Copyright ©The Histochemical Society, Inc.


REVIEW

Ultrastructural Studies of Human Basophils and Mast Cells

Ann M. Dvorak

Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts

Correspondence to: Ann M. Dvorak, MD, Department of Pathology/East Campus, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. E-mail: advorak{at}bidmc.harvard.edu


    Summary
 Top
 Summary
 Introduction
 Identity
 Secretion
 Vesicles
 Recovery
 Synthesis
 Literature Cited
 
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.

(J Histochem Cytochem 53:1043–1070, 2005)

Key Words: basophil • mast cell • ultrastructure • secretion • vesicle transport • piecemeal degranulation • recovery from degranulation • synthesis • granules • lipid bodies


    Introduction
 Top
 Summary
 Introduction
 Identity
 Secretion
 Vesicles
 Recovery
 Synthesis
 Literature Cited
 
DURING THE PAST 35 years, I have used the electron microscope as my principle tool to examine the biology of mast cells and basophils (reviewed in Dvorak 1989Go,1991Go,2005Go). These studies include their normal morphology and developmental, functional, and pathological circumstances that impact their morphology in multiple species. In this review, human basophils (HBs) and human mast cells (HMCs) are discussed in detail with some reference to our studies in guinea pigs and mice. The topics of presentation include five major categories as follows: Identity, Secretion, Vesicles, Recovery, and Synthesis. We begin with identity because these two metachromatic cells have been regularly misidentified. Secretion follows because our earliest studies identified a new form of release from these granulated secretory cells, a process we called piecemeal degranulation (PMD). We follow this with the identification of vesicular transport as the mechanism for effecting PMD. Next, the fate of secretory mast cells and basophils is analyzed in recovery studies—studies leading to the suggestion that site-specific synthesis may be possible in unlikely subcellular organelles such as granules and lipid bodies (Dvorak et al. 2000aGo,2003Go). Altogether, we think that these findings prepare the way for future studies using subcellular fractions and imaging methods such as ultrastructural tomography to more perfectly understand the cell biology of mast cells and basophils.


    Identity
 Top
 Summary
 Introduction
 Identity
 Secretion
 Vesicles
 Recovery
 Synthesis
 Literature Cited
 
The ultrastructural anatomy of HMCs has been described in numerous tissues (reviewed in Dvorak 1989Go). HMCs are readily recognized by electron microscopy (Figure 1A). The primary cell with which they have been confused are basophils (Figure 1B). Criteria for identification of mast cells in human tissues include a monolobed nucleus; surface architecture composed of narrow, elongated folds; the presence of typical cytoplasmic granules; and the absence of cytoplasmic glycogen aggregates. The cytoplasm also contains mitochondria, free ribosomes, intermediate filaments, and lipid bodies. Golgi areas are small in mature cells, and membrane-bound ribosomes are rare in mature and immature cells.



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Figure 1

Ultrastructure of human mast cells (HMCs) and human basophils (HBs). (A) Human lung mast cells (HLMCs) after isolation and culture for 36 hr show electron-dense secretory granules, a monolobed nucleus, and numerous narrow surface folds. (B) HB isolated from the peripheral blood has electron-dense secretory granules and a polylobed nucleus. (A) x10,000; (B) x17,000.

 
Mature mast cell granules are smaller, vary more in shape, are more numerous, and generally contain more complex substructural patterns than the granules of basophils. Most observers have described several of the prevalent mast cell granule patterns in their individual works. Examination of published micrographs as well as HMCs from nearly all tissue sites (Dvorak AM, unpublished data) provides a working description of granule patterns. Different terms have been used by various authors to identify identical granule patterns. General agreement exists, however, among the published micrographs (reviewed in Dvorak 1989Go). Thus, four basic granule patterns of HMCs include scrolls, crystals, particles, and mixed (Figure 2). A small number of mast cells have been found to have homogeneously dense granules. Other granule patterns have been described in small numbers. Among these are reticular granules that contain thick, interwoven, irregular, dense threads, and granules with finely or coarsely granular material.



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Figure 2

High-magnification micrographs of granules from isolated HLMCs (A,B,D) and from a mucosal mast cell in an ileal biopsy (C). The cells in (B,D) were cultured for 6 hr and 18 hr, respectively. (A) Scroll granules are illustrated in cross-section. Note that variable numbers of individual scrolls are present in membrane-bound granules. The centers of the scroll sometimes contain dense particles. (B) Two granules filled with particles are illustrated. (C) A regular crystalline pattern, viewed in cross-section, is seen. (D) A mixed granule contains particles and scrolls. (A) x51,500; (B) x56,000; (C) x88,500; (D) x49,000. With permission, (A) Dvorak 1989; (B–D) Dvorak 1991.

 
Scroll-containing granules consist of regularly arranged lamellae that form parallel, straight, or curved figures, or multilayered figures. The centers of scrolls can be empty or contain granular, particulate, or crystalline material. Crystal-containing granules display extremely regular arrays with basically two periods—120 Å and 60 Å. These parallel structures can be oriented in several different directions in individual granules. When seen in cross-section, they present a hexagonal array. Particle-containing granules are a normal granule pattern in HMCs. Particle-filled granules are the type of granule most often not described in the literature, as they are often referenced by different terminology (e.g., coarsely granular, reticular). They are also the granule type most often contributing to confusion with basophils because the major substructural granule pattern of HBs is that of particles. Individual HMCs contain granules that are predominantly of the scroll, crystal, particle, or mixed types. Mixed granule refers to a majority of the granules in the individual cell that display mixtures of the basic three patterns—scrolls, particles, and crystals.

Lipid bodies are non-membrane-bound dense structures that do not display evidence of substructural granule patterns. These organelles are generally larger than mast cell granules and are often found encased in large numbers of intermediate filaments.

Basophilic leukocytes can be distinguished from HMCs by ultrastructural criteria (Figure 1B) (reviewed in Dvorak 1989Go,1991Go,2005Go). These diagnostic features include polylobed nuclei with a condensed chromatin pattern; surface architecture that consists of irregular, broad, cytoplasmic protrusions; cytoplasmic glycogen; and granules. In mature basophils, the Golgi apparatus is inconspicuous, and membrane-bound ribosomes are rare. Free ribosomes, mitochondria, cytoplasmic vesicles, and filaments are present. Lipid bodies can be found in basophils. Basophil granules are larger and less numerous than their counterparts in mast cells. These membrane-bound structures are filled with dense particles that vary in the density of packing within granules. Characteristic Charcot-Leyden crystals (CLCs) are sometimes embedded within the dense intragranular particles or enlarge to virtually completely fill these membrane-bound secretory granules. Some granules contain focal collections of membranes that may enclose granule particle contents. These membrane collections sometimes resemble scroll patterns found in HMCs. When all nuclear and cytoplasmic criteria are considered, HMCs (Figure 1A) can, however, be readily distinguished from HBs (Figure 1B).


    Secretion
 Top
 Summary
 Introduction
 Identity
 Secretion
 Vesicles
 Recovery
 Synthesis
 Literature Cited
 
Ultrastructural analyses of degranulation mechanisms, which HMCs and HBs use to effect the release of cellular products involved in inflammatory processes, reveal two basic morphological patterns that may represent separate points along a degranulation continuum important to the function of these two similar cells. These two basic patterns have been termed anaphylactic degranulation (AND) and piecemeal degranulation (PMD) (reviewed in Dvorak 1991Go,1993Go,2005Go). Both release patterns are found in multiple circumstances. Such circumstances include those stimulated in partially purified samples of basophils from peripheral blood (Figure 1B) or mast cells from lung (Figure 1A) or skin, those stimulated in skin in vivo, those appearing in rhIL-3- or rhIL-5-containing cultures of cord blood derived-basophils, those appearing in tissue mast cells and basophils in vivo in a wide variety of pathologies, and those occurring in basophils present in peripheral blood or body fluids in vivo (all reviewed in Dvorak 1993Go).

Regulated secretion from basophils and mast cells occurs when one of a variety of secretogogues is used to stimulate the cells. Ultrastructural studies have been performed in which either basophils or mast cells have been stimulated by one of these triggers (reviewed in Dvorak 1993Go). Some of these ultrastructural studies include multiple samples obtained at time points preceding and including the peak release of histamine, as measured in replicate samples. Such studies yield significant new information about the ultrastructural kinetics of these examples of regulated secretion.

Specific stimuli that induce PMD are less well known than those that stimulate AND. PMD, however, is the single most frequently seen event in basophils and mast cells participating in several diseases in vivo (reviewed in Dvorak 1992Go). It seems likely that triggers of this form of secretion will be identified among the wide variety of cellular- and pathogen-associated products present in diseased tissues.

