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Interaction of the European genotype porcine reproductive and respiratory syndrome virus (PRRSV) with sialoadhesin (CD169/Siglec-1) inhibits alveolar macrophage phagocytosis.

De Baere MI, Van Gorp H, Delputte PL, Nauwynck HJ - Vet. Res. (2012)

Bottom Line: Similar results were obtained using inactivated PRRSV (LV), showing that initial interaction of the virion with the cell is sufficient to reduce phagocytosis, and that no productive infection is required.When macrophages were incubated with sialoadhesin- (Sn) or CD163-specific antibodies, two entry mediators of the virus, only Sn-specific antibodies downregulated the phagocytic capacity of PAM, indicating that interaction with Sn, but not CD163, mediates the inhibitory effect of PRRSV on phagocytosis.If similar events occur in vivo, this interaction may be important in the development of PRDC, as often seen in the field.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Virology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820, Merelbeke, Belgium. Hans.Nauwynck@Ugent.be.

ABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus that shows a restricted in vivo tropism for subsets of porcine macrophages, with alveolar macrophages being major target cells. The virus is associated with respiratory problems in pigs of all ages and is commonly isolated on farms with porcine respiratory disease complex (PRDC). Due to virus-induced macrophage death early in infection, PRRSV hampers the innate defence against pathogens in the lungs. In addition, the virus might also directly affect the antimicrobial functions of macrophages. This study examined whether interaction of European genotype PRRSV with primary alveolar macrophages (PAM) affects their phagocytic capacity. Inoculation of macrophages with both subtype I PRRSV (LV) and subtype III PRRSV (Lena) showed that the virus inhibits PAM phagocytosis. Similar results were obtained using inactivated PRRSV (LV), showing that initial interaction of the virion with the cell is sufficient to reduce phagocytosis, and that no productive infection is required. When macrophages were incubated with sialoadhesin- (Sn) or CD163-specific antibodies, two entry mediators of the virus, only Sn-specific antibodies downregulated the phagocytic capacity of PAM, indicating that interaction with Sn, but not CD163, mediates the inhibitory effect of PRRSV on phagocytosis. In conclusion, this study shows that European genotype PRRSV inhibits PAM phagocytosis in vitro, through the interaction with its internalization receptor Sn. If similar events occur in vivo, this interaction may be important in the development of PRDC, as often seen in the field.

