Table 1.
Overview of Chi3L3 mRNA levels, BAL eosinophil counts and presence of eosinophilic crystals in BAL cell fluid.
Figure 1.
Overview of the observed CNT-cell interactions.
Each interaction has a descriptive sketch, an example image of how the interaction was expressed in CNTSmall and CNTLarge exposed samples, and a description of the differences and progression of the interaction. White arrowheads indicate the presence of CNTs in TEM images.
Figure 2.
Representative images of the samples 1 day (a-b), 3 days (c-d), and 28 days (e-f) post exposure for CNTSmall (a,c,e) and CNTLarge (b,d,f).
(a-b) Shows CNTSmall and CNTLarge, respectively, on day 1, where CNTs were mainly found in the alveolar lumen or in the interface between cells. (c-d) On day 3 CNTs were increasingly found in vesicles inside what appeared to be alveolar macrophages, but some CNTs were observed outside cells. (e) On day 28, CNTSmall was found inside vesicles and in the cytosol of cells in large cell agglomerations, but were also found inside vesicles in singular cells. (f) On day 28, there were fewer vesicles containing CNTLarge and the CNTs were more individually spaced in the cytosol. In addition, immune cells were found in large agglomerations indicating inflammation at this stage. Arrowheads points to CNTs.
Figure 3.
TEM (a-c) and SEM (d-f) images of crystalline bodies observed on day 28 in CNTLarge samples.
(a) Low magnification TEM image showing large crystals, some up to 40 μm long. (b) Image showing scattered crystalline bodies and CNTs in clusters. (c) Ordered crystalline bodies in the cytosol. (d) SEM image of the ultramicrotomed block revealing the underlying cells prior to milling. The image also shows a few protruding CNTs (white arrowhead) [27]. (e) FIB-SEM image of crystals in a cell. (f) 3D representation of the FIB-SEM stack where a few of the crystals have been traced (yellow), revealing their plate-like structure. Black arrowheads indicate ECP crystals while white arrowheads indicate the CNTs.
Figure 4.
Representative image of stained eosinophilic crystals in BAL from mice exposed to the highest dose (162 μg) of CNTLarge 28 days post exposure.
Large amounts of crystals were also observed for the two lower doses (18 μg and 54 μg), and similarly for Mitsui-7 samples on day 28. Black arrowheads point to crystals, whereas white arrowheads indicate CNT agglomerations or cells containing CNTs.
Figure 5.
Model showing CNT interactions with lung tissue over time in vivo.
At day 1, most CNTs were found in the alveolar spacing, while a few CNTs were found freely inside cells apparently having entered the cells using a non-endocytotic pathway. At day 3, CNTs are found in the alveolar spacing and a larger number of CNTs are found within vesicles, indicating an endocytotic-pathway. At day 28, most of the CNTs were either taken up by cells or in close relation to cells. Compared to post exposure day 3, the two larger CNTs had more CNTs outside vesicle bounds, indicating CNT escape from vesicles.