1 Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium
2 Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany
Correspondence
Hans Nauwynck
hans.nauwynck{at}rug.ac.be
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ABSTRACT |
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INTRODUCTION |
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PRV-infected monocytes express viral envelope proteins in their plasma membrane (Favoreel et al., 1999). Antibodies bind to these viral glycoproteins and should induce antibody-dependent lysis of the infected cells (Sissons & Oldstone, 1980
). Apparently, this does not happen efficiently in PRV-infected monocytes. Earlier, it has been shown that addition of PRV-specific antibodies to PRV-infected monocytes results in aggregation of the membrane-bound viral glycoproteins, followed by internalization of glycoproteinantibody aggregates. This antibody-dependent internalization is fast and efficient, is mediated by viral proteins gB and gD (Favoreel et al., 1999
, 2002
; Van de Walle et al., 2001
) and protects infected monocytes from efficient antibody-dependent cell lysis in vitro (G. R. Van de Walle, H. W. Favoreel, H. J. Nauwynck & M. B. Pensaert, unpublished observations). This mechanism may help to explain how infected monocytes survive in the blood of vaccinated animals. How these infected monocytes with internalized viral cell surface proteins (immune-masked monocytes) are then able to transmit PRV to the foetuses in the presence of neutralizing antibodies remains unclear. As a first step to reach the foetuses, PRV has to cross the endothelial cell barrier of the maternal blood vessels. A likely scenario of how this happens is that PRV-infected monocytes adhere to the endothelial cells and, subsequently, PRV crosses the endothelial barrier in the presence of virus-neutralizing antibodies. At least two hypotheses as to how the latter occurs can be put forward: on the one hand, immune-masked PRV-infected monocytes may cross the endothelial cell barrier by means of diapedesis; on the other hand, immune-masked PRV-infected monocytes may fuse with the vascular endothelial cells, thereby transmitting virus to these cells. In both cases, transmission of virus to endothelial cells is the very first step required for PRV to reach the foetuses. Although the subsequent steps are still to be unravelled, it has been shown before that all these steps can occur through direct cell-to-cell spread of the virus. Indeed, monocytes infected with a gDnull PRV mutant, which can only spread through direct cell-to-cell transport (Rauh & Mettenleiter, 1991
; Peeters et al., 1992a
), still can induce abortion in vaccinated sows (Nauwynck, 1997
).
Diapedesis of leukocytes at sites of inflammation is a well-known process (reviewed by Muller & Randolph, 1999) and in general requires the action of chemokines (Baggiolini, 1998
). The hypothesis of diapedesis as a means of PRV transmission through the endothelial cell barrier can certainly not be ruled out as a possible means of PRV transmission to foetal tissues and may be worth investigating in more detail. However, in the case of PRV-induced abortion, there are no indications of inflammation or local production of chemokines at the placental site or other triggers that could induce diapedesis of PRV-infected monocytes. Therefore, the aim of the current study was to evaluate the second hypothesis, comprising the adhesion and subsequent fusion of immune-masked PRV-infected monocytes with endothelial cells. In this context, it is noteworthy that contact between infected macrophages or neutrophils and endothelial cells has been suggested to be implicated in the dissemination of human cytomegalovirus (HCMV) from and to endothelial cells (Waldman et al., 1995
; Grundy et al., 1998
). Spread of HCMV to the embryo or foetus is well documented and is probably mediated by infected trophoblasts, macrophages and endothelial cells (Fisher et al., 2000
).
Alphaherpesviruses are well known for their cell-to-cell spread and the roles of different viral glycoproteins in PRV cell-to-cell spread have been studied extensively by the use of PRV deletion mutants. Viral glycoproteins gB and gH/gL have been shown to be essential for cell-to-cell spread, whereas gE and gD are modulatory (Klupp et al., 1997; Peeters et al., 1992a
, b
; Rauh & Mettenleiter, 1991
; Zsak et al., 1992
). Recently, it was suggested that the betaherpesvirus HCMV spreads from infected endothelial cells to leukocytes via microfusion events (Gerna et al., 2000
).
The aim of the present study was to investigate in vitro if immune-masked PRV-infected monocytes can transmit PRV to endothelial cells via adhesion and fusion in the presence of virus-neutralizing antibodies and if so, what the underlying mechanism is.
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METHODS |
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Antibodies.
