Biochemical and microscopic evidence for the internalization of drug-containing mast cell granules by macrophages and smooth muscle cells.
During mast cell degranulation the soluble component of the granule is released into extracellular fluid, whereas two neutral proteases and heparin proteoglycans form the extracellular granule remnants. These structures are negatively charged and bind with high affinity LDL and other basic molecules. In this study we show that granule remnants expelled into extracellular fluid are able to bind the aminoglycoside antibiotic gentamicin and the anticancer agent doxorubicin in a dose-dependent manner. In addition, granule remnants loaded with the two basic substances are subsequently phagocytosed by macrophages. Indeed, when cells are incubated for 24 h with 1 mg/ml gentamicin, the intracellular concentration of the drug, which in basal conditions is extremely low, increases significantly in the presence of degranulating mast cells (from 5.1 +/- 1.0 to 25.4 +/- 2.5 microg/mg protein) and a good correlation between histamine release and gentamicin uptake is evident. The antineoplastic agent doxorubicin can penetrate cells by passive diffusion; however, when mast cells are added to macrophage monolayer, incubated for 30 min with 50 microM of the antineoplastic agent, a significant increase in intracellular doxorubicin concentration is observed (from 3.5 +/- 0.2 to 4.7 +/- 0.2 microg/mg protein). Internalization of granule remnants carrying gentamicin or doxorubicin is also evident in smooth muscle cells of the synthetic phenotype. In particular, when smooth muscle cells are incubated for 24 h with 1 mg/ml gentamicin, addition of isolated granules increases the uptake from 2.4 +/- 0.2 to 4.8 +/- 0.4 microg/mg protein. Similar results are obtained in smooth muscle cells incubated for 4 h with doxorubicin 50 microM (from 3.3 +/- 0.2 to 4.8 +/- 0.5 microg/mg protein). Data are confirmed by microscopic experiments by means of fluorescence microscopy and electron microscopic studies. The study demonstrates that basic substances can enter phagocytic cells when loaded to granule remnants. The phenomenon can be of particular interest for substances like the aminoglycosides that do not cross biological membranes; indeed, the storage of these antibiotics in phagocytic cells could have important consequences on their antibacterial activity in vivo. Macrophages and smooth muscle cells can also act as a reservoir for doxorubicin. High concentrations of the antineoplastic agent in these cells could be responsible for toxicity, as well as play an important role in the transport of the drug to tumor cells.
Decorti
G
G
Department of Biomedical Sciences, University of Trieste, I-34100 Trieste, Italy.
Klugmann
F B
FB
Crivellato
E
E
Malusà
N
N
Furlan
G
G
Candussio
L
L
Giraldi
T
T
D000818:Animals; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004317:Doxorubicin / Q000493:pharmacokinetics; D005839:Gentamicins / Q000493:pharmacokinetics; D017737:Macrophages, Peritoneal / Q000502:physiology* / Q000648:ultrastructure; D008297:Male; D008407:Mast Cells / Q000378:metabolism*; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000648:ultrastructure; D010587:Phagocytosis; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar
D005839:Gentamicins; D004317:Doxorubicin