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Antibody binding to sialoadhesin, but not CD163, downregulates phagocytosis. Flow cytometric analysis of the effect of Sn- or CD163-specific antibodies on macrophage phagocytosis. The effect of the Sn-specific mAb 41D3 (Black circle), the CD163-specific mAb 2A10 (Dark grey circle) or an isotype-matched control mAb 13D12 (Light grey circle) on phagocytosis of beads by viable macrophages, expressed as the percentage of viable cells that have beads associated with them (A) or the MFI per viable cell (B), which is a measure for the amount of beads per cell. PAM were incubated with the indicated amount of mAb for 1 h, after which a phagocytosis assay was performed. As a control, non-treated PAM were included in the study. In addition, a phagocytosis assay was performed at 4°C (dotted line) with non-treated cells, to assess the percentage of macrophages that have beads bound to their surface and their MFI. Data represent the mean ± SEM of 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.
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Figure 3: Antibody binding to sialoadhesin, but not CD163, downregulates phagocytosis. Flow cytometric analysis of the effect of Sn- or CD163-specific antibodies on macrophage phagocytosis. The effect of the Sn-specific mAb 41D3 (Black circle), the CD163-specific mAb 2A10 (Dark grey circle) or an isotype-matched control mAb 13D12 (Light grey circle) on phagocytosis of beads by viable macrophages, expressed as the percentage of viable cells that have beads associated with them (A) or the MFI per viable cell (B), which is a measure for the amount of beads per cell. PAM were incubated with the indicated amount of mAb for 1 h, after which a phagocytosis assay was performed. As a control, non-treated PAM were included in the study. In addition, a phagocytosis assay was performed at 4°C (dotted line) with non-treated cells, to assess the percentage of macrophages that have beads bound to their surface and their MFI. Data represent the mean ± SEM of 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Mentions: Since inactivated virus also downregulates phagocytosis, the process causing this downregulation is not a replicative viral process, and probably results from a cellular process initiated upon interaction of the virus with the cell. Therefore we investigated whether the downregulation of phagocytosis could be explained by the interaction of the virus with its entry mediators Sn and/or CD163. To do so, another phagocytosis assay was performed. PAM were incubated with different concentrations of an Sn-specific mAb, a CD163-specific mAb or an isotype-matched control mAb for 1 h, after which the effect on phagocytosis was studied (Figure 3). Incubation of PAM with the Sn-specific mAb caused a downregulation of phagocytosis, starting from the lowest dose tested, affecting both the percentage of macrophages phagocytosing beads (Figure 3a) and the number of beads taken up per PAM (Figure 3b). PAM phagocytic capacity was further reduced when higher doses of the Sn-specific mAb were administered, up to 1.5 μg/mL, at which point phagocytosis was reduced down to the level observed when the assay was performed at 4°C, for both the percentage of PAM phagocytosing beads and the number of beads taken up per PAM. Further increasing the antibody dose did not further reduce the phagocytic capacity of PAM. This suggests that the phagocytic capacity of PAM is completely blocked when 1.5 μg/mL of the Sn-specific mAb is administered. Interestingly, the CD163-specific mAb 2A10 had no effect on phagocytosis, even at concentrations 30 times higher than the dose at which the Sn-specific mAb showed a maximum effect on phagocytosis. Similarly, experiments performed with a polyclonal antibody against CD163 also showed no effect on PAM phagocytosis (data not shown). In both cases, the effect of the CD163-specific antibodies was comparable to the effect of the isotype-matched control mAb treated or non-treated cells. Our data show that binding of the Sn-specific mAb to its receptor Sn leads to a similar inhibitory effect on PAM phagocytosis as observed after PAM inoculation with European genotype PRRSV, which suggests the involvement of Sn in the previously observed downregulation of phagocytosis by European genotype PRRSV. Our findings indicate that the observed downregulation of phagocytosis upon European genotype PRRSV inoculation is due to the interaction of the virus with its receptor Sn, but not CD163.


Interaction of the European genotype porcine reproductive and respiratory syndrome virus (PRRSV) with sialoadhesin (CD169/Siglec-1) inhibits alveolar macrophage phagocytosis.

De Baere MI, Van Gorp H, Delputte PL, Nauwynck HJ - Vet. Res. (2012)

Antibody binding to sialoadhesin, but not CD163, downregulates phagocytosis. Flow cytometric analysis of the effect of Sn- or CD163-specific antibodies on macrophage phagocytosis. The effect of the Sn-specific mAb 41D3 (Black circle), the CD163-specific mAb 2A10 (Dark grey circle) or an isotype-matched control mAb 13D12 (Light grey circle) on phagocytosis of beads by viable macrophages, expressed as the percentage of viable cells that have beads associated with them (A) or the MFI per viable cell (B), which is a measure for the amount of beads per cell. PAM were incubated with the indicated amount of mAb for 1 h, after which a phagocytosis assay was performed. As a control, non-treated PAM were included in the study. In addition, a phagocytosis assay was performed at 4°C (dotted line) with non-treated cells, to assess the percentage of macrophages that have beads bound to their surface and their MFI. Data represent the mean ± SEM of 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3403922&req=5