Unlabelled and FITC-labelled protein A-purified polyclonal IgG antibodies were used, derived from PRV (89V87)-inoculated pigs, originating from a PRV-negative farm (Nauwynck & Pensaert, 1995b). Unlabelled anti-PRV IgG antibodies with a titre of 512, as determined by a serum neutralization test (Andries et al., 1978
), were used in culture medium to inhibit virus transmission via extracellular medium. FITC-labelled anti-PRV IgG antibodies were used to induce internalization of viral cell surface proteins in PRV-infected monocytes and were used at a concentration of 0·33 mg ml-1 (Favoreel et al., 1999
). To visualize cellular adhesion molecules CD15, CD11a, CD11b and CD18 on porcine monocytes, monoclonal antibodies (mAbs) DU-HL60-3 (Sigma; Whyte & Binns, 1994
), BL1H8, 2F4/11 and BA3H2 (Alvarez et al., 2000
), respectively, were used.
Isolation of porcine arterial endothelial cells.
Endothelial cells were isolated as described previously by Nauwynck & Pensaert (1995b), with some modifications. Briefly, endothelial cells were obtained from the aorta of 5- to 6-week-old piglets by flushing several times with 2·5 mg trypsin ml-1 (Sigma) at 37 °C. The collected cell fractions were centrifuged for 10 min at 260 g and resuspended in endothelial growth medium, based on Dulbecco's MEM (Gibco-BRL), and supplemented with 10 % foetal bovine serum (FBS), 0·6 mg glutamine ml-1, 100 U penicillin ml-1, 0·1 mg streptomycin ml-1, 0·1 mg kanamycin ml-1, 1 mM sodium pyruvate and 1 % non-essential amino acids (100x; Gibco-BRL). Cells were seeded in Corning cell culture flasks (Costar). Purity of endothelial cells was analysed by fluorescence staining with DiI-Ac-LDL (Biomedical Technologies; Voyta et al., 1984
). Purity was always
90 %. Cells from the first to third passages were harvested by trypsinization, seeded on a 4-well multidish (Nunc) in endothelial growth medium and used upon confluency.
Isolation of blood monocytes.
PRV-negative pigs were used as blood donors. Blood was collected from the vena jugularis on heparin (15 U ml-1) (Leo). Blood mononuclear cells were separated on FicollHypaque (Amersham Pharmacia) following the manufacturer's instructions. Mononuclear cells were then resuspended in medium (A), based on RPMI-1640 (Gibco-BRL), and supplemented with 10 % FBS, 0·3 mg glutamine ml-1, 100 U penicillin ml-1, 0·1 mg streptomycin ml-1, 0·1 mg kanamycin ml-1, 1 mM sodium pyruvate, 1 % non-essential amino acids (100x; Gibco-BRL) and 10 U heparin ml-1. Afterwards, cells were seeded on 58 mm Petri dishes with cell culture coating (Nunc) at a concentration of 2x106 cells ml-1 and cultivated at 37 °C with 5 % CO2. After 48 h, non-adhering lymphocytes were removed by washing the Petri dishes three times with RPMI-1640.
Infection of blood monocytes.
After the removal of lymphocytes, adherent cells, consisting of 70 % of monocytes, as assessed by flow cytometry after indirect immunofluorescence staining with the monocyte marker 74.22.15 (Pescovitz et al., 1984
), were infected with the different PRV strains at an m.o.i. of 20 in 0·5 ml medium A without heparin. Cells were incubated further at 37 °C with 5 % CO2. For all strains used and for all experiments, between 80 and 90 % of the monocytes were infected.
Incubation of PRV-infected monocytes with porcine anti-PRV polyclonal antibodies (pAbs).
At 13 h post-inoculation (p.i.) with PRV, monocytes were centrifuged for 10 min at 500 g, washed three times and resuspended in medium A. Cells were incubated for 1 h at 37 °C with FITC-labelled PRV pAbs (as described by Favoreel et al., 1999). Cells were washed two times with RPMI-1640 and then used in the adhesion/fusion assay. As a negative control, 50 µg genistein ml-1 (Sigma) was added 45 min before and also during antibody incubation. Genistein inhibits tyrosine kinase activity and therefore antibody-induced internalization of viral glycoproteins (Favoreel et al., 1999
). This concentration of genistein had no effect on cell viability, as determined earlier (Favoreel et al., 1999
), and also had no effect on adhesion or fusion processes (data not shown).
Definition of viral glycoprotein distribution.
The distribution of viral glycoproteins was scored as no internalization when the fluorescence label exhibited a homogeneous or patched cell surface cover but were considered internalized when viral glycoproteins were located in vesicles inside the cell.
Adhesion/fusion assay.
