression

We have previously shown that placental trophoblasts can release TNF{alpha} [31], that TNF{alpha} stimulates ICAM-1 expression in ST-like cultures [16], and that CMV-IE protein expression stimulates TNF{alpha} release [31]. Consequently, we examined whether HCMV-induced ICAM-1 was mediated by TNF{alpha}. Excess neutralizing antibody to TNF{alpha} was added to cultures at the time of HCMV infection or CMV-IE transfection, and cell surface ICAM-1 expression was measured 24 h later by fluorescence emissions (Fig. 3). The neutralizing TNF{alpha} antibody inhibited approximately 75% of CMV and CMV IE1-72-inducible ICAM-1 expression (total minus control) (Fig. 3). After HCMV infection or IE expression plasmid transfection, these cultures contain approximately 10 pg/ml TNF{alpha} [31]. Antibody to TNF{alpha} at 20 µg/ml is able to completely inhibit HCMV or IE-protein-induced apoptosis mediated by this concentration of TNF{alpha} [31] and is able to neutralize apoptosis induced by 5 ng/ml of exogenous TNF{alpha} (data not shown). We therefore conclude that 25% and 50% of HCMV and HCMV IE-induced ICAM-1 expression is independent of paracrine induction by TNF{alpha}.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 3. The effect of neutralizing anti-TNF{alpha} antibody and anti-IL1ß antibody on HCMV and CMV-IE protein induced ICAM-1 expression in trophoblasts as measured by fluorescence emissions. ST-like cultures were not treated (control), infected with HCMV (MOI 10) or transfected with HCMV IE1-72, cultured with or without 20 µg/ml of neutralizing antibody to TNF{alpha} or IL1ß for 24 h, then assessed for cell surface ICAM-1 expression by immunofluorescence. Depicted are the average ± SD of emissions (expressed as arbitrary units) from three independent experiments carried out on two different placental preparations. Emission units in the different cultures have been normalized to the number of nuclei (determined by DAPI staining) per image. Experimental groups within each panel labeled with different letters (a, b, or c for uninfected; {alpha} or ß for HCMV-infected; A or B for IE72-transfected) are significantly different (P < 0.05)

HCMV-induced ICAM-1 expression in endothelial cells is partly mediated by secretion of IL1ß [24, 33]. Consequently, we next examined whether IL1ß accounts for the HCMV-induced ICAM-1 independent of TNF{alpha}. Excess neutralizing antibody to IL1ß was added to the cultures during the time of challenge, and ICAM-1 surface expression was measured 24 h later by fluorescence emissions (Fig. 3). The addition of neutralizing IL1ß antibody did not have any significant effect, although a slight decrease was observed. Adding IL1ß antibody in combination with TNF{alpha} antibody was marginally more effective than adding TNF{alpha} antibody alone; however, this again was not significant.

Although fluorescence emission gives an indication of the levels of ICAM-1 in the culture as a whole, it does not convey an impression of the distribution of ICAM-1. This can be determined by analyzing the photomicrographs for the percent area of high ICAM-1 expression (higher than control cultures). As with fluorescence emission, the presence of neutralizing IL1ß antibody did little to reduce the area of high ICAM-1 expression during an HCMV infection (Fig. 4). However, unlike the fluorescence emission, IL1ß antibody together with TNF{alpha} antibody was more effective than TNF{alpha} antibody alone at reducing the area of high ICAM-1 expression (Fig. 4). Taken in combination, the data suggest that TNF{alpha} is the major mediator of paracrine HCMV-induced ICAM-1 expression, whereas IL1ß plays a minor role.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 4. The effect of neutralizing anti-TNF{alpha} antibody and anti-IL1ß antibody on HCMV-induced ICAM-1 expression on trophoblasts as measured by percent area of high ICAM-1 expression in the culture. ST-like cultures were not treated (control), infected with HCMV (MOI 10), or cultured with or without 20 µg/ml of neutralizing antibody to TNF{alpha} or IL1ß for 24 h, then assessed for the percent surface area of high ICAM-1 expression in the cell culture. Depicted are the average ± SD percent area of high ICAM-1 expression from three independent experiments carried out on two different placental preparations. Experimental groups within each panel labeled with different letters (a, b, c, d, or e) are significantly different (P < 0.05)

Neutralizing antibody was added in large excess, yet we were unable to completely block HCMV upregulation of ICAM-1 as determined by both fluorescence emission (Fig. 3) and high expression area (Fig. 4). The involvement of another cytokine is possible; however, images show large colocalization of ICAM-1 and viral replication, suggesting a direct viral effect (Fig. 5D).

