Figure 1.
Immunohistochemistry of CD90/Thy-1 in murine precision cut lung slices.
A) The anti-CD90/Thy-1 antibody (green) stains a vascular system (arrowheads) in murine precision cut lung slices that is distributed throughout the lung. Red staining shows immunoreactivity for α-smooth muscle actin. AW: airways. B) Initial CD90/Thy-1-immunoreactive capillaries (arrowheads) in the alveolar region. C) The CD90/Thy-1-immunoreactive vascular network is interconnected. D) A CD90/Thy-1-immunoreactive valve (arrowheads). E) Only close to the hilum, α-smooth muscle actin-immunoreactive cells (red, arrow) were found on CD90/Thy-1-immuoreactive vessels.
Figure 2.
Preembedding staining for CD90/Thy-1 in murine precision cut lung slices.
A) Intense immunostaining for CD90/Thy-1 is found in vascular structures. B) Boxed area in A, is shown as semi thin section. C) Boxed area in B, is shown as low magnification electron micrograph. D) Boxed area in C, is shown in higher magnification showing reaction product on an endothelial cell (arrowheads) that exhibited intimate contact to collagen fibers. E) Boxed area in D. Black arrowheads in A–E: immunoreactive lymph vessel, red arrowheads in A, B: pulmonary artery, arrows in D, E: collagen fibers.
Figure 3.
Double-labeling of CD90/Thy-1 with established lymph vessel markers.
A–C) Cryostat section. Podoplanin and LYVE-1 not only label lymph vessels (arrowheads) but also other cell types in the murine lung. D–F) Precision cut lung slice. Double-labeling of LYVE-1 with CD90/Thy1 shows that a lymph vessel (arrowheads) is labeled by both antibodies but staining of other cells prevents identification of lymph vessels in the alveolar region (arrow). G–I) Precision cut lung slice. Co-labeling with antibodies against VEGF R3 and CD90/Thy-1 shows colocalization in lymph vessels in the alveolar region (arrowheads) but the antibody to VEGF R3 binds to other structures in the alveolar region.
Figure 4.
Immunostaining for CD90/Thy-1 in C57BL/6 mice and FVB mice.
(A, B) The anti-CD90/Thy-1 antibody used that is directed against the CD90.2 variant labels cells in precision cut lung slices in C57BL/6 mice (A) that express CD90.2 but not in FVB mice (B) that express the CD90.1 variant. C, D) Conversely, the anti-CD90.1/Thy-1.1 antibody used, does not label lymph vessels in C57BL/6 mice (C) that express CD90.2 but labels lymph vessels in FVB mice (D) expressing CD90.1. Arrowheads = labeled lymph vessels.
Figure 5.
Identification of airways, arteries, and veins by α-smooth muscle actin staining in murine precision cut lung slices.
A) Precision cut lung slice stained with antibodies against α-smooth muscle actin (red) and CD90/Thy-1 (green). Based on the location and their characteristic architecture of smooth muscle cells, airways, arteries, and veins can be distinguished. B) Boxed area in A labeled with B. An airway (AW) is accompanied by a pulmonary artery (PA). The airway is identified by smooth muscle cells that are oriented perpendicular to course of the airway lumen and exhibit regular gaps between them. C) Boxed area in A labeled with C. Pulmonary arteries are identified by their continuous layer of α-smooth muscle actin stained smooth muscle cells and their proximity to airways. Smaller intraacinar arteries (IA) branch from the pulmonary artery, accompany alveolar ducts (not visible here) and subsequently lose their coating of α-smooth muscle actin-immunoreactive cells. D) Boxed area in A labeled D. Pulmonary veins (V) are identified by their coating of irregularly formed α-smooth muscle actin-immunoreactive cells that form a lose mesh around the vessel. They do not accompany airways or alveolar ducts and lie separately.
Figure 6.
Distribution of lymph vessels in the murine lung with respect to blood vessels and airways.