After incubation with FITC-labelled PRV pAbs in the presence or absence of genistein (as described above), PRV-infected monocytes were washed, counted and resuspended in endothelial growth medium, supplemented with PRV-neutralizing IgG antibodies in the presence or absence of 50 µg genistein ml-1. To each well of endothelial cells, approximately 1·5x105 monocytes were added, followed by centrifugation for 2 min at 44 g. At different time-points of co-cultivation (0, 30, 60 and 120 min), wells were washed two times thoroughly with endothelial growth medium and fixed for 10 min with 2 % formaldehyde and 0·2 % glutaraldehyde (Merck). The 4-well multidishes, consisting of monocytes and endothelial cells, were then stained for -galactosidase activity with X-Gal, following the manufacturer's instructions (Invitrogen). After 2 h, cells were mounted in a glycerin/PBS (0·9 : 0·1, vol/vol) solution with DABCO (Janssen). Quantitative results were obtained by examining the X-Gal signal (showing adhesion/fusion) by light microscopy and the distribution of fluorescence (showing internalization/no internalization) by fluorescence microscopy. Cells were excited with an Osram HBO 50 W bulb using a I3 filter and observed under a Leitz DM RBE microscope (Wild Leitz). Images were obtained using a Sony colour video camera (model DXC-9100P) linked to a Macintosh computer.
The percentage of adhesion of monocytes to endothelial cells was calculated as follows: (number of adhered monocytes at time-point x/number of monocytes added per well)x100. To determine the percentage of monocytes fused with endothelial cells, the following formula was used: (number of LacZ-positive endothelial cells at time-point x/number of adhered monocytes at time-point x)x100. All assays were run independently at least three times.
Some wells were, instead of being fixed, cultivated further at 37 °C with 5 % CO2 in the presence of virus-neutralizing antibodies and controlled for plaque formation at 30 h of co-cultivation.
Indirect immunofluorescence staining of cellular adhesion proteins on the cell surface of PRV-infected monocytes before and after antibody-induced internalization of viral cell surface proteins.
Monocytes (isolated as described above) were seeded on 4-well-chambered coverslips (Nunc) at a concentration of 2·5x106 cells ml-1 and inoculated with PRV. Monocytes, at 13 h p.i. with PRV, were incubated with 0·33 mg FITC-labelled PRV pAbs ml-1 and fixed for 10 min with 3 % formaldehyde at two time-points post-antibody addition (0 min, before internalization; 60 min, after internalization). Cells were washed two times with RPMI-1640 and incubated for 1 h at 37 °C with mAbs DU-HL60-3 (dilution 1 : 50), BL1H8 (dilution 1 : 10), 2F4/11 (dilution 1 : 30) or BA3H2 (dilution 1 : 30). Cells were again washed two times with RPMI-1640 and subsequently incubated for 1 h at 37 °C with Texas red-conjugated, goat anti-mouse antibodies (dilution 1 : 50) (Molecular Probes). Finally, cells were washed thoroughly, mounted in a glycerin/PBS solution and analysed by confocal microscopy.
Adhesion-blocking assay.
Experiments were performed as described for the adhesion/fusion assay, with some modifications. After incubation for 1 h at 37 °C with FITC-labelled PRV pAbs in the absence of genistein (to allow internalization of viral cell surface proteins), PRV-infected monocytes were washed and subsequently incubated for 30 min at 37 °C with medium A, supplemented with mAbs BL1H8, 2F4/11 and/or BA3H2 (dilution 1 : 10). After washing and counting, monocytes were added to wells of endothelial cells, fixed at 0, 30 and 60 min of co-cultivation and stained for -galactosidase activity with X-Gal (as described above). As a control, no antibodies were supplemented to medium A.
Confocal laser scanning microscopy.
Double-stained samples of viral glycoproteins and cellular adhesion molecules were examined with a Leica TCS SP2 laser scanning spectral confocal system (Leica) and linked to a DM IRB inverted microscope (Leica). Argon and HeNe laser light were used to excite FITC (488 nm line) and Texas red (543 nm line) fluorochromes. Extended focus images were obtained with Leica confocal software.