HCMV-Induced ICAM-1 Expression Is also a Direct Effect of Virus

HCMV-induced ICAM-1 upregulation in human fibroblasts is direct and not mediated by inflammatory cytokine release [25, 34]. In fibroblasts, HCMV transactivators IE2-86 and pp71 synergistically induce ICAM-1 through the Sp1 binding site [35]. In order to determine whether only cells positive for HCMV replication express ICAM-1 after treatment with neutralizing antibodies, we carried out the infection experiments with gfp-CMV for 4 days, then examined whether there was any high ICAM-1 expression at a distance from GFP-positive cells (Fig. 5). Cultures not treated with neutralizing TNF{alpha} and IL1ß antibodies exhibited areas of high ICAM-1 expression away from (red) and at (yellow) sites of viral replication. The presence of TNF{alpha} antibody reduced ICAM-1 expression not colocalized with GFP (Fig. 5B), whereas antibody to IL1ß had little effect (Fig. 5C). The combination of both antibodies was slightly more effective at neutralizing paracrine ICAM-1 expression than TNF{alpha} antibody alone (Fig. 5D).

We next quantitated the high ICAM-1 staining on cells infected (ICAM-1+/GFP+ [yellow]) and not infected (ICAM-1+/GFP– [red]) with HCMV. If the yellow (ICAM-1+/GFP+) area was unaffected by antibodies to TNF{alpha} and IL1ß, then the upregulation of ICAM-1 was due to direct effects of the virus. Alternatively, if the antibodies decrease this compartment, it would suggest that HCMV upregulates ICAM-1 expression in infected cells largely by autocrine release of TNF{alpha} or IL1ß. We found that approximately 15% of the cell culture surface of ST-like cultures were positive for high ICAM-1 expression and HCMV, and that neutralizing antibodies did not change this percentage (Fig. 6A). Thus, ICAM-1 expression in infected cells was independent of the concentration of TNF{alpha} and IL1ß in the supernatant. However, when the area of ICAM-1 not associated with infection (ICAM-1+/GFP–) was examined, it was reduced by the addition of antibodies (Fig. 6B). Antibodies to TNF{alpha} and IL1ß both reduced ICAM-1 expression (the former greater than the latter), and together, they almost completely reversed the paracrine effect of HCMV. This showed that the ICAM-1 upregulation on uninfected cells was almost entirely due to the release of TNF{alpha} and IL1ß by the infection.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 6. The effect of neutralizing anti-TNF{alpha} antibody and anti-IL1ß antibody on HCMV-induced ICAM-1 expression colocalized (ICAM-1+/ GFP+) or not colocalized (ICAM-1+/GFP–) to sites of viral replication (GFP expression). ST-like cultures were either untreated (control) or challenged with gfp-HCMV (MOI 10) for 4 days in the presence of 20 µg/ml of anti-TNF{alpha} antibody, anti-IL1ß antibody, or both. Depicted are the average ± SD of the percent area of ICAM-1+/GFP+ (A) and ICAM-1+/ GFP– (B) from three independent experiments carried out on two different placental preparations. Experimental groups within each panel labeled with different letters (a, b, or c) are significantly different (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We hypothesized that ICAM-1 would be strongly expressed by the ST after HCMV infection. In this study, villous CT (by the criterion of absent MHC class I, MHC class II, and CD9) were differentiated to an ST-like culture with EGF [29]. Until induction with HCMV (and IFN{gamma}), ST-like cultures expressed little ICAM-1. However, after induction with the virus, they expressed high levels on their apical surfaces within 24 h of exposure. Thus, HCMV infection of the villous ST can initiate an inflammatory cascade that could lead to enhanced monocyte binding to the ST surface, and thereby induce ST damage [16, 17]. This is the first report showing that HCMV infection induces ICAM-1 expression in primary trophoblast cultures.

