Mast Cell Phagocytosis of Red Blood Cells S. S. Spicer, MD, J. A. V. Simson, PhD, and J. E. Farrington, BS
The prevalence of mast cells infiltrating bone marrow of different rats varied widely. as did the staining properties and size of their cytoplasmic granules. Bone marrow mast cells from several rats revealed large membrane-limited inclusions which stained metachromatically or orthochromatically and resembled inclusions in some macrophages. Ultrastructurally, mast cells varied widely in content of uniform dense granules or enlarged granules with less dense. fine grained content. Some of the large inclusions observed ultrastructurally in mast cells were heterophagic vacuoles which contained ervthrocvtes or reticulocytes, or remnants from other phagocytized cells, possibly neutrophils or unidentified homogeneous material. Smaller bodies. interpreted as fragments of ervthrocytes, lay extracellularly near mast cells and occupied small, membrane-limited, heterophagic vacuoles in some mast cells. In other mast cells. communal vacuoles enclosed several specific cytoplasmic granules in various stages of disruption. The communal vacuoles occasionally opened to the extracellular space. A few large indeterminate vacuoles in mast cells contained amorphous flocculent matter which apparently derived either from coalescence of cvtoplasmic granules through fusion of granule membranes or from endocytosis. (Am J Pathol 80:481-498, 1975)
EVIDEN-CE HAS ACCU-NIULATED that. in addition to secreting their granules or the contents thereof.1 6 mast cells may also endocytose extrinsic material.7- Antigen binding to immuinoglobulin E (IgE) on the surface of mast cells 10 initiates release of vasoactive amines that mediate inflammatorv responses."-13 Mast cells have been shown to incorporate a varietv of particulates into their cvtoplasmic grantules. converting them to heterophagosomes.78 The presence of ferritin particles in rat mast cells closely adjacent to hemosiderin-laden phagocytes supports the interpretation that the cells are endocytic.9 In the present studvy mast cells in rat bone marrow and lymph nodes were examined tultrastnrcturallv for evidence of endocvtic activitv. The material examined provided several examples of phagocytosis of ervthroid cells as well as of other unidentified bodies by mast cells in some rats. A preliminary account of this studv has appeared." Materials and Methods Twentx- one adult albino rats of the Sprague-Dawlev. Fischer, or Wistar strains were obtained from commercial souirces and houised for several w-eeks to months in the local From the Department of Pathologv. Medical University of Souith Carolina. Charleston. Sou,th Carolina Supported by Grants AM-10956 and ANI-11028 from the National Institutes of Health and Training Grant TR-1681 from the Veterans Administration, Accepted for puiblication Mav 20. 19753 Address reprint requests to Dr S S Spicer. Department of Pathologv. Medical UnisersitN- of Souith Carolina. 80 Barre Street. Charleston. SC 29401 481
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animal facility. Rats 1 to 8 and 16 to 21 were maintained on a standard pellet diet. Rats 9 to 12 and 13 to 15 were Fischer rats maintained in a separate experiment on a Remington or Evans low iodine diet, respectively. The rats were sacrificed uinder anesthesia. Femoral marrow from Rats 1 to 15 was minced promptly in 2 to 6% gluitaraldehyde, buiffered with 0.1 M cacodylate at pH 7.2, and fixed for 90 minuites at 4 C in this soluition. Cervical lymph nodes from Rats 4 and 16 to 21 were fixed similarly. A portion of the bone marrow from Rat 2 was also fixed for 90 minutes in a 4% formaldehyde 2.5% glutaraldehyde mixture,15 and rinsed in buffered sucrose. The latter specimen was incubated for 30 minutes at 25 C in 3,3'-diaminobenzidine-H202 substrate medium for demonstration of peroxidase16 as a possible means of identifying erythroid cells by their content of peroxidase. Control tissues were incubated in suibstrate medium lacking the diaminobenzidine. All of the specimens were then postfixed for 1 hour at 24 C in 2% osmium tetroxide, buffered at pH 7.2 with 0.1 M cacodylate. This was followed by brief rinsing with water, routine dehydration through alcohols, and embedment in Epon. Thick epoxy sections of several blocks of each specimen were stained with alkaline ethanolic toluidine blue for light microscopic examination. Thin sections were prepared of bone marrow from Rats 1 to 6 and of lymph nodes from Rats 16 to 21. The sections of specimens processed for morphologic examination were stained with a uranyl acetate-lead citrate sequence and examined in an AEI-6B electron microscope. Thin sections of the portion of the specimen from Rat 2, incubated for peroxidase, were examined with and without uranyl acetate-lead citrate staining.
