Laboratory of Virology, Faculty of Veterinary Medicine, Ghent UniversitySalisburylaan 133, 9820 Merelbeke, Belgium
Correspondence
Hans J. Nauwynck
hans.nauwynck{at}rug.ac.be
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ABSTRACT |
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MAIN TEXT |
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Following infection of cells with enveloped viruses, viral glycoproteins are incorporated into cellular membranes. Binding of virus-specific antibodies to glycoproteins present in the plasma membrane makes infected cells recognizable for the classical complement pathway, phagocytes and natural killer cells, leading to lysis of the cell (Harper, 1994). Several herpesviruses have developed strategies to avoid antibody-dependent cell lysis. For pseudorabies virus (PRV)-infected monocytes, addition of PRV-specific antibodies results in clearance of viral glycoproteins from the plasma membrane by antibody-induced internalization (Favoreel et al., 1999
). Clearance of the plasma membrane renders infected monocytes significantly less susceptible towards antibody-dependent, complement-mediated lysis (G. R. Van de Walle, H. W. Favoreel, H. J. Nauwynck and M. B. Pensaert, unpublished results). In human cytomegalovirus (HCMV)-infected monocyte-derived macrophages, transport of viral glycoproteins to the plasma membrane is prevented due to the destruction of the microtubule network (Fish et al., 1996
). The absence of HCMV glycoproteins on the cell surface may be another strategy of avoiding recognition by antibody-dependent immune responses. Finally, several herpesviruses are known to encode proteins that interfere with the activation of the complement cascade (reviewed by Favoreel et al., 2000
).
The main purpose of the present study was to investigate how EHV-1-infected PBMCs are able to avoid recognition and destruction by antibody-dependent immune responses.
PBMCs were isolated from infection-immune horses by density centrifugation on FicollHypaque. After isolation, PBMCs were incubated for 24 h in medium supplemented with 0·5 µM ionomycin (IONO) and 10 nM phorbol dibutyrate (PDB) (Sigma) to favour EHV-1 replication (van der Meulen et al., 2001). Rabbit kidney (RK13) and equine embryonic lung (EEL) cells were maintained in minimal essential medium supplemented with 5 % foetal bovine serum, 100 U penicillin ml-1, 0·1 mg streptomycin ml-1, 0·1 mg kanamycin ml-1 and 0·3 mg glutamine ml-1, until trypsinization and inoculation. All cells were inoculated with EHV-1 strain 97P70 at an m.o.i. of 10 (van der Meulen et al., 2000
). After 1 h of incubation, the cells were washed and cultured at 37 °C. To ensure that the percentages of infected PBMCs obtained were not confounded by a possible reactivation in or reinfection of the horses during the sampling period, we always included stimulated, but non-inoculated, PBMCs. The expression of viral antigens was never detected in these control samples.
Cell surface and intracellular expression of EHV-1 antigens in PBMCs were examined at 0, 6, 9, 12 and 24 h post-inoculation (p.i.). PBMCs were fixed with 3 % paraformaldehyde and viral proteins on the cell surface were stained with polyclonal, FITC-labelled horse anti-EHV-1 IgGs. Intracellular viral proteins were stained upon permeabilization with 0·1 % saponin, using polyclonal, biotinylated anti-EHV-1 IgGs and a streptavidinAlexa Fluor 350 substrate (Molecular Probes). Anti-EHV-1 IgGs were raised by the hyperimmunization of a horse. An infection-immune horse was inoculated intranasally with EHV-1 strain 97P70. After 2 weeks, a second intramuscular immunization was performed using the same virus strain mixed with adjuvants (Suvaxyn Aujeszky im o/w, Fort Dodge). After 2 weeks, serum was collected. IgGs were purified on a protein G column and labelled with FITC (Becton Dickinson) or biotinylated (Amersham Pharmacia), following the manufacturers' instructions. Cells were analysed using an inverted fluorescence microscope (Leica DM IRBE). Cells were considered for further examination on possible surface expression only if intracellular expression of viral antigens was present. Total percentages of infected cells were 2·0±1·3 % at 6 h p.i., 4·3±1·9 % at 9 h p.i., 6·6±1·2 % at 12 h p.i. and 14·2±4·3 % at 24 h p.i. Of these infected PBMCs, 4·0±0·8 % showed cell surface expression at 6 h p.i., 14·4±3·7 % at 9 h p.i., 26·8±6·9 % at 12 h p.i. and 31·4±4·5 % at 24 h p.i. Thus, only a minority of EHV-1-infected PBMCs showed expression of viral antigens on their surface. Among these, two-thirds of the PBMCs expressed antigens on part of the cell surface only, designated focal expression, whereas the remaining cells expressed EHV-1 antigens on the entire surface, designated general expression. No differences were observed in the ratio of these expression patterns between the various time-points p.i. Confocal images of an EHV-1-infected PBMC without surface expression (Fig. 1a), with focal expression (Fig. 1b
) and with general expression (Fig. 1c
) are shown. These images were obtained using a Bio-Rad Radiance 2000 MP confocal laser scanning system attached to an inverted microscope (Eclipse TE300, Nikon).
