In situ characterization of mast cells in the frog Rana esculenta

The number, distribution, and ultrastructural characteristics of mast cells were assessed in the tongue, heart, and kidney of the frog Rana esculenta. The density of tongue mast cells (253±45 mast cells/mm²) was significantly higher than that of the heart (5.3±0.4/mm²) and kidney (15.3±1.4 /mm²). A striking feature of this study was the remarkable association of frog mast cells to nerves. The ultrastructural study of the mast cell/nerve association demonstrated that mast cells were closely apposed to or even embedded in nerves. Mast cells were also physically associated with melanocytes even in the heart. Mast cells were Alcian blue⁺/safranin⁺ in the tongue and in the peritoneum, whereas in the heart and in the kidney they were Alcian blue^–/safranin⁺. The mast cells in the lamina propria of the gastrointestinal tract were Alcian blue⁺/safranin^–. The cytoplasm of frog mast cells was packed with numerous heterogeneous, membrane-bound granules. The ultrastructure of these cytoplasmic granules was unique, being totally unlike any other previously described granules in other animal species as well as in man. The histamine content/frog mast cell (≈0.1 pg/cell) was approximately 30 times lower than that of human mast cells isolated from different tissues (≈3 pg/cell). A monoclonal anti-histamine antibody was used to confirm the ultrastructural localization of histamine in secretory granules in frog mast cells.     

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.     

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.     

Cutaneous mast cell depletion and recovery in murine graft-vs-host disease

Mast cells as studied by light microscopy with metachromatic staining, have been noted to "disappear" from the skin of mice with chronic graft-vs-host disease (GVHD) produced across minor histocompatibility barriers. This mast cell disappearance is accompanied by ultrastructural evidence of loss of granule contents. In this study, we followed cutaneous mast cells in chronic GVHD over 9 mo by three methods: Light microscopy of toluidine blue-stained sections showed that mast cells not seen at day 42 reappeared between days 94 and 125, were supramaximal at days 146 and 164, and returned to normal levels at days 195 and 280. Double immunofluorescent staining of mast cells for the presence of surface IgE receptors and cytoplasmic granules (avidin) revealed IgE receptor-bearing cells that lacked avidin-binding granules at the time when mast cells were not apparent on light microscopy. By electron microscopy, reappearing mast cells have the morphology of immature dermal mast cells. Ultrastructural abnormalities of mast cells persist some 150 days after GVHD induction. The possible relationship of these mast cell changes to the development of dermal fibrosis in chronic GVHD is discussed.     

IgE by Itself Affects Mature Rat Mast Cell Preformed and De Novo-Synthesized Mediator Release and Amplifies Mast Cell Migratory Response

Background

Immunoglobulin E (IgE) binds to high affinity receptor FcεRI numerously expressed on mast cells. Recent findings have revealed that IgE by itself may regulate various aspects of mast cell biology, however, detailed data is still limited.

Methodology/Findings

Here, we have examined the influence of IgE alone, used at different concentrations, on mast cell activity and releasability. For the study we have employed in vivo differentiated mature tissue mast cells isolated from rat peritoneal cavity. Mast cells were exposed to IgE alone and then the release of preformed and de novo-synthesized mediators, surface FcεRI expression and mast cell migratory response were assessed. IgE by itself was found to up-regulate FcεRI expression and activate mast cells to degranulation, as well as de novo synthesis and release of cysteinyl leukotrienes and TNF. We have provided evidence that IgE alone also amplified spontaneous and CCL5- or TNF-induced migration of mast cells. Importantly, IgE was effective only at concentrations ≥ 3 µg/mL. A molecular basis investigation using an array of specific inhibitors showed that Src kinases, PLC/PLA₂, MAP kinases (ERK and p38) and PI3K were entirely or partially involved in IgE-induced mast cell response. Furthermore, IgE alone stimulated the phosphorylation of MAP kinases and PI3K in rat mast cells.

Conclusion

Our results clearly demonstrated that IgE by itself, at higher concentrations, influences mast cell activity and releasability. As there are different conditions when the IgE level is raised it might be supposed that in vivo IgE is one of the important factors modulating mast cell biology within tissues.     

