DIFFERENTIATION AND PROLIFERATION OF EMBRYONIC MAST CELLS OF THE RAT

Histochemical reactions and radioautography were used to investigate the sequence of mast cell development in rat embryos. Mast cells arise ubiquitously in and are confined to the loose connective tissue in the embryo. The alcian blue-safranin reaction distinguishes between weakly sulfated and strongly sulfated mucopolysaccharides by a shift from alcian blue to safranin staining. Based on this reaction and morphologic characteristics, four stages were identified. Stage I mast cells are lymphocyte-like cells with cytoplasmic granules which invariably stain blue with the alcian blue-safranin reaction. In Stage II cells the majority of granules are alcian blue-positive, but some safranin-positive granules have appeared. Stage III mast cells are distinguished by a majority of safranin-positive cytoplasmic granules; some alcian blue-positive granules still remain. Stage IV cells contain only safranin-positive granules. Thymidine-H³ uptake and identification of mitotic figures indicates that mast cells in Stages I and II comprise a mitotic pool while those in Stages III and IV are mitotically inactive. The pattern of S³⁵O₄ incorporation and the sequence of appearance of histochemically identifiable mast cell constituents corroborates division of the proliferation and differentiation of embryonic mast cells into the four stages described above. The process of formation of mast cell granules is interpreted as reflecting the synthesis and accumulation of a heparin precursor in alcian blue positive granules followed by the synthesis and accumulation of highly N-sulfated heparin along with mast cell chymase and finally histamine in safranin-positive granules.     

Mucosal mast cells of the rat intestine: a re-evaluation of fixation and staining properties, with special reference to protein blocking and solubility of the granular glycosaminoglycan

Mucosal mast cells of the gastrointestinal tract constitute a separate cell line within the mast cell system of the rat, differing in several respects from the classical connective tissue mast cells and, unlike the latter, requiring special fixation techniques for their demonstration. We have examined some histochemical properties of mucosal mast cells of the duodenum and compared them with connective tissue mast cells of the tongue or skin. The results indicate that the structural integrity of the granules of both types of mast cell is partly dependent on ionic linkages between glycosaminoglycan and protein. The so far unidentified glycosaminoglycan of mucosal mast cells appears to be more soluble than the heparin of connective tissue mast cells. The strongly fluorescent binding of Berberine to the granules of connective tissue mast cells and, depending on their content, of heparin is absent from mucosal mast cells, confirming previous findings which suggested that they contain a glycosaminoglycan with a lower degree of sulphation. Aldehyde fixation by routine procedures reversibly blocks the cationic dye binding of mucosal mast cell granules. The dye binding groups may be unmasked by trypsination or by long staining times of the order of several days. The results suggest that the blocking of staining by aldehydes is caused by a diffusion barrier of a protein nature. Mucosal and connective tissue mast cells thus differ with respect to the spatial arrangement of glycosaminoglycan and protein in their granules. As a result of the study a modified method for the demonstration of mucosal mast cells in tissue sections is described, based on normal formaldehyde fixation and staining in Toluidine Blue for a long time. It has some advantages over previous methods and preserves the structure of mucosal and connective tissue mast cells equally well.     

Staining Mast Cells for Morphometric Evaluation on an Image Analysis System

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.     

Immortalization of murine connective tissue-type mast cells at multiple stages of their differentiation by coculture of splenocytes with fibroblasts that produce Kirsten sarcoma virus

Mature connective tissue mast cells (CTMC) have not been previously available as a cell line from any species. Here we describe 15 novel mast cell lines (KiSV-MC) that were derived by coculturing murine splenocytes with fibroblasts that produce a Ki-ras-containing murine sarcoma virus. Some of the KiSV-MC lines are similar to CTMC in that they synthesize predominantly heparin proteoglycans, and contain up to 35 micrograms of histamine and 2.2 units of carboxypeptidase A/10(6) cells in secretory granules which stain red with Safranin. Other cell lines display phenotypic characteristics intermediate to CTMC and mucosal-like mast cells in being predominantly Safranin-, having lower amounts of histamine and carboxypeptidase A, and in synthesizing chondroitin sulfate E proteoglycans in preference to heparin proteoglycans. When the individual KiSV-MC lines were compared, a linear relationship was found between the number of Safranin+ granules, the cellular contents of histamine and carboxypeptidase A, and the biosynthesis of heparin relative to chondroitin sulfate E proteoglycans. Upon sensitization with monoclonal IgE and exposure to hapten-specific antigen, the cells exocytose the contents of their secretory granules. Thus, these immortalized cells provide the first source of CTMC-like lines for chemical and functional analysis and illustrate that murine mast cells can express a continuum of phenotypes.     