AND is the general term used to describe the rapid, regulated secretory events of which basophils and mast cells are capable. It is equivalent to the coordinated secretion of granule mediators, accompanied by the visible extrusion, or solubilization within specially constructed intracytoplasmic degranulation chambers, of typical secretory granules stimulated by IgE-mediated mechanisms. Thus, this is an explosive and rapid secretory event that is completed within minutes of stimulation. AND, then, is a special type of regulated secretion, of which all granule-containing secretory cells, including basophils and mast cells, are capable. Important anatomical findings associated with AND in appropriately stimulated HBs (Figure 3A) include extrusion of CLCs and dense concentric membranes in concert with granule particulate contents through multiple pores in the cell membrane; shedding of multiple membranes and processes; surface amplification by externalization of granule containers; formation of intracytoplasmic degranulation chambers or sacs by fusion of multiple granule membranes; decreased granule numbers; decreased numbers of cytoplasmic vesicles; and resultant completely degranulated, viable basophils (reviewed in Dvorak 1993Go).



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Figure 3

Secretory patterns from HBs (A,B) and mast cells (C,D) include anaphylactic degradation (AND) (A,C) and piecemeal degranulation (PMD) (B,D). Peripheral blood basophils stimulated with formyl methionyl leucyl phenylalanine (FMLP) for 20 sec show extrusion of secretory granules to the cell exterior (arrows) with subsequent amplification of surface architecture, typical of AND (A) and numerous enlarged empty granules and smooth surface architecture, typical of PMD (B). HMSCs stimulated in vivo with the cytokine, recombinant methionyl-human stem cell factor (rhSCF) (C), or present in an ileal biopsy of a patient with Crohn's disease (D) show extrusion of granules to the cell exterior (arrows) typical of AND (C) and enlarged empty and partially empty granule containers typical of PMD (D). (A) x12,500; (B,C) x17,000; (D) x11,500.

 
AND in HMCs (Figure 3C) is also a rapid event when stimulated by IgE-mediated mechanisms. It results in the coordinated release of histamine and placement of granule matrix contents into elaborate cytoplasmic degranulation chambers or through the cell membrane into the adjacent space. In either case, granule-stored mediators are effectively in the extracellular milieu after degranulation channels, formed by granule membrane fusions, or individual granules, fuse to the plasma membrane, and open through multiple pores to the cell surface.

Isolated preparations of HMCs stimulated to undergo AND by anti-IgE were examined from two organs: lungs and skin. We observed several differences in IgE-mediated secretion between lung and skin mast cells. For example, human lung mast cells (HLMCs) more generally formed intracytoplasmic degranulation chambers within which altered granule matrices dissolved before release through channel-plasma membrane pores than did human skin mast cells (HSMCs). By contrast, HSMCs more generally directly extruded individual altered membrane-free granule matrices through multiple plasma membrane pores than did HLMCs. In each cell population, both AND patterns did, however, occur (reviewed in Dvorak 1993Go).

Sequential samples of stimulated HLMCs suggested sequential morphological events associated with AND. That is, the earliest visible changes were those of swelling and alteration of granule matrix density and patterns, followed by granule membrane fusions to form degranulation channels. Extracellular tracers indicated that this process generally anteceded fusion of channels to plasma membranes. After pore formation and release of channel contents, flow of channel membranes to the cell surface created extensively amplified complex surface folds. Some of these were shed with granules and membranous debris, leaving small process-free completely degranulated cells; others were recycled into cells as canalicular structures (reviewed in Dvorak 1993Go).

Some of these events associated with AND in HMCs have been identified in vivo. For example, biopsies of normal or mast cell-rich (urticaria pigmentosa) human skin obtained by mechanical or chemical (complement, antigen) stimulation produced visible evidence of multiple granule extrusions by skin mast cells. Unstimulated samples of urticaria pigmentosa skin also showed intracytoplasmic degranulation channels with altered granule contents (reviewed in Dvorak 1993Go). Stem cell factor, the c-kit receptor ligand, can induce mast cell secretion. We performed an ultrastructural analysis of human skin biopsies from patients who received daily subcutaneous (SC) dosing with recombinant methionyl-human stem cell factor (rhSCF) (reviewed in Dvorak 2005Go). The biopsies were obtained at sites of SC administration of rhSCF, within ~1 to 2 hr of rhSCF injection. SC dosing with rhSCF in these subjects induced the local development of a wheal-and-flare response, which was associated with mast cell degranulation. The electron microscopic analysis revealed that all biopsies of swollen, erythematous rhSCF-injected sites exhibited AND of HMCs (Figure 3C). Together, these in vivo observations of AND in HSMCs are similar to those observed in isolated preparations of HSMCs (reviewed in Dvorak 2005Go).

Biopsy samples of human tissues from two other organs have also revealed AND in HMCs in vivo. These include heart biopsies from several patients with poorly defined hypokinetic heart disorders in the absence of all well-known causes of heart disease and ileal tissues of patients with inflammatory bowel disease (IBD). In each instance, both intracytoplasmic channel formation with released granules contained therein and extrusion of membrane-free granules to the extracellular tissues were documented (reviewed in Dvorak 2005Go).

PMD is a term introduced to explain the ultrastructural finding of partially and completely empty granule containers, in the absence of intergranule fusions or granule fusions to the plasma membrane and subsequent extrusion of granule contents to the microenvironment. It occurs in HBs (Figure 3B) participating in large numbers in experimentally induced and sequentially biopsied contact allergy lesions in human skin (reviewed in Dvorak and Dvorak 1975Go). These mature basophils are also characterized by large numbers of cytoplasmic vesicles, some of which are attached to granules (Figure 4A). Visible particles, like those in granules, homogeneously dense contents, or apparently empty (electron-lucent) interiors prevail among these smooth membrane-bound small cytoplasmic vesicles. PMD, simply stated, defines the release of granule materials, in the absence of typical granule extrusion, from basophils and mast cells.



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Figure 4

High magnification micrographs of vesicles attached to HB (A) and HMC (B) granules. Dense glycogen particles are in contact with a focal constriction of the vesicle budding from the HB granule in a 3-day contact dermatitis reaction to dinitrochlorobenzene shown by arrow in (A). Arrow shows an electron-dense particle-filled vesicle protruding from the mast cell granule (B) in a biopsy of a fibrous histiocytoma in the skin. (A) x60,000; (B) x73,500. With permission, (A) Dvorak et al. J Immunol 116:687–695, 1976; (B) Dvorak and Kissel. J Leukoc Biol 49:197–210, 1991.

 
We examined the kinetics of morphological change induced by stimulation of HBs with formyl methionyl leucyl phenylalanine (FMLP) using partially purified cells from normal donors (reviewed in Dvorak 1993Go). Supernatants were collected at 30 and 60 min and assayed for histamine. Samples of basophils were prepared for electron microscopy at 0, 10, 20, and 30 sec and 1, 2, 5, and 10 min poststimulation with FMLP.

We found that FMLP, a bacterial peptide, induced a unique sequence of morphologic events that included morphologies we have previously identified and termed PMD in HBs in situ as well as those induced by IgE mechanisms ex vivo and termed AND, thus supporting our previously suggested general degranulation model for basophils and mast cells (Dvorak and Dvorak 1975Go). In addition to this degranulation continuum, we found that chambers of releasing granules underwent extraordinary increases in size as they emptied their contents and before their resolution by extrusion (Figure 3B). The enlarging granule chambers accumulated numerous concentric dense membranes, vesicles, and CLCs. These early changes generally preceded half-maximum histamine release, whereas the later extrusion of full granules, emptied granules, and their membranous contents coincided with half-maximum histamine release. Shedding of membranes from several sources accompanied extrusion of granules and intragranular CLCs. These membrane sources included the expanded granule membranes from empty granules, granule membranes from full granules, collections of intragranular concentric dense membranes and vesicles, and surface membranes and processes. These extraordinary membrane shifts were generated and persisted over the 10-min period examined and coincided with the later time frame within which leukotriene C4 (LTC 4) was generated and released from HBs stimulated by FMLP (reviewed in Dvorak 2005Go). Viable basophils, completely free of both full and empty granules, showed morphologic evidence of recovery of granule products by 10 min after stimulation with FMLP.

