Daily Archives: August 30, 2017

Silicone oil liquid breathing, one macrophage

Electron microgaphs of lung, Legacy Perfluorocarbons, Ultimate order, the cell

Here is a not-so-hot electron micrograph of an alveolar macrophage, but the inclusions of silicone oil are kind of interesting. By 72 hours after just one hour of breathing silicone oil, show droplet inclusions that look a lot like they are doing the Oswald ripening thing, but also havn’t stimulated quite the lysosomal enzyme thing that macrophages that have phagocytoses perfluorochemicals show.  There are also bizarre areas of the lysosomes which are not typical of the PFC inclusions.  So differences – noted!

One of the more interesting differences (well two) is that there is almost no discernible trilaminar membrane around the inclusions (LE/SiO droplets) and there is certainly not a double outline (one is the trilaminar membrane of the lysosome, the other the protein coat as seen on PFC droplets (see previous posts)), so those two differences in ultrastructure plus the lack of a definite enzyme overload in those particles in alveolar macrophages that contain silicone oil droplets, and the sloppy separation (or ripening) of the individual droplets of silicone oil vs PFC droplets, makes for nice contrast between the two.

electron micrograph of alveolar macrophage from animal that breathed silicone oil
Here on the left is an enlargement of silicone oil inclusions in an alveolar (1 hr breathing 72 hr recovery) macrophage and on the right, E2 inclusions 3 hr breathing 48 hour recovery (biasing the E2 in the direction of less organization, but still showing much more organization and lysosomal enzyme content than the silicone oil).

comparison of ultrastructure of silicone oil droplets and E2 in alveolar macrophage

Protein coat? in phagocytosed E2 inclusions in alveolar macrophages?

Electron microgaphs of lung, Legacy Perfluorocarbons, The cell: images, Ultimate order, the cell

The bounding electron dense coat of proteins on the perfluorochemical droplets, whether phagocytosed from a neat liquid as inhaled from liquid breathing, or provided to the blood stream as artificial blood, it seems to be a fixture of the morphology.  I have scanned the edge of a single alveolar macrophage for evidence for “point of entry” of a droplet of E2 into said cell.  It is pretty clear even from this slightly too thick, thin section of a mouse lung (1349 4840 E2 3hr 48hr recovery) that the electron dense coat around droplets exists in lung of liquid breathing mice before they are phagocytosed by alveolar macrophages.  In this case the proteins likely to be lipid molecules derived from surfactant.

I suppose it is also likely that the slight lipophilicity of perfluorocarbons means that the surfactant lipids (such as phosphatidylcholine and phosphatidylglycerol) and the amphipathic proteins in surfactant (like SP-B) might contribute in some way to this droplet coat (as derived from surfactant already in the alveolus) reflecting its contribution shown in vitro studies suggesting an ability uptake of bacteria from the alveolar space (the leap is to E2 droplets) (ref Yang et al below)

There are at least 4 whole E2 droplets, and two cut off (left hand side of micrograph) droplets of E2 in this image. The tiny dotted periodicity were shown by lines in the droplet at the middle right, the green arrow points to the electron dense bounding layer of “some lipid and protein components from the alveolar space, likely from surfactant”, and the black arrow points to the space in the plasma membrane where the particle is likely being engulfed by the alveolar macrophage. Ribosome size is judged by the upper right corner inset with a portion of RER enlarged identically with the larger micrograph.  X marks an inclusion type that I have seen before but do not recognize. Any suggestions from the audience are welcome (millermn – ucmail dot uc dot edu).

transmission electron micrograph of alveolar macrophage and E2 perfuorocarbon droplets

Surfactant protein B and lipids look like a good candidates for the electron dense cover for E2 droplets — reading the article (Hawgood et al) and Yang et al  specifically referring to SP-B and from which I quote: “In no rank order these activities include  membrane binding, membrane lysis, membrane fusion, promotion of lipid adsorption to air–liquid surfaces, stabilization of monomolecular surface films, and respreading of films from collapse phases.”

SP-B has some interesting properties as referenced in many articles in PubMed which suggest that it has saposin like properties. Wikipedia identifies saposins thusly: “Saposins are small lysosomal proteins that serve as activators of various lysosomal lipid-degrading enzymes”. It is interesting that the huge lysosomal response that is induced by liquid breathing both in alveolar macrophages (and also when perfluorochemical emulsions when given IV) might be seen in the types of lysosomal bodies found, the length of time required to degrade/and/or reassociate the enzymes that appear to cover the droplets in the alveolar space, and concentrate (add more? or recycle more?  possibly be reflected in the fine granularity of the electron micrographic views of these MVB/LE/PFC organelles. The amount of re-emulsivication of phagocytosed E2 in alveolar macrophages appears to be time dependent, and would fit with the tim required to increase in production of some lysosomal enzymes and become associated with the E2 droplets.