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RESULTS |
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Adhesion of immune-masked PRV-infected monocytes to endothelial cells
PRV Kaplan gG-infected monocytes, at 13 h p.i., were incubated with FITC-labelled PRV-specific antibodies at 37 °C for 1 h, as described in Methods. This antibody incubation resulted in 63·5±2·5 % of monocytes with internalized viral cell surface proteins (immune-masked monocytes) and 36·5±1·2 % of cells without internalized viral cell surface proteins (for images of cells without internalized viral cell surface proteins, see Fig. 3A
; for images of cells with internalized viral cell surface proteins, see Fig. 3B
). The mixture of monocytes with and without internalized viral cell surface proteins was then added to a monolayer of endothelial cells at 37 °C in the presence of virus-neutralizing antibodies and adhesion kinetics of both types of monocytes (with and without internalized viral glycoproteins) were determined. Fig. 1
(A) shows (i) that adhesion of monocytes to endothelial cells reaches its plateau at 60 min of co-cultivation and (ii) that immune-masked PRV-infected monocytes (with internalized viral cell surface proteins) adhere much more efficiently to the endothelial cells than PRV-infected monocytes without internalization. To confirm the latter, experiments were repeated in the presence of genistein, a reagent known to inhibit antibody-induced internalization (Favoreel et al., 1999
). Adding genistein before and during antibody addition (as described in Methods) resulted in a population of 94·3±1·5 % of PRV-infected monocytes without internalization. Adhesion efficiency of these monocytes to endothelial cells was low (Fig. 1A
). It was reported previously that the PRV Kaplan mutants
gB and
gD have reduced antibody-induced internalization efficiencies (Favoreel et al., 1999
; Van de Walle et al., 2001
). Monocytes inoculated with these strains for 13 h and incubated with PRV-specific antibodies for 1 h showed reduced adhesion efficiencies compared to the reference strain (Fig. 1B
). Reduction in adhesion efficiency of the
gB and
gD mutants compared to the wild-type virus corresponded fairly close to their reduction in endocytosis efficiency, suggesting further that, in the presence of neutralizing antibodies, endocytosis of viral cell surface proteins correlates directly with an increase in adhesion efficiency of infected monocytes to endothelial cells. Taken together, these results show that efficient internalization of antibodyantigen complexes from the cell surface of infected monocytes is essential to allow efficient adhesion of PRV-infected monocytes to endothelial cells in the presence of virus-neutralizing PRV-specific antibodies.
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Fusion is mediated by the virus
To investigate if fusion of immune-masked PRV-infected monocytes with endothelial cells is mediated by viral proteins, we inoculated monocytes with the PRV mutant gH. This PRV mutant carries the
-galactosidase fusion gene (lacZ) (Babic et al., 1996
). Furthermore, PRV gH is known to be essential for viruscell fusion and cell-to-cell spread of PRV (Peeters et al., 1992b
) and is not important for the antibody-induced internalization process (Favoreel et al., 1999
; Van de Walle et al., 2001
). Since PRV
gH has no effect on efficient antibody-induced internalization, using PRV
gH had no effect on the percentage of adhesion of PRV-infected monocytes to endothelial cells compared to the
gG reference strain (Fig. 4
A). Fig. 4(B)
shows that
gH-infected immune-masked monocytes are unable to fuse with endothelial cells (P<0·01, one way ANOVA). This implies that fusion is mediated by the virus, probably by a mechanism similar to viruscell fusion.
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DISCUSSION |
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In order for PRV to be able to reach the pregnant uterus in vaccinated animals, these immune-masked PRV-infected blood monocytes should be capable of transmitting the virus, in the presence of neutralizing antibodies, to the endothelial cells of the placental blood vessel walls. Here, we showed in an in vitro assay that, in the presence of virus-neutralizing antibodies, immune-masked monocytes can adhere efficiently to endothelial cells, followed by transmission of virus through fusion of the monocyte with the endothelial cell.
PRV-infected monocytes with internalized viral cell surface proteins (immune-masked monocytes) adhered much more efficiently to endothelial cells than PRV-infected monocytes without internalization. These results indicate that, in the presence of virus-neutralizing antibodies, antibody-induced internalization of viral cell surface proteins is important for efficient adhesion of PRV-infected monocytes to endothelial cells. Why are PRV-infected monocytes without internalized viral cell surface proteins unable to adhere efficiently to the endothelial monolayer? PRV-infected cells without internalized viral cell surface proteins, in contrast to immune-masked monocytes, are covered by PRV-specific antibodies, which may possibly result in (sterically) hindered access of the adhesion molecules on the monocytes (cellular as well as viral, such as gC and gD) to adhesion receptors on the underlying endothelial cells. This is consistent with earlier results showing that PRV-infected cells, covered with PRV-neutralizing antibodies, are unable to adhere to non-infected cells (Hanssens et al., 1993).
