Jan L. E. Ericsson

Mechanisms of Intralysosomal Degradation with Special Reference to Autophagocytosis and Heterophagocytosis of Cell Organelles

Publisher Summary This chapter describes the mechanisms of intralysosomal degradation with a special reference to autophagocytosis and heterophagocytosis of cell organelles. Autophagy is the process of sequestration of intracellular components and their subsequent degradation by the lysosomes. The process of degradation is of fundamental importance in cell function because under steady-state conditions subcellular components are broken down and resynthesized many times during the life span of the cells. Autophagy contributes to the turnover of cell constituents during physiological cell conditions. Autophagy is induced by numerous treatments, conditions, or agents that cause cell dysfunction. Biological components from the extracellular space can also enter the lysosomal compartment and become degraded through the process of heterophagy. The chapter discusses an experimental model in which intracellular organelles are introduced into the lysosomal apparatus of Kupffer cells by means of heterophagy. The study of the heterophagic model has advantages over induced autophagy because the degradation of each membrane constituent or cell organelle can be separately studied. The heterophagy model is also useful to evaluate the capacity of lysosomes to degrade various membrane components in vivo .

Morphologic and histochemical studies on the differing radiosensitivity of ductular and acinar cells of the rat submandibular gland

SummaryThe submandibular glands of male rats were exposed to 50 Gy X-irradiation as a single dose, with or without pre-treatment with either alpha-adrenergic agonists (noradrenaline, phenylephrine) or a cholinergic antagonist (atropine). The effects were analyzed by morphometric, cytochemical and biophysical methods. When X-irradiated without drug pre-treatment, many serous epithelial cells of the intralobular convoluted ducts displayed morphologic evidence of irreversible radiation damage, in contrast to neighbouring mucous and other cells which were unaffected. The effect was maximal 96 h after irradiation. Serous cells from animals irradiated after pre-treatment with atropine showed much more wide-spread injury than those of animals exposed to X-irradiation only. In contrast, serous cells suffered considerably less damage if their secretory granules had been depleted 1 or 2 h before irradiation with either noradrenaline or phenylephrine. Other epithelial cells showed no modulation of their slight radioresponsiveness by these drugs. The observations were substantiated by morphometry of three cell types: (a) mucous cells, (b) non-granulated serous and intralobular striated duct cells, and (c) granulated serous cells. The findings suggest that the striking radiosensitivity of salivary gland serous epithelial cells is linked to their content of secretory granules. These granules are rich in heavy metals, as demonstrated cytochemically with the sulphide silver method (SSM). Using particle-induced X-ray emission (PIXE) spectroscopy, the principal metals were shown to be Zn, Mn and Fe. It is conceivable that membranes which enclose organelles rich in metals with the ability to form redox systems (e.g. Fe2+ ai Fe3+) show enhanced sensitity to radiation damage due to the metal-catalyzed induction of lipid peroxidation by ionizing radiation. Disruption of secretory granules would be expected to release lytic enzymes into the cell sap, resulting in autolysis. This hypothesis is supported by the findings that atropine-which increases the number of granulated serous cells-enhances radiosensitivity, while noradrenaline and phenylephrine-which cause degranulation of serous cells-decrease radiosensitivity.

An electron microscopic study of the post-partum involution of the rat uterus

SummaryDuring pregnancy the increase in mass of the rat uterus has been accounted for mainly by hypertrophy rather than hyperplasia of the smooth muscle cells and by an increase in collagen content. Following parturition the rat uterus regains its non-pregnant weight within little more than a week. Autophagy is a likely mechanism for the reduction in size of the smooth muscle cells. However, many previous investigations have failed to demonstrate an extensive lysosomal apparatus in uterine smooth muscle cells, a morphologically detectable prerequisite for augmented autophagy. The superfluous collagen has been assumed to be digested by macrophages.In the present electron microscopical study the involution of the rat uterus was followed at various time points post-partum. The results indicated that three pathways were involved namely, autophagocytosis, heterophagocytosis and crinophagy. Autophagocytosis was noted in smooth muscle cells — apparently a means to reduce their size. In some instances extensive autophagy appeared to lead to cell death. Macrophages and also to some extent fibroblasts have the ability to endocytose collagen and cell debris including split off portions of smooth muscle cell cytoplasm. The evidence obtained suggested that at the time of parturition the (pro)collagen synthesized but not yet secreted was retained within the fibroblasts and degraded by means of crinophagy.