Tissue engineering: in vitro embryonal nidation in a murine endometrial construct

The epithelial and mesothelial cellular components of organs can be obtained as dissociated cells using adequate procedures of enzymatic digestion followed by pycnotic separation on density gradients. Using a specially developed procedure for tissue dissociation, the epithelial and connective tissue components of endometria from pseudopregnant mice were grown in culture using a combination of three dimensional culture of connective tissue components in collagen gel, with the superimposition of epithelial components in liquid medium on the surface of the gells. After a few days of growth, when the cultures became dense, murine blastocysts obtained on postcoital day 4.5 by fallopian flushing of hormonally primed and mated mice, were transferred onto the imitated endometria. The blastocysts hatched and grew on the endometrial epithelium as spherical coherent conglomerates of cells quite different from hatched blastocysts grown on the surface of a petri dish, in which the presumtive trophoblasts spred around the central mass. Light and electronmicroscopy of resin embeded sections (2 days after nidation on the simulated endometria) revealed that at least two populations of cell types were recognisable as layers. This is interpreted as an early sign of morphogenesis and the first visible steps of differentiation. The presence of mitotic figures indicates viability and continuing growth. Electronmicroscopy of cell types grown under conditions simulating in vivo tissue architectonics showed overtly less cytopathology and better cell function. Simulated endometria may, therefore, serve as an attractive model for studying early mammalian embryogenesis and the effects of toxic agents.     

Inhibition of the growth of IL-3-dependent mast cells from murine bone marrow by recombinant granulocyte macrophage-colony-stimulating factor

The mouse mast cell line PT-18 demonstrates [3H] thymidine uptake in the presence of either mouse IL-3 or mouse recombinant granulocyte-macrophage CSF (rGM-CSF). Experiments were thus undertaken to determine whether rGM-CSF would affect IL-3-dependent growth of mast cells from mouse bone marrow cells (BMC). BMC placed in liquid culture containing 50 U/ml of IL-3 gave rise to cultures containing up to 95% mast cells by 2 to 3 wk. The rise in percentage of mast cells was accompanied by an increase in total cell-associated histamine. In contrast, BMC grown in the presence of 50 U/ml of rGM-CSF gave rise to cultures containing primarily macrophages and granulocytes with less than 1% mast cells. The addition of increasing amounts of rGM-CSF to BMC cultures grown in the presence of IL-3 resulted in a decrease in the number of mast cells present in culture at 2 to 3 wk. Cells other than mast cells in these cultures consisted principally of granulocytes and macrophages. The rGM-CSF-related inhibition of mast cell growth was not abrogated by the addition of indomethacin to cultures. Granulocyte-macrophage cell populations added to IL-3-containing cultures did not inhibit mast cell growth. The suppressive effect of rGM-CSF on IL-3-dependent mast cell growth may indicate an important role for GM-CSF in the down-regulation of mast cell proliferation in tissues.     

Morphological and functional characteristics of peritoneal mast cells from young rats

To study why neonatal and young rats are resistant to the effects of some secretagogues, such as compound 48/80 and 2.5-S nerve growth factor, we examined peritoneal mast cells from 14–15-day-old rats (young rats) and compared them to peritoneal mast cells from adults. Peritoneal mast cells from young rats contain approximately one-tenth of the amount of histamine observed in adult peritoneal mast cells. However, both cell populations contained similar low levels of the mucosal mast cell-associated protease rat mast cell protease II. Histochemical analysis of peritoneal mast cells from young rats using safranin O and berberine sulphate suggested that only a portion of the granules of these cells contained heparin. At an ultrastructural level the young rat peritoneal mast cell contains relatively few granules. The majority of mast cells from young rats have a bilobed or indented nucleus which is only rarely observed in adult cells. Functionally, the young rat peritoneal mast cell demonstrates a significantly reduced histamine release in response to the connective tissue mast cellspecific secretagogues compound 48/80 and 2.5-S nerve growth factor. In contrast, the percent histamine release in response to the neurotransmitter substance P, which degranulates both connective tissue mast cells and intestinal mucosal mast cells, was similar in the adult cells and the young rat cells. This study demonstrates substantial differences between the young rat and adult peritoneal mast cells which may explain the ability of very young animals to withstand large doses of certain secretagogues.     

