Purification and characterization of human skin mast cells. Evidence for human mast cell heterogeneity

Our previous studies of human lung and intestinal mast cells failed to show the heterogeneity found among mast cells in murine species. Recently, we and others have developed techniques for the enzymatic dispersion of human neonatal skin mast cells. In addition, we are now able to make single cell suspensions of mast cells from adult skin and to purify these cells to near homogeneity. Comparative studies of mast cells from these several sources have uncovered several major differences among them. Adult and neonatal skin mast cells themselves differ in that the former are 10-fold less sensitive to goat anti-human IgE, with maximal release occurring at 3.0 and 0.3 microgram/ml, respectively. Skin mast cells also differ in optimal temperature for release: adult mast cells respond maximally at 23 to 30 degrees C and neonatal cells at 37 degrees C. Skin mast cells from both sources are dramatically different from lung and intestinal mast cells in two aspects. First, skin mast cells are quite responsive to several stimuli--morphine sulfate (10(-4) to 10(-6) M), substance P (10(-5) to 10(-7) M), compound 48/80 (10 to 0.1 microgram/ml), f-Met peptide (10(-6) M), and C5a (10(-8) M)--to which the other mast cells fail to respond. Second, although stimulated skin mast cells produce prostaglandin D2, little leikotriene C4, if any, is generated, unlike lung or intestinal mast cells. These differences in inflammatory potential among human mast cells from various sites have important implications for the management of allergic and inflammatory responses.     

Calcium release associated with discharge of specific granule contents from human neutrophils

Neutrophil discharge of calcium into the ambient medium was measured, using an ion-sensitive electrode, after cells were stimulated with phorbol myristate acetate. Dose-dependent calcium efflux was observed over the same range of stimulus concentrations associated with specific granule marker release. Calcium efflux was preserved when cells were treated with vanadate to inhibit the plasma membrane calcium ATPase. However, less calcium was released than in previous studies employing a complete secretagogue to discharge both specific and azurophil granule contents. These studies suggest that calcium is stored in both the azurophil and the specific granules of the neutrophil, and can be mobilized from both sites during degranulation.     

Evidence for control of mast cell granule protease in situ by low pH

The second order rate constant, k2, for the inhibition of mast cell protease I by phenylmethanesulfonyl fluoride (PMSF) is lower for intact mast cells and isolated granules with intact membranes than for granules stripped of their membranes and suspended in medium at pH 7.1. In order to test the hypothesis that the decreased activity of the protease in intact granules is attributable to low pH, two agents capable of lowering pH in intracellular compartments similar to mast cell granules were tested. Ammonium chloride increased k2 of the protease in isolated granules with intact membranes and mast cells and wash out of the salt partially reversed this effect. Treatment of cells with nigericin also substantially increased the rate of protease inactivation by PMSF. These results are consistent with the proposal that the observed k2 is determined in whole or part by a low pH of the granule in situ or isolated with intact membranes. If the low k2 in situ is solely dependent on low pH, then the rate of protease inhibition can be utilized as an endogenous probe of granule pH. On this basis we have estimated the pH of the intracellular granule as 5.2 and that of the isolated granule with its membrane intact as 6.0. The value for the pH of granules in situ is lower than that previously estimated, and we have considered possible bases for this discrepancy.     

Serglycin proteoglycan is required for secretory granule integrity in mucosal mast cells

SG (serglycin) PGs (proteoglycans) are strongly implicated in the assembly of MC (mast cell) granules. However, this notion has mainly been on the basis of studies of MCs of the connective tissue subtype, whereas the role of SG PG in mucosal MCs has not been explored. In the present study, we have addressed the latter issue by using mice with an inactivated SG gene. Bone marrow cells were differentiated in vitro into the mucosal MC phenotype, expressing the markers mMCP (mouse MC protease) -1 and -2. Biosynthetic labelling experiments performed on these cells revealed an approximately 80% reduction of 35SO4(2-) incorporation into PGs recovered from SG-/- cells as compared with SG+/+ counterparts, indicating that SG is the dominating cell-associated PG of mucosal MCs. Moreover, the absence of SG led to defective metachromatic staining of mucosal MCs, both in vivo and in the in vitro-derived mucosal MCs. Ultrastructural analysis showed that granules were present in similar numbers in SG+/+ and SG-/- cells, but that their morphology was markedly affected by the absence of SG, e.g. with electron-dense core formation only seen in SG+/+ granules. Analysis of the MC-specific proteases showed that mMCP-1 and mMCP-7 were completely independent of SG for storage, whereas mMCP-2 showed a partial dependence. In contrast, mMCP-4 and -6, and carboxypeptidase A were strongly dependent on SG for storage. Together, our data indicate that SG PG is of crucial importance for assembly of mature mucosal MC granules, but that the specific dependence on SG for storage varies between individual granule constituents.     