Effect of iron on the stability of macrophage lysosomes

SummaryMouse peritoneal macrophages exposed to 60 μM Fe3+ in culture showed a pronounced decline in survival rate and an enhanced capacity for lipid peroxidation, as estimated by the formation of malondialdehyde. The iron-exposed cells contain secondary lysosomes loaded with iron as demonstrated by transmission electron microscopy, energy dispersive X-ray micro-analysis and the sulphide-silver method.The macrophages concentrated within their lysosomes the weak base acridine orange (AO) when the latter was added to the medium in a low concentration (1:105). When cells were viewed under continuous illumination with short wave blue light in a fluorescence microscope, AO-exposed cells initially showed a brilliant red granular fluorescence and subsequently a progressive redistribution of AO into the cell sap and the nucleus with resultant green fluorescence. This redistribution of AO was associated with the disappearance of the normal granular reaction product pattern of the lysosomal marker enzyme acid phosphatase (AP), as demonstrated with a Gomori-type reaction, and later with cell death. The redistribution of AO and AP from the lysosomal vacuome to the cell sap under the influence of blue light occurred markedly more rapidly in macrophages containing iron loaded lysosomes than in control cells.The findings indicate that the presence of iron in lysosomes decreases the proton-retaining ability of their bordering membranes, possibly by way of enhanced metal (iron) catalyzed lipid peroxidation. It appears that leakage of AO signifies destabilization of the lysosomal membrane.

Giant cell tumor of bone

The fine structural localization of nonspecific alkaline phosphatase was elucidated in two giant cell tumors of bone using lead as capturing ion and Β-glycerophosphate as substrate in the incubation solution. Lead phosphate precipitate — indicating presence of alkaline phosphatase — was demonstrated on the plasma membranes, and the membranes bordering vesicles and vacuoles of presumed endocytotic nature, in giant cells and type 1 stromal cells (fibroblast-like cells). The findings support the view that stromal cells type I and giant cells are histogenetically related.

Morphological studies on endocytosis of chondroitin sulphate proteoglycan by rat liver endothelial cells

SummaryEndocytosis via the hyaluronic acid/chondroitin sulphate receptor of rat liver endothelial cells was studied ultrastructurally, by use of a probe consisting of chondroitin sulphate proteoglycan attached to 15-nm gold particles. The probe bound to the surface of the cells exclusively in coated regions of the plasma membrane. Internalization at 37° C took place in less than one minute during which time interval the bound probe was transferred to coated vesicles. Further transfer to lysosomes was delayed in association with an accumulation of probes in a prelysosomal compartment consisting of large vacuoles in which probes lined the inner aspect of the membrane. Transport to lysosomes occurred only after a lag phase of at least 40–60 min at 37° C.

Macrophage radiosensitivity in culture as a function of exposure to ionic iron

SummaryThe effects of various concentrations of Fe3 + (30–125 μM) and various doses of X-irradiation (10 to 30 Gy) on the survival of mouse peritoneal macrophages in culture was tested and compared with the effect of combinations of both Fe3 + and X-irradiation.Exposure of the cells to 30 μM Fe3 + or 10 Gy did not significantly alter the survival pattern, as compared with control cells. Greater dosages of iron or X-irradiation caused increasing cellular degeneration and death. When the combined effect of 30 μM Fe3 + and 10 Gy was tested a pronounced decrease in survival time was noted. The production of malondialdehyde (MDA) was studied in cells cultured with and without iron and after exposure to X-irradiation. The results indicated an increasing peroxidative capacity in parallel with increasing dose and extent of exposure to iron and irradiation. Combinations of 30 μM Fe3 + and 10 Gy resulted in enhanced MDA production as compared with these treatments alone (in which levels of MDA did not differ from those in controls).The findings support the theory that cells containing iron-loaded lysosomes are abnormally sensitive to X-irradiation due to the catalytic activity of the Fe2 +⇌ Fe3 + redox system on lipid peroxidation. The postulated mechanism involves disruption of lysosomal membranes as a consequence of increased lipid peroxidation, resulting in leakage of potent lytic enzymes into the cell sap causing cellular degeneration and death.

