Regulation of Maedi-visna virus and cytokine gene expression in machrophages

Macrophages are proposed to play a central role in Maedi-visna virus (MVV) infection as target cells. However, the level of MVV load in alveolar macrophages (AMs) and its correlation with pulmonary lesions is unknown. Also it is not yet clear whether cytokine expression is dysregulated in macrophages infected with MVV. In addition, the effects of exogenous cytokines on MVV replication in macrophages have not been documented. The aims of this study were to determine 1. the level of MVV DNA in AMs compared with blood monocytes in natural infection and assess the correlation of viral burden and replicative status to the lung lesions related to MVV infection; 2. the differential expression of cytokine genes in macrophages infected with MVV in vivo and in vitw, 3. the effect of GM-CSF, TGF-(3 and IFN-y on MVV replication in macrophage in vitro.

Quantitative competitive-PCR for MVV pol was developed to quantify accurately the level of MVV DNA load in alveolar macrophages (AMs). This first required constructing a competitive template which bears the same recognition site as target template to be quantified, but truncated by a 25 bp sequence. The quantitative evaluation of the PCR signal clearly shows that the overall levels of MVV DNA load in AMs was significant higher than that in blood monocytes (P<0.05). Changes of viral load in AMs differed in relation to the histopathological lesions in the lungs. Relatively high viral load was found in AMs from the lung with histopathological lesions. Furthermore, MVV replication did not occur in AMs isolated from sheep without histopathological lesions in the lung, but could stimulated in vitro, suggesting that the levels of MVV DNA load reflects the pathological manifestation in the site of disease. In addition, MVV replication in AMs in vivo may require the availability of certain factors to activate viral replication and these may be important in disease progression. Such factors could be cytokines produced by macrophages present or recruited in the site of disease.

Determination of cytokine expression in response to MVV infection in vivo and in vitro show a significant increase in the expression of IL-6, IL-10, GM-CSF and TGF- ß mRNA in AMs isolated from sheep naturally infected with MVV. The level of GMCSF mRNA was found to be much higher in AMs isolated from sheep with lung lesion when compared with those without lung lesions (P<0.05), but the levels of IL10 and IL-6 mRNA expression in AMs had no association with histopathological findings in the lung. TNF-a and TGF-ß mRNA level were not altered significantly in AMs. Blood monocytes showed a similar pattern of cytokine transcripts to AMs. In lymph nodes, the levels of IL-10, IL-2, and IL-6 mRNA were found to be higher than those in seronegative controls, whereas the levels of GM-SCF mRNA werenot significantly altered.IFN-γ mRNA was undetectable in both groups. Interestingly, IL10 mRNA expression in monocyte-derived macrophages (MDMs) was altered in response to MVV infection in vitro. This result raised a possibility that direct infection of MDMs with MVV may be not sufficient to stimulate detectable levels of IL-10 mRNA in these cells. In analysing heterogeneity of cytokine function in MVV infection increased expression of IL-6 was detected in MDMs exposed to GM-CSF but not to MVV, suggesting that interaction of MVV with the target cells is not essential for the expression of IL-6. In determining the effects of GM-CSF, TGF-ß and IFN-γ on the replication of MVV in MDMs, the enhancing effect of GM-CSF and inhibitory effects of TGF-P on MVV replication in MDMs was observed whereas IFN-γ was unable to demonstrate any apparent enhanced or inhibitory effects of IFNγ on MVV replication in MDMs. Cytokines modulation of surface antigens expression on macrophages was also investigated IFN-γ had an enhanced effect on the expression of MHC class I and II on MDMs whereas GM-CSF was showed to have an enhanced effects on MHC class I but not on MHC class II. Both cytokines did no significantly increased the expression of CD 14, CD45 and CD 11c, and had no influence on the expression of CDlb and CDllb on MDMs and MVV-infected MDMs.

These results may have important implications in understanding of regulation of MVV in macrophages as target cell for MVV and the balance between stimulatory and inhibitory cytokines that may play a critical role in controlling MVV replication and expression and subsequently in the clinical progression of MVV infection.