REVIEW |
Ultrastructural Studies of Human Basophils and Mast Cells
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
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Summary |
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(J Histochem Cytochem 53:10431070, 2005)
Key Words: basophil mast cell ultrastructure secretion vesicle transport piecemeal degranulation recovery from degranulation synthesis granules lipid bodies
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Introduction |
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Identity |
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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 1989,1991
,2005
). 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).
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Secretion |
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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 1993). 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 1992). 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 1993).
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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 1993).
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 1993).
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 1993). 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 2005
). 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 2005
).
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 2005).
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 1975). 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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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 1975). 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 2005
). 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 1993, 2005
). 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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TPA, on the other hand, elicits histamine release slowly (reviewed in Dvorak 1993). 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 absenta 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 1992, 1993
). 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 1992
,1993
). 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 2005). All surgical biopsies were obtained from uninvolved sites of patients with either one of two IBDsulcerative colitis (UC) or Crohn's diseaseand 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 2005
).
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.
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Vesicles |
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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 2005). 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 1989) and a newly developed enzyme affinity gold method to image histamine in both HBs (Figure 9) and HMCs (Dvorak et al. 1993
, Dvorak 1998b
).
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We also examined IgE-mediated AND in isolated HLMCs with the ultrastructural method to detect histamine (Figure 13) (reviewed in Dvorak 1998b). 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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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 1975). We postulated that closely coupled endocytoticexocytotic traffic of small, smooth membrane-bound vesicles effected the emptying of secretory granule containers, in the absence of granule fusion and extrusiona 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 membraneevents 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) 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 2005
). 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)
.
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Recovery |
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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 1998b). 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 1998b). 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 locationsbeneath 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 1998,1999
). 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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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 1991). 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. 1996
; Dvorak and Morgan 1999
).
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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 2005). 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.
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Synthesis |
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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 2005). 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 1991
,2005
).
Ultrastructural observations identified close associations of ribosomes, granules, and lipid bodies in developing HMCs, in resting mature HMCs and in HMCs recovering from secretionassociations that suggest non-traditional sites for protein synthesis in secretory cells poorly endowed with rough endoplasmic reticulum and Golgi structures (Dvorak 2002; Dvorak et al. 2000a
,2003
). 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 1989
). 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 2002
; Dvorak et al. 2000a
), 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 1981,1989
) to explore the possible relationship(s) of HMC granules, lipid bodies, and RNA (Dvorak and Morgan 1998
,1999
). 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 1981
,1989
).
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. 2003). 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. 2003
). Thus, the R-G method identified a new subcellular location for RNA in HMCsin lipid bodies (Figure 19) (Dvorak et al. 2003
).
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Whereas secretory granules and lipid bodies have only recently been implicated in organelle-specific RNA metabolism (reviewed in Dvorak 2005), 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 2005
).
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 2005). 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. 1990) and our communications regarding HMC granules and lipid bodies (Dvorak et al. 2000a
,2003
). Polar granules in germ cells of Caenorhabditis elegans are said to contain embryonic RNAs that are poly(A) positive (Seydoux and Fire 1994
), and poly(A)-binding proteins have been localized in the same polar granules (Kawasaki et al. 1998
) as well as in the dual function, i.e., secretory-lysosome, granules of cytolytic lymphocytes (Tian et al. 1991
).
Similar parallels to our studies (Dvorak et al. 2000a,b
,2003
; Dvorak and Morgan 2000a
,b
,2001
) exist in the realization that site-specific synthesis plays a role in neurobiology (Steward and Levy 1982
; Merlie and Sanes 1985
; Davis et al. 1987
; Garner et al. 1988
; Jirikowski et al. 1990
,1992
; Knowles et al. 1996
; Martone et al. 1996
). Initially, morphologic studies recorded preferential localization of polyribosomes under the base of dendritic spines of neuronal cells and at synaptic sites (Steward and Levy 1982
), despite the fact that most organelles responsible for protein synthesis are remotely located in neuronal cell bodies (Peters et al. 1976
). 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 1985
; Davis et al. 1987
; Martone et al. 1996
). Protein-specific mRNAs were then localized at synaptic sites (Garner et al. 1988
; Jirikowski et al. 1990
,1992
), and direct visualization of RNA motility in living neurons suggested the existence of a cellular trafficking system for RNA targeting (Knowles et al. 1996
).
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.
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Acknowledgments |
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Expert technical assistance was provided by Ellen Morgan, Rita Monahan-Earley, Kathryn Pyne, and Tracey Sciuto.
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Footnotes |
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Literature Cited |
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