Periodic,multimodal distribution of granule volumes in mast cells

The areas of 2327 mast cell granules in transmission electron micrographs of sections of peritoneal mast cells from adult rats were measured by digitized planimetry. A histogram constructed using equivalent volumes calculated from the measured areas assuming approximation of the granules to spheres showed a periodic multimodal distribution in which the modes fell at volumes that were successively larger integral multiples of the volume at the first mode. Application of a moving-bin technique to the data confirmed the presence of the modes. We propose a mechanism of fusion of unit sized granules to account for the multimodal distribution. The presence of pear- and dumbbell-shaped granules in mast cells is consistent with this mechanism.     

Distribution of chymase-containing mast cells in human bronchi.

Mast cell chymase stimulates secretion from cultured airway gland serous cells and hydrolyzes bronchoactive peptides in vitro. To explore the likelihood of these interactions occurring in situ, we examined the distribution and concentration of chymase-containing mast cells near glands and smooth muscle of major human bronchi from eight individuals without known airway disease. Total airway mast cells and the subset of mast cells containing chymase were detected by staining for methylene blue metachromasia and chloroacetate esterase activity, respectively. The percentage of chymase-containing mast cells was found to differ strikingly among bronchial tissue compartments. Near glands, for example, the concentration of chymase-positive mast cells (640 +/- 120 cells/mm3) was 73 +/- 9% that of total mast cells (910 +/- 130 cells/mm3), whereas in smooth muscle the concentration of chymase-positive mast cells (450 +/- 200 cells/mm3) was only 14 +/- 4% that of total mast cells (2920 +/- 620 cells/mm3). Of all chymase-containing mast cells in the airway subepithelium, 30 +/- 4% were located within 20 microns of submucosal glands. Although the percentage of chymase-containing cells varied, the absolute concentration of chymase-containing mast cells was similar in all compartments. These results reveal a differential distribution of mast cell subpopulations in human airway and suggest that mast cells containing chymase are near gland and smooth muscle targets.     

Characterization of rat mast cell granule proteins.

Mast cell granules free of membranes were isolated by differential centrifugation of water-lysed cells. The granules were extracted sequentially with 0.5, 1.0, and 2.0 m KCl. The 0.5 m fraction contained 95% of the N-acetyl-β-glucosaminidase activity; this enzyme probably accounts for no more than 1% of the total granule protein. The 1.0 m fraction contained more than 80% of the granule chymotrypsin-like activity; the chymotrypsin-like enzyme was calculated to represent at least 15% of total granule protein. Heparin was found largely in the 1.0 m extract and in the residue after 2.0 m extraction. The heparin in both fractions had a molecular weight by gel exclusion chromatography considerably in excess of commercial porcine heparin. Acrylamide-gel electrophoresis of granules dissolved in 1% sodium dodecyl sulfate and reduced with dithiothreitol demonstrated four major protein bands. The 1.0 m fraction contained the most prominent, rapidly migrating band. The more slowly migrating, higher molecular weight bands appeared in greater proportion in the 2.0 m and residue fractions. Autodigestion of the 1.0 m extract permitted purification of the mast cell chymotrypsin-like enzyme to specific activities as high as that of crystallized bovine pancreatic α-chymotrypsin. The mast cell chymotrypsin-like enzyme purified in this way migrated on dodecyl sulfate-gel electrophoresis as a single major band with an estimated molecular weight of 29,000.     

Patch-clamp measurements reveal multimodal distribution of granule sizes in rat mast cells

Using patch-clamp techniques, we have followed the attributes of the secretory granules of peritoneal mast cells obtained from rats of different ages. The granule attributes were determined by following the step increases in the cell surface membrane area caused by the exocytosis of the granules in GTP gamma S stimulated mast cells. Our data show that the amount of granule membrane available for exocytosis depends exponentially on the weight (age) of the donor rat, reaching a maximum at approximately 300 g. The data are consistent with an exponential growth in the number of granules contained by mast cells of maturing animals. Histograms of the sizes of the step increases in surface area caused by exocytosis of the granules showed at least four equally spaced peaks of similar variance where the position of the first peak and the spacing between peaks averaged 1.3 +/- 0.4 micron2. In all cells recorded, no more than seven peaks could be found, the higher order peaks having a lower probability of occurrence. The distribution of granule sizes did not change measurably between young and adult animals. This study suggests that at least two separate steps may determine the size of a secretory granule: granule to granule fusion that may account for the subunit composition of granule sizes and traffic of microvesicles through the maturing granules that may account for the variance observed in the granule sizes. This study also demonstrates a novel way to study granulo-genesis in living cells.     

Differences between basophils and mast cells: failure to detect Charcot-Leyden crystal protein (lysophospholipase) and eosinophil granule major basic protein in human mast cells

Human eosinophils contain several distinctive proteins including eosinophil granule MBP and the membrane-associated CLC protein (lysophospholipase). Human basophils also contain these proteins, indicating biochemical similarities between eosinophils and basophils. To determine whether MBP or CLC protein is present in connective tissue mast cells, we studied human lung and cutaneous mast cells by immunofluorescence by utilizing specific antibodies to CLC and MBP. Cytocentrifuge slides of enriched lung mast cells and mast cells in sections of formalin-fixed, paraffin-embedded cutaneous tissue from urticaria pigmentosa lesions were stained for CLC and MBP. Neither pulmonary nor cutaneous mast cells stained for CLC protein or MBP. In contrast, lung and cutaneous eosinophils in the same preparations showed bright staining for both proteins. The failure to find CLC protein and MBP in mast cells provides additional evidence of dissimilarity between mast cells and basophils, and an immunochemical means to distinguish between them.     

Histochemical heterogeneity of dispersed human lung mast cells.

Cytocentrifuge preparations of enzymatically dispersed human lung parenchymal mast cells were examined by light microscopy after fixation in either Mota's basic lead acetate or 10% neutral buffered formalin followed by toluidine blue staining at pH 0.5. Fixation in Mota's basic lead acetate allowed detection of all mast cells. However, after formalin fixation only 10.8 +/- 1.3%, range 4.7 to 17%, n = 8 remained detectable (i.e., formalin "resistant"). Therefore, the vast majority of human lung mast cells lose their metachromatic staining after formalin fixation (i.e., are formalin "sensitive"). Mast cells were then separated on the basis of diameter by countercurrent elutriation and on the basis of density by discontinuous Percoll gradients. Histochemically distinct populations of mast cell types emerged in all lungs studied. The proportion of formalin-resistant mast cells increased as a function of diameter: less than 5% at diameters of less than or equal to 11 mu and densities less than or equal to 1.063 g/ml, to 30 to 40% in cells of diameters greater than or equal to 16 mu and densities greater than or equal to 1.100 g/ml. Maximum anti-IgE challenge of nearly homogeneous formalin-sensitive mast cells (94.3 +/- 2.1% purity, n = 6) caused the generation of both leukotriene C4 (64.6 +/- 26.4 pg/mast cell) and PGD2 (114.8 +/- 37.5 pg/mast cell). Six- to eight-fold enrichment of formalin-resistant mast cells did not significantly alter the histamine release response or profiles of arachidonate metabolites. Similar results were obtained for the nonimmunologic stimulus ionophore A23187. We conclude that two histochemically distinct subpopulations, of mast cells are present in human lung suspensions. Although formalin-sensitive cells account for almost 90% of lung mast cells, formalin-resistant cells are separable by their large diameters and higher densities. Both subtypes show similar histamine release responses and arachidonate oxidation profiles.     

Ultrastructural studies of human basophils and mast cells.

Ultrastructural studies of human mast cells (HMCs) and basophils (HBs) are reviewed. Sources of HMCs include biopsies of tissue sites and in situ study of excised diseased organs; isolated, partially purified samples from excised organs; and growth-factor-stimulated mast cells that develop de novo in cultures of cord blood cells. Sources of HBs for study include partially purified peripheral blood basophils, basophils in tissue biopsies, and specific growth factor-stimulated basophils arising de novo from cord blood cells. The ultrastructural studies reviewed deal with identity, secretion, vesicles, recovery, and synthesis issues related to the biology of these similar cells.     

Electrophysiological properties of human oviduct smooth muscle cells in dissociated cell culture.

Intracellular recordings were made from human oviduct smooth muscle maintained in cell culture. Solitary cells isolated from one another and cells in contact with one another retained electrical properties of smooth muscle in vivo. Membrane potential of solitary cells and connected cells was -35 mV. Connected cells formed electrotonic junctions which transmitted current from one cell to another. This current spread was responsible for differences in input resistance and time constant in solitary cells, 66 Momega and 96 msec, compared to connected cells, 26 Momega and 56 msec. All cells expressed delayed rectification to depolarizing current pulses. Some cells generated action potentials spontaneously or in response to intracellular current pulses. Action potentials were abolished by cobalt or by EGTA. Slow wave potentials, 5 . 20 mV in amplitude, occurred continuously once every 15 to 45 seconds in connected cells.     

Evidence for a novel Kit adhesion domain mediating human mast cell adhesion to structural airway cells

Background

Human lung mast cells (HLMCs) infiltrate the airway epithelium and airway smooth muscle (ASM) in asthmatic airways. The mechanism of HLMC adhesion to both cell types is only partly defined, and adhesion is not inhibited by function-blocking anti-Kit and anti-stem cell factor (SCF) antibodies. Our aim was to identify adhesion molecules expressed by human mast cells that mediate adhesion to human ASM cells (HASMCs) and human airway epithelial cells.

Methods

We used phage-display to isolate single chain Fv (scFv) antibodies with adhesion-blocking properties from rabbits immunised with HLMC and HMC-1 membrane proteins.

Results

Post-immune rabbit serum labelled HLMCs in flow cytometry and inhibited their adhesion to human BEAS-2B epithelial cells. Mast cell-specific scFvs were identified which labelled mast cells but not Jurkat cells by flow cytometry. Of these, one scFv (A1) consistently inhibited mast cell adhesion to HASMCs and BEAS-2B epithelial cells by about 30 %. A1 immunoprecipitated Kit (CD117) from HMC-1 lysates and bound to a human Kit-expressing mouse mast cell line, but did not interfere with SCF-dependent Kit signalling.

Conclusion

Kit contributes to human mast cell adhesion to human airway epithelial cells and HASMCs, but may utilise a previously unidentified adhesion domain that lies outside the SCF binding site. Targeting this adhesion pathway might offer a novel approach for the inhibition of mast cell interactions with structural airway cells, without detrimental effects on Kit signalling in other tissues.     

Alterations in granule matrix and cell surface of focal adhesion kinase-deficient mast cells

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that plays an important role in many cellular processes and is tyrosine phosphorylated after FcepsilonRI aggregation in mast cells. In mice, null mutation of the fak gene results in a lethal phenotype in which the embryos fail to develop past day 8.5 of gestation. To study the role of FAK in these mast cells, 8.5-day embryos were isolated and placed in culture with IL-3 and stem cell factor (SCF). Although FAK was not required for the development of mast cells in culture, the FAK(-/-) embryo-derived mast cells had several distinct characteristics. Compared with the controls, the mast cells that lack FAK were less metachromatic and by electron microscopy had granules that appeared largely electron lucid, although their histamine content was unchanged. The FAK-deficient mast cells had a reduction in the content of chondroitin/dermatan sulfate, the major glycosaminoglycan component of the granular matrix. The FAK-deficient cells had fewer microvilli that were fused with each other, giving the cell surface a ruffled appearance. There was also a 3-fold increase in the number of cells highly expressing beta(7) integrin. However, signal transduction from the high affinity IgE receptor for the secretion of histamine was similar in the wild-type, heterozygote, and the FAK-deficient cells. The FcepsilonRI-induced tyrosine phosphorylation of paxillin, Crk-associated tyrosine kinase substrate (CAS), and mitogen-activated protein kinase proteins was independent of FAK. These results indicate that FAK plays a role in regulating the glycosaminoglycan content of the secretory granules and influences the cell surface morphology of mast cells.     

