Figure 1.
Phenotype of tumor cells in naturally occurring OPA.
Immunofluorescence of lung tumor sections from sheep affected by OPA. Panel A–C show tumor sections analyzed by confocal microscopy using antibodies towards SP-C (showed in red), the JSRV Env (showed in green) and the appropriate secondary antibodies as described in Materials and Methods. Panels D–F show tumor sections analyzed by confocal microscopy using antibodies towards CC10 (showed in red), and the JSRV Env (showed in green). Nuclei were stained with DAPI and are shown in blue. Arrows in panel E indicates a CC10+ cells where JSRV Env expression is not detectable. The insert in panel E shows a larger magnification of the area indicated by the arrows. Scale bars: A,F = 75 µm, B = 43 µm, C = 34 µm, D = 89 µm, E = 29 µm.
Figure 2.
JSRV infection in lambs and adult sheep.
(A) Schematic diagram of the sheep lungs. Experimental inoculations were performed administering JSRV by bronchoscopy directly into the accessory bronchus. Tissue samples (1 for each lamb and 2 for each adult sheep) were collected from eight areas (delimited by red lines dotted lines in the panel) of the right cranial lobe. (B) Graph showing the mean number of JSRV Env+ clusters per animal as detected by immunohistochemistry in four lambs and four adult sheep (error bars indicate ± SD) 10 days post-infection. (C–E) Immunohistochemistry of JSRV Env+ cells in lung sections of adult sheep (C) and lambs (D–E) 10 days post infection. Env expression (characterized by the intra cytoplasmic dark brown colour) was detected in all experimentally infected lambs but not in adult sheep. (F–I) Phenotype of JSRV infected cells in experimentally infected lambs after 10 day post-infection. Panels F–G show lung sections analyzed by confocal microscopy using antibodies towards SP-C (showed in red) and the JSRV Env (showed in green). Nuclei were stained with DAPI and are shown in blue. Arrows indicate JSRV Env+ cells. Panel H–I show lung sections analyzed by confocal microscopy using antibodies towards CC10 (showed in red) and the JSRV Env (showed in green). Inserts show a larger magnification of the area indicated by the arrows. Scale bars in C = 200 µm; D–E = 100 µm; F–G = 47 µm; H = 30 µm; H = 75 µm.
Figure 3.
Number of proliferating SP-C+ and CC10+ cells in healthy lambs and adult sheep.
(A) Analysis of proliferating type 2 pneumocytes was performed by counting SP-C/Ki67 double positive cells in 2–4 day-old lambs (n = 4) and adult sheep (n = 2) by confocal microscopy as described in Materials and Methods. 10 sections for each animal were analysed using by confocal microscopy and numbers of double positive cells were normalized to the sectioned area. Results shown are the average numbers of SP-C+/Ki67+ (± SD) per section for both groups of animals. (B) Representative image of a lung section from a 2 day old lamb analyzed by confocal microscopy using antibodies towards SP-C (showed in red) and Ki67 (showed in green). Nuclei were stained with DAPI and are shown in blue. Note that Ki67 is a nuclear marker and therefore positive signal appears in turquoise in the merged image. Arrows indicate SP-C+/Ki67+ cells. (C) Analysis of proliferating Clara cells was performed by counting the number of CC10+/Ki67+ cells in 100 terminal bronchioli per each animal as indicated in Panel A. Results shown are the average numbers of CC-10+/Ki67+ (± SD) per 100 terminal bronchioli for both groups of animals. (D) Representative image of a lung section from a 2 day old lamb analyzed by confocal microscopy using antibodies towards CC-10 (showed in green) and Ki67 (showed in red). Nuclei were stained with DAPI and are shown in blue. Arrows indicate CC-10+/Ki67+ cells.
Figure 4.
Mild lung injury model in adult sheep.