HCMV exposure to vascular endothelial cells upregulates ICAM-1 [23, 24, 27, 35, 36]. However, whether the upregulation is due to virus alone or is mediated by paracrine release of IL1ß is controversial [23, 24, 36]. Because villous trophoblasts are also vascular cells and upregulation of ICAM-1 on these cells can be induced by TNF{alpha}, IL1ß, and IFN{gamma} [16], we examined whether the upregulation of ICAM-1 by HCMV was paracrine (i.e., mediated by cytokines) or direct. We found that <15% of the cells were infected, but that >30% of the surface area expressed elevated levels of surface ICAM-1. Adding antibody to TNF{alpha} reduced the surface area that expressed ICAM-1 by greater than half. When examining cells not infected with HCMV, we determined that IL1ß antibody could reproducibly reduce the expression of ICAM-1 alone, and in combination with antibody to TNF{alpha}, it almost completely reversed paracrine induction. This indicated that HCMV upregulation of ICAM-1 on cells not infected with HCMV took place by paracrine release of TNF{alpha} and IL1ß. What this means in vivo is complicated by the ST being a single, continuous cell. The infected portion of the ST could secrete TNF{alpha} and IL1ß, which in turn, could act at a distance to induce ICAM-1. However, this remains to be proven and it is unclear how much this mechanism contributes to ICAM-1 expression in vivo.

In contrast, our results showing that high ICAM-1 expression on infected cells was significant and unaffected by antibody to TNF{alpha} and IL1ß provide an unambiguous way for HCMV-1 to upregulate ICAM-1 on intact ST. Presumably, the autocrine contributions of the cytokines are subsumed by the direct effect of HCMV infection on ICAM-1 expression. A direct effect of HCMV on ICAM-1 expression is in accord with earlier observations in fibroblasts of HCMV IE2-86 and pp71, inducing ICAM-1 through the SP1 site [35]. However, this is the first report that HCMV both stimulates expression in a paracrine manner via cytokine release and directly in infected cells.

The rapid nature of HCMV-induced ICAM-1 expression suggests that an early event is responsible. Virus entry is the earliest event in the virus life cycle and has been shown to regulate transcription of a number of cellular genes in the absence of viral replication [11, 37–41] via cellular signaling receptors such as toll-like receptor (TLR)-2 [42]. Binding of the virus upregulates a number of transcription factors, including Sp1 [37], which is known to regulate the promoter of ICAM-1 [35]. However, we have found that UV-inactivated virus does not alone stimulate significant surface ICAM-1, suggesting that viral gene transcription and translation are required. We have also shown that transfection with the viral IE genes IE-72, IE-55, and IE-86 upregulates ICAM-1 expression. This indicates that virus gene transcription to the IE stage is necessary and sufficient for ICAM-1 induction.

HCMV infection of placental trophoblast is associated with villous inflammation (villitis) [4, 5, 12, 13], which is characterized by the focal loss of villous trophoblast [4]. We have previously suggested a mechanism whereby HCMV infection stimulates TNF{alpha} release and, subsequently, the rapid induction of apoptosis in uninfected neighboring cells [31]. This loss of the trophoblast would contribute to HCMV-related placental villitis. However, in this communication, we suggest an alternative, and perhaps complementary, mechanism. There is an accumulation of mononuclear phagocytes within placental lesions associated with HCMV [14, 43] and we have shown that binding of monocytes to cultured ST via ICAM-1 leads to TNF{alpha}-mediated ST damage [17]. Infection would induce ICAM-1 expression on the ST, and this would bind monocytes to the ST. Thus, induction of ICAM-1 on the villous ST by HCMV infection could be an early step in trophoblast loss, which is characteristic of villitis.

Our current studies also have major implications for possible mechanisms of in utero transmission of HCMV from mother to fetus across the placenta. Infected villous trophoblasts release less than 5% of progeny virus [8]. Furthermore, progeny virus from productively infected polarized ST-like membrane cultures is released from the apical surface (toward the maternal circulation) [44]. Taken together, these observations do not support an infection and release model of vertical transmission. An alternative model is one in which vertical transmission occurs when the ST barrier has been damaged and thus compromised. Release of TNF{alpha} due to HCMV infection can directly deplete the underlying CT that has not yet been infected [31]. In this study we show that infection of ST-like cultures leads to ICAM-1 expression, which in turn, could lead to monocyte binding [16] and localized loss of the ST [17]. The combination of ST loss by monocyte binding and CT loss by release of TNF{alpha} can lead to local rupture of the villous trophoblast by HCMV. Such a model suggests that vertical transmission is secondary to villous trophoblast damage and is consistent with correlations between in utero transmission and placental villitis [45].