Since monocytes with internalized viral cell surface proteins possess only very few, if any, viral proteins on their plasma membrane (Favoreel et al., 1999), adhesion of immune-masked monocytes to endothelial cells is most likely mediated by cellular, and not viral, adhesion molecules on the cell surface. Physiological adhesion requires the interaction of integrins, such as CD11a/CD18 (also designated LFA-1) and CD11b/CD18 (also designated MAC-1) on monocytes with the Ig superfamily (ICAM-1, ICAM-2 and VCAM-1) on endothelial cells (Stewart et al., 1995
; Bullido et al., 1996
). However, it has been shown before that during internalization of viral cell surface proteins induced by PRV-specific antibodies, at least certain, unidentified, cellular proteins undergo co-internalization with the cell surface proteins (Favoreel et al., 1999
). Determining if and which cellular adhesion molecules remain on the plasma membrane of immune-masked PRV-infected monocytes was therefore essential as a first step to examine the process of adhesion of immune-masked monocytes to endothelial cells. Double immunofluorescence staining before and after antibody-induced internalization of viral cell surface proteins showed that significant amounts of the adhesion molecules CD11b and CD18 remained on the plasma membrane after antibody-induced internalization, whereas CD15 and CD11a were no longer visually present on the cell surface, suggesting that the adhesion complex CD11bCD18, but not CD15 and CD11aCD18, may be involved in the adhesion of immune-masked monocytes to endothelial cells. The CD11bCD18 complex was confirmed to be involved in the adhesion process, since pre-incubation of immune-masked PRV-infected monocytes with mAbs against these adhesion molecules, but not against CD11a, significantly reduced adhesion. Similarly, the CD18 molecule has been shown before to be involved in the adhesion of peripheral blood leukocytes to HCMV-infected endothelial cells (Span et al., 1991
). A possible hypothesis for the very efficient adhesion of the immune-masked monocytes to endothelial cells via cellular adhesion molecules could be that PRV induces an upregulation of certain adhesion molecules upon infection, as has already been described for many viruses, including herpesviruses. For example, for herpes simplex virus type 1, it has been shown that virus-infected blood monocytes adhere much more efficiently to human endothelial cells compared to non-infected monocytes, due to the production of certain cytokines, leading to an upregulation of cellular adhesion molecules on endothelial cells (Larcher et al., 2001
). Furthermore, HCMV has been shown to induce an upregulation of adhesion molecules in infected endothelial cells, resulting in an increase in adhesion of the endothelial cells to peripheral blood leukocytes (Shahgasempour et al., 1997
). Based on the present study, no indications were found for a similar upregulation of adhesion molecules in PRV-infected monocytes. However, the potential of such a putative upregulation to increase the chances for PRV to cross the barrier of endothelial cells may make it worth investigating in more detail.
Following adhesion, immune-masked monocytes were shown to fuse with endothelial cells. The rate of fusion appeared slower than the rate of adhesion, possibly suggesting that upon adhesion, expression/transport of viral proteins (e.g. gB, gH/gL) to the cell surface was necessary to induce fusion. Indeed, fusion was shown to be virus mediated, since inoculating monocytes with a lacZ-carrying gH mutant abolished the fusion capacity of the immune-masked monocytes (without affecting internalization and adhesion efficiencies). Fusion was shown to result in virus transmission to endothelial cells by the appearance of plaques. Only about 50 % of adhered and infected monocytes fused with the endothelial cells, even after 240 min of co-cultivation of monocytes with endothelial cells (data not shown), indicating that a significant fraction of immune-masked monocytes are incapable of fusing with endothelial cells. The reason for this inability to fuse is speculative but one explanation could be that, upon adhesion to the endothelial cells, not all monocytes are triggered efficiently to resume expression of viral proteins on the cell surface
Adhered leukocytes, either during immune cell-to-cell contact or during contact with epithelial/endothelial cells, are generally polarized (Sanchez-Madrid & del Pozo, 1999; Bromley et al., 2001
). Such polarization may support efficient virus spread, since it may result in predominant transport of viral cell surface proteins (implicated in cell fusion) or even virions, to the contact area between both cells. Such unidirectional deposition of virions at the contact site between infected leukocytes and contact cells has already been described for human immunodeficiency virus (Pearce-Pratt et al., 1994
; Fais et al., 1995
) and may be worth investigating during the interaction of immune-masked PRV-infected monocytes with endothelial cells.
In conclusion, it can be stated that immune-masked PRV-infected monocytes efficiently adhere to and subsequently fuse with endothelial cells in the presence of virus-neutralizing antibodies in vitro. The adhesion process is mediated by cellular adhesion molecules CD11b and CD18 and subsequent fusion is mediated by the virus. This adhesion and fusion process gives a potential explanation as to how PRV-infected monocytes transmit virus to vascular endothelial cells in vaccinated animals, as a first step to reach internal organs such as the pregnant uterus.
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ACKNOWLEDGEMENTS |
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Received 26 August 2002;
accepted 1 November 2002.