Figure 3: Antibody binding to sialoadhesin, but not CD163, downregulates phagocytosis. Flow cytometric analysis of the effect of Sn- or CD163-specific antibodies on macrophage phagocytosis. The effect of the Sn-specific mAb 41D3 (Black circle), the CD163-specific mAb 2A10 (Dark grey circle) or an isotype-matched control mAb 13D12 (Light grey circle) on phagocytosis of beads by viable macrophages, expressed as the percentage of viable cells that have beads associated with them (A) or the MFI per viable cell (B), which is a measure for the amount of beads per cell. PAM were incubated with the indicated amount of mAb for 1 h, after which a phagocytosis assay was performed. As a control, non-treated PAM were included in the study. In addition, a phagocytosis assay was performed at 4°C (dotted line) with non-treated cells, to assess the percentage of macrophages that have beads bound to their surface and their MFI. Data represent the mean ± SEM of 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.
Mentions: Since inactivated virus also downregulates phagocytosis, the process causing this downregulation is not a replicative viral process, and probably results from a cellular process initiated upon interaction of the virus with the cell. Therefore we investigated whether the downregulation of phagocytosis could be explained by the interaction of the virus with its entry mediators Sn and/or CD163. To do so, another phagocytosis assay was performed. PAM were incubated with different concentrations of an Sn-specific mAb, a CD163-specific mAb or an isotype-matched control mAb for 1 h, after which the effect on phagocytosis was studied (Figure 3). Incubation of PAM with the Sn-specific mAb caused a downregulation of phagocytosis, starting from the lowest dose tested, affecting both the percentage of macrophages phagocytosing beads (Figure 3a) and the number of beads taken up per PAM (Figure 3b). PAM phagocytic capacity was further reduced when higher doses of the Sn-specific mAb were administered, up to 1.5 μg/mL, at which point phagocytosis was reduced down to the level observed when the assay was performed at 4°C, for both the percentage of PAM phagocytosing beads and the number of beads taken up per PAM. Further increasing the antibody dose did not further reduce the phagocytic capacity of PAM. This suggests that the phagocytic capacity of PAM is completely blocked when 1.5 μg/mL of the Sn-specific mAb is administered. Interestingly, the CD163-specific mAb 2A10 had no effect on phagocytosis, even at concentrations 30 times higher than the dose at which the Sn-specific mAb showed a maximum effect on phagocytosis. Similarly, experiments performed with a polyclonal antibody against CD163 also showed no effect on PAM phagocytosis (data not shown). In both cases, the effect of the CD163-specific antibodies was comparable to the effect of the isotype-matched control mAb treated or non-treated cells. Our data show that binding of the Sn-specific mAb to its receptor Sn leads to a similar inhibitory effect on PAM phagocytosis as observed after PAM inoculation with European genotype PRRSV, which suggests the involvement of Sn in the previously observed downregulation of phagocytosis by European genotype PRRSV. Our findings indicate that the observed downregulation of phagocytosis upon European genotype PRRSV inoculation is due to the interaction of the virus with its receptor Sn, but not CD163.

Bottom Line: Similar results were obtained using inactivated PRRSV (LV), showing that initial interaction of the virion with the cell is sufficient to reduce phagocytosis, and that no productive infection is required.When macrophages were incubated with sialoadhesin- (Sn) or CD163-specific antibodies, two entry mediators of the virus, only Sn-specific antibodies downregulated the phagocytic capacity of PAM, indicating that interaction with Sn, but not CD163, mediates the inhibitory effect of PRRSV on phagocytosis.If similar events occur in vivo, this interaction may be important in the development of PRDC, as often seen in the field.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Virology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820, Merelbeke, Belgium. Hans.Nauwynck@Ugent.be.

ABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus that shows a restricted in vivo tropism for subsets of porcine macrophages, with alveolar macrophages being major target cells. The virus is associated with respiratory problems in pigs of all ages and is commonly isolated on farms with porcine respiratory disease complex (PRDC). Due to virus-induced macrophage death early in infection, PRRSV hampers the innate defence against pathogens in the lungs. In addition, the virus might also directly affect the antimicrobial functions of macrophages. This study examined whether interaction of European genotype PRRSV with primary alveolar macrophages (PAM) affects their phagocytic capacity. Inoculation of macrophages with both subtype I PRRSV (LV) and subtype III PRRSV (Lena) showed that the virus inhibits PAM phagocytosis. Similar results were obtained using inactivated PRRSV (LV), showing that initial interaction of the virion with the cell is sufficient to reduce phagocytosis, and that no productive infection is required. When macrophages were incubated with sialoadhesin- (Sn) or CD163-specific antibodies, two entry mediators of the virus, only Sn-specific antibodies downregulated the phagocytic capacity of PAM, indicating that interaction with Sn, but not CD163, mediates the inhibitory effect of PRRSV on phagocytosis. In conclusion, this study shows that European genotype PRRSV inhibits PAM phagocytosis in vitro, through the interaction with its internalization receptor Sn. If similar events occur in vivo, this interaction may be important in the development of PRDC, as often seen in the field.

Show MeSH
Related in: MedlinePlus