Differences in the behavior of cytoplasmic granules and lipid bodies during human lung mast cell degranulation.

Abstract

We used a morphometric and autoradiographic approach to analyze changes in specific cytoplasmic granules and cytoplasmic lipid bodies associated with human lung mast cell degranulation. Mast cells were dissociated from lung tissue by enzymatic digestion and were then enriched to purities of up to 99% by countercurrent centrifugation elutriation and recovery from columns containing specific antigen bound to Sepharose 6 MB. Degranulation was induced by goat anti-lgE. At various intervals after stimulation, parallel aliquots of cells were recovered for determination of histamine release or were fixed for transmission electron microscopy. We found that lipid bodies, electron-dense structures that lack unit membranes, were present in both control and stimulated mast cells. Autoradiographic analysis showed that lipid bodies represented the major repository of 3H-label derived from [3H]arachidonic acid taken up from the external milieu. By contrast, the specific cytoplasmic granules contained no detectable 3H-label. In addition, lipid bodies occurred in intimate association with degranulation channels during mast cell activation, but the total volume of lipid bodies did not change during the 20 min after stimulation with anti-lgE. This result stands in striking contrast to the behavior of specific cytoplasmic granules, the great majority of which (77% according to aggregate volume) exhibited ultrastructural alterations during the first 20 min of mast cell activation.These observations establish that mast cell cytoplasmic granules and cytoplasmic lipid bodies are distinct organelles that differ in ultrastructure, biochemistry, and behavior during mast cell activation.

Development of human mast cells in vitro.

“…Narrow surface folds were regularly distributed over the cell surface. In the cultured cells, previously reported substructural granule patterns were evident (22,23). These patterns included crystalline arrays of several periodicities, spirals and scrolls, particles, and homogeneously dense areas (Fig.…”

Ultrastructural Autoradiographic Analysis of RNA in Isolated Human Lung Mast Cells during Secretion and Recovery from Secretion

“…Comparisons of the nucleus compartment and the granule compartment for these samples showed that nucleus label exceeded granule label in all of them (p < 0.001). [Previously, we calculated that the volume fraction of human mast cell nuclei does not change in similar experiments but that granule volume fractions decrease in actively secreting cells [25]. The observed reduction in granule volume may be reflected in a larger proportion of isotope incorporation into stable nuclear volumes than in diminishing granule volumes.…”

“…Mast cell release experiments similar to those previously reported [25, 26]were repeated. Thus, mast cells were stimulated to degranulate by exposure to 3 or 5 µg/ml goat anti-human IgE in buffer (25 m M piperazine- N,N ′-bis-2-ethane, sulfonic acid, 140 m M NaCl, 6 m M KCl, 0.003% human serum albumin and 0.1% glucose) plus 1 m M CaCl 2 at 37°C [27].…”

“…The ultrastructural morphology of HLMCs in vivo and/or ex vivo after short-term culture, after stimulation to degranulate by exposure to anti-IgE and recovering in short-term cultures after stimulated secretion have all been reported in detail [21, 25, 26, 29, 33, 37, 38, 39, 40, 41]. Similar samples were studied in this report following incubation in (5,6- 3 H)-uridine to determine the ultrastructural site(s) of RNA in resting, degranulating and recovering HLMCs.…”

“…These were prepared for autoradiography by established methods [25, 31, 32, 33, 34, 35, 36]. Ilford L4 photographic emulsion (Polysciences, Worthington, Pa., USA) was used to place a wire loop full of liquid emulsion over thin sections on copper grids.…”