We also examined the ultrastructural kinetic morphology associated with stimulation of human basophils with tetradecanoyl phorbol acetate (TPA)—a tumor-promoting phobol diester known to elicit histamine (but not LTC 4) release (reviewed in Dvorak 1993Go, 2005Go). Partially purified HBs were prepared for electron microscopy and examined either after control incubations in buffer alone or at 0 time, 1, 2, 5, 10, 30, and 45 min after TPA stimulation. Standard morphology and ultrastructural quantitation of vesicles and granules and contents of vesicles or alteration of granules was done. Like biochemical studies that have determined that TPA is a unique secretogogue for HBs, the morphology stimulated by TPA and associated with histamine release was also unique. For example, very few images of AND were evident. A far greater number of PMD images were seen. PMD was associated with ~50% alteration of cytoplasmic granules by 45 min after TPA stimulation. This evidence of empty granules was associated with, and preceded by, a rapid, extensive, and sustained increase in particle-containing cytoplasmic vesicles (Figures 5A and 5B), as compared with buffer controls (p<0.001 for each TPA stimulation time compared with unstimulated basophils).



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Figure 5

HBs stimulated for 2 min with TPA show increased particle-filled vesicles (closed arrows) adjacent to granules and plasma membranes. One empty granule (B) has empty vesicles (open arrow) nearby. (A) x50,500; (B) x47,000. With permission, Dvorak et al. Am J Pathol 1992; 141:1309–1322.

 
An ultrastructural analysis of HBs stimulated with anti-IgE, or with the cytokine recombinant histamine-releasing factor (rHRF), or monocyte chemotactic protein-1 (MCP-1) was performed (reviewed in Dvorak 2005Go). Partially purified peripheral blood basophils were prepared for electron microscopy at time points known to precede histamine release and at half-maximum histamine release times for each secretogogue. Activation morphologies associated with stimulation included granule–vesicle attachments (GVAs) (Figures 6A–6D), PMD, AND, and uropod formation. These features were qualitatively similar in the stimulated samples. Quantitative differences were evident, however, when stimulated samples were compared with controls or at different time points after stimulation with a single agent or when individual secretogogues were compared (Figure 7). All stimulated samples differed quantitatively from the control samples. Rank orders for morphologic activation events revealed that the most effective trigger for AND was anti-IgE, whereas the most effective trigger for uropod formation was rHRF. Rank orders for PMD and GVA were the same, and the most effective trigger was MCP-1. Important relationships among these anatomic events reveal that the development of motile configurations is not associated with the development of secretion morphologies, GVA and PMD are associated, and PMD precedes and is inversely related to AND in stimulated samples.



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Figure 6

Glycogen-rich granule-vesicle attachments (arrows) are shown in HBs stimulated for 7 min with recombinant histamine-releasing factor (rHRF) (A,B) or 30 sec with monocyte chemotactic protein-1 (MCP-1) (C,D). Granule–vesicle attachments (GVAs) are encased with electron-dense glycogen aggregates; focal piecemeal losses of granule contents near GVAs are present (B,C). (A) x52,500; (B) x42,000; (C) x54,500; (D) x51,000. With permission, Dvorak et al. J Allergy Clin Immunol 98:355–370, 1996.

 


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Figure 7

Kinetic and quantitative relationships of four activated morphologic features induced in HBs by anti-IgE, rHRF, or MCP-1 compared with unstimulated basophils. With permission, Dvorak et al. J Allergy Clin Immunol 98:355–370, 1996.

 
Quantitation of total granule numbers, altered (by piecemeal losses of granule matrix) granule numbers, and total vesicle numbers was done for two disparate HBs secretogogues, FMLP and TPA (reviewed in Dvorak 1993Go). These two secretogogues, FMLP with rapid biochemical release kinetics and TPA with slow biochemical release kinetics, also showed different ultrastructural quantitative correlates of secretion. For example, large numbers of cytoplasmic vesicles were present with either stimulus, and in the FMLP-stimulation model, vesicle numbers increased in conjunction with acquisition of the morphology of PMD and preceded the development of the morphology of AND, characterized by granule extrusions. Vesicle numbers decreased dramatically in cells displaying the morphology of AND, defined by the extrusion of full granules, the membranes of granules previously emptied by PMD, and intragranular CLCs. These events coincided with the half-maximum histamine release reported for this model at 1.3 min poststimulation. Granule-free cells were prevalent between 20 and 120 sec. Thus, a degranulation continuum of morphologic change occurred when HBs were stimulated with FMLP that was coincident with the rapid release of histamine (reviewed in Dvorak 1993Go).

TPA, on the other hand, elicits histamine release slowly (reviewed in Dvorak 1993Go). Unlike the rapid kinetics associated with IgE-mediated histamine release (15 min) or FMLP-mediated histamine release (2 min), the histamine release stimulated from HBs by the phorbol ester TPA reaches a maximum by 1 hr. By electron microscopic evaluation, extensive PMD was evident in multiple samples, achieving ~50% granule alteration by 45 min poststimulation. This evidence of empty granules was associated with, and preceded by, a rapid, extensive, and sustained elevation in particle-containing cytoplasmic vesicles (Figure 5). There was minimal classical exocytosis, and this was not associated with significant reductions in HB granule numbers over a 45-min period. Completely granule-free cells were absent—a feature that is regularly present at peak histamine release times after FMLP stimulation of replicate samples from the same donors. There was extensive PMD, characterized by a change in the ratio of altered to unaltered granules, from 1 to 4 (in controls) to 1 to 2 by 45 min after exposure to TPA. In concert with these findings, a 5-fold increase in the number of cytoplasmic vesicles containing particles occurred in TPA samples, compared with unstimulated cells at multiple times sampled, including 45 min after TPA. At no time did electron-lucent, empty vesicles increase to levels observed in FMLP-stimulated samples from the same donors or did extensively enlarged empty granule containers appear. The total number of vesicles (after TPA) was stable, indicating balanced vesicular traffic between the cell surface and granules, accompanying PMD and extending to 45 min after stimulation of basophils. Thus, a coordinate secretion of histamine was associated with the morphology of PMD in this model.

HMCs undergo PMD in multiple organ sites in human disease (Figure 3D) (reviewed in Dvorak 1992Go, 1993Go). Initially, we noted the characteristic ultrastructural morphology of empty and partially empty granules in HMCs in bowel samples of patients with Crohn's disease (CD), a chronic IBD of unknown etiology (Figure 3D). Other inflammatory and neoplastic diseases are accompanied by PMD of mast cells (Figure 4B) (reviewed in Dvorak 1992Go,1993Go). In particular, we noted extensive PMD of HSMCs in vivo in bullous pemphigoid and melanoma with subsequent ultrastructural evidence of recovery of granule contents.

In a large study, 117 coded intestinal biopsies were examined by electron microscopy (reviewed in Dvorak 2005Go). All surgical biopsies were obtained from uninvolved sites of patients with either one of two IBDs—ulcerative colitis (UC) or Crohn's disease—and from patients with preneoplastic and neoplastic diseases (adenocarcinoma, rectal polyp, familial polyposis). Biopsy sites included normal ileum, colon, and rectum as well as conventional ileostomies and continent pouches constructed from the ileum. This large sample of coded biopsies was evaluated for ultrastructural evidence of mast cell secretion in vivo. Sixty percent of the biopsies had such evidence. Mast cell secretion was evident in control biopsies, many of which were obtained from uninvolved tissues of patients with IBD. Biopsies of inflamed continent pouches from UC patients showed more mast cell secretion than non-inflamed UC pouch biopsies. This evidence of mast cell secretion supports work that documents high constitutive levels of histamine in jejunal fluids of Crohn's disease patients and suggests a proinflammatory role for mast cells in inflammation associated with pouchitis (reviewed in Dvorak 2005Go).

The primary ultrastructural form of secretion from human gastrointestinal mast cells in this study was PMD (Figure 3D) typified by variable losses of dense content from granules (rarely, typical images of AND were also seen). Granule losses of PMD (Figure 3D) were either focal within single granules, complete losses of single granule contents, or partial to complete losses of dense material from variable numbers of, to sometimes all, cytoplasmic granules. The end result of such granule losses was the presence of non-fused, empty granule containers in undamaged mast cells. Some of these containers were larger than granules; most were of similar size.


    Vesicles
 Top
 Summary
 Introduction
 Identity
 Secretion
 Vesicles
 Recovery
 Synthesis
 Literature Cited
 
The ultrastructural morphologic evidence supports vesicular transport as the mechanism for effecting PMD from HBs in vivo and in vitro (reviewed in Dvorak and Dvorak 1975Go; Dvorak 1988Go,1992Go,1993Go,1998aGo,cGo). Thus, visual evidence of vesicles fused to secretory granules, free in the cytoplasm and in the peripheral cytoplasmic area and fused to plasma membranes, has been published for HBs (Figure 4A, Figure 5, and Figure 6).