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To examine the effect of antibody and complement on the viability of EHV-1-infected PBMCs with and without surface expression, PBMCs were infected for 24 h and incubated subsequently for 1 h at 37 °C with various concentrations of the FITC-labelled, anti-EHV-1 antibodies described previously. Afterwards, different concentrations of unheated EHV-1-negative horse serum were added for 1 h at 37 °C as a source of complement. Ethidium monoazide bromide (EMA) (Molecular Probes) was then added for 30 min at 4 °C to stain the nucleus of lysed cells. No photo-crosslinking of EMA was performed, since it reduced the intensity of fluorescence of the FITC-labelled antibodies and did not affect the observed percentages of lysed cells when compared to non-photo-crosslinked samples (data not shown). Finally, intracellular viral proteins were stained upon fixation and permeabilization as described earlier. In order to visualize surface expression in PBMCs incubated in the absence of FITC-labelled antibodies during the first step of the assay, an additional labelling step was included in between fixation and permeabilization. For this, the polyclonal, FITC-labelled anti-EHV-1 IgG was used. PBMCs were analysed using an inverted fluorescence microscope (Leica DM IRBE). A minimal number of 100 cells was scored per data-point. Statistical analysis was based on analysis of variance (rejection level 0·05). Table 1 presents the percentages of lysed PBMCs when using varying antibody and complement concentrations. In non-infected PBMCs, between 1·2 and 2·2 % of the cells were lysed and the addition of antibodies and complement had no effect on viability. For infected PBMCs without cell surface expression, percentages of lysed cells were similar to those in non-infected PBMCs. For infected PBMCs showing focal surface expression, the percentage of lysed cells was only significantly higher compared to non-infected PBMCs when antibody concentrations of 1·6 mg ml-1 and serum concentrations of 20 % were used (8·3 %). For infected PBMCs showing general surface expression, between 19·4 and 31·2 % of the cells were lysed in the presence of antibodies and complement, which was significantly higher compared to non-infected cells. When complement was added in the absence of antibodies, 10·5 % of PBMCs showing general surface expression were lysed, whereas the addition of antibodies in the absence of complement resulted in 5·4 % lysed cells. In contrast to infected PBMCs without and with focal surface expression, PBMCs showing general surface expression are apparently destroyed by three different immune mechanisms. Binding of antibodies to the surface of these infected PBMCs in the absence of complement triggered destruction, most likely via antibody-dependent, cell-mediated cytotoxicity. Lysis by complement in the absence of antibodies most likely occurred via the antibody-independent, complement-mediated pathway, whereas the addition of antibodies increased the percentage of lysed cells by activation of the antibody-dependent, complement-mediated pathway.
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The present study describes a potential strategy of immune evasion for EHV-1-infected PBMCs. We demonstrate that the majority of EHV-1-infected PBMCs lacks cell surface expression of viral antigens. Absence of surface expression protects infected PBMCs from lysis by equine complement.
Infected PBMCs showing focal cell surface expression could escape from complement-mediated lysis as well. Circumvention of antibody-dependent, complement-mediated lysis may be explained by the fact that efficiency of lysis depends on the amount of antibody bound per infected cell (Joseph et al., 1975; Perrin et al., 1976
; Sissons et al., 1979
). In our study, the extent of antigen expression on the surface of infected PBMCs showing focal expression varied from only a small area of expression to an almost complete surface expression. It is possible that only cells with more extensive antigen expression may bind enough IgGs to induce cell lysis. How infected PBMCs without surface expression and with focal surface expression escape antibody-independent, complement-mediated lysis seems less obvious. Analogous to the antibody-dependent pathway, the extent of antigen expression on the surface of infected cells may be important in turning the scale for either prevention or activation of the antibody-independent, complement-mediated pathway.
The maximal percentage of lysed PBMCs showing general surface expression was 31·2 %. This suggests that the efficiency of equine complement to lyse EHV-1-infected PBMCs is rather low. A low efficiency of equine complement, both of the antibody-independent and the antibody-dependent pathway, compared to other mammalian species has already been described by Ish et al. (1993). Low efficiency of complement in our study may also result from virus interference with the complement cascade. For herpes simplex virus (HSV), varicella-zoster virus (VZV) and PRV, it has been demonstrated that the viral glycoprotein complex gEgI displays Fc-receptor activity, which interferes with efficient antibody-dependent complement activation (Johnson et al., 1988
; Frank & Friedman, 1989
; Litwin et al., 1992
; Favoreel et al., 1997
; Nagashunmugam et al., 1998
; G. R. Van de Walle, H. W. Favoreel, H. J. Nauwynck and M. B. Pensaert, unpublished results). Homologues of gE and gI are also expressed by EHV-1 (Audonnet et al., 1990
; Elton et al., 1991
; Telford et al., 1992
) but whether they exert Fc-receptor activity is not known yet. Glycoprotein gC of HSV, VZV, EHV-1 and -4, PRV and bovine herpesvirus-1 is capable of binding with complement factor C3, a pivotal component of complement activation, which may result in inhibition of the antibody-independent pathway (
Friedman et al., 1984; Harris et al., 1990; Huemer et al., 1992
, 1993
, 1995
).
In conclusion, at least two-thirds of EHV-1-infected PBMCs lack cell surface expression of viral antigens. Absence of surface expression was observed independently of the subtype of PBMCs and protects infected cells from antibody-dependent and antibody-independent lysis by equine complement.
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ACKNOWLEDGEMENTS |
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Received 27 September 2002;
accepted 3 October 2002.