Results Light Microscopy
The abuindance of mast cells varied widely in the different bone marranging from 0.3 to 12.3 cells per oil immersion field (Table 1).The staining and morphologic characteristics of the granuiles differed markedly in the individual mast cells of some specimens and varied from one rows
Table 1-The Variable Prevalence and Structure of Mast Cells in Rat Bone Marrow Percent of mast cells With With over With over No. of mast 20% enlarged inclusions Mast cells 80% small cells/oil >2 u granules Rat granules observed immersion field 97 6.6 3 1 30 0.5 6.3 55 45 2 251 1.5 14 3.6 3.7 86 111 3 21.3 11 89 4 61 0.3 1 4.8 6.2 99 1335 5 3.8 17 83 4.2 264 6 17.6 75 17 25 7 0.3 62 38 6.9 0.5 29 8 1 19.0 99 12.3 530 9 14.4 1 99 5.1 382 10 17.5 33 171 5.3 66 11 5.3 18 82 10.4 12 785 8.3 20 80 528 8.6 13 6.5 94 6 9.9 14 596 5.2 34 66 4.9 228 15
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specimen to another. Many cells contained excluisivelv or predominantlv a population of abundant, fairlv uniform, rouind granuiles which resembled granules in mast cells of rat connective tissuies in that they were uiniform in size and stained dark purple with toluidine bluje. Nuimerouis cells possessed a variable proportion of granules which were moderately enlarged, stained relatively lightly, and were highly metachromatic. There was no obviouis correlation between the abuindance of mast cells in bone marrow and the extent to which mast cells in a given sample contained enlarged granules (Table 1). In addition to classic granuiles, the bone marrow mast cells occasionally disclosed incluisions (Table 1) which were rouighly two to five times the size of normal granules and either stained gray-bluie like neighboring ervthrocvtes or, more often, exhibited a metachromatic pink coloration. Occasional macrophages in these specimens enclosed gray-bluie inclusions like some in mast cells. Electron Microscopy
Many of the mast cells in the specimens examined uiltrastnrctuirallv contained predominantly uniform granuiles which were very dense, roughly spherical, and crowded together in the cvtoplasm (Figuire 1). Other mast cells contained such granules admixed with a variable proportion of enlarged granules whose content was often less dense and finelv grained. The latter granules apparently corresponded with the lighter staining, more metachromatic granules in toluiidine-bluie-stained thick sections. The proportion of cells with mostly small, dense granuiles varied in different specimens. Small vesicles containing sphenrles of densitv similar to that of the cytoplasmic granules often lay near Golgi zones in the latter cells (Figure 2). Some of the mast cells from each rat showed one or more unusual ultrastructural features (Table 2). In Rats 1 and 2, mast cells of the bone marrow appeared to lie in frequent and close approximation to ervthroblasts, reticulocytes, or erythrocytes over a variable distance (Figures 3 and 4). Erythroid cells in extensive contact with these mast cells often had an irregular profile characterized by long curved processes which conformed to the surface of the mast cell (Figure 4). Processes from suich irreguilar erythroid cells on occasion deeply indented the surface of some mast cells (Figures 5 and 6). Dense or filaimentous material occasionally extended inward from the blurred plasmalemma of erythroid cells where they contacted mast cells (Figure 3, Inset). The cytoplasmic granules in such cells varied widely in densitv. A profile of a mast cell from a lymph node of Animal 4 disclosed an irregular erythrocyte within a vacuole-like space which was delimited peripherally
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Table 2-Inclusions Observed Ultrastructurally in Mast Cells of Rat Bone Marrow and Lymph Nodes
Small
Rat
multiple Large heteorphagic heterophagic vacuoles vacuoles Homogeneous content consistent with Hetero- Of probable erythroid Communal geneous erythroid vacuoles content origin cells ++ + +++
Indeterminate vacuoles
Tissue 1 Bone marrow ++ ++ + ++ 2 Bone marrow + 3 Bone marrow 4 + + Bone marrow + Lymph node 5 Bone marrow 6 Bone marrow Lymph 16 node + to +++ = Estimated relative prevalence of structure, - = not encountered in terial examined.