Proof of principle that PMD is effected by vesicular transport of loaded vesicles requires visualization and kinetic analyses of granule protein-loaded, 80- to 100-nm vesicles in stimulated basophils and/or mast cells. For the greater part of the past 10 years we have pursued this goal. This pursuit required the development of numerous tools and resources. Chief among these were the isolation and purification of circulating basophils, identification of specific growth factors to increase the supply of this rare granulocyte, understanding of secretogogue mechanisms and reliable analyses of secreted basophil products, and the development of ultrastructural preparations allowing imaging of small vesicles and quantifiable electron-dense tags for granule materials in small vesicles (reviewed in Dvorak 2005Go). Applications of these tools to well-defined models of basophil (and mast cell) secretion have provided substantial proof of principle for the effector function of vesicular transport in PMD.

Electron-dense tags for the identification of granule contents in transit from secretory HMCs and HBs include an immunogold method to visualize the CLC protein in HBs (Figure 8) (Dvorak and Ackerman 1989Go) and a newly developed enzyme affinity gold method to image histamine in both HBs (Figure 9) and HMCs (Dvorak et al. 1993Go, Dvorak 1998bGo).



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Figure 8

HBs stimulated with tetradecanoyl phorbol acetate (TPA) for 1 min (A), 2 min (B), 5 min (C), 10 min (D,E), and 15 min (F) show Charcot-Leyden crystal (CLC)-protein-gold-labeled vesicles in the cytoplasm (arrows). Some vesicles are electron lucent; others contain particles similar to the particulate matrix of adjacent granules. CLC protein is also localized by gold particles in the nuclear matrix and nuclear membrane (C), the cytosol (A–F), the plasma membrane (A,E), a homogeneously dense primary granule (D), a formed CLC within the particulate matrix of a granule (E), and the granule membrane of an empty granule chamber (F). The arrowhead in (D) shows an example of the small minor granule population of HBs that does not contain label for CLC protein. (A) x66,000; (B) x63,000; (C) x41,000; (D) 42,000; (E) x44,500; (F) x81,000. With permission, Dvorak et al. Lab Invest 74:969–974, 1996.

 


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Figure 9

TPA-stimulated HBs prepared with diamine oxidase-gold (DAO-gold) to detect histamine studied at 0 time (A), 2 min (B–D), 5 min (E–G), 10 min (H,I), 15 min (J), 30 min (K), and 45 min (L) after activation. In (A), at 0 time, the basophil granule (G) is labeled with DAO-gold. In (B), at 2 min, three cytoplasmic vesicles near the cell surface (open arrowhead) are labeled for histamine. One gold-labeled vesicle is electron lucent and is encased in electron-dense glycogen particles (solid arrowhead). Another gold-labeled vesicle also contains granule particles (long arrow), and a third gold-labeled vesicle is electron lucent (short arrow). In (C), at 2 min, note the gold-labeled vesicle filled with granule particles adjacent to the labeled granule (G). In (D), also at 2 min, the electron-lucent vesicle contains DAO-gold. In (E–G), at 5 min, the cell surface is indicated by open arrowheads. Note in (E,F) the granule particle-filled, DAO-gold labeled perigranular vesicles (open arrows) adjacent to the cell surfaces. The adjacent granules (G) also label for histamine; the granule in (F) has a focal electron-lucent region (arrow) typical for PMD. Cytoplasmic glycogen particles shown by arrows in (E) are electron dense and generally larger than the ~20-nm gold label. In (G) at 5 min, a DAO-gold labeled vesicle is fused to the cell surface (open arrowhead). In (H,I), at 10 min, electron-lucent (H) and particle-filled (I) cytoplasmic vesicles (arrows) contain histamine. In (H), a large empty granule (EG) devoid of gold label and granule particles is typical for PMD. In (I), the cell surface is coated with cationized ferritin (used in cell processing for electron microscopy) and is indicated by the open arrowhead. The granule particle-filled cytoplasmic vesicles are labeled with DAO-gold (arrows) at 15 (J), 30 (K), and 45 (L) min after TPA activation. The cell surface is indicated by open arrowheads in (K,L); an empty granule (EG) typical for PMD is present at 45 min poststimulation in (L). (A,B) x55,000; (C) x117,500; (D) x69,000; (E,K) x71,500; (F) x61,000; (G) x80,000; (H) x67,500; (I,J) x113,000; (L) x54,500. With permission, Dvorak et al. Blood 88:4090–4101, 1996.

 
We evaluated the eyelid lesions that develop in IL-4 transgenic mice using light microscopy of alkaline–Giemsa-stained plastic sections, routine transmission electron microscopy, and enzyme affinity-gold electron microscopy to detect histamine (reviewed in Dvorak 1998bGo). We found that the tissue mast cells exhibiting PMD in the eyelid lesions had greatly diminished staining of granules with diamine oxidase-gold (DAO-gold) (Figure 10), results that indicate that this morphologic expression of mast cell secretion is associated with secretion of histamine in vivo. These results in vivo in a mouse mast cell model of inflammatory eye disease, together with similar findings of PMD in HMCs in vivo in human IBD (Figure 11), represent the first direct evidence for histamine secretion by mast cells in vivo.



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Figure 10

(A,B) Light microscopy of alkaline-Giemsa-stained plastic 1-µm sections of eyelids in IL-4 transgenic mice shows PMD of mast cells. (A) A mast cell with no altered granules is shown to compare with one showing staining alterations of virtually all (B) of the cytoplasmic granules. The mast cell undergoing PMD (B) shows many cytoplasmic granules that stain pink, in contrast to the dark blue staining exhibited by the granules in the normal-appearing mast cell in (A). However, the granules with altered staining remain within the cytoplasm. (C,D) Electron micrographs of DAO-gold preparations of inflamed eyelids in IL-4 transgenic mice. Electron-dense secretory granules in mast cells undergoing PMD are gold labeled, indicating the presence of histamine in the unaltered cytoplasmic granules of these mast cells, shown by arrowhead in (D). However, there is little or no gold labeling of the swollen granules, which exhibit greatly diminished electron density and an altered granule matrix (D). In one mast cell (C), two immature granules show less gold label (arrowhead) than do the mature, electron-dense granules nearby. (A,B) x2,200; (C) x48,000; (D) x24,000. With permission, Dvorak et al. Blood 83:3600–3612, 1994).

 


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Figure 11

Electron micrographs of DAO-gold staining of ileal biopsies of patients with IBD show gold-labeled histamine in electron-dense granules in mast cells (A). One granule shows focal, piecemeal loss of dense material and absence of gold label for histamine in this area. Residual electron-dense reticular arrays contain small amounts of gold label for histamine in otherwise electron-lucent, empty, histamine-free granules of a mast cell undergoing PMD (B). (A) x37,000; (B) x42,000. With permission, Dvorak and Morgan. J Allergy Clin Immunol 99:812–820, 1997.

 
Application of ultrastructural labeling methods for histamine to several HMC models of AND provide information about the release of histamine during AND of HLMCs ex vivo and HSMCs in vivo (reviewed in Dvorak 1998bGo). We took advantage of the extensive mast cell degranulation that was present at skin sites of rhSCF injection to assess the ultrastructural localization of histamine in HMCs that were undergoing classical AND in vivo (Figure 12). Although there is no question that mast cells store histamine in their granules and that this biogenic amine can be released by mast cells in response to agents that induce mast cells to degranulate, until recently it has not been possible to study this process morphologically at the ultrastructural level. The newly developed DAO-gold ultrastructural enzyme-affinity technique to localize histamine (Dvorak et al. 1993Go) can be used in conventionally prepared ultrastructural samples, for this purpose. It is based on the affinity of the enzyme, diamine oxidase (histaminase), for its substrate, histamine.



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Figure 12

HMCs in skin biopsies obtained after subcutaneous (SC) injections of rhSCF in patients who had received daily SC injections of rhSCF for 13 days. Reactivity for histamine is indicated by DAO-gold labeling in the cytoplasmic granules of a mast cell that exhibits no morphologic evidence of secretory activity (A); DAO-gold label is absent when the grid containing a section of the specimen was digested with DAO before staining with DAO-gold (B). Granules that have been released by AND (C) have diminished gold label indicating loss of histamine. In a sample that was stained with DAO-gold that had been absorbed by passage over histamine–agarose, the released, membrane-free granule does not bind the DAO-gold reagent (D). (A) x67,500; (B) x75,000; (C) x30,000; (D) x64,500. With permission, (A,B) Dvorak et al. Blood 90:2893–2900, 1997).