Human mast cells release metalloproteinase-9 on contact with activated T cells: juxtacrine regulation by TNF-alpha

Mast cells, essential effector cells in allergic inflammation, have been found to be activated in T cell-mediated inflammatory processes in accordance with their residence in close physical proximity to T cells. We have recently reported that mast cells release granule-associated mediators and TNF-alpha upon direct contact with activated T cells. This data suggested an unrecognized activation pathway, where mast cells may be activated during T cell-mediated inflammation. Herein, we show that this cell-cell contact results in the release of matrix metalloproteinase (MMP)-9 and the MMP inhibitor tissue inhibitor of metalloproteinase 1 from HMC-1 human mast cells or from mature peripheral blood-derived human mast cells. The expression and release of these mediators, as well as of beta-hexosaminidase and several cytokines, were also induced when mast cells were incubated with cell membranes isolated from activated, but not resting, T cells. Subcellular fractionation revealed that the mature form of MMP-9 cofractionated with histamine and tryptase, indicating its localization within the secretory granules. MMP-9 release was first detected at 6 h and peaked at 22 h of incubation with activated T cell membranes, while TNF-alpha release peaked after only 6 h. Anti-TNF-alpha mAb inhibited the T cell membrane-induced MMP-9 release, indicating a possible autocrine regulation of MMP release by mast cell TNF-alpha. This cascade of events, whereby mast cells are activated by T cells to release cytokines and MMP-9, which are known to be essential for leukocyte extravasation and recruitment to affected sites, points to an important immunoregulatory function of mast cells within the context of T cell-mediated inflammatory processes.     

Compartmentalized mast cell degranulations in the ovarian hilum, fat pad, bursa and blood vessel regions of the cyclic hamster: relationships to ovarian histamine and blood flow.

Ovaries from hamsters on each day of the oestrous cycle at 09.00 h were observed for the number of mast cells, the pattern of mast cell degranulation, histamine concentration and blood flow. On day 4 (pro-oestrus), ovaries were also observed at 9.00, 15.00 and 21.00 h. Mast cell degranulation was evaluated by 3 criteria: (1) no degranulation = less than 5 granules dispersed from the cell; (2) moderate degranulation = 5 or more granules dispersed but less than 15, and (3) extensive degranulation = 15 or more granules released. Blood flow was determined using radio-active microspheres in anaesthetized animals. Mast cells were observed in fat pad (beyond 2 mm of the bursal mesothelium), bursa (within 2 mm of the bursal mesothelium), hilum and near ovarian blood vessels (these 4 regions are collectively called the ovarian complex). The distribution of ovarian mast cells was not uniform. Most mast cells were near ovarian blood vessels (42.2%) and in the fat pad (37.2%). A moderate number of cells were in the bursal wall (20%) and only a few cells were observed in the hilum (0.64%). Mast cell number remained unchanged on days 1-4 of the cycle in each ovarian compartment. However, summation of the number of mast cells in the entire ovarian complex revealed a significant decline in number at 15.00 h on pro-oestrus. Alterations in mast cell degranulation were primarily restricted to 2 periods of the cycle (pro-oestrus and di-oestrus). An increase in moderate but not extensive degranulation was observed in only the fat pad and bursa on day 2 when compared with day 1 values. In most ovarian compartments on pro-oestrus, degranulation was higher than on any other day of the cycle. At 15.00 h on pro-oestrus, extensive degranulation in bursa, fat pad and blood vessel regions (but not hilum) coincided with an increase in ovarian histamine and decline in number of mast cells; ovarian blood flow also increased at the time but remained unchanged the remainder of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mast Cells Contain Large Quantities of Secretagogue-Sensitive N-Acetylaspartate

Abstract: Mast cells play a central role in both immediate allergic reactions and inflammation. A functional nerve-mast cell interaction has been proposed, given the morphological association between mast cells and neuropeptide-containing peripheral nerves. We now show that purified rat peritoneal mast cells contain large quantities of N -acetylaspartate (NAA; 747.50 nmol/mg of protein). Mast cell levels of NAA were rapidly reduced, by 64.0 and 86.4%, following treatment with compound 48/80 and mastoparan, respectively. These secretagogues strongly decreased mast cell histamine content over the same time period, suggesting also that NAA is stored in secretory granules. The data are the first to show that NAA is present in an immune effector cell type. Because NAA may be involved in myelin synthesis and glutamyl peptide metabolism, NAA released from mast cells following nervous or other stimuli could participate in neuroimmune interactions. Mast cells in multiple sclerosis plaques may contribute to the reported elevations in brain NAA in this disease.     