+ +
-
+ + +
the ma-
by thin cytoplasmic processes (Figuire 7). Several grantules in the periphery of the latter mast cell were slightly enlarged and decreased in density, and appeared to be in a process of discharge from the cell (Figure 7, Inset). Mast cells from the bone marrow of Rats 1 to 6 and lymph nodes of Rats 4 and 16 occasionally contained one or more membrane-limited, rolundto-somewhat-irreguilar vactuoles measuiring 2 to 6 ,u in diameter. Suich vacuioles varied in abuindance, size, and content in different specimens (Table 2) and were interpreted as the uiltrastruictuiral couinterpart of the large metachromatic and orthochromatic inclutsions observed in thick sections. These mast cell incluisions couild be classified into folur basic types: a) large heterophagic (endocytic) vacuioles, b) small, muiltiple, heterophagic vacuoles, c) commuinal vactioles with coalesced grantules, or d) vacuioles of indeterminate natuire.
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Large Heterophagic Vacuoles
The large heterophagic vactuoles measuired tup to 5.0 g or more in diameter and contained material that obviouLslv had been endocvtosed (Figures 8-14). Occasional mast cells of Rats 1, 3, and 4 disclosed one to three of these vacuoles. The material differed in the vactuoles of different or even the same cell, varving in density and degree of heterogeneitv. Some of the vacuoles enclosed a large stnrctuire resembling an ervthrocvte (Figure 8) or smaller bodies consistent with an ervthroid derivation (Figure 11). Other such vacuoles enclosed similar bodies which contained infrequient vesicles and peripheral dense material consistent with degenerated mitochondria and ribosomes of reticuilocvtes (Figures 9, 10, 14). Infrequent vacuoles, generally bordering the nucleuis, possessed fairlv homogeneous material of high density (Figuires 12 and 14). Vacuioles encountered in a few marrow cells of Rats 1, 2, and 4 contained pleomorphic or heterogeneous material (Figure 13) reminiscent of that occasionallv encountered in macrophage vacuoles containing phagocytized leukocytes with their abundant granules. Occasional cells contained lip to three vacuolar profiles which sometimes resembled one another buit often differed in content (Figure 14). A few moderatelv dense, small granuiles measuring abouit 0.1 yI in diameter sometimes bordered the large heterophagic inclusions (Figures 9 and 10). Small Heterophagic Vacuoles
Small heterophagic vacuoles measuired tip to 2.5 u in diameter and contained endocytosed material. These vacuioles occturred in varving numbers in bone marrow mast cells of Rat 2 (Figuires 15-18). Thev generally exceeded neighboring cytoplasmic granuiles in size and were less dense than the granules, especiallv in specimens processed for peroxidase (Figures 15-18). In the latter, the incluisions resembled neighboring erythrocytes in density and textutre. Extracelluilar bodies similar in densitv and texture to both the incluisions and nearby ervthrocytes also bordered these mast cells (Figuires 15, 16, and 18) and occulpied invaginations of the cell periphery, apparently in an incipient stage of endocvtosis (Figuires 16-18). The small bodies in the vicinity of mast cells appeared to be fragments of erythrocytes which, after engulfment, became the muiltiple small mast cell inclusions. Incluisions observed in rouitinelv processed bone marrow from Rat 4 resembled the small heterophagic vactuoles of Rat 2 except that thev were less clearly distinguishable from the equiallv dense buit generallv smaller and more spherical cvtoplasmic granuiles (Figuire 19). Cytoplasmic granules in cells with small heterophagic vacuiloes were small
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and dense and disclosed no evident alteration. Other struictuires occulpying surface-connected spaces in the periphery of rat mast cells appeared rather to be grantules altered following exocytosis (Figuire 21). Inclusions in neighboring macrophages often resembled some of the heterophagic vacuoles of the mast cells. The uiniformly very dense macrophage inclusions, known to arise from ingestion of erythroblast nuclei, displayed striking similarity to some paranuiclear incluisions in mast cells (compare Figures 20 and 12). Communal Vacuoles
Communal vacuoles consisted of membrane-limited spaces which were partially filled by cytoplasmic granuiles in stages of degeneration and apparently resulted from fusion of the limiting membrane of several granules (Figures 22-25). These inclusions were encouintered in marrow cells of Rats 1, 2 and 5 (Table 2). The commuinal vacuioles occuipied extensive areas within otherwise unaltered mast cells. In a few instances, the vacuoles appeared continuouis with the extracelluilar space (Figulre 22). A mast cell profile in the marrow of Rat 1 enclosed a rouind, membranelimited vacuole containing crowded residuies interpreted as decomposing granuiles. At an adjacent level, the content of this vacuiole incluided also an' eccentric area composed of flocculent material like that in the adjoining disintegrated granules (Figuire 25). The floccuilent material in this instance was apparently formed from granule coalescence. It resembled the content in some of the vacuioles in the following category (compare Figuire 25 with Figuires 26-28). Vacuoles of Indeterminate Origin