 
We used this method to detect histamine in skin biopsies obtained from patients with breast carcinoma who were receiving daily SC injections of rhSCF (reviewed in Dvorak 1998bGo). We examined control biopsies obtained at sites remote from rhSCF injection as well as biopsies of rhSCF-injected skin that were obtained within 2 hr and 30 min of the SC injection of rhSCF at that site. The rhSCF-injected sites (which clinically exhibited a wheal-and-flare response), but not the control sites, contained mast cells undergoing regulated secretion by granule extrusion. The DAO-gold-affinity method detected histamine in electron-dense granules of mast cells in control and injected skin biopsies (Figure 12); however, the altered matrix of membrane-free, extruded mast cell granules was largely unreactive with DAO-gold (Figure 12). These findings represent the first morphologic evidence of histamine secretion by classical granule exocytosis in HMCs in vivo.

We also examined IgE-mediated AND in isolated HLMCs with the ultrastructural method to detect histamine (Figure 13) (reviewed in Dvorak 1998bGo). HLMCs that were stimulated with antibody to IgE and sampled 5 and 20 min later were stained. Specificity controls for the technique were negative. DAO-gold labeled electron-dense, unaltered cytoplasmic granules adjacent to degranulation channels in anti-IgE-stimulated mast cells (Figure 13). Completely electron-lucent cytoplasmic degranulation channels were devoid of gold particles, indicating the absence of histamine in them (Figure 13). When residual wisps of altered granule matrix materials were visible in degranulation channels as well as in the process of extrusion from them, small numbers of gold particles labeled this material, indicating some residual histamine association.



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Figure 13

A HLMC at 20 min poststimulation with anti-IgE shows degranulation channels (D) that contain a few residual wisps of electron-dense matrix with some DAO-gold label indicating histamine. The degranulation channel is open to the cell surface (arrowhead). The unaltered granules remaining are heavily labeled for histamine. x32,000. With permission, Dvorak et al. J Leukoc Biol 59:824–834, 1996.

 
To label granule contents in vesicles trafficking from emptying granules to plasma membrane in cells undergoing PMD, we used a morphologic, kinetic analysis of HBs, ex vivo, because we could manipulate this system to image vesicles (Figure 5 and Figure 6) (reviewed in Dvorak 1998aGo,cGo,2005Go). Initially, we imaged the CLC protein by a postembedding immunogold method (Figure 8) (Dvorak and Ackerman 1989Go). With time, after stimulation of human basophils either with FMLP or TPA, the percentage of total cytoplasmic vesicles that were gold-loaded (indicating the presence of CLC protein) changed (Figure 14). CLC protein-loaded vesicles were significantly increased at 10 and 20 sec, 1 and 2 min, compared with unstimulated cells when HBs were stimulated with FMLP. The developmental kinetics of CLC protein-loaded vesicles in basophils stimulated by TPA showed progressively increased levels of gold-labeled vesicles that were significantly greater than in unstimulated cells at 2, 5, and 10 min after stimulation. Like in FMLP-stimulated cells, levels of CLC protein-loaded vesicles decreased at later stimulation times (30 and 45 min). The mechanism of decline differed, however, for these two secretogogues. For example, a decreased percent of gold-loaded vesicles indicating CLC protein in FMLP-stimulated cells was associated with reconstitution of large granule stores of CLC protein and, for TPA-stimulated cells, was associated with depletion of virtually all granule stores of CLC protein in emptied granule containers, as well as in remaining particle-filled granules.



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Figure 14

Percentage gold-labeled vesicles of the total vesicle population (% VG/V) in human peripheral blood basophils either unstimulated (UN) or stimulated with FMLP (A,C) or TPA (B,D), recovered for electron microscopy at the indicated times and prepared with a postembedding enzyme affinity-gold method to detect histamine (A,B) or an immunogold method to detect CLC protein (C,D). Significance values for each panel (compared with unstimulated cells) are as follows: (A) *p<0.001; (B) *p<0.25, **p<0.05, ***p<0.001; (C) *p<0.005, **p<0.001; (D) *p<0.05, **p<0.01, ***p<0.001. Replicate samples examined for FMLP-stimulated histamine release (A,C) released 56% at 60 min, and for TPA-stimulated histamine release (B,D) released 70% at 60 min. Adapted with permission from Dvorak et al. Lab Invest 74:967–974, 1996; Dvorak et al. Blood 88:4090–4101, 1996; Dvorak et al. Int Arch Allergy Immunol 113:465–477, 1997.

 
Histamine transport in vesicles developing over the kinetic release reactions induced by FMLP and TPA in HBs was also quantitated (Figure 9 and Figure 14) (reviewed in Dvorak 1998aGo–c,2005). For the rapid releaser, FMLP, histamine-loaded vesicles exceeded those of unstimulated cells at all times examined. For the slow releaser, TPA, histamine-loaded vesicles significantly exceeded those of unstimulated cells at 1, 2, 10, 30, and 45 min. Thus, for each secretogogue, histamine-loaded vesicles increased and remained elevated, compared with unstimulated cells, for the duration of the time examined. The mechanism for the persistence of histamine transport in vesicles differed, however, between these two different basophil secretogogues. Continued vesicular transport of histamine in FMLP-stimulated cells was associated with endocytosis of gold-labeled histamine from the plasma membrane in completely degranulated, agranular basophils, and recovering basophils—phenotypes that populate the kinetic samples obtained between 20 sec and 10 min. The continued vesicular transport of histamine in TPA-stimulated cells at late times in the kinetic sequence was associated with ongoing evidence of PMD, virtually no ultrastructural evidence of secretion by AND, and no evidence of recovery.

In 1975, we proposed a degranulation model to explain progressive losses, occurring over days, of granule contents from HBs in experimentally induced contact allergy skin lesions (Dvorak and Dvorak 1975Go). We postulated that closely coupled endocytotic–exocytotic traffic of small, smooth membrane-bound vesicles effected the emptying of secretory granule containers, in the absence of granule fusion and extrusion—a process characterized by the retention of granule containers of undiminished size in the cytoplasm. We postulated further that this steady-state secretion would be altered in an important way if either the rate or the amount of vesicular traffic was changed. For example, we envisioned that a faster rate of vesicular traffic would result in fusions of vesicles that would create channels between granules and plasma membrane, thus producing the anatomy of regulated secretion or AND. The cytoplasmic channels that form could contain multiple membrane-free granules (degranulation sacs or channels) in situ as well as provide communication between a single granule and the plasma membrane—events necessary for exocytosis directly through membrane pores to the external milieu.

We now present the evidence developed since the degranulation model was proposed by Dvorak and Dvorak (1975)Go in support of vesicular transport as a mechanism for effecting secretion from HBs. The evidence was collected in three ways: (a) direct inspection, (b) quantitation, and (c) direct labeling of expected vesicular cargo. By direct inspection, the existence of large numbers of vesicles of appropriate size was documented in basophils; and, in certain circumstances, fusion and/or budding of vesicles with/from large cytoplasmic secretory granules, termed granule-vesicle attachments, was also documented (reviewed in Dvorak 2005Go). Quantitation allowed documentation of rapidly changing numbers and contents of vesicles in HBs stimulated with different secretogogues over time. Direct labeling of expected vesicular cargo was accomplished with ultrastructural immunogold and enzyme affinity-gold methods, which label the CLC protein and histamine, respectively. Quantitation of gold-loaded vesicular carriers in stimulated HBs directly confirmed that releasing basophils transported these granule materials in cytoplasmic vesicles, as predicted by the degranulation model proposed by Dvorak and Dvorak (1975)Go.


    Recovery
 Top
 Summary
 Introduction
 Identity
 Secretion
 Vesicles
 Recovery
 Synthesis
 Literature Cited
 
Basophils, like other granulocytes, are generally viewed as end-stage cells with no potential for renewal or prolonged functional life, particularly after migration to tissues from the peripheral blood. Basophils are secretory cells characterized by synthetic product storage in prominent cytoplasmic granules and regulated secretion of these products by exocytosis of granules. Many secretory cells with similar granule storage sites and secretory programs (i.e., numerous endocrine and exocrine cells) are not end-stage cells and characteristically replenish secreted materials as needed. Evidence that basophils also recover from regulated degranulation by granule reconstitution was acquired through electron microscopic studies of guinea pig basophils in short-term cultures following secretion (reviewed in Dvorak 1991Go). Mast cells (cells with functional similarity to basophils) of two species (rat and human) are also known to reconstitute granules by several mechanisms following their regulated secretion by exocytosis (reviewed in Dvorak 1991Go).