   ACKNOWLEDGMENTS
 
We thank the Tissue Collection Core of the CIHR Group in Perinatal Health and Disease and the delivery room staff at the Royal Alexandra Hospital in Edmonton for their assistance in timely collection of placentas for this study, Bonnie Lowen for trophoblast isolation, and Mary Pat Gibson for secretarial assistance.


   FOOTNOTES
 
1 Correspondence: Larry J. Guilbert, Department of Medical Microbiology and Immunology, 6-25 HMRC, University of Alberta, Edmonton, Alberta T6G 2S2 Canada. FAX: 780 492 9828; larry.guilbert{at}ualberta.ca Back

Received: 5 February 2004.

First decision: 25 February 2004.

Accepted: 3 May 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kumar ML, Prokay SL. Experimental primary cytomegalovirus infection in pregnancy: timing and fetal outcome. Am J Obstet Gynecol 1983 145:56-60[Medline]
  2. Stagno S, Pass RF, Dworsky ME, Henderson RE, Moore EG, Walton PD, Alford CA. Congenital cytomegalovirus infection: the relative importance of primary and recurrent maternal infection. N Engl J Med 1982 306:945-949[Abstract]
  3. Yow MD, Williamson DW, Leeds LJ, Thompson P, Woodward RM, Walmus BF, Lester JW, Six HR, Griffiths PD. Epidemiologic characteristics of cytomegalovirus infection in mothers and their infants. Am J Obstet Gynecol 1988 158:1189-1195[Medline]
  4. Benirschke K, Kaufmann P. Pathology of the Human Placenta. New York: Springer; 2000
  5. Sinzger C, Muntefering H, Loning T, Stoss H, Plachter B, Jahn G. Cell types infected in human cytomegalovirus placentitis identified by immunohistochemical double staining. Virchows Arch A Pathol Anat Histopathol 1993 423:249-256[CrossRef][Medline]
  6. Sachdev R, Nuovo GJ, Kaplan C, Greco MA. In situ hybridization analysis for cytomegalovirus in chronic villitis. Pediatr Pathol 1990 10:909-917[Medline]
  7. Muhlemann K, Miller RK, Metlay L, Menegus MA. Cytomegalovirus infection of the human placenta: an immunocytochemical study. Hum Pathol 1992 23:1234-1237[CrossRef][Medline]
  8. Hemmings DG, Kilani R, Nykiforuk C, Preiksaitis J, Guilbert LJ. Permissive cytomegalovirus infection of primary villous term and first trimester trophoblasts. J Virol 1998 72:4970-4979[Abstract/Free Full Text]
  9. Halwachs-Baumann G, Wilders-Truschnig M, Desoye G, Hahn T, Kiesel L, Klingel K, Rieger P, Jahn G, Sinzger C. Human trophoblast cells are permissive to the complete replicative cycle of human cytomegalovirus. J Virol 1998 72:7598-7602[Abstract/Free Full Text]
  10. Furnari BA, Poma E, Kowalik TF, Huong SM, Huang ES. Human cytomegalovirus immediate-early gene 2 protein interacts with itself and with several novel cellular proteins. J Virol 1993 67:4981-4991[Abstract/Free Full Text]
  11. Yurochko AD, Kowalik TF, Huong SM, Huang ES. Human cytomegalovirus upregulates NF-kappa B activity by transactivating the NF-kappa B p105/p50 and p65 promoters. J Virol 1995 69:5391-5400[Abstract]
  12. Garcia AG, Fonseca EF, Marques RL, Lobato YY. Placental morphology in cytomegalovirus infection. Placenta 1989 10:1-18[Medline]
  13. Bernstein PS, Divon MY. Etiologies of fetal growth restriction. Clin Obstet Gynecol 1997 40:723-729[CrossRef][Medline]
  14. Greco MA, Wieczorek R, Sachdev R, Kaplan C, Nuovo GJ, Demopoulos RI. Phenotype of villous stromal cells in placentas with cytomegalovirus, syphilis, and nonspecific villitis. Am J Pathol 1992 141:835-842[Abstract]
  15. Altemani AM. Immunohistochemical study of the inflammatory infiltrate in villitis of unknown etiology. A qualitative and quantitative analysis. Pathol Res Pract 1992 188:303-309[Medline]
  16. Xiao J, Garcia-Lloret M, Winkler-Lowen B, Miller R, Simpson K, Guilbert LJ. ICAM-1-mediated adhesion of peripheral blood monocytes to the maternal surface of placental syncytiotrophoblasts: implications for placental villitis. Am J Pathol 1997 150:1845-1860[Abstract]