CYTOPLASMIC EFFECTS OF X-IRRADIATION ON CULTURED CELLS IN A NON-DIVIDING STAGE

Cultured human glial cells were blocked in interphase (G1) by 24 h of serum starvation and subsequently subjected to 200 Gy, 8 MV X-radiation. Immediately following irradiation the cultures were transferred to serum-containig medium. Time-lapse cinemicrography performed during the next 48 h showed a profoundly disturbed motility with decreased ability for polarization and locomotion of the irradiated cells. Specimens fixed 24 and 48 h after irradiation and investigated by immunofluorescence with tubulin-antibodies and DNase/DNase-antibodies, and by whole cell transmission electron microscopy showed derangements in the distribution of microfilament bundles related to the cytoplasmic ramification of the irradiated cells, but otherwise no detectable alterations in structure or distribution of either microtubules or microfilaments. It is suggested that the alteration in the arrangement of filament bundles is of importance to the impaired locomotion of the irradiated cells.

Levels of malondialdehyde production in rat liver following loading and unloading with iron

SummaryRats were given daily injections of an iron sorbitol citric acid complex in a total dose of 50 mg Fe3 + /100 g of body weight and either killed immediately after iron loading, or investigated 2 months later. Among the latter animals, one group was subjected to weekly phlebotomies in order to mobilize iron from the stores, while another group was not further treated. Quantitation of iron and malondialdehyde production was performed on homogenates of liver, kidney and spleen from controls and rats in the different experimental groups, and the distribution of iron in granular form was studied in the livers by means of electron microscopy. The results showed substantially increased amounts of iron in the organs studied after iron-loading and also augmented malondialdehyde production in the liver and kidney (but not in the spleen). A decreased malondialdehyde production was recorded two months after iron-loading in the kidney and spleen of non-bled animals; this decrease was exaggerated in the same organs from bled animals. The production of malondialdehyde as well as the iron content in the livers of both bled and non-bled rats 2 months after iron loading was higher than in the controls. The evidence obtained suggested that the accumulation of iron in the liver was causally related to increased lipid peroxidation. Judging from the morphological appearances this change did not result in cell damage, the only pertinent morphologic alteration being the occurrence of iron particles in the lysosomal vacuome and the cell sap.

Human osteogenic sarcoma: fine structure of the fibroblastic type.

The fine structure of representative regions of nine fibroblastic osteogenic sarcomas was studied. As judged by light microscopic criteria, the tumors represented both highly malignant (grades 3-4) and less malignant (grade 2) varieties. By electron microscopy, six basic cell types were found in the selected regions (fibroblastlike, histiocytelike, and myofibroblastlike cells, along with xanthoma cells, multinucleated giant cells, and undifferentiated cells). In addition, occasional osteoblastlike cells were encountered. Fibroblastlike cells in general, and especially in grade 2 tumors, showed a fine structure, enabling differentiation from osteoblastlike cells. Multinucleated giant cells were of two morphologic types, and the fine structure appeared to be related to the malignancy potential and differentiation of the tumors. Many multinucleated giant cells in grade 2 tumors had a ruffled border and appeared to be highly active in digestive events (especially phagocytosis of whole cells and portions of cells). Accumulation of variable amounts of lipid in droplet form was common in the various types of cells present in the tissues. The observations were discussed with particular emphasis on the interrelationships and functional roles of the cells.