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Human bone marrow cells grown in liquid culture in the presence of conditioned medium from concanavalin A stimulated peripheral blood mononuclear cells, from mixed lymphocyte reactions, or from an osteogenic sarcoma tumor line gave rise to basophils which were maximal in number at 1-3 weeks. Basophils had a multilobed nucleus, contained large cytoplasmic granules of variable size which stained metachromatically with acid, but not neutral toluidine blue, were negative for chloroacetate esterase, and did not contain human mast cell tryptase. In long-term cultures, mast cells were detected after 5-6 weeks. Mast cells had cytoplasmic granules that stained with acid and neutral toluidine blue, were positive for chloroacetate esterase, and contained human mast cell tryptase. It, therefore, appears that in liquid cultures even in the absence of feeder layers, human bone marrow cell cultures can give rise initially to basophils, and then to mast cells.

Cultured rat embryonic skin fibroblasts phagocytosed rat mast cell granules added to the medium or released from co-cultured mast cells by rabbit anti-rat IgE or Compound 48/80. Electron microscopy of fibroblasts incubated with mast cell granules revealed that granules adjacent to the plasmalemma were engulfed by long, thin cytoplasmic processes. Internalization proceeded to fusion of encircling processes and formation of phagosomes. Microtubules and 60 A microfilaments became closely associated with the phagosomal membrane to which small vesicles and cisternae of endoplasmic reticulum fused. The rate of uptake of mast cell granules by fibroblasts was dependent upon temperature and granule concentration. Cytochalasin B inhibited granule uptake whereas colchicine and nocodazole had little effect. Phagocytosis was not influenced by actinomycin D and cycloheximide, was partially inhibited by fluoride, and was markedly inhibited by cyanide, azide, and 2,4-dinitrophenol. Supernatants from fibroblast cultures incubated with mast cell granules for 24 and 48 hr, during which period phagocytosis occurred, contained elevated levels of collagenase and beta-hexosaminidase, but normal levels of lactate dehydrogenase and superoxide dismutase. These results support the concept that immediate hypersensitivity reactions are in part terminated by phagocytosis of biologically active discharged mast cell granules by resident connective tissue fibroblasts. Further, it is suggested that a consequence of this process is an alteration in fibroblast behavior, providing a unique link between immediate hypersensitivity reactions and connective tissue responses to inflammation.

Incubation of [35S]heparin-containing mast cell granules with cultured bovine endothelial cells was followed by the appearance of 35S-granule-associated radioactivity within the endothelial cells and a decrease in radioactivity in the extracellular fluid. These changes occurred during the first 24 hours of incubation and suggested ingestion of the mast cell granules by the endothelial cells. Periodic electron microscopic examination of the monolayers confirmed this hypothesis by demonstrating apposition of the granules to the plasmalemma of endothelial cells, which was followed by the engulfment of the granules by cytoplasmic projections. Under light microscopic examination, mast cell granules within endothelial cells then appeared to undergo degradation. The degradation of [35S]heparin in mast cell granules was demonstrated by a decrease in the amount of intracellular [35S]heparin proteoglycan after 24 hours and the appearance of free [35S]sulfate in the extracellular compartment. Intact endothelial cells were more efficient at degrading [35S]heparin than were cell lysates or cell supernatants. These data provide evidence of the ability of endothelial cells to ingest mast cell granules and degrade native heparin that is presented as a part of the mast cell granule.

Human skin mast cells were degranulated in vivo by intradermal injection of antigen. Biopsy specimens of control and stimulated skin were obtained at 5 minutes, again at 10 to 15 minutes, and analyzed by electron microscopy. Degranulation, defined as dissolution of granule contents and swelling in at least 20% of secretory granules of mast cells was observed in one third of mast cells at 5 minutes and in nearly two thirds of mast cells at 10 to 15 minutes, but was absent in biopsy specimens of control subjects. Anaphylactic degranulation was characterized by extensive fusion of granules to form degranulation channels, fusion of channels with the cell membrane to form large pores, and exocytosis of amorphous or fibrillar granule matrix into the connective tissue. Extruded secretory granules of skin mast cells persist for at least 15 minutes in the connective tissues, in apparent contrast to the process of in vivo degranulation in nasal and lung mast cells.