We examined subcellular histamine localizations in purified HBs (Figure 9) that were stimulated to degranulate with FMLP, using an ultrastructural enzyme-affinity technique (reviewed in Dvorak 1998bGo). Basophils were collected at early (0, 20 sec, 1 min) and late (10 min to 6 hr) time points poststimulation and were prepared for routine ultrastructural and DAO-gold cytochemical analysis. Histamine was present in unaltered cytoplasmic secretory granules (30.77 gold particles/µm2; p<0.001 as compared with background); specificity controls (histamine absorption, DAO digestion) abrogated granule label for histamine. Altered granules in stimulated cells were not significantly labeled for histamine, as compared with background (p=not significant); unaltered granules in the same cells contained more histamine than altered granules (p<0.05).

During recovery times spanning 10 min to 6 hr, granules again appeared electron dense and contained histamine (33.49/µm2; p=not significant, as compared with unaltered granules in 1 min FMLP-stimulated cells, and p<0.05, compared with altered granules in 1 min FMLP-stimulated samples) (reviewed in Dvorak 1998bGo). Other structures devoid of histamine in actively secreting cells included extruded granules and intragranular and extruded CLC crystals. Recovering basophils displayed morphologic evidence of material and membrane conservation, granule content condensation, and biosynthesis. Subcellular histamine-rich sites in actively recovering basophils included condensing granules and collections of cytoplasmic vesicles in three locations—beneath the plasma membrane, adjacent to granules, and in the Golgi region.

HMCs are a rich and unique source of heparin, which is stored in cytoplasmic secretory granules and accounts for metachromasia, a staining property used to identify mast cells by light microscopy. We used a labeling method for heparin, which depends on the well-known property of RNase inhibition by heparin, to image subcellular sites of heparin in HLMCs (Figure 15) (Dvorak and Morgan 1998Go,1999Go). HLMCs were isolated, partially purified, either stimulated or not stimulated to secrete with anti-IgE, and recovered 20 min later for routine electron microscopy. Histamine secretion was also determined on replicate samples. A previously developed postembedding, enzyme affinity-gold electron microscopic technique to image RNA with RNase-gold (R-G), which also binds to the enzyme inhibitor heparin, was employed to determine the subcellular locations of heparin in non-secretory and secretory mast cells. Specificity controls for the novel use of this method and quantitation of granule labeling in these controls were performed.



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Figure 15

HMCs stained with RNase-gold (R-G) (A,C) or gold alone (B,D) show extensive binding to secretory granule heparin (A,C); gold does not bind to cytoplasmic granules (B,D). (A) x56,000; (B) x26,000; (C) x103,000; (D) x57,000. With permission, Dvorak and Morgan. Clin Exp Allergy 29:1118–1128, 1999.

 
Heparin was labeled by R-G in electron-dense granules within non-secretory HLMCs (Figure 15) and in electron-dense granules that persisted in secretory HLMCs at the maximum histamine secretion time (20 min). Specificity controls showed that gold alone did not label HLMCs, and that absorption with heparin significantly reduced or abrogated HLMC granule staining with R-G, but that RNA absorption did not. Heparin stores were absent in newly formed, electron-lucent intracytoplasmic degranulation channels in secretory HLMCs. Electron-dense granule matrices in the process of extrusion to the cell exterior still retained heparin at the instant of cellular secretion.

We also examined recovering HLMCs held in short-term cultures (3, 6, 18, and 24 hr) following stimulation of AND. The ultrastructural morphology of these events has been reviewed (Dvorak 1991Go). Using the new ultrastructural probes for histamine and heparin, we localized these mast cell products in recovering cells (Figure 16 and Figure 17) (Dvorak et al. 1996Go; Dvorak and Morgan 1999Go).



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Figure 16

HLMC stained with R-G 6 hr after stimulation with anti-IgE shows resolving degranulation channels (D) with variable electron-dense contents and R-G stain indicating heparin. x30,500.

 


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Figure 17

Condensing granule materials in a resolving degranulation channel (D) of a HLMC 3 hr after stimulation with anti-IgE are variably labeled with DAO-gold indicating histamine. Separation of some smaller domains with more electron density and gold label is evident in this recovering HLMC. x33,500. With permission, Dvorak et al. J Leukoc Biol 59:824–834, 1996.

 
Recovery of HLMCs from AND in vitro proceeded primarily by two mechanisms: (a) conservation by condensation, 3–6 hr (reviewed in Dvorak 1991Go); (b) synthesis, 18–24 hr (reviewed in Dvorak 1991Go). In the early recovery period (3–6 hr), DAO-gold labeled condensing granule domains that appeared within degranulation chambers (Figure 17). As separation of progressively more electron-dense granule domains enclosed within membranes occurred, the appearance of scroll foci were noted, and these structures were stained with DAO-gold with corresponding increased label density. Later recovery times (18–24 hr) showed a continuation of conservation mechanics as well as the appearance of large numbers of vesicles, vacuoles, progranules, and Golgi structures. DAO-gold labeled most of these structures, whether located in Golgi areas or in the peripheral cytoplasm. Also, gold particles prevailed in electron-lucent vesicles and vacuoles as well as in electron-dense or scroll-containing progranules.

These images document the presence of histamine in synthetic organelles of HLMCs recovering from AND but do not rule out re-uptake of previously released histamine. We suggest that the DAO-gold method is revealing synthesis of new histamine during recovery of HLMCs from degranulation, but that re-uptake of histamine may also be occurring. Re-uptake of extruded granule materials that were labeled with DAO-gold was visible in our study of HBs recovering from AND (reviewed in Dvorak 2005Go). HBs generally release each granule through separate degranulation pores during AND, and each granule is not solubilized but is available to be internalized by macropinocytosis. Also, portions of granules can be internalized by micropinocytosis. This process of recovery (conservation) is somewhat analogous in principle to conservation of retained granule materials in HLMC degranulation chambers that condense and reform histamine-rich granule domains.

The distribution of heparin stained with R-G in cells that have primarily used conservation for their recovery is of interest. Retained cytoplasmic degranulation channels developed increased amounts of electron-dense material that contained heparin (Figure 16). Condensing degranulation channels with heparin-containing dense material were never evident in non-secretory mast cells or in secretory mast cells examined at 20 min after stimulation. Electron-lucent degranulation channels did not contain heparin; these chambers underwent progressive partitioning with internal membranes and development of rounded, electron-dense granule domains that did contain heparin (Figure 16). Ultimately, condensing, electron-dense, heparin-rich crystalline arrays developed within granule-sized, membrane-bound containers that were derived from these channels as they resolved.

HLMCs that primarily extruded individual granules and that resolved their newly formed degranulation channels by inserting granule and channel membranes into the plasma membrane compartment ultimately resolved this rapid and extensive cell surface expansion by internalizing cell processes into cytoplasmic structures, termed canaliculi. HLMCs that were recovering from stimulated secretion and that utilized this mechanism of membrane conservation demonstrated well-formed, newly developed, heparin-rich granules in their cytoplasm. The canalicular structures were entirely devoid of heparin.

Also of interest was the distribution of heparin in HLMCs that primarily utilized a synthetic recovery mechanism as opposed to that of channel resolution. Synthetic HLMCs generally were those that had released granules and their membranes in their entirety and did not retain cytoplasmic degranulation channels, necessitating resolution by conservation. Such synthetic cells were characterized by the presence of large numbers of cytoplasmic vesicles and vacuoles. These structures were electron lucent or contained electron-dense material and, together with single scrolls, were scattered throughout the cytoplasm. Electron-dense, heparin-containing vesicles and progranules were evident in expanded Golgi areas. Peripheral cytoplasmic areas in synthetic mast cells also contained large numbers of newly formed, small scroll granules, which were heavily labeled for heparin.


    Synthesis
 Top
 Summary
 Introduction
 Identity
 Secretion
 Vesicles
 Recovery
 Synthesis
 Literature Cited
 
Cytoplasmic organelles in mature HMCs are dominated by storage-secretory granules, whereas clusters of free ribosomes and strands of rough endoplasmic reticulum are minor cytoplasmic constituents. HMC secretory granules have been primarily conceptualized as storage organelles for the synthetic products of the cell. Routine ultrastructural studies of HMCs suggested the possibility of another function for their prominent secretion-storage granules. Many HMC granules of all types displayed irregularly shaped electron-dense particles often identical in appearance and size to free perigranular ribosomes nearby (Figure 18) (Dvorak 2002Go). We found these granule-associated, ribosome-like particles in variable amounts, but we particularly noticed their increased numbers in cells actively undergoing synthesis during maturation, after granule losses mediated by PMD in vivo and recovering from stimulated AND ex vivo (Dvorak 2002Go). These morphologic observations fueled our pursuit of a possible synthetic role for HMC cytoplasmic granules.



