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Vet. Res.
Volume 31, Number 1, January-February 2000
Page(s) 42 - 43
How to cite this article Vet. Res. (2000) 42-43
Vet. Res. 31 (2000) 42-43

Phenotypical and functional analysis of macrophages infected with PRRS virus

L. López-Fuertesa, E. Camposa, N. Doménecha, A. Ezquerraa, J.M. Castrob, J. Domíngueza and F. Alonsoa

a  CISA-INIA, Valdeolmos 28130 Madrid, Spain
b  Dept. Patología Animal I. Facultad de Veterinaria, UCM. 28015 Madrid, Spain

Abstract - The PRRS virus shows a specific tropism for cells of the porcine monocyte/macrophage lineage. These cells play important roles both in innate and acquired immunity, performing a great variety of functions that include phagocytosis, scavenging at sites of tissue injury, killing of microbes and tumor cells, processing and presentation of antigens to lymphocytes and cytokine production. The aim of this study was to investigate the effect of the virus on different functional capacities of these cells. We have analysed the effect of the virus on the expression of different pro-inflammatory cytokines synthesised by the macrophage and the effect of the virus on the respiratory burst. We have also analysed the effect of TNF- $\alpha$ on virus replication and the modulation of different macrophage and monocyte cell surface molecules by the virus. Porcine alveolar macrophages (PAM) obtained from 3-week old healthy piglets were activated with 10 ng/mL of phorbol 12-myristate-1, 3-acetate (PMA) in the presence or absence of a European isolate (5710) of the PRRS virus (MOI = 1). Two and six hours later, the RNA from 10 7 cells/sample were extracted and a RT-PCR was carried out with specific primers for TNF- $\alpha$, IL-1 and MIP-1 $\beta$. In other experiments, PAM were treated under different conditions with porcine recombinant TNF- $\alpha$ (100 ng/ml) and infected with 10-fold dilutions of the PRRS virus, starting at a MOI of 1. Seven days later the cytopathic effect was recorded. Oxidative burst. After being infected or not with the PRRS virus (MOI = 1) for 16 h the PAM were activated with PMA and the hydrogen peroxide concentration was measured by flow cytometry, using the di-chlorofluorescein method. The African swine fever and Classical swine fever viruses were included as controls. The modulation of the expression of different leukocyte surface antigens (CD11a and CD11b, CD45, SLA-I, SLA-II, SWC3, 3F7/11, 3B11/11 and 2A10/11) by the PRRS virus was studied by two-colour staining, using monoclonal antibodies (mAbs) to these molecules labeled in red and, as a marker of viral infection, a mAb to the viral nucleoprotein labeled in green. These analyses were performed on infected and on non-infected PAM. The PRRS virus strongly reduced the TNF- $\alpha$ mRNA expression, dependent of PMA activation (46% inhibition) in PAM. There was only a slight difference between cells activated for 2 or 6 hours. The production of IL-1 mRNA under the same conditions, was inhibited only after 6 hours of incubation (50% inhibition). Its production after 2 hours was not affected. Regarding MIP-1 $\beta$ expression, the presence of the virus inhibited its expression by 37% after 2 hours of incubation; this inhibition increased up to 83% after 6 hours. Pre-treatment of PAM for 24 hours with 100 ng/mL of recombinant porcine TNF- $\alpha$ resulted in a reduction of 0.8 log in the virus output. When TNF- $\alpha$ was maintained in culture, a 2 log reduction was obtained. PAM stimulated with PMA showed an increase in their production of hydrogen peroxide, that was reduced when cells were infected by the virus for 16 hours. However, when these cells were infected by the African swine fever or Classical swine fever in similar conditions, this inhibitory effect was not seen. Infection of PAM did not show an effect on the expression of adhesion molecules such as CD11a and CD11b, nor CD45, SLA-class II molecules or SWC3. However, a clear down modulation of SLA-class I molecules was observed when infected and non-infected cells were compared. Molecules recognised by mAbs 3F7/11 and 3B11/11 were also down modulated. Finally, only cells expressing the 2A10 molecule more intensely were infected. Our results show an important disability of infected macrophages to generate an inflammatory response. The PRRS virus interferes with the production of proinflammatory cytokines. In addition, we have found that the virus is sensitive to TNF- $\alpha$. In this respect, it would be interesting to investigate the synergistic effect of this cytokine with other antiviral cytokines, such as IFN, which also seems to be affected by the virus. The reduction of hydrogen peroxide production in PAM stimulated with PMA after being in the presence of the virus, also reflects an alteration of the oxidative burst metabolism as a consequence of virus activity. The virus also causes a slight reduction in the expression of SLA-class I antigens that may be important in the recognition of infected cells by cytotoxic T lymphocytes. Other surface receptors (3B11/11 and 3F7/11) also appear down regulated after infection. However, at the present we do not know their function nor the significance of this reduction in macrophage activity. All these effects may provide the virus with some evasion strategies from the immune response, facilitating its spreading. On the contrary, the disfunction of macrophages caused by the virus may increase host susceptibility to secondary infections.

Corresponding author: F. Alonso Fax: (34) 91 620 2247;

© INRA, EDP Sciences 2000