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.     

Mast cells in the developing avian eye

Mast cells are present in the eye of Gallus domesticus, appearing in the anterior uvea in embryos at stage 39 HH (13th day). In hatching and adult birds, they are present in the sclera, uvea, pectinate ligament, and conjunctiva. Mast cells are absent in the cornea, retina, and pecten oculi. Maturing mast cells in the anterior eye segment appear as round cells having eccentric nuclei and a few cytoplasmic metachromatic granules, whose fluorescence increases during development. Mature cells are more numerous in late development, and their cytoplasm is rich in metachromatic and intensely fluorescent granules. Ultrastructurally, maturing mast cells display progranules and a few electron dense and homogeneous granules on one side of the cell. Mast cells of adult birds possess homogeneous cytoplasmic granules, some of which display protuberances that penetrate hollows of adjoining granules. Heterogeneous granules exhibiting latticed and mottled patterns are also present. The existence of mast cells in the anterior eye segment indicates that these cells might perform a physiological role during development and in aqueous humor outflow. They might modulate exchanges between blood and aqueous humor through chemical mediators present in their granules. © 1996 Wiley-Liss, Inc.     

Polyamines are present in mast cell secretory granules and are important for granule homeostasis

Background

Mast cell secretory granules accommodate a large number of components, many of which interact with highly sulfated serglycin proteoglycan (PG) present within the granules. Polyamines (putrescine, spermidine and spermine) are absolutely required for the survival of the vast majority of living cells. Given the reported ability of polyamines to interact with PGs, we investigated the possibility that polyamines may be components of mast cell secretory granules.

Methodology/Principal Findings

Spermidine was released by mouse bone marrow derived mast cells (BMMCs) after degranulation induced by IgE/anti-IgE or calcium ionophore A23187. Additionally, both spermidine and spermine were detected in isolated mouse mast cell granules. Further, depletion of polyamines by culturing BMMCs with α-difluoromethylornithine (DFMO) caused aberrant secretory granule ultrastructure, impaired histamine storage, reduced serotonin levels and increased β-hexosaminidase content. A proteomic approach revealed that DFMO-induced polyamine depletion caused an alteration in the levels of a number of proteins, many of which are connected either with the regulated exocytosis or with the endocytic system.

Conclusions/Significance

Taken together, our results show evidence that polyamines are present in mast cell secretory granules and, furthermore, indicate an essential role of these polycations during the biogenesis and homeostasis of these organelles.     

Formation of mast cell colonies in methylcellulose by mouse peritoneal cells and differentiation of these cloned cells in both the skin and the gastric mucosa of W/Wv mice: evidence that a common precursor can give rise to both "connective tissue-type" and "mucosal" mast cells

We investigated the issue of mast cell heterogeneity by cloning mast cell colonies from peritoneal cells in methylcellulose, injecting the cloned cells into the skin and stomach of mast cell-deficient (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mice, and staining the mast cells that developed in these sites with Berberine sulfate, a fluorescent dye that identifies heparin-containing mast cells. When peritoneal cells of nontreated WBB6F1-+/+ mice were plated in methylcellulose containing pokeweed mitogen-stimulated spleen cell conditioned medium, pure mast cell colonies developed. In contrast, the peritoneal cavity of genetically mast cell-deficient WBB6F1-W/Wv mice lacked the progenitor cells that made mast-cell colonies. The clonal nature of the mast cell colonies was determined by using the giant granules of C57BL/6-bgJ/bgJ mice as a marker: even when mixture of peritoneal cells of C57BL/6-bgJ/bgJ mice and C57BL/6-+/+ mice were plated, all of the resulting colonies consisted of either bgJ/bgJ-type mast cells alone or +/+-type mast cells alone. Individual mast c 11 colonies of WBB6F1-+/+ mouse origin were divided into two parts; one part was directly injected into the wall of the glandular stomach of a WBB6F1-W/Wv mouse, and another part was injected into the skin of the same W/Wv mouse. Injections of 14 of 46 such colonies resulted in development of mast cells in both the "connective tissues" (skin or stomach muscle or both) and the stomach mucosa. Mast cells in the connective tissues were stained with Berberine-sulfate, indicating that they contained heparin, whereas mast cells in the stomach mucosa were not. These results suggest that a single precursor cell can give rise to both "connective tissue-type" and "mucosal" mast cells.     

