Abstract
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.