Analysis of pure and mixed murine mast cell colonies

Mast cells have been proposed to originate from diverse sources, including connective tissues, macrophages, T lymphocytes, and hemopoietic cells. Evidence for a hemopoietic origin of mast cells includes the presence of mast cell precursors in spleen colonies and the presence of mast cells in hemopoietic colonies in culture. Here we report a detailed analysis of mouse spleen mixed hemopoietic colonies containing mast cells. All of the colonies in cultures plated at low cell densities were individually removed for analysis by May-Grunwald-Giemsa staining on day 15 of culture. Examination of five dishes which contained a total of 82 colonies showed 16 pure mast cell colonies and 36 mixed mast cell colonies. Sixteen different combinations of cell types were seen and were not distinguishable from each other in situ. The most diverse type of mixed colony contained macrophages (m), neutrophils (n), eosinophils (e), mast cells (Mast), megakaryocytes (M), erythroid cells (E), and blast cells. The clonal origin of mixed mast cell colonies was established by the replating of single cells obtained from blast cell colonies. Individual cells were removed with a micromanipulator, replated, and allowed to grow for 15 days. Cytospin preparations of 10 such colonies showed diverse combinations of cell lineages which were seen in the different types of mixed mast cell colonies described above. Replating studies of mixed mast cell colonies were carried out and a high incidence of replating was seen. Approximately one half of these colonies formed only mast cell colonies upon replating. Further studies showed that pure mast cell colonies could be serially replated four to five times. The replating efficiency of cells in the primary mast cell colonies varied over a wide range (2.5–44%) with an average replating efficiency of 13%. The data also revealed that cells containing metachromatic granules possess significant proliferative capacity. From these studies of pure and mixed mast cell colonies, we concluded (1) that mast cells are in wide variety of types of mixed colonies and that the in situ identification of mixed colonies is unreliable, (2) that mast cells are derived from pluripotent hemopoietic stem cells, and (3) that mast cells with metachromatic granules can have a high proliferating ability.     

Gut-associated lymphoid tissue (GALT) of the goldfish,Carassius auratus

The head kidney and spleen are major sites of haemopoiesis in fish; a secondary center is found in loose connective tissue of the intestine. In this study we determined the nature of gut-associated haemopoietic tissue in the goldfish, Carassius auratus, using light and electron microscopy. This tissue is a loose stroma of reticular cells and fibers vascularized by capillaries, venules, and arterioles. The cellular population includes lymphoblasts, small and medium-sized lymphocytes, plasmocytes, macrophages, and various granulocytes. The most abundant granulocyte is the mast cell, whose large granules stain with Alcian blue and toluidine blue. Heterophils are found in the intestinal connective tissue as well as two other granulocytes: one with ovoid granules having dense parallel lamellae and another with granules containing crystalline inclusions. Immature forms of both granulocytes were also noted. Macrophages containing phagocytosed debris were often located close to the epithelium; they were observed forming clusters with lymphocytes. The epithelium contained a number of migrating leucocytes including lymphocytes and lymphoblasts, macrophages, and heterophils. Although many granulocytes were found in the connective tissue, granulopoiesis does not seem to be a major function. Gut-associated haemopoietic tissue in goldfish resembles diffuse lymphoid tissue and may be involved in intestinal immune responses.     

Intracellular localization of serotonin in mast cells of the colon in normal and colitis rats

Intracellular localization of serotonin (5-HT) in the mast cells of two phenotypes in normal rat colon and dextran sodium sulphate-induced colitis was studied by immunoelectron microscopy with a quantitative analysis of the distribution of immunogold labelling. Mucosal mast cells in normal rats contained round shape secretory granules with varying electron density. Immunogold labelling for 5-HT was concentrated over the secretory granules. In mucosal mast cells from colitis rats, vacuolated granules without 5-HT labelling were frequently observed and immunogold labelling over the secretory granules was significantly increased compared to controls. On the other hand, connective tissue mast cells in normal rats contained oval shape secretory granules with homogeneous electron density. Their immunogold labelling was diffusely scattered over the secretory granules as well as over the cytoplasm. In connective tissue mast cells from colitis rats, secretory granules with high electron density were increased and the immunogold labelling over the secretory granules was much higher than that in controls. The present results suggest that intracellular localization of 5-HT is different in two phenotypes of mast cells and they may release 5-HT in a different manner. Mucosal mast cells may release 5-HT by a degranulation or exocytosis, while connective tissue mast cells may release 5-HT by a diacrine manner of secretion.     

Intramucosal lymphocytes of the gut: Lyt-2 and thy-1 phenotype of the granulated cells and evidence for the presence of both T cells and mast cell precursors

The gut mucosa contains lymphocyte-like cells, a proportion of which contain a small number of granules that resemble those of mast cells in that they contain histamine and stain metachromatically. It has been suggested that these granulated lymphocytes represent transitional forms in the differentiation of T cells into mast cells. We used monoclonal antibodies and the fluorescence-activated cell sorter to analyze the expression of Thy-1 and Lyt-2 antigens on gut intramucosal lymphocytes with particular emphasis on the granulated cells. A minority of the granulated cells (10 to 20%) expressed Thy-1 antigen at high levels equivalent to those on cortical thymocytes. A much higher proportion of the granulated cells (about 90%) expressed readily detectable levels of Lyt-2 antigen and the most prevalent phenotype of the granulated lymphocytes (60 to 70%) was Lyt-2+, Thy-1-. Two operationally specific preparations of growth factors, one maintaining the proliferation of T cells and containing T cell growth factor, and the other containing a factor stimulating the growth of persisting (P) cells that are probably mast cell progenitors, were tested on lymphocytes from the gut mucosa. By using the appropriate preparation of growth factors, both T and P cells could be grown readily from the preparation of gut intramucosal lymphocytes. Estimates of the frequency of P cell precursors among these cells indicated a minimum of one in 300 could give rise to cells resembling mast cells. Fractions of Lyt-2+ cells that were enriched in granulated cells had few detectable P cell precursors, an observation lessening the likelihood that the granulated cells were progenitors of the P cells. The precise relationship of the granulated lymphocytes (mainly Lyt-2+, Thy-1-) to T cells remains to be established.     

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.     

Stimulation of mouse connective tissue-type mast cells by hemopoietic stem cell factor, a ligand for the c-kit receptor.

The proliferative capacity of mouse connective tissue-type mast cells (CTMC) was analyzed by using a newly discovered c-kit ligand, termed stem cell factor (SCF). More than 90% of CTMC in the peritoneal cavity responded to recombinant rat SCF (rrSCF) and were able to give rise to pure mast cell colonies in methylcellulose culture. Serial observation (mapping) of growth of individual CTMC in culture containing rrSCF confirmed their striking proliferative ability. No serum but accessory cells (non-CTMC cells) in the peritoneal population were required for the clonal growth of CTMC induced by rrSCF in our methylcellulose culture of whole peritoneal cells. The rrSCF-induced mast cell colony formation from peritoneal CTMC was completely inhibited by the addition of anti-c-kit antibody, which can block the binding of SCF to c-kit, to the culture. When IL-3 was combined with rrSCF, mast cell colonies dramatically increased in size. Mapping studies revealed that the combination of the two factors augmented the proliferative rate of CTMC. Approximately 60% of the constituent cells of the mast cell colonies which were formed from peritoneal CTMC in the culture containing rrSCF alone were stained with berberine sulfate, which is a characteristic of CTMC. However, most mast cells which were induced by rrSCF+IL-3 from peritoneal CTMC contained berberine(-)-safranin(-)-Alcian blue(+) granules. Although IL-4 exhibited little synergism with rrSCF in the induction of CTMC proliferation, the addition of IL-4 to the culture containing rrSCF+IL-3 resulted in an increase in mast cells which retained CTMC characteristics.     

The mast cell-committed progenitor. I. Description of a cell capable of IL-3-independent proliferation and differentiation without contact with fibroblasts

We have identified a late, committed stage in the differentiation of the mast cell progenitor just before granulation. Mast cell committed progenitors (MCCP) are nongranulated cells with a density of 1.060 to 1.070 g/ml which can be harvested from the mesenteric lymph node of mice infected with Nippostrongylus brasiliensis. Mast cell-committed progenitors are able to proliferate and differentiate in the absence of IL-3 or IL-4 when cultured on a monolayer of embryonic skin or 3T3 fibroblasts and can form colonies in methylcellulose supplemented with fibroblast conditioned medium. Fibroblast conditioned medium appears to contain a soluble MCCP proliferation factor that maintains biologic activity when heated to 56 degrees C for 45 min but is destroyed by incubation with either trypsin or chymotrypsin. It can be selectively precipitated with 60 to 70% saturated ammonium sulfate. The factor is not absorbed by immobilized antibodies to nerve growth factor. The MCCP proliferation activity of the factor could not be mimicked by IL-1, IL-2, IL-4, granulocyte-macrophage-CSF, granulocyte-CSF, macrophage-CSF, IFN-alpha/beta, IFN-gamma, nerve growth factor, epidermal growth factor, serum fibronectin, heparin, or a number of glycosaminoglycans. At high salt concentrations, the factor passes through a 50-kDa membrane and can be concentrated above a 5-kDa membrane. MCCP acquire a connective tissue phenotype when cultured on a fibroblast monolayer and a mucosal phenotype when cloned in the presence of conditioned medium from PWM-stimulated spleen cells. When cultured in the absence of IL-3 on a monolayer of embryonic skin or 3T3 fibroblasts, mast cell-committed progenitors produce mast cells which stain with berberine sulfate suggesting a connective tissue phenotype; however, the mast cells that develop when mast cell-committed progenitors are cultured in the presence of IL-3 or conditioned media from PWM-stimulated spleen cells do not stain with berberine sulfate. MCCP intercalate into monolayers of embryonic skin or 3T3 fibroblasts, but T cells are not able to associate with the monolayer and can be completely washed away. Attempts to enrich mast cell-committed progenitors by intercalation and elution from embryonic skin monolayers proved unsuccessful, but some enrichment of mast cell-committed progenitors could be achieved by discontinuous Percoll gradients. Thus, we have identified a way to obtain late-stage, mast cell-committed progenitors in an environment that is virtually uncontaminated with other hematopoietic progenitors.     

Mast Cells Produce a Unique Chondroitin Sulfate Epitope

The granules of mast cells contain a myriad of mediators that are stored and protected by the sulfated glycosaminoglycan (GAG) chains that decorate proteoglycans. Whereas heparin is the GAG predominantly associated with mast cells, mast cell proteoglycans are also decorated with heparan sulfate and chondroitin sulfate (CS). This study investigated a unique CS structure produced by mast cells that was detected with the antibody clone 2B6 in the absence of chondroitinase ABC digestion. Mast cells in rodent tissue sections were characterized using toluidine blue, Leder stain and the presence of mast cell tryptase. The novel CS epitope was identified in rodent tissue sections and localized to cells that were morphologically similar to cells chemically identified as mast cells. The rodent mast cell-like line RBL-2H3 was also shown to express the novel CS epitope. This epitope co-localized with multiple CS proteoglycans in both rodent tissue and RBL-2H3 cultured cells. These findings suggest that the novel CS epitope that decorates mast cell proteoglycans may play a role in the way these chains are structured in mast cells.     

Development of rat mast cells in vitro. I. Differentiation of mast cells from thymus cells.

Mast cells were differentiated by long-term culture of rat thymus cells on rat embryonic fibroblasts monolayers. Mature mast cells obtained in the culture were morphologically similar to normal peritoneal and thoracic mast cells and possessed specific receptors for IgE on their surface. In culture, blast cells appeared on the monolayer several days after seeding of thymus cells. These cells developed into young mast cells in the monolayer and became free in the culture medium with maturation. Receptors for IgE were detected on the surface of mastoblasts which contained a small amount of metachromatic granules. Evidence was obtained which suggested that the number and/or affinity of the receptors for IgE increases with maturation of mast cells. It was found that some mast cells differentiated from monolayers of embryo cells without seeding thymus cells. The present experiments, however, clearly showed that mast cells can be differentiated from thymus cell culture without monolayer. It appears that both thymus and embryo tissues contain precursors of mast cells.     

Comparison of histochemical staining techniques for detecting mast cells in oral lesions

ABSTRACT

Mast cells are large cells with granular cytoplasm that participate in wound healing, angiogenesis and defense against pathogens. They also contribute to inflammation by initiating innate and acquired immunity. The granules of these cells exhibit characteristic staining properties. We investigated toluidine blue, astra blue, Alcian blue-pyronin Y and May-Grunwald Giemsa stains for mast cells in various oral lesions and assessed the efficacy of each for identifying mast cells. Sections were obtained from 10 each of diagnosed cases of inflammatory fibrous hyperplasia, periapical cyst, mild dysplasia, oral submucous fibrosis and oral squamous cell carcinoma and stained using the stains listed above. Mast cells were assessed for their presence, contrast of the mast cell in the connective tissue background and number. We found that May-Grunwald Giemsa stain was the best for identification of mast cells, although toluidine blue staining is less time-consuming. Overall we obtained better results using May-Grunwald Giemsa and toluidine blue for staining mast cells.     

Characteristics of mast cells in Chediak-Higashi mice: light and electron microscopic studies of connective tissue and mucosal mast cells

The beige mouse, a homologue of the Chediak-Higashi syndrome in man, possesses abnormally large granules in many tissue cells. The granules in the mucosal mast cells (MMC) of the small intestine of beige and littermate C57BL/6J mice were examined after infecting the mice with the intestinal parasite, Nippostrongylus brasiliensis. MMC in both beige and littermate mice had irregular granules which contained paracrystalline substructures embedded in an amorphous matrix. Granules were not observed in fusion with the cell membrane. Instead, in late-stage mast cells, the granule membrane broke down, the granule contents were spread throughout the cytoplasm, and the cell organelles disintegrated. Unlike connective tissue mast cells, MMC were poorly demonstrated with formalin fixation and toluidine blue staining.     

Eosinophils and mast cell homogeneity of the guinea pig eyelid skin, conjunctiva, and ileum

Mast cell heterogeneity has been described on the basis of differential staining reactions, light microscopic morphology, anatomic location, degranulation after polyamines, biochemical contents, growth requirements, and reactions to lymphokines. We have demonstrated typical "connective-tissue mast cells" by using anatomic criteria, histological staining reactions, electron microscopy, and reaction to compound 48/80 in the guinea pig conjunctiva, eyelid skin, and ileum. A second, much larger population of cells in the ileal mucosa and the conjunctiva, and rarely in the eyelid skin stained reddish-blue with acid toluidine blue in tissue fixed in ethanol-acetate-lead subacetate (BLA) and with alkaline Giemsa in formaldehyde-fixed tissue, did not stain with ethanolic or acid toluidine blue in formaldehyde-fixed tissue or with alkaline Giemsa in BLA-fixed tissue, and did not degranulate after 48/80 treatment. These are features of the rat intestinal "mucosal mast cells"; however, ultrastructural and light microscopic studies with the orcein Giemsa stain demonstrated these cells in the guinea pig to be eosinophils. Tissue culture, biochemical, and immunological studies indicate the existence of a second type of mast cell (bone-marrow-derived mast cell), ultrastructurally almost indistinguishable from the connective tissue mast cell. Our studies demonstrate only one mast cell type in the guinea pig and support the contention that other forms of mast cells are immature forms or variants of the connective-tissue mast cell.     

Biochemical and morphological characterization of basophilic leukocytes from two patients with myelogenous leukemia

Basophilic leukocytes from two patients with myelogenous leukemia were enriched to a purity of 10 to 45% by density gradient centrifugation. Ultrastructurally, these basophilic leukocytes contained segmented nuclei and granules with reticular patterns resembling those of normal basophils, and other granules with scroll and grating patterns resembling those of normal connective tissue mast cells. The 35S-labeled macromolecules isolated from these cells were approximately 140,000 m.w. Pronase-resistant proteoglycans bearing approximately 15,000 m.w. glycosaminoglycans. On incubation with chondroitinase ABC, nitrous acid, and heparinase, the 35S-labeled proteoglycans were degraded 50 to 84%, 16 to 43%, and 8 to 37%, respectively, indicating the presence of both chondroitin sulfate and heparin. As assessed by high performance liquid chromatography, the 35S-labeled chondroitin sulfate disaccharides liberated by chondroitinase ABC treatment were approximately 95% monosulfated chondroitin sulfate A and approximately 5% disulfated chondroitin sulfate E. The presence of heparin was confirmed by two-dimensional cellulose acetate electrophoresis of the 35S-labeled glycosaminoglycans. Cell preparations, enriched to 75% basophilic leukocytes by sorting for IgE+ cells, also synthesized 35S-labeled proteoglycans containing chondroitin sulfate and heparin. In one experiment, treatment of the cells with 1 microM calcium ionophore A23187 resulted in a 12% net release of both chondroitin sulfate and heparin containing 35S-labeled proteoglycans, a 57% net release of histamine, and the de novo generation of 8, 8, and 0.16 ng of immunoreactive equivalents of prostaglandin D2, leukotriene C4, and leukotriene B4, respectively, per 10(6) cells. Because only mast cells have been found to contain Pronase-resistant heparin proteoglycans, to generate PGD2 on cell activation, and to contain granules with scroll and grating patterns, these findings indicate that in some patients with myelogenous leukemia there are basophilic cells that possess properties of tissue mast cells.