Growth of dissociated beating human heart cells in tissue culture

Fetal human heart cells have been dissociated by trypsinization and successfully grown in tissue culture on two occassions. The characteristics of the growth and mitotic activity were quite similar to those previously found for rodent heart cells. The maximum beating rates seen in the cultures from each specimen were 33 and 75 per minute.     

Involvement of mast cells in monocrotaline-induced pulmonary hypertension in rats

Background

Mast cells (MCs) are implicated in inflammation and tissue remodeling. Accumulation of lung MCs is described in pulmonary hypertension (PH); however, whether MC degranulation and c-kit, a tyrosine kinase receptor critically involved in MC biology, contribute to the pathogenesis and progression of PH has not been fully explored.

Methods

Pulmonary MCs of idiopathic pulmonary arterial hypertension (IPAH) patients and monocrotaline-injected rats (MCT-rats) were examined by histochemistry and morphometry. Effects of the specific c-kit inhibitor PLX and MC stabilizer cromolyn sodium salt (CSS) were investigated in MCT-rats both by the preventive and therapeutic approaches. Hemodynamic and right ventricular hypertrophy measurements, pulmonary vascular morphometry and analysis of pulmonary MC localization/counts/activation were performed in animal model studies.

Results

There was a prevalence of pulmonary MCs in IPAH patients and MCT-rats as compared to the donors and healthy rats, respectively. Notably, the perivascular MCs were increased and a majority of them were degranulated in lungs of IPAH patients and MCT-rats (p < 0.05 versus donor and control, respectively). In MCT-rats, the pharmacological inhibitions of MC degranulation and c-kit with CSS and PLX, respectively by a preventive approach (treatment from day 1 to 21 of MCT-injection) significantly attenuated right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH). Moreover, vascular remodeling, as evident from the significantly decreased muscularization and medial wall thickness of distal pulmonary vessels, was improved. However, treatments with CSS and PLX by a therapeutic approach (from day 21 to 35 of MCT-injection) neither improved hemodynamics and RVH nor vascular remodeling.

Conclusions

The accumulation and activation of perivascular MCs in the lungs are the histopathological features present in clinical (IPAH patients) and experimental (MCT-rats) PH. Moreover, the accumulation and activation of MCs in the lungs contribute to the development of PH in MCT-rats. Our findings reveal an important pathophysiological insight into the role of MCs in the pathogenesis of PH in MCT- rats.     

The last exon of SNAP-23 regulates granule exocytosis from mast cells

SNAP-25 and its ubiquitous homolog SNAP-23 are members of the SNARE family of proteins that regulate membrane fusion during exocytosis. Although SNAP-23 has been shown to participate in a variety of intracellular transport processes, the structural domains of SNAP-23 that are required for its interaction with other SNAREs have not been determined. By employing deletion mutagenesis we found that deletion of the amino-terminal 18 amino acids of SNAP-23 (encoded in the first exon) dramatically inhibited binding of SNAP-23 to both the target SNARE syntaxin and the vesicle SNARE vesicle-associated membrane protein(VAMP). By contrast, deletion of the carboxyl-terminal 23 amino acids (encoded in the last exon) of SNAP-23 does not affect SNAP-23 binding to syntaxin but profoundly inhibits its binding to VAMP. To determine the functional relevance of the modular structure of SNAP-23, we overexpressed SNAP-23 in cells possessing the capacity to undergo regulated exocytosis. Expression of human SNAP-23 in a rat mast cell line significantly enhanced exocytosis, and this effect was not observed in transfectants expressing the carboxyl-terminal VAMP-binding mutant of SNAP-23. Despite considerable amino acid identity, we found that human SNAP-23 bound to SNAREs more efficiently than did rat SNAP-23. These data demonstrate that the introduction of a "better" SNARE binder into secretory cells augments exocytosis and defines the carboxyl terminus of SNAP-23 as an essential regulator of exocytosis in mast cells.