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Figure 18

Recovering HLMC prepared for electron microscopy with standard methods following anti-IgE stimulated AND. Note ribosomes, electron-dense irregular particles, attached to the nuclear membrane, free in the cytoplasm and in secretory granules. x103,500. With permission, Dvorak and Morgan. Prog Histochem Cytochem 37:229–320, 2002.

 
During the experiments designed to establish a role for HMC granules in RNA biology, we noted that another prominent cytoplasmic organelle, lipid bodies, also had close associations with ribosomes (Dvorak et al. 2003Go). These were even more difficult to discern than those associated with granules in routine preparations because HMC lipid bodies are characterized by intense osmophilia, which often effectively obscures internal structures and structures associated with the phospholipid-rich shell encasing lipid bodies. Are lipid bodies, like secretory granules in HMCs, also subcellular sites/organelles that are important in RNA biology? Studies using a multimodal, ultrastructural approach serve to implicate these organelles in RNA biology in HMCs (Dvorak et al. 2003Go).

In aggregate, the new studies indicate HMC secretory granule and lipid body associations with ribosomes (the protein synthetic machine of cells), with ribosomal proteins, with RNA, with poly(A)-positive mRNA, and with various long-lived, or short-lived, uridine-rich and poly(A)-poor RNA species with key roles in RNA processing and splicing (reviewed in Dvorak 2005Go). These studies indicate that secretory-storage granules and lipid bodies in HMCs are also equipped for a synthetic role, and they considerably augment our vision of the role of secretory-storage granules in the cell biology of all granulated secretory cells and of lipid bodies generally in mammalian cells, where they are known to occur (reviewed in Dvorak 1991Go,2005Go).

Ultrastructural observations identified close associations of ribosomes, granules, and lipid bodies in developing HMCs, in resting mature HMCs and in HMCs recovering from secretion—associations that suggest non-traditional sites for protein synthesis in secretory cells poorly endowed with rough endoplasmic reticulum and Golgi structures (Dvorak 2002Go; Dvorak et al. 2000aGo,2003Go). The observed intragranular particles approximated the size, shape, and electron density of ribosomes in the perigranular and perilipid body cytoplasm of HMCs (Figure 18). Particulate content within mixed granules and a subset of granules, termed particulate granules, in HMCs (Figure 2B) could be a source of interpretive confusion regarding the presence of ribosomes within granules (Dvorak 1989Go). However, the particles in the mixed and particle granules are larger and more uniformly shaped electron-dense structures than the ragged, ~25-nm electron-dense ribosomes. The latter intragranular structures also decreased and increased in recovery from functional, and during developmental, processes in HMCs (Dvorak 2002Go; Dvorak et al. 2000aGo), much as ribosome numbers do in other functionally and developmentally engaged cells. Another difficulty in the recognition of granule or lipid body-associated electron-dense ribosomes in electron microscopic preparations of HMCs is that the granules, lipid bodies, and ribosomes are often equally electron dense, making visualization of the small particles (~25 nm) within the larger granules and lipid bodies (~1 µm) difficult at best. These observations informed our decision to search further for granule- and lipid body-ribosome relationships in the biology of RNA and HMCs.

Initially we used an enzyme affinity-gold method to detect RNA based on the affinity of a gold-labeled enzyme, RNase, for its substrate, RNA (Bendayan 1981Go,1989Go) to explore the possible relationship(s) of HMC granules, lipid bodies, and RNA (Dvorak and Morgan 1998Go,1999Go). This method provided excessive granule labeling that qualitatively was diminished when appropriate controls for RNA specificity were done. These studies did not, however, reach statistical significance. We established that the "noise" in this system did reach statistical significance for heparin, the proteoglycan specific for HMC granules and known to inhibit RNase activity (Figure 15). Thus, a new ultrastructural probe based on affinity-gold detection of an enzyme (RNase) inhibitor (heparin) was established, allowing for accurate detection of heparin in resting, functional, and recovering HMCs (Figure 15 and Figure 16) but disallowing detection of RNA stores in HMC granules with this approach (Bendayan 1981Go,1989Go).

In samples of HMCs prepared similarly with R-G staining, another prominent organelle (cytoplasmic lipid bodies) was stained. Unlike the extensive studies showing that this staining indicated heparin in granules (Figure 15 and Figure 19, lipid bodies appeared to bind the R-G by virtue of contained RNA (Figure 19) (Dvorak et al. 2003Go). That is, prior absorption of the reagent with heparin removed granule staining (e.g., heparin stores) but not lipid body staining, indicating RNA (Figure 19) (Dvorak et al. 2003Go). Thus, the R-G method identified a new subcellular location for RNA in HMCs—in lipid bodies (Figure 19) (Dvorak et al. 2003Go).



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Figure 19

Control HLMC in culture for 6 hr prepared with R-G staining after absorption of the R-G reagent with heparin Sepharose. The electron-dense lipid body is labeled with gold (e.g., not removed with heparin absorption) indicating the presence of RNA whereas virtually all of the gold label (see Figure 15) in the scroll granule is absent after this absorption indicating the usefulness of this technique for labeling HMC granule heparin. x76,000.

 
Uridine, an RNA precursor, was demonstrated in HMC granules and lipid bodies by two disparate ultrastructural methods (Dvorak and Morgan 2000aGo; Dvorak et al. 2000bGo,2003Go). The first method demonstrated the ex vivo incorporation of [3H]-uridine into granules (Figures 20A–20C) and lipid bodies (Figure 20D) in living cells by ultrastructural autoradiography (Dvorak et al. 2000aGo,bGo,2003Go). The second method identified the presence of intragranule and intralipid body uridine by postembedding immunogold stains (Figure 21) (Dvorak and Morgan 2000aGo; Dvorak et al. 2003Go).



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Figure 20

HLMCs stimulated with anti-IgE and recovered 6 hr (A,D) or 18 hr (B,C) later after exposure to [3H]-uridine for 2 hr (A), 6 hr (D) or 18 hr (B,C) and prepared for EM autoradiography with a chemical (B,D) or physical (A,C) developer. Recovery 6 hr after degranulation shows recovering channels (C) filled with electron-dense materials that are labeled for RNA after exposure to [3H]-uridine (A). Recovery 18 hr after degranulation shows a labeled granule in (B) that contains only particles; two single, unlabeled scrolls (arrows) are present in the cytoplasm immediately adjacent to the labeled granule. Each scroll encloses dense particles centrally. (C) The physical developer has produced small, round silver grains that do not obscure underlying details in granules. One granule has three silver grains, well-formed scrolls, and numerous ribosomes identical to the cytoplasmic ribosomes that surround this granule. One such cluster is labeled with a silver grain, as is the center of a longitudinally oriented scroll in an adjacent granule. (D) This 6-hr recovery sample shows a silver grain-labeled lipid body indicating RNA. (A) x40,000; (B) x42,000; (C) x66,000; (D) x55,000. With permission, (A–C) Dvorak et al. Int Arch Allergy Imunol 122:124–136, 2000; (D) Dvorak et al. Histol Histopathol 18:943–968, 2003.

 


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Figure 21

Six-hr control cultured HLMCs show gold-labeled immunoreactive uridine in granule scrolls and adjacent cytoplasmic ribosomes in (A) (note that the ribosomes and the 20-nm gold particles are of similar size) and in a lipid body in (B) indicating the presence of RNA in these sites. (A) x98,000; (B) x80,000. With permission, (A) Dvorak and Morgan. Histochem J 32:685–696, 2000; (B) Dvorak et al. Histol Histopathol 18:943–968, 2003.

 
Ribosome-associated ribonuclear protein complexes (RNPs) were imaged in and adjacent to HMC granules and lipid bodies, likewise by two disparate ultrastructural techniques (Figure 22) (Dvorak et al. 2000aGo,2003Go; Dvorak and Morgan 2000aGo,2001Go). The first of these was an EDTA chelation regressive staining method (Figure 22A) (Dvorak et al. 2000aGo,2003Go; Dvorak and Morgan 2001Go), and the second was an immunogold method that detects ribosome-specific RNPs with human autoimmune sera (P0, P1, and P2) (Figure 22B) (Dvorak and Morgan 2000aGo, Dvorak et al. 2003Go); both of these methods yielded identical localization results in HMCs (Dvorak et al. 2000aGo,2003Go; Dvorak and Morgan 2000aGo,2001Go).