(A–B) Histology of lung sections of sheep treated with 3 methylindole (3MI) (A) and mock-treated controls (B). Treatment with 3MI is characterized by edema and haemorrhagic areas. (C–D) Representative images of lung sections from adult sheep treated with 3MI (C) or mock control (D) analyzed by confocal microscopy using antibodies towards CC10 (showed in green) and Ki67 (showed in red) and the appropriate fluorescent conjugates as described in Materials and Methods. Nuclei were stained with DAPI and are shown in blue. Note in panel C the extensive injury of bronchiolar epithelium (absence of CC10 staining) and proliferation of toxicant resistant Clara cell progenitors (indicated by arrows). Note in both panels C and D the terminal bronchioli (Tb). (E–F) Representative images of lung sections from adult sheep treated with 3MI (E) or mock-treated (F) analyzed by confocal microscopy using antibodies towards SP-C (showed in red) and Ki67 (showed in green). Note in panel E the presence of several SP-C+/Ki67+ cells. Nuclei were stained with DAPI and are shown in blue. Arrows indicate SP-C+/Ki67+ cells. (G) Graph representing the quantification of the data analysed by confocal microscopy. Results shown are the average numbers of SP-C+/KI-67+ (± SD) per section for both groups of animals. (H) Analysis of proliferating Clara cells was performed by counting the number of CC10/Ki67 double-positive cells in 100 terminal bronchioli per each animal. Results shown are the average numbers of CC-10+/Ki67+ (± SD) per 100 terminal bronchioli for both groups of animals. Scale bars, A–B = 200 µm; E–F = 47 µm; C–D = 89 µm.
Figure 5.
Induction of mild lung injury renders adult sheep susceptible to JSRV infection.
(A) Schematic diagram of the study design. Ten adult sheep were randomly allocated in two groups. Sheep in Group I (red bar) were dosed 3MI (red arrow) as described in Materials and Methods while Group II served as control. After 2 days all animals in both groups were infected with JSRV (black arrow) and euthanized 10 days post infection. (B) Graph showing the mean number of JSRV Env+ clusters per animal as detected by immunohistochemistry in sheep of Group I and Group II 10 days post-infection (± SD). (C) Graph showing the number of cells forming each cluster of JSRV Env+ cells in sheep of Group I. (D–F) Immunohistochemistry of lung sections of adult sheep of group II (D) and group I (E–F, pre-treated with 3MI before JSRV infection) 10 days post-infection as described in Materials and Methods. Env expression (characterized by the intra cytoplasmic dark brown colour) was detected in sheep of Group I but not in sheep of Group II. Scale bars, D = 200 µm; E–F = 100 µm.
Figure 6.
Phenotype of JSRV infected cells in adult sheep with lung injury.
Representative images of lung sections from adult sheep pre-treated with 3MI before experimental JSRV infection. (A–C) Sections were analyzed by confocal microscopy using antibodies towards SP-C (showed in red) and the JSRV Env (showed in green). Nuclei were stained with DAPI and are shown in blue. Arrows indicate autofluorescent red blood cells (RBC). (D–F) Sections analyzed as above using antibodies towards CC10 (showed in red) and the JSRV Env (showed in green). Nuclei were stained with DAPI and are shown in blue. Scale bars, A–B = 47 µm; C = 89 µm; D = 26 µm; E = 33 µm and F = 25 µm.
Figure 7.
Infectivity of JSRV in proliferating and mitotic arrested cell.
(A) Schematic representation of the experimental design. A JSRV-based vector was derived by transfecting 293T with pJS-EFGFP-MC, pGPP-MX-4CTE and pC-MLV-JSenv as described in the materials and methods. The resulting vector, JS-eGFP, was then used to infect synchronized choroid plexus cells (CP) cultures in the presence or absence of aphidicolin. (B) Histograms showing the DNA content of CP cells with or without Aphidicolin (5 µg/ml for 24 hours). The DNA content was measured by 7AAD staining and flow-cytometry analysis and provides an indication of the cell cycle. The x and y axis represent the relative DNA content and the cell counts. (C) Graph showing the transduction efficiency (expressed as fluorescence forming foci/ml) of JS-eGFP in proliferating or mitotic arrested CP as described in Materials and Methods.