  17. Garcia-Lloret MI, Winkler-Lowen B, Guilbert LJ. Monocytes adhering by LFA-1 to placental syncytiotrophoblasts induce local apoptosis via release of TNF-alpha. A model for hematogenous initiation of placental inflammations. J Leukoc Biol 2000 68:903-908[Abstract/Free Full Text]
  18. Jutila MA. Leukocyte traffic to sites of inflammation. APMIS 1992 100:191-201[Medline]
  19. Marlin SD, Springer TA. Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1). Cell 1987 51:813-819[CrossRef][Medline]
  20. Dustin ML, Singer KH, Tuck DT, Springer TA. Adhesion of T lymphoblasts to epidermal keratinocytes is regulated by interferon gamma and is mediated by intercellular adhesion molecule 1 (ICAM-1). J Exp Med 1988 167:1323-1340[Abstract/Free Full Text]
  21. Gaffuri B, Vigano P, Nozza A, Gornati G, Di Blasio AM, Vignali M. Expression of intercellular adhesion molecule-1 messenger ribonucleic acid and protein in human term placental cells and its modulation by pro-inflammatory cytokines (interleukin-1beta and tumor necrosis factor alpha). Biol Reprod 1998 58:1003-1008[Abstract/Free Full Text]
  22. Sugiyama T, Cuevas LE, Bailey W, Makunde R, Kawamura K, Kobayashi M, Masuda H, Hommel M. Expression of intercellular adhesion molecule 1 (ICAM-1) in Plasmodium falciparum-infected placenta. Placenta 2001 22:573-579[CrossRef][Medline]
  23. Burns LJ, Pooley JC, Walsh DJ, Vercellotti GM, Weber ML, Kovacs A. Intercellular adhesion molecule-1 expression in endothelial cells is activated by cytomegalovirus immediate early proteins. Transplantation 1999 67:137-144[CrossRef][Medline]
  24. Dengler TJ, Raftery MJ, Werle M, Zimmermann R, Schonrich G. Cytomegalovirus infection of vascular cells induces expression of pro-inflammatory adhesion molecules by paracrine action of secreted interleukin-1beta. Transplantation 2000 69:1160-1168[CrossRef][Medline]
  25. Craigen JL, Grundy JE. Cytomegalovirus induced upregulation of LFA-3 (CD58) and ICAM-1 (CD54) is a direct viral effect that is not prevented by ganciclovir or foscarnet treatment. Transplantation 1996 62:1102-1108[CrossRef][Medline]
  26. Grundy JE, Downes KL. Upregulation of LFA-3 and ICAM-1 on the surface of fibroblasts infected with cytomegalovirus. Immunology 1993 78:405-412[Medline]
  27. Shahgasempour S, Woodroffe SB, Garnett HM. 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. Microbiol Immunol 1997 41:121-129[Medline]
  28. Watanabe M, Ito M, Kamiya H, Sakurai M. Adherence of peripheral blood leukocytes to cytomegalovirus-infected fibroblasts. Microbiol Immunol 1996 40:519-523[Medline]
  29. Guilbert LJ, Winkler-Lowen B, Sherburne R, Rote NS, Li H, Morrish DW. Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments. Placenta 2002 23:175-183[CrossRef][Medline]
  30. Yui J, Garcia-Lloret M, Wegmann TG, Guilbert LJ. Cytotoxicity of tumour necrosis factor-alpha and gamma-interferon against primary human placental trophoblasts. Placenta 1994 15:819-835[Medline]
  31. Chan G, Hemmings DG, Yurochko AD, Guilbert LJ. Human cytomegalovirus-caused damage to placental trophoblasts mediated by immediate-early gene-induced tumor necrosis factor-alpha. Am J Pathol 2002 161:1371-1381[Abstract/Free Full Text]
  32. Isomura H, Stinski MF. The human cytomegalovirus major immediate-early enhancer determines the efficiency of immediate-early gene transcription and viral replication in permissive cells at low multiplicity of infection. J Virol 2003 77:3602-3614[Abstract/Free Full Text]
  33. Woodroffe SB, Garnett HM, Danis VA. Interleukin-1 production and cell-activation response to cytomegalovirus infection of vascular endothelial cells. Arch Virol 1993 133:295-308[CrossRef][Medline]