Mast cells express tyrosine hydroxylase and store dopamine in a serglycin-dependent manner

Here we show that mast cells contain dopamine and that mast cell activation causes dopamine depletion, indicating its presence within secretory granules. Dopamine storage increased during mast cell maturation from bone marrow precursors, and was dependent on the presence of serglycin. Moreover, the expression of tyrosine hydroxylase, the key enzyme in dopamine biosynthesis, was induced during mast cell maturation; histidine decarboxylase and tryptophan hydroxylase 1 were also induced. Mast cell activation caused a robust induction of histidine decarboxylase, but no stimulation of tyrosine hydroxylase or tryptophan hydroxylase 1 expression. The present study points toward a possible role of dopamine in mast cell function.     

Role of Mast Cells in Inflammatory Bowel Disease and Inflammation-Associated Colorectal Neoplasia in IL-10-Deficient Mice

Background

Inflammatory bowel disease (IBD) is hypothesized to result from stimulation of immune responses against resident intestinal bacteria within a genetically susceptible host. Mast cells may play a critical role in IBD pathogenesis, since they are typically located just beneath the intestinal mucosal barrier and can be activated by bacterial antigens.

Methodology/Principal Findings

This study investigated effects of mast cells on inflammation and associated neoplasia in IBD-susceptible interleukin (IL)-10-deficient mice with and without mast cells. IL-10-deficient mast cells produced more pro-inflammatory cytokines in vitro both constitutively and when triggered, compared with wild type mast cells. However despite this enhanced in vitro response, mast cell-sufficient Il10 ^−/− mice actually had decreased cecal expression of tumor necrosis factor (TNF) and interferon (IFN)-γ mRNA, suggesting that mast cells regulate inflammation in vivo. Mast cell deficiency predisposed Il10 ^−/− mice to the development of spontaneous colitis and resulted in increased intestinal permeability in vivo that preceded the development of colon inflammation. However, mast cell deficiency did not affect the severity of IBD triggered by non-steroidal anti-inflammatory agents (NSAID) exposure or helicobacter infection that also affect intestinal permeability.

Conclusions/Significance

Mast cells thus appear to have a primarily protective role within the colonic microenvironment by enhancing the efficacy of the mucosal barrier. In addition, although mast cells were previously implicated in progression of sporadic colon cancers, mast cells did not affect the incidence or severity of colonic neoplasia in this inflammation-associated model.     

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.     

Morphological characteristics of mastocytes in the infertile human testicle]

Mast cells, basophilic elements of the connective tissue, have been studied in numerous researches carried out both in man and animals. Previous studies showed that mast cells have been found in increased number in testis affected by pathology. In the present research morphologic characteristics of the mast cells in human infertile testis have been studied. Testicular biopsies obtained from 49 subjects, aged from 21 to 61 years, have been treated according to the current techniques for transmission electron microscopy. Results showed that it is possible to distinguish at least two types of mast cells: 1) "Interstitial" mast cells, large, round shaped, with great content of large characteristic granules, in relation with the loose connective tissue surrounding capillary vessels and Leydig cells; 2) "Peritubular" mast cells, flattened and relatively poor in granules, trapped in the conspicuous peritubular collagenic layers. These two types of mast cells differ from one another as concerns not only localization, but also general morphological characteristics and number and ultrastructure of cytoplasmic granules. Probably it is possible to establish a relationship between peritubular mast cells trapping and the increased deposition of fibrous connective tissue in the peritubular layers; such a deposition characterizes a lot of testicular pathologies.     

Mast Cells and Gastrointestinal Dysmotility in the Cystic Fibrosis Mouse

Background

Cystic fibrosis (CF) has many effects on the gastrointestinal tract and a common problem in this disease is poor nutrition. In the CF mouse there is an innate immune response with a large influx of mast cells into the muscularis externa of the small intestine and gastrointestinal dysmotility. The aim of this study was to evaluate the potential role of mast cells in gastrointestinal dysmotility using the CF mouse (Cftr^tm1UNC, Cftr knockout).

Methodology

Wild type (WT) and CF mice were treated for 3 weeks with mast cell stabilizing drugs (ketotifen, cromolyn, doxantrazole) or were treated acutely with a mast cell activator (compound 48/80). Gastrointestinal transit was measured using gavage of a fluorescent tracer.

Results

In CF mice gastric emptying at 20 min post-gavage did not differ from WT, but was significantly less than in WT at 90 min post-gavage. Gastric emptying was significantly increased in WT mice by doxantrazole, but none of the mast cell stabilizers had any significant effect on gastric emptying in CF mice. Mast cell activation significantly enhanced gastric emptying in WT mice but not in CF mice. Small intestinal transit was significantly less in CF mice as compared to WT. Of the mast cell stabilizers, only doxantrazole significantly affected small intestinal transit in WT mice and none had any effect in CF mice. Mast cell activation resulted in a small but significant increase in small intestinal transit in CF mice but not WT mice.

Conclusions

The results indicate that mast cells are not involved in gastrointestinal dysmotility but their activation can stimulate small intestinal transit in cystic fibrosis.     

THE REACTION OF RAT MAST CELLS TO POLYLYSINE

The effects of a single intraperitoneal injection of polyamino acids (lysine, glutamic, aspartic) on mast cells of the rat are described. In vitro interaction of mast-cell components with these polyamino acids is also explored. Poly-DL-lysine (but not the acidic amino acids) has both immediate (minutes-hours) and long-term (days-weeks) effects on mast cells. At the dosage selected, some cells evidence rapid fusion of granules and degranulation, but without concomitant swelling; most display intracellular changes only. Neither degranulation nor granule fusion appears to be lethal. Rather, these spur the cell to greater synthetic activity which involves first the Golgi apparatus and subsequently also the endoplasmic reticulum. Early involvement of macrophages and eosinophils is described. Sequential studies after polylysine injection support the following concepts: (a) mast-cell granules exist as "physiological sets," several being confined to a common membranous "sac;" (b) each set can respond independently to applied stimuli; (c) each set can connect directly to the extracellular milieu; (d) poly-DL-lysine binds directly to the granules and stabilizes them; it is not readily digested; (e) mast-cell granules are produced directly; they do not arise by intake of exogenous polysaccharides. It is hypothesized that mast-cell granules are topologically outside the cell while held intimately within extensive cytoplasmic folds and recesses. Mast cells may function by causing intercellular connective tissue fluids to percolate over their granules which may process this fluid in some as yet undefined way(s).     

Conjugated avidin binds to mast cell granules

The glycoprotein, avidin, conjugated either to the enzyme horseradish peroxidase, or to the fluorochrome dyes, fluorescein or rhodamine, identifies the granules of mast cells in both tissues and cell suspensions. In the absence of prior fixation, mast cells were not identified with conjugated avidin; however, granules released from these cells were stained with this labeled glycoprotein. The specificity of avidin for mast cells was confirmed by the absence of conjugated avidin-positive cells in the skin of mice (S1/S1d) deficient in mature dermal mast cells. Electron microscopic studies confirmed that avidin binds specifically to individual mast cell granules rather than to other cellular structures. Rodent and human mast cells were readily stained with avidin conjugated to horseradish peroxidase or to either of the fluorochrome dyes. The conjugated avidin staining technique is a reliable and simple method for identifying rodent and human mast cells, one that is useful as both an investigative and a clinical tool.