Micro-autoradiographic assessment of cell types contributing to 2-deoxy-2-[(18)F]fluoro-D-glucose uptake during ventilator-induced and endotoxemic lung injury

Abstract

Purpose: The aim of the study was to use micro-autoradiography to investigate the lung cell types responsible for 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) uptake in murine models of acute lung injury (ALI).

Procedures: C57/BL6 mice were studied in three groups: controls, ventilator-induced lung injury (VILI), and endotoxin. VILI was produced by high tidal volumes and zero end-expiratory pressure and endotoxin ALI, by intranasal administration. Following FDG injection, the lungs were processed and exposed to autoradiographic emulsion. Grain density over cells was used to quantify FDG uptake.

Results: Neutrophils, macrophages, and type 2 epithelial cells presented higher grain densities during VILI and endotoxin ALI than controls. Remarkably, cell grain density in specific cell types was dependent on the injury mechanism. Whereas macrophages showed high grain densities during endotoxin ALI, similar to those exhibited by neutrophils, type 2 epithelial cells demonstrated the second highest grain density (with neutrophils as the highest) during VILI.

Conclusions: In murine models of VILI and endotoxin ALI, FDG uptake occurs not only in neutrophils but also in macrophages and type 2 epithelial cells. FDG uptake by individual cell types depends on the mechanism underlying ALI.

Fig. 1.

Representative autoradiographic views of different cell types labeled with 2-deoxy-2[¹⁸F]fluoro-D-glucose: Top panel: a Hematoxylin–eosin histological sample of lung tissue from instilled endotoxin ALI model, at ×40. Region demarcated by rectangle is amplified in b. b Inset view at ×100 depicting the presence of significant numbers of grains within the neutrophil. Bottom panel: c Lung parenchyma from VILI model at ×40. d Inset view of box from c at ×100 view. Grains are observed within an alveolar macrophage in VILI model. e Inset view of dashed box at ×100 view. Black arrow indicates a type 2 epithelial cell, and blue arrows show type 1 epithelial cells. Overall, these images exemplify the predominance of grains overlying lung parenchyma.

Fig. 2.

Grain density of 2-deoxy-2-[¹⁸F]fluoro-D-glucose normalized by injected FDG in micro-autoradiographic samples. Comparisons among ALI models and controls for epithelial cells type 1 (EC1) and type 2 (EC2), macrophages, and neutrophils. ALI produced an increase in normalized grain density for several cell types as compared to controls. Type 2 epithelial cells demonstrate significantly higher normalized grain density during VILI compared with instilled endotoxin and control conditions. Macrophages demonstrate the highest normalized grain density during endotoxin-induced lung injury. Triple asterisks indicate p<0.001.

Fig. 3.

Percent contribution of studied cell types to the total number of 2-deoxy-2-[¹⁸F]fluoro-D-glucose grains on cells in micro-autoradiographic samples. Comparisons among ALI models and controls for epithelial cells type 1 (EC1) and type 2 (EC2), macrophages, and neutrophils. Neutrophils demonstrate a higher contribution during ALI than during control conditions. Macrophages, conversely, demonstrate a higher contribution during control conditions than during ALI. Double asterisks indicate p<0.01, and triple asterisks indicate p<0.001.

Fig. 4.

Immunohistochemical staining of endothelial cells and 2-deoxy-[³H]-D-glucose micro-autoradiography from endotoxin-induced lung injury. a Phase contrast image of HDG grains at ×40 magnification. The area in the white rectangle is magnified at ×100 in b–e. b Grains of HDG. Dark granules indicate presence of HDG (yellow arrows). c Endothelial cells identified with P-selectin. d Nuclear counter-staining with Hoëchst. e Merged image of b–d. Note that grains of HDG and endothelial cells (indicated by black arrows) are colocalized.

Fig. 5.

Immunohistochemical staining of neutrophils 2-deoxy-[³H]-D-glucose micro-autoradiography on tissue sections from endotoxin-induced lung injury. a Phase contrast image of HDG grains (yellow arrows) at ×100 magnification. b Neutrophils stained with Alexa-488-conjugated anti-Gr-1 antibody (green arrows). c Nuclear counter-staining with Hoëchst (blue arrows). d Merged image of a–c. Grains of HDG and neutrophils are colocalized.

Fig. 6.

Immunohistochemical staining of type 2 epithelial cells and 2-deoxy-[³H]-D-glucose micro-autoradiography on tissue sections from ventilator-induced lung injury. a Hematoxylin–eosin image with HDG grains (arrows) and type 2 epithelial cells indicated (red asterisk) as confirmed by SP-C immunofluorescence in b (×60 magnification). b Type 2 epithelial cells stained with rabbit anti-SP-C and Alexa Fluor 488 goat anti-rabbit antibody (red asterisk). The nuclei were counter-stained blue with 4′,6-diamidino-2-phenylindole. c Merged image of a–b (and inverted to permit visualization of grains). Grains of HDG (arrows) and type 2 epithelial cells (red asterisk) are colocalized.