François Guiguen

In vivo activation of alveolar macrophages in ovine lentivirus infection

Sheep infected by visna-maedi virus, a lentivirus related to the human immunodeficiency virus, develop a chronic interstitial lung disease. Since monocyte/macrophages are known to be specifically infected by visna-maedi virus, we investigated the role of macrophages in the appearance of pulmonary lesions in animals with naturally occurring disease. Alveolitis in maedi leads to a doubling in bronchoalveolar lavage total cell counts and of macrophages as compared to normal sheep. A significant increase in the relative percentage of neutrophils was also observed, accompanied by an increased spontaneous release of neutrophil chemotactic activity by alveolar macrophages of diseased animals, suggesting that they may be activated. Macrophage activation is also demonstrated by the observation of a significant (x3) increase of spontaneous fibronectin release by alveolar macrophages from maedi lungs, and furthermore by the high level expression of major histocompatibility complex class II antigens on most of these cells. Thus viral infection, although restricted to a small population of macrophages, is able to modulate extensive activation of macrophages in the lung. Activated macrophages release mediators likely to play a role in the development of the alveolitis and the parenchymal desorganization. These findings may be relevant to our understanding of the mechanisms by which human immunodeficiency virus infection leads to pulmonary disease other than that caused by opportunistic infections.

Characterization of the lymphocytic alveolitis in visna‐maedi virus‐induced interstitial lung disease of sheep

In order to investigate the contribution of lymphocytes to interstitial lung disease in animals with visna‐maedi infection, we studied in parallel bronchoalveolar cells and lung tissue from slaughterhouse animals (n= 29) and from colostrum‐deprived lambs transtracheally inoculated with field isolates of visna‐maedi virus (n= 9) or saline (n = 6). Lymphocyte subpopulations were identified in bronchoalveolar lavagc by immunofluorescence and flow cylomctry analysis and in lung tissue using indirect immunohistochemistry. In infected animals a lymphocytic alveolitis containing CD4 and CD8 lymphocytes was observed. Pcribronchovascular lymphoid nodules comprise mostly CD4 lymphocytes. Alveolar lymphocytes of both subsets displayed increased expression of MHC class II antigens in animals with naturally occurring maedi but not in experimentally infected ones. A sequential process of lymphocyte attraction and activation is likely to occur in vivo as part of the alveolitis.

Visna-maedi virus-induced expression of interleukin-8 gene in sheep alveolar cells following experimental in vitro and in vivo infection

Visna-maedi virus is a lentivirus which causes inflammatory disorders in sheep, including a chronic interstitial lung disease resembling that observed in human immunodeficiency virus type 1 (HIV 1) infection. In view of our previous demonstration of the production of neutrophil chemotactic activity by alveolar macrophages, and given the lymphocytic and neutrophilic nature of the alveolar cell infiltrate in both naturally and experimentally infected animals, we hypothesized that interleukin-8 (IL8) could be a candidate for at least part of the chemotactic activity we described. In this study, we investigated IL8 mRNA expression following visna-maedi virus infection. Northern analysis of total RNA using an ovine IL8-specific probe demonstrated that the IL8 gene is upregulated in alveolar macrophages as a consequence of in vitro infection and in alveolar cells from experimentally infected animals. Using a semi-quantitative RT-PCR method, we showed that various levels of IL8 mRNA are expressed by alveolar cells from infected animals and that they correlate with the intensity of the lesions. In conclusion, visna-maedi virus is able to induce IL8 mRNA expression in sheep alveolar cells. Results from in vivo infected animals suggest that IL8 could play a role in the early build-up of visna-maedi virus-induced lesions.

Flow cytometric analysis of goat milk lymphocytes: subpopulations and adhesion molecule expression.

Cytometric analysis of cells in milk from healthy goats was achieved after elimination of interfering signals from debris and dead cells by irreversible staining with ethidium monoazide. Some 61% of milk lymphocytes are CD8+ T cells, 17% are CD4+ and about 20% of these express class II antigens: less than 4% are B cells. Compared with blood, the CD4/CD8 ratio was inverted and fewer lymphocytes expressed CD4SR or L-selectin. Monoclonal antibodies to ovine integrins recognised the caprine alpha 4 chain on most lymphocytes from milk or blood, while beta 1 was more frequent on milk cells.

Increased expression of tissue factor mRNA and procoagulant activity in ovine lentivirus-infected alveolar macrophages

To link ovine lentivirus infection to lung tissue damage, we studied the procoagulant response in alveolar macrophages from experimentally infected lambs and in in vitro infected alveolar macrophages. We cloned ovine tissue factor cDNA and analysed its in vitro expression by Northern blotting. Visna-maedi virus induced tissue factor mRNA. In order to correlate this mRNA induction with its cellular function, we analysed macrophage procoagulant activity after in vitro and in vivo infection. The procoagulant activity was increased by interaction with the virus in both cases. Thus, visna-maedi virus-induced expression of tissue factor mRNA was associated with enhanced macrophage procoagulant activity. These findings indicate an active role of alveolar macrophages in the pathogenesis of these inflammatory lung lesions.