It has been suggested that patients who present with episodes of unexplained anaphylaxis (UEA) or unexplained flushing (UEF) have systemic mastocytosis (SM), a proposal believed to be supported by the presence of excess mast cell (MC) numbers in the skin of these individuals. To examine this hypothesis, we determined the number and distribution of MCs in the skin of nine normal subjects, nine patients with UEA/UEF, six patients with urticaria pigmentosa (UP), and 14 patients with SM. Skin biopsy specimens of normal subjects contained 38.4 +/- 4 (mean +/- SEM) MCs per square millimeter. Biopsy specimens of patients with UEA/UEF contained 71.8 +/- 13 MCs per square millimeter. Although the numbers were significantly different from numbers in skin of normal subjects (p less than 0.05), similar modest increases in MC numbers are observed in a number of skin conditions. In marked contrast, lesional biopsy specimens of patients with UP contained 596.5 +/- 278 MCs per square millimeter (p less than 0.05, n = 6, compared to MC numbers in the skin of normal subjects), and patients with SM had 720.6 +/- 176 MCs per square millimeter in lesional skin (p less than 0.01, n = 12, compared to normal skin). Patients with UP or SM also had increased MC numbers in nonlesional skin compared to normal skin (168.0 MCs per square millimeter, p less than 0.05, n = 5, and 184.4 MCs per square millimeter, p less than 0.01, n = 10, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

It has long been recognized that mast cells occur throughout connective tissues. Histologic studies have revealed that such cells release their granules into the surrounding environment upon exposure to both immunologic and nonimmunologic stimuli. By microscopy these extracellular granules appeared to be phagocytosed by fibroblasts and by blood-borne phagocytic cells as they entered the site of mast cell degranulation. Such in vivo observations led to the suggestion that mast cells both altered connective tissue components and influenced fibroblast function through these discharged granules. Recent in vitro studies using cultured fibroblasts and isolated mast cells and mast cell granules have confirmed both these hypotheses. In addition, such studies have also documented that fibroblasts degrade ingested mast cell granules. Such studies document that a number of critical interactions may occur between mast cells and connective tissue components.

Mucosal mast cells in human nasal turbinates obtained at surgery were studied by electron microscopy before and after exposure en bloc to a degranulating stimulus. Mast cells occurred as solitary cells and in islets typically containing one mast cell and one to three mononuclear cells. All islets and most solitary mast cells were associated with thin cytoplasmic processes of distinctive, branched stromal cells that also phagocytosed debris from pyknotic mast cells. Mast cells were grouped into three categories based largely on published ultrastructural criteria. Resting (nondegranulating) mast cells possessed secretory granules that typically packed the cytoplasm and contained a densely stained amorphous material and scroll-like crystalline profiles. Granules in some resting cells were larger, more polymorphic, and appeared nearly homogeneous. Secretory granules of degranulating mast cells possessed crystalline, scroll-like, or reticular constituents predominantly with interspersed amorphous material. Granules were fewer in number and were often concentrated in the peripheral cytoplasm. Few degranulating mast cells exhibited labyrinth formation or exocytosis of granule contents. Largely degranulated mast cells had few if any typical granules but possessed small vacuoles containing recognizable granule remnants. Resting and degranulating mast cells possessed very long cell surface projections that, in places, interdigitated to form stacks of parallel folds above the plasmalemma, resulting in a threefold increase in cell-surface area. The surface membrane of the folds appeared to be continuous with that lining intracytoplasmic channels extending from the cell surface to the Golgi zone.

Two histamine-containing granulated cell lines were cloned from mouse bone marrow by using sequential soft agar and limiting dilution techniques. Concanavalin A-stimulated mouse spleen cell supernatants were required for cell proliferation. Cloned cell lines were Lyt-1.2, Lyt-2.2, Thy-1.2, surface Ig negative, I-A positive, and failed to ingest latex particles. Cells expressed 6.4 to 9.0 X 10(4) IgE high affinity receptors and contained approximately 1 to 2 pg of histamine per cell. Electron microscopy revealed granule and surface membrane features characteristic of mast cells. The cloned histamine-containing granulated cell lines synthesized a proteoglycan containing glycosaminoglycan side chains with an estimated average m.w. of 40,000. The glycosaminoglycan side chains consisted only of chondroitin sulfates identified by charge characteristics and through the use of selective polysaccharidases. These cells degranulated to immunologic stimuli and the calcium ionophore A23187 but not to compound 48/80. Taken together, the above findings suggest that these histamine-containing granulated cell lines that are cloned from bone marrow are mucosal (atypical) mast cells.