Figure 1.

Pulmonary intravascular lung monocytes are retained during lung perfusion. The location and phenotype of monocyte subsets in the lungs were determined by dual compartment, intra-vascular and intra-alveolar leukocyte staining. Mice were injected intravenously (i.v.) with anti-CD45 (PE-CF594) before anaesthetic overdose, followed by intra-tracheal (i.t.) instillation of anti-CD45.2 (APC). To analyse mononuclear leukocytes within the lung cell suspensions, extra-alveolar leukocytes were first identified as CD45.2− and CD11b+ cells (A). In these cell populations, monocytes/macrophages were further identified as F4/80+ and subdivided into Ly6C^High and ^Low subsets (B). Intravascular monocytes were identified as anti-CD45 PE-CF594+ (C and D). MHCII was used as a marker for interstitial CD11b+, F4/80+ macrophages. To evaluate intravascular monocyte removal during perfusion, lungs were flushed with open circuit perfusion for 15 min after compartmental antibody staining. Only partial reduction in intravascular monocyte numbers was observed following washout (black diamonds) compared with non-surgical controls (white boxes) with both intravascular Ly6C^High (E) and Ly6C^Low (F) populations. Numbers of interstitial Ly6C^Low, MHCII+ macrophages (G) were not significantly changed, confirming their extravascular location. These numbers of intravascular monocyte subsets far exceeded those expected in an unlikely situation when residual blood within the pulmonary vasculature (estimated ∼50 µL as maximum) was not washed out, and totally preserved within the lung despite this extended perfusion, and hence can be ascribed to a marginated pool. Data are displayed as mean±SD, and analysed by t test. n=4–6, *p<0.05, ***p<0.001.