  34. Ito M, Watanabe M, Ihara T, Kamiya H, Sakurai M. Increased expression of adhesion molecules (CD54, CD29 and CD44) on fibroblasts infected with cytomegalovirus. Microbiol Immunol 1995 39:129-133[Medline]
  35. Kronschnabl M, Stamminger T. Synergistic induction of intercellular adhesion molecule-1 by the human cytomegalovirus transactivators IE2p86 and pp71 is mediated via an Sp1-binding site. J Gen Virol 2003 84:61-73[Abstract/Free Full Text]
  36. Scholz M, Cinatl J, Gross V, Vogel JU, Blaheta RA, Freisleben HJ, Markus BH, Doerr HW. Impact of oxidative stress on human cytomegalovirus replication and on cytokine-mediated stimulation of endothelial cells. Transplantation 1996 61:1763-1770[CrossRef][Medline]
  37. Yurochko AD, Hwang ES, Rasmussen L, Keay S, Pereira L, Huang ES. The human cytomegalovirus UL55 (gB) and UL75 (gH) glycoprotein ligands initiate the rapid activation of Sp1 and NF-kappaB during infection. J Virol 1997 71:5051-5059[Abstract]
  38. Zhu H, Cong JP, Mamtora G, Gingeras T, Shenk T. Cellular gene expression altered by human cytomegalovirus: global monitoring with oligonucleotide arrays. Proc Natl Acad Sci U S A 1998 95:14470-14475[Abstract/Free Full Text]
  39. Boyle KA, Pietropaolo RL, Compton T. Engagement of the cellular receptor for glycoprotein B of human cytomegalovirus activates the interferon-responsive pathway. Mol Cell Biol 1999 19:3607-3613[Abstract/Free Full Text]
  40. Browne EP, Wing B, Coleman D, Shenk T. Altered cellular mRNA levels in human cytomegalovirus-infected fibroblasts: viral block to the accumulation of antiviral mRNAs. J Virol 2001 75:12319-12330[Abstract/Free Full Text]
  41. Simmen KA, Singh J, Luukkonen BG, Lopper M, Bittner A, Miller NE, Jackson MR, Compton T, Fruh K. Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B. Proc Natl Acad Sci U S A 2001 98:7140-7145[Abstract/Free Full Text]
  42. Compton T, Kurt-Jones EA, Boehme KW, Belko J, Latz E, Golenbock DT, Finberg RW. Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2. J Virol 2003 77:4588-4596[Abstract/Free Full Text]
  43. Kohut KG, Anthony MN, Salafia CM. Decidual and placental histologic findings in patients experiencing spontaneous abortions in relation to pregnancy order. Am J Reprod Immunol 1997 37:257-261
  44. Hemmings DG, Lowen B, Sherburne R, Sawicki G, Guilbert LJ. Villous trophoblasts cultured on semi-permeable membranes form an effective barrier to the passage of high and low molecular weight particles. Placenta 2001 22:70-79[CrossRef][Medline]
  45. Benirschke K, Mendoza GR, Bazeley PL. Placental and fetal manifestations of cytomegalovirus infection. Virchows Arch B Cell Pathol 1974 16:121-139[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
G. Chan, E. R. Bivins-Smith, M. S. Smith, P. M. Smith, and A. D. Yurochko
Transcriptome Analysis Reveals Human Cytomegalovirus Reprograms Monocyte Differentiation toward an M1 Macrophage
J. Immunol., July 1, 2008; 181(1): 698 - 711.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
E. A. V. Ferro, J. R. Mineo, F. Ietta, N. Bechi, R. Romagnoli, D. A. O. Silva, G. Sorda, E. Bevilacqua, and L. R. Paulesu
Macrophage Migration Inhibitory Factor Is Up-Regulated in Human First-Trimester Placenta Stimulated by Soluble Antigen of Toxoplasma gondii, Resulting in Increased Monocyte Adhesion on Villous Explants
Am. J. Pathol., January 1, 2008; 172(1): 50 - 58.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/3/797    most recent
biolreprod.104.028118v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chan, G.
Right arrow Articles by Guilbert, L.J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chan, G.
Right arrow Articles by Guilbert, L.J.
Agricola
Right arrow Articles by Chan, G.
Right arrow Articles by Guilbert, L.J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS