Detection and partial characterization of a human mast cell carboxypeptidase

Using a high performance liquid chromatography assay that detects the cleavage of the C-terminal leucine from angiotensin I, we have identified a carboxypeptidase activity in mast cells from human lung and in dispersed mast cell preparations from human skin. The enzyme activity was detected in a preparation of dispersed human mast cells from lung of greater than 99% purity and was released with histamine after stimulation with goat anti-human IgE. In nine preparations of dispersed human mast cells from lung of 10 to 99% purity, net percentage of release of carboxypeptidase correlated with the release of histamine, localizing carboxypeptidase to mast cell secretory granules. The enzyme activity was also detected in preparations of dispersed human mast cells from skin and in extracts of whole skin. The inhibitor profile and m.w. of carboxypeptidase activity from preparations of dispersed mast cells from skin was similar to that from dispersed mast cells from lung. Mast cell carboxypeptidase had a m.w. on gel filtration of 30,000 to 35,000. The enzyme in crude lysates of dispersed mast cell preparations had optimal activity between pH 8.5 and 9.5 and was inhibited by potato inhibitor, which distinguished it from carboxypeptidase in cultured human foreskin keratinocytes and adult fibroblasts, and from other proteolytic mast cell enzymes. The enzyme activity was also inhibited by EDTA, o-phenanthroline, and, to a small extent, by 8-OH quinoline, but not by Captopril, soybean trypsin inhibitor, or pepstatin. These findings demonstrate that human mast cell secretory granules contain carboxypeptidase in addition to tryptase and chymase. It appears that mast cells from skin may have a higher content of carboxypeptidase than do mast cells from lung.     

Human uterine mast cells. Isolation, purification, characterization, ultrastructure, and pharmacology.

As part of an ongoing investigation of human mast cell heterogeneity, we have isolated, partially purified, and characterized the uterine mast cell and compared it with mast cells isolated from other organs. The average histamine content of myometrium and leiomyofibroma obtained from hysterectomies was 2.1 +/- 0.3 (mean +/- SEM) microgram/g of tissue (n = 10), and the histamine content of the two tissues did not differ significantly. A mild collagenase, hyaluronidase, and DNase digestion was used to disperse the uterine mast cells, with an average yield of 9.5% (range, 0 to 21%). The average histamine/uterine mast cell was 2.1 +/- 0.2 pg (n = 3), and 61 +/- 7% (n= 3) of the uterine mast cells survived overnight culture. Early purification efforts with Percoll gradients have yielded up to 80% pure uterine mast cells, with an average of 27 +/- 10% (n = 5). Uterine mast cells released histamine in response to the secretogogues anti-IgE and A23187 but did not respond to substance P or to the basophil secretogogues FMLP, C5a, and 12-O-tetradecanoylphorbol-13-acetate. After 1 microgram/ml anti-IgE stimulation, the uterine mast cell appeared to make significant quantities of PGD2 (89 +/- 26 ng/10(6) cells, n = 6) (p less than 0.05), as assayed by RIA. Simultaneously, leukotriene C4 release was 45 +/- 15 ng/10(6) cells, (n = 6) (p less than 0.05), as assayed by RIA. Combined gas-chromatography mass spectroscopy analysis of anti-IgE-stimulated cell supernatants confirmed the production of PGD2. In pharmacologic studies, isobutyl-methylxanthine and isoproterenol blocked anti-IgE-induced histamine release. The uterine mast cell is similar to the lung mast cell in terms of response to secretogogues and release of arachidonic acid metabolites. Ultrastructurally, the uterine mast cell contains scroll granules, crystal granules, combined granules, homogeneously dense granules, and large lipid bodies, many with focal lucencies within them. Particle granules, most frequently present in gut mast cells of mucosal origin, were absent from uterine mast cells. Although certain features are analogous to the ultrastructure of skin or lung mast cells, the combination of structures is distinctive for uterine mast cells.     

Co-culture of human lung-derived mast cells with mouse 3T3 fibroblasts: morphology and IgE-mediated release of histamine, prostaglandin D2, and leukotrienes

Human mast cells, dispersed from lung tissue by proteolytic treatment and enriched to a purity of 23 to 68% by density-gradient centrifugation, were maintained ex vivo for up to 13 days when co-cultured with mouse skin-derived 3T3 fibroblasts in RPMI 1640 containing 10% fetal calf serum. The human mast cells were adherent to the fibroblast cultures within 2 to 4 hr after seeding, and after 7 days of co-culture were localized between the layers of fibroblasts. The cell surfaces of the mast cells and the fibroblasts did not form tight junctions, but rather approached within 20 nm of each other. The co-cultured mast cells did not divide; they maintained their cellular content of histamine and TAMe esterase and resembled in vivo mast cells in that their secretory granules exhibited scroll patterns and their nuclei were oval. Both the freshly isolated and the co-cultured mast cells responded to activation with anti-human IgE by exocytosing histamine and generating and releasing arachidonic acid metabolites. When freshly isolated mast cells were activated immunologically, they exocytosed 38 +/- 8% of their total histamine content and released 28 +/- 1.9 ng (mean +/- range, n = 2) of immunoreactive equivalents of prostaglandin D2 (PGD2) per microgram of total cellular histamine, but did not generate significant amounts of either leukotriene C4 (LTC4) or leukotriene B4 (LTB4). The 1-wk co-cultured mast cells, on the other hand, exocytosed 43 +/- 2.4% of their total histamine content, and released 86 +/- 10, 43 +/- 20, and 5.2 +/- 5.2 ng (mean +/- SD, n = 4) of immunoreactive equivalents of PGD2, LTC4, and LTB4, respectively, per microgram of histamine. Thus, human lung-derived mast cells can be maintained ex vivo when co-cultured with fibroblasts, and, when treated with anti-IgE, they metabolize arachidonic acid via both the cyclooxygenase and the 5-lipoxygenase pathways.     

Secretion in dissociated human pulmonary mast cells. Evidence for solubilization of granule contents before discharge

Mast cells were enzymatically dissociated from human lung fragments that had been sensitized with serum from human allergic to ragweed and were enriched by isopyknic and velocity gradient sedimentation. Electron microscope examination showed that the mast cells were well preserved at the end of the dissociation and isolation and that the majority of their secretory granules contained crystalline structures. These structures exhibited three patterns--scrolls, gratings, and lattices--which all could be found in the same granule. The period of crystalline structures was found to be bimodal, with maxima at 150 and 75 A. Both periods were observed in gratings that had been tilted and in scrolls that had been cut obliquely, indicating that the various gross patterns are composed of the same basic substructure. After the mast cells were stimulated by rabbit anti-human IgE to release histamine, the contents of the granule were transformed from a crystalline to an amorphous state, and only granules with amorphous contents were seen discharging from the cell. Clusters of intermediate filaments were present around the granules with amorphous contents, both deep in the cytoplasm and discharging at the cell surface. Discharge occurred both by fusion of granule membranes with the plasma membrane and by fusion of granule membranes with other granule membranes that ultimately were continuous with the plasma membrane. After discharge, the granule residue was fibrillar. Cells challenged with anti-human IgE in calcium-free medium neither released histamine nor demonstrated morphologic changes in their granules. We conclude that the crystalline state represents a storage form for materials that are solubilized before fusion of the granule membrane with the plasma membrane and discharge.     

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.     

Acid-phosphatase activity of reticular cells and macrophages in the lymph node of the rat after ingestion of mast-cell granules

Acid phosphatase (ACPase) was ultracytochemically demonstrated in the lymph-node sinus reticular cells and macrophages of rats. After the uptake of horseradish peroxidase (HRP), marked ACPase activities were seen in both reticular cells and macrophages, although only sparse ACPase activity was detected in the reticular cells of the control. After the injection of HRP into the footpad, the mast cells in the regional lymph node became degranulated, and the released granules were taken up by reticular cells and macrophages. In macrophages, these taken-up mast-cell granules exhibited ACPase reaction products, whereas none of the granules taken up by reticular cells showed ACPase activity. The heparin-protamine complex was also engulfed by reticular cells and macrophages, and ACPase activity was demonstrable in the complex taken up by both types of cell. It is probable that, as is the case in macrophages, reticular cells in the lymph-node sinuses take up and digest foreign substances through the formation of phagolysosomes, but they do not digest granules originating from the mast cells in the lymph node of the same animal.     

Acid-phosphatase activity of reticular cells and macrophages in the lymph node of the rat after ingestion of mast-cell granules

Summary Acid phosphatase (ACPase) was ultracytochemically demonstrated in the lymph-node sinus reticular cells and macrophages of rats. After the uptake of horseradish peroxidase (HRP), marked ACPase activities were seen in both reticular cells and macrophages, although only sparse ACPase activity was detected in the reticular cells of the control. After the injection of HRP into the footpad, the mast cells in the regional lymph node became degranulated, and the released granules were taken up by reticular cells and macrophages. In macrophages, these taken-up mast-cell granules exhibited ACPase reaction products, whereas none of the granules taken up by reticular cells showed ACPase activity. The heparin-protamine complex was also engulfed by reticular cells and macrophages, and ACPase activity was demonstrable in the complex taken up by both types of cell. It is probable that, as is the case in macro-phages, reticular cells in the lymph-node sinuses take up and digest foreign substances through the formation of phagolysosomes, but they do not digest granules originating from the mast cells in the lymph node of the same animal.     

Separation of epithelial cells from suspensions of cells from the hamster parotid gland in an isokinetic density gradient of Ficoll in tissue culture medium.

Epithelial cells were separated from suspensions of hamster parotid cells by velocity sedimentation in an isokinetic gradient and by isopycnic sedimentation. Epithelial cells were 48.1 ± 18.0% of the cells in the starting sample suspensions of cells from the disaggregated hamster parotid glands. The purest gradient fractions following velocity sedimentation in a previously described isokinetic gradient contained 98.8 ± 1.8% epithelial cells. The purest fractions obtained from isopycnic sedimentation contained 99.9 ± 0.2% epithelial cells. Purification of parotid epithelial cells by velocity sedimentation in the isokinetic gradient seems preferable to purification using isopycnic centrifugation because a larger proportion of the epithelial cells are obtained in the zone of the gradient which contains highly purified epithelial cells and because velocity sedimentation requires lower centrifugal forces for a shorter period of time.     

Alterations of the electrophoretic mobility distribution of rat mast cells after immunologic activation.

Changes in the electrophoretic mobility distributions of rat serosal mast cells after immunologic activation have been measured using the laser Doppler technique of electrophoretic light scattering. Rat serosal mast cells of 98% purity isolated by isopycnic and velocity gradient sedimentation had a highly negative electrophoretic mobility which was unaffected by incubation with normal rabbit serum or, at 4 degrees C or in the absence of Ca+2, with rabbit anti-rat E(ab')2 antiserum. Immunologic activation of the cells with this antiserum in the presence of Ca+2 at 37 degrees C resulted in a dose- and time-dependent increase in the electrophoretic mobility. Thus at a 1:25 dilution of anti-F(ab')2 the mean and mode electrophoretic mobilities of the mast cell population increased 25 and 21%, respectively. The width of the electrophoretic mobility distribution also increased with activation, indicating a heterogeneous response of the mast cells in the population. The increase in electrophoretic mobility after immunologic activation is not diminished by treatment of the cells with 1 M NaCl to solubilize adsorbed mast cell granule or heparin.     

Mast cell heterogeneity in higher animals: a comparison of the properties of autologous lung and intestinal mast cells from nonhuman primates

The issue of mast cell heterogeneity has been investigated in nonhuman primates by a comparative examination of lung and intestinal mast cells. These cells were obtained in parallel from the respective tissues of individual monkeys by an identical enzymatic dispersion technique. Mast cells derived from the lungs differed from those derived from the intestine in that the majority of the former cell type could be stained with toluidine blue at pH 4 to 5, whereas the intestinal mast cells in the dispersed preparations required a more acidic pH (less than 1) to display metachromasia. In addition, the lung cells exhibited an increased content of the mast cell mediator histamine. Nonhuman primate lung mast cells were also quantitatively more responsive to an immunologic challenge than their intestinal counterparts in that they released a higher percentage of cellular histamine and generated more leukotriene C4 on stimulation. Considerable inter-animal variation was observed between the magnitude of mediator release from both mast cell types after anaphylactic activation, but evidence for the presence in nonhuman primates of the phenomenon of releasability was not obtained. The responsiveness of both cell types to a range of potential nonimmunologic secretagogues and anti-allergic agents, including compound 48/80, substance P, theophylline, and isoprenaline, was essentially similar. We conclude that mast cell heterogeneity in higher animals may be reflected more by cytochemical rather than by functional differences between mast cell classes.     

Simultaneous detection of histamine release and lactate production in rat mast cells induced by compound 48/80 using 1H NMR.

1H NMR spectroscopy was used to evaluate histamine release and lactate production in intact mast cells isolated from rats. The resonance lines of the aromatic histamine protons in mast cells, detected by the selective spin-excitation technique, were broader and located in a lower magnetic field than those in free histamine solution. When exocytosis of mast-cell granules was induced by compound 48/80, free histamine appeared, with a corresponding decrease in the amount of histamine in the mast cells; the lactate signal was also detected in the spectrum. On the addition of compound 48/80, there was a further release of histamine from mast cells, accompanied by further production of lactate. This result indicates that the mechanisms which induce the exocytosis of granules, and/or the events following exocytosis, activate glycolysis.     

Simultaneous detection of histamine release and lactate production in rat mast cells induced by compound 48/80 using H NMR

¹H NMR spectroscopy was used to evaluate histamine release and lactate production in intact mast cells isolated from rats. The resonance lines of the aromatic histamine protons in mast cells, detected by the selective spin-excitation technique, were broader and located in a lower magnetic field than those in free histamine solution. When exocytosis of mast-cell granules was induced by compound 48/80, free histamine appeared, with a corresponding decrease in the amount of histamine in the mast cells; the lactate signal was also detected in the spectrum. On the addition of compound 48/80, there was a further release of histamine from mast cells, accompanied by further production of lactate. This result indicates that the mechanisms which induce the exocytosis of granules, and/or the events folowing exocytosis, activate glycolysis.     

Signal transduction pathways in mast cell granule-mediated endothelial cell activation

BACKGROUND: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8. AIMS: The objective of the present study was to identify candidate molecules and signal transduction pathways involved in the synergy between mast cell granules and lipopolysaccharide on endothelial cell activation. METHODS: Human umbilical vein endothelial cells were incubated with rat mast cell granules in the presence and absence of lipopolysaccharide, and IL-6 production was quantified. The status of c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 activation, nuclear factor-kappaB translocation and intracellular calcium levels were determined to identify the mechanism of synergy between mast cell granules and lipopolysaccaride. RESULTS: Mast cell granules induced low levels of interleukin-6 production by endothelial cells, and this effect was markedly enhanced by lipopolysaccharide. The results revealed that both serine proteases and histamine present in mast cell granules were involved in this activation process. Mast cell granules increased intracellular calcium, and activated c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2. The combination of lipopolysaccharide and mast cell granules prolonged c-Jun amino-terminal kinase activity beyond the duration of induction by either stimulant alone and was entirely due to active proteases. However, both proteases and histamine contributed to calcium mobilization and extracellular signal-regulated kinase 1/2 activation. The nuclear translocation of nuclear factor-kappaB proteins was of greater magnitude in endothelial cells treated with the combination of mast cell granules and lipopolysaccharide. CONCLUSIONS:Mast cell granule serine proteases and histamine can amplify lipopolysaccharide-induced endothelial cell activation, which involves calcium mobilization, mitogen-activated protein kinase activation and nuclear factor-kappaB translocation.     

Demonstration of the origin of human mast cells from CD34+ bone marrow progenitor cells.

It has been established that murine mast cells are derived from a pluripotent bone marrow stem cell. In humans, the corresponding pluripotent cell is included in the CD34+ bone marrow population. To determine whether human mast cells arise from CD34+ human progenitor cells, enriched CD34+ cells were cultured over agarose surfaces (interphase cultures) or cocultured with mouse 3T3 fibroblasts in the presence of recombinant human (rh) IL-3. The presence of both mast cells and basophils was determined using a variety of histochemical and immunohistologic techniques, including immunogold labeling for IgE receptors and mast cell tryptase. Mast cells and basophils continued to appear in cultures when T cell, B cell, macrophage, and eosinophil committed progenitor cells were removed, but were not seen in cultures from which CD34+ cells were removed. CD34+ cells layered over agarose in the presence of rhIL-3 were shown to give rise to cultures that contained mast cells (1 to 5%) and basophils (25 to 40%). Cultures supplemented with rhIL-4 showed no additional increase in mast cells or basophils. CD34+ cells cocultured with 3T3 fibroblasts in the presence of rhIL-3 gave rise to mast cells within the fibroblast monolayer, which by 6 wk comprised up to 46% of the monolayer. CD34-cells on 3T3 fibroblasts gave rise to few mast cells (2% of the monolayer). Mast cell granules from interphase cultures contained homogeneous electron-dense material. In contrast, mast cells within 3T3 monolayers at 6 wk contained a variety of granule morphologies, including scroll, mixed, reticular, dense core, or homogeneous patterns. We conclude that both human mast cells and basophils arise from CD34+ human progenitor cells.     

Effect of histamine and divalent cations on the activity and stability of tryptase from human mast cells

Tryptase from human mast cells is stabilized by negatively charged macromolecules such as heparin and is not affected by the protein inhibitors of serine proteinases normally present in human extracellular fluids. The current study demonstrated inhibition of tryptase-catalyzed cleavage of tosyl-Gly-Pro-Lys-p-nitroanilide by histamine and calcium, and destablization only by calcium. Calcium-mediated inhibition was competitive with a Ki of 30 mM. Cooperation of calcium with other extracellular cations or concentrations of calcium possible within cells or granules may permit calcium-mediated inhibition to occur in vivo. In contrast, only 5 mM calcium is needed to cause an irreversible 50% loss of tryptase activity after 60 min at room temperature. Histamine and N-methyl histamine concentrations of 2 mM to 10 mM inhibited tryptase activity by a different mechanism than calcium, resulting in sigmoid rather than hyperbolic kinetics. Whether this reflects cooperative binding of histamine to tryptase or conformational alterations of tryptase is not known. These concentrations of histamine are most relevant to those in mast cell secretory granules estimated at 100 mM, where tryptase is stored fully active and where histamine may play a role in attenuating tryptase activity.     

鸡中枢淋巴器官肥大细胞的组织化学与形态学

对哺乳动物的,特别是啮齿动物和人类肥大细胞已有了比较深入的研究, 但关于家禽肥大细胞的研究很少.本研究旨在阐明鸡中枢淋巴器官中肥大细胞的组织化学与形态学特征.本研究证实Carnoy 氏液是鸡肥大细胞的优良的固定液,而中性缓冲福尔马林(NBF) 却阻断了大多数肥大细胞的着染力.甲苯胺蓝和阿尔新蓝是鸡肥大细胞的良好的染料,但阿尔新蓝能使更多的肥大细胞着染,虽然其也可使杯状细胞着染.作者的一种新的染色法, 长时间阿尔新蓝染色(LAB-S)可用于NBF固定的组织中肥大细胞的染色,因为其着染的细胞数与Carnoy 氏液固定甲苯胺蓝染色的细胞数无显著差异(P<0.001).在胸腺髓质中见有大量的肥大细胞,而胸腺皮质仅可见个别肥大细胞位于血管周围及小叶间结缔组织中.腔上囊的皮质与髓质中很少见有肥大细胞.肥大细胞有血管周围分布的倾向,但一个有趣的发现是血管内偶尔也有个别肥大细胞.电镜下可见肥大细胞的胞浆颗粒内充满无定形的颗粒状基质,但其电子密度有的较高,有的较低.少数胞浆颗粒内有旋涡状及网状亚微结构.但未见有人类肥大细胞胞浆颗粒内特征性的晶格状和卷轴状的亚微结构,也未见到在绵羊肥大细胞中描述过的特殊亚微结构.     

Failure to detect IL-3-binding sites on human mast cells

IL-3, a pleiotropic lymphokine, has been termed mast cell growth factor because it promotes growth and differentiation of murine mast cells. Murine mast cells, in turn, express cell surface receptors for IL-3. Human rIL-3 has been shown to induce proliferation and differentiation of human basophils and to activate basophils via high affinity binding sites. To investigate whether human mast cells express IL-3R, binding studies with 125I-radiolabeled human rIL-3 were performed on HMC-1, a novel human mast cell line, and on pure populations (i.e., 93 to 99% purity) of human tissue mast cells obtained with mAb and C from dispersed lung (n = 2). Unexpectedly, neither enriched human lung mast cells nor HMC-1 cells bound radiolabeled human rIL-3 specifically. Moreover, human rIL-3 failed to promote uptake of [3H]thymidine, synthesis of histamine, histamine releasability, or changes in expression of mast cell differentiation Ag (YB5B8, CD54/ICAM-1, CD9/p24, CD33/gp67) on either human lung mast cells or HMC-1 cells. It is hypothesized that the fundamental difference in the biologic response to IL-3 between human and murine mast cells is due to a loss during evolution of mast cell high affinity IL-3 binding sites.     

Enzymatic dispersion of mast cells from human sinus mucosa: characterization of histamine release and its comparison with chopped fragments of the tissue.

Functional characteristics of mast cells in chopped fragments from sinus mucosa, which was dissected from patients with chronic sinusitis, were compared with those from dispersed cells prepared by enzymatic treatment. The results obtained in this study were the following. (1) Both chopped fragments and dispersed cells released histamine in a dose-dependent manner when incubated with anti-IgE. However, higher histamine release was always observed in dispersed cells. (2) Although no differences in the ability to reduce histamine release with salbutamol or forskolin could be observed between chopped fragments and dispersed cells, staurosporin and p-bromophenacyl bromide were more active on dispersed mast cells than chopped fragments. (3) Passive sensitization of dispersed cells with an allergic serum containing IgE to mite could be achieved only after elution of IgE on the cells with lactic acid.     

Mode of action of protamine sulfate on histamine secretion in the rat mast cells

Protamine sulfate, known for a long time as a histamine releaser, was labeled with a fluorescent dye (FITC). This conjugate was shown to stain selectively the mast cell fraction of rat peritoneal cells. Within a few seconds, the protamine was found inside the cells. Although the cells had lost their histamine completely, no granules were found outside the cells. In the electron microscope, the protamine treated mast cells showed a loss of the electron density of their granules, a vacuolization, and other signs of histamine release. Evidence for a direct connection between the vacuoles and the extracellular fluid was gained by incubating mast cells in FITC-labeled human serum albumin followed by the addition of unlabeled protamine. After washing, the fluorescence was found to be located inside the cells, demonstrating an influx of the FITC-HSA under the influence of protamine. The protamine-induced release reaction is increased after addition of Ca2+, reduced by lowering the temperature, addition of 2-deoxyglucose, or cytochalasin B. Disodium cromoglycate also diminished the histamine release in a dose dependent manner. Protamine did not induce a loss of lactate dehydrogenase from the mast cells. The release reaction is mediated by the cell membrane, as shown by the releasing activity of insolubilized protamine. We conclude that the protamine-induced release is a non-cytotoxic reaction, fulfilling some criteria of the anaphylactic histamine release.     

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.