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Figure 22

Control HLMCs after 6 hr in culture and prepared with Bernhard's EDTA stain (modified) (A) or with immunogold to detect ribosomal P antigen (B). In (A), scroll granules are surrounded by electron-dense ribosomes, many of which are attached to granule surfaces; some extend into longitudinally oriented granule scrolls. In (B), a scroll granule and a lipid body are gold-labeled, indicating the presence of immunoreactive ribosomal P antigen. (A) x92,000; (B) x76,000. With permission, Dvorak et al. J Histochem Cytochem 48:1–12, 2000.

 
We used direct binding and ultrastructural in situ hybridization (ISH) of general polyuridine [poly(U)] probes, prepared in diverse ways, to detect polyadenine [poly(A)]-positive mRNA stores in human mast cells (Dvorak et al. 2000aGo,2003Go; Dvorak and Morgan 2000bGo). These methods demonstrated that poly(A)-positive mRNA species were contained not only in the expected nuclear and cytoplasmic sites but also in the secretory granules and lipid bodies of human mast cells (Figure 23). RNA processing in the nucleus involves splicing of precursor mRNA, polyadenylation of pre-mRNAs, and transport of specific mRNAs from nucleus to cytoplasmic sites—mRNAs that associate with cytoplasmic ribosomes to direct specific protein syntheses (reviewed in Dvorak 2005Go). The electron microscopic ISH localizations of granule and lipid body stores of poly(A)-containing mRNAs, as well as nuclear stores of poly(A)-containing precursors of mRNA, indicate that site-specific localization of mRNAs in cytoplasmic secretory granule and lipid bodies (in addition to cytoplasmic ribosomes) exist.



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Figure 23

Ultrastructural methods to detect mRNA performed on HMCs with a variety of poly(U)-labeled probes show labeled granules (A,C,D,E) and lipid bodies (B,F,G). In situ hybridization or direct binding methods used [3H] poly(U) (A,B), poly(U)-gold (C), BSA-poly(U)-gold (D,E), photobiotinylated poly(U) (F), or poly(U)-anti-uridine (G). (A) x85,000; (B) x52,000; (C) x112,000; (D,E) x103,500; (F) x79,000; (G) x62,000. With permission, (A,C,D,E) Dvorak et al. J Histochem Cytochem 48:1–12, 2000; (B,F,G) Dvorak et al. Histol Histopathol 18:943–968, 2003.

 
We investigated the presence of individual RNA species in HMCs using specificity-proven autoimmune sera from humans with autoimmune disorders and performing postembedding immunogold staining (Dvorak et al. 2000aGo,2003Go; Dvorak and Morgan 2000aGo). These sera allowed us to visualize granule and lipid body sites of several RNA species (Figure 24). These included ribosomal RNP (see earlier), small nuclear RNPs [snRNPs (RNP and Sm)], which associate with uridine-rich, poly(A)-negative snRNAs that are either essential for the splicing of mRNA precursors (U1) or associated with splicing events in RNA processing (U2, U4–6). Additionally, granule and lipid body sites for a small cytoplasmic RNP, Ro (thought to operate in translation of mRNA subsets and in ribosome synthesis, assembly, and transport), and La (representative of 5 S ribosomal RNA and certain transfer RNAs), were found in HMCs. La also is postulated to function widely in RNA biology, including in protein synthesis, mRNA translation, biogenesis and nuclear retention of polymerase III-transcribed RNAs, and biogenesis of ribosomes and has been localized to nucleus, cytoplasm, rough endoplasmic reticulum, polyribosomes, and to signal recognition particle RNA in cells (Dvorak and Morgan 2000aGo; Dvorak et al. 2003Go; Dvorak 2005Go).



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Figure 24

Immunogold preparations of HMCs using autoimmune human sera with specificity for RNP (A), La (B), or Ro (C) show gold-labeled lipid bodies (A–C) and granules (A). (A) x74,000; (B) x60,000; (C) x70,000.

 
Granule and lipid body stores of mRNA could reflect en site synthetic capacities that define a new role(s) for secretory granules and lipid bodies in general. A large body of evidence has recently defined RNA transport and localization as a mechanism for facilitating site-specific synthesis in various subcellular organelles and compartments in cells of diverse origin from multiple species (reviewed in Dvorak 2005Go). Such RNA has been intimately associated with all three major cytoskeletal elements, i.e., microfilaments, intermediate filaments, and microtubules (reviewed in Dvorak 2005Go), and specific zip codes for localizing individual mRNAs at subcellular sites have been postulated (reviewed in Dvorak 2005Go). Secretory granules and lipid bodies have not been considered as such sites, but the evidence that we present suggests that they must also be included in the travels and localizations of mRNA (Dreyfuss et al. 1988Go; Wilhelm and Vale 1993Go; Knowles et al. 1996Go) in mast cell biology.

Whereas secretory granules and lipid bodies have only recently been implicated in organelle-specific RNA metabolism (reviewed in Dvorak 2005Go), RNA transport, targeting, localization, and compartment-specific synthesis to localize protein products where desired (or to prevent protein products from accumulating where not desired) have been proposed in a number of circumstances (reviewed in Dvorak 2005Go).

RNA sorting in mammalian cells has become a hot topic in cell biology with the recognition that focal accumulations of mRNA with certain organelles, portions of organelles, and cytoplasmic sites exist (reviewed in Dvorak 2005Go). In many instances, the identification of these localizations was preceded by ultrastructural visualization of ribosomes in these sites. These sightings, coupled with determinations that mRNA and, in some cases, their specific proteins are colocalized in areas of potential need, have suggested that specific localization of mRNAs may function to concentrate proteins locally where their function occurs, and/or to prevent product localization where they are of no use.

Few of all these reports have implicated mRNA accumulation in classic storage granules of secretory cells and in lipid bodies generally. Some reports do suggest such a relationship, one in granular vesicles of the hypothalamus of lactating rats (Jirikowski et al. 1990Go) and our communications regarding HMC granules and lipid bodies (Dvorak et al. 2000aGo,2003Go). Polar granules in germ cells of Caenorhabditis elegans are said to contain embryonic RNAs that are poly(A) positive (Seydoux and Fire 1994Go), and poly(A)-binding proteins have been localized in the same polar granules (Kawasaki et al. 1998Go) as well as in the dual function, i.e., secretory-lysosome, granules of cytolytic lymphocytes (Tian et al. 1991Go).

Similar parallels to our studies (Dvorak et al. 2000aGo,bGo,2003Go; Dvorak and Morgan 2000aGo,bGo,2001Go) exist in the realization that site-specific synthesis plays a role in neurobiology (Steward and Levy 1982Go; Merlie and Sanes 1985Go; Davis et al. 1987Go; Garner et al. 1988Go; Jirikowski et al. 1990Go,1992Go; Knowles et al. 1996Go; Martone et al. 1996Go). Initially, morphologic studies recorded preferential localization of polyribosomes under the base of dendritic spines of neuronal cells and at synaptic sites (Steward and Levy 1982Go), despite the fact that most organelles responsible for protein synthesis are remotely located in neuronal cell bodies (Peters et al. 1976Go). Specialized techniques to localize RNA precursors and poly(A)-positive mRNA followed and provided further evidence of site-specific protein synthesis at synapses (Merlie and Sanes 1985Go; Davis et al. 1987Go; Martone et al. 1996Go). Protein-specific mRNAs were then localized at synaptic sites (Garner et al. 1988Go; Jirikowski et al. 1990Go,1992Go), and direct visualization of RNA motility in living neurons suggested the existence of a cellular trafficking system for RNA targeting (Knowles et al. 1996Go).

The accumulating evidence of synthetic machinery in secretory-storage granules and lipid bodies suggests a more comprehensive role for these organelles in secretion-synthetic processes than previously recognized. In mast cells, this role may also implicate them in the regulation of mRNA levels to rid cells of excessive or redundant mRNAs concomitant with secretion of granules in toto by AND or their contents in part by PMD. This mechanism may complement, or function as an alternative to, mRNA degradation (to lower mRNA levels) as needed. Thus, mRNA sorting to secretory granules in mast cells may serve to regulate protein supplies spatially and temporally.


    Acknowledgments
 
These studies were supported by National Institutes of Health Grant AI-33372.

Expert technical assistance was provided by Ellen Morgan, Rita Monahan-Earley, Kathryn Pyne, and Tracey Sciuto.


    Footnotes
 
Received for publication January 26, 2005; accepted February 22, 2005


    Literature Cited
 Top
 Summary
 Introduction
 Identity
 Secretion
 Vesicles
 Recovery
 Synthesis
 Literature Cited
 

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