A–D and F) Double-labeling of precision cut lung slices with anti-CD90/Thy-1 antibody (green) and anti-α-smooth muscle actin antibody (red). A) CD90/Thy-1-immunoreactive lymph vessels are found around veins (V) and B) around muscularized airways (AW). C) Intraacinar arteries (IA) are not accompanied by lymph vessels. D, E) Lymph vessels are found frequently in the connective tissue between pulmonary arteries and airways. E) Paraffin section of murine lung stained with Masson Goldner stain. F) Frequently, accumulations of CD90/Thy-1-immunoreactive cells with lymphocyte morphology (arrows) are found around lymph vessels. Arrowheads = lymph vessels.
Figure 7.
Two separate routes for lymphatics can be identified in murine lungs.
Double-labeling of precision cut lung slices with anti-CD90/Thy-1 antibody (green) and anti-α-smooth muscle actin antibody (red). A–C) Lymphatic capillaries (arrowheads) that follow veins (V) begin either in the parenchyma (A, B) or directly on veins (C). D, E) Lymphatic capillaries that follow airways (AW) either begin directly on airways (D) or (E) in the connective tissue between arteries (labeled A in E) and airways.
Figure 8.
Other cell types are CD90/Thy-1-immunoreactive.
Double-labeling of precision cut lung slices with anti-CD90/Thy-1 antibody (green) and anti-α-smooth muscle actin antibody (red). A–C) In addition to lymph vessels, cells with fibroblast morphology (A, arrows) and boxed area in A, nerve fibers (B, arrows) and cells with lymphocyte morphology (C, arrows) exhibit CD90/Thy-1-immunoreactivity.
Figure 9.
Characterization of CD90/Thy-1-immunoreactive cells with lymphocyte morphology.
A–C) Double-labeling of a precision cut lung slices with antibodies against CD90/Thy-1 (green) and CD45 (red), and subsequent quantitative analysis (D) shows that more than 96% of CD90/Thy-1-immunoreactive cells with lymphocyte morphology are also stained with CD45 (white arrowheads, cells positive for CD90/Thy-1and CD45; red arrow, cell immunoreactive for CD90/Thy-1 only). In each animal, 90 to 150 CD90/Thy-1-immunoreactive cells were analyzed. E–G). Double-labeling with antibodies against CD90/Thy-1 (green) and CD3 (red) indicates that most CD90/Thy-1-labeled cells with lymphocyte morphology are also CD3-immunoreactive.
Figure 10.
Identification of lymph vessels in the living trachea by ex vivo staining with an anti-CD90/Thy-1 antibody.
Projection of a z-stack in a living murine trachea ex vivo recorded by multiphoton microscopy. Preincubation with an anti-CD90/Thy-1 antibody coupled to FITC stains a lymph vessel (white arrow = lymph vessel valve) and a cell with fibroblast morphology (red arrow) in a living trachea ex vivo. Other structures of the tissue are visualized by detection of tissue autofluorescence.
Figure 11.
Identification of lymph vessels in murine lungs after house dust mite sensitization and challenge.
Precision cut lung slices from house dust mite sensitized and stimulated mice stained with antibodies against CD90/Thy-1 (green) and α-smooth muscle actin (red). A–F) Maximum intensity projections of z-stacks of confocal sections (A, C, E) and single confocal sections (B, D, F) from the z-stacks used to generate projections in A, C, E. While projections (A, C, E) show many T cells that are located around lymph vessels (arrowheads) and can mask them, a single confocal section (B, D, F) allow the unambiguous identification of lymph vessels (arrowheads) and the localization of T cells around and within (arrow in F) lymph vessels. V = vein, AW = airway, PA = pulmonary artery.
Figure 12.
T cell distribution in the murine lung after house dust mite sensitization and challenge.
A, B) CD90/Thy-1-immunoreactive T cells are found (A) around veins (V) and (B) around intraacinar arteries (IA). C) Accumulations of T cells around arteries are continuous from intraacinar arteries to pulmonary arteries (PA). D) Around airways (AW), T cells preferentially accumulate around pulmonary arteries.
Figure 13.
Scheme of lymph vessels and model of cell exit from the murine lung tissue.
Two separate routes for lymph vessels exist. One begins in the parenchyma and leaves the lung via veins and the other begins around airways or in the connective tissue between airways and arteries and follows the airways to leave the lung. Cells that have left the vasculature in the alveolar region can enter the lymphatic system by migrating to lymph vessels around veins or by migration to lymph vessels around airways, possibly by using intraacinar arteries as guide.