Vacuoles of indeterminate origin encouintered in occasional mast cells in marrow of Rats 1, 2, and 4 (Figuires 26 and 27) and lymph node of Rat 16 (Figuire 28) afforded little evidence as to their origin. These moderately large, membrane-limited struictures contained finely partictulate, fairly homogenouis material of rather low density. Discussion
Althouigh mast cells have been fouind to incorporate exogenouis particles of widely varying size uinder experimental conditions,4 relatively little evidence has accumuilated for endocytic activity in the abuindant mast cells infiltra.ting variouis normal tisstues. These cells are often associated closely with phagocytes containing lipid and hemosiderin, especially in older animals.17 Such siderophage-associated mast cells have been shown histochemically to possess diminished content of acid muicosuibstance and
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stronglv basic protein."8 The content of ferritin in cytoplasm and granuiles of mast cells intimatelv bordering siderophages described previouislv9 attested to in vivo exchange between macrophage and mast cell and microendocvtic activity in the latter. The present findings demonstrate macroendocvtic activitv (i.e., uiptake of particles > 2000 A in diameter) by mast cells in bone marrow. Observation of stages of mast cell ingestion of ervthroid cells and of large mast cell incluisions composed of a generally uiniform material similar to that of erythroid cells implies that these cells are capable of endocvtosing whole ervthrocvtes. Some inclusions contained dense material at the border and membranouis internal struictures, interpreted as ribosomes and mitochondria of reticulocytes. That reticuilocvtes as well as ervthrocvtes may be engulfed was fuirther indicated by the intimate fuill approximation of reticulocytes to the mast cell surface and protrusion of reticulocyte processes into mast cells. Several inclusions in mast cells resembled some in macrophages in their uniform high density and frequient paranuiclear location. Such inclusion bodies appear similar to those which have been observed frequently in normal bone marrow and have been interpreted as ingested nuclei extruded from erythroblasts. In addition, occasional mast cells contained heterogenous structures, some of which were reminiscent of leukocvte granules (Figure 3). These heterophagic vactuoles suiggest that mast cells, on occasion, phagocvtose leuikocytes as well as red blood cells. The varied content of mast cell vacuoles possiblv reflects stages of digestion of ervthrocvtes or other endocvtosed material bv hvdrolases known to exist in mast cells."9 Suich digestion conceivably couild transform the endocvtosed component into material like that in indeterminate vactuoles. However, little evidence was obtained for depletion of the specific granuile population throuigh fuision with the heterophagic vacuioles in the way that neutrophil leuikocvte granuiles fuise with and admit hydrolases into phagocytic vacuioles. The granuiles in cells with muiltiple small inclusions appeared normal in nuimber and stnrctture and those in cells with large inclusions were apparentlv normal in abtindance, althouigh often altered in struieture. Transport of matrix content from cytoplasmic granuiles into phagocvtic vacuioles conceivablv couild occuir here without degranuilation. Loss of granule content withouit secretion or exocvtosis of granules apparently transpires in some hypersensitive states.5 6 However, cell mechanisms have not been described for transport of granuile content to phagocytic vacuioles or extracelluilar space withouit fuision between granuile membrane and the vacuiolar membrane or plasmalemma. The metachromatic staining of some of the large incluisions indicates they contain acid mucosuibstance which they presuimably acquiire bv some means
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from the heparin-rich granules. Alternatively, hydrolases may be transported to the vacuoles by Golgi-derived vesicles or the small dense bodies seen bordering vacuioles (Figures 9-11). Suich a souirce, i.e., primary lysosomes other than specific granuiles, was previouisly considered for the acid phosphatase demonstrable in mast cell granuiles that had been converted to heterophagosomes by tuptake of colloidal gold.20 That the mast cell grantules converted to heterophagosomes may acquiire enzymes which the unaltered specific granules lack was indicated by demonstration of acid phosphatase in the converted buit not in the tunaltered granuiles in endocytosing cells. Some mast cells showed smaller incluisions which differed from mast cell granules. The inclusions resembled extracelltular bodies suiggestive of fragmentated erythrocytic cells. Fragmentation of red blood cells conceivably cotuld occtur as a spontaneouis event restulting from an abnormality of the cells, as a consequence of the release of lysosomal hydrolases into intercelltular space, or as a restult of stepwise cleavage of parts of erythrocytes by mast cells or other endocytes. Fragmentation of red blood cells and ingestion of the fragments by macrophages has been described previously. 21 The erythrocytes observed in bone marrow mast cells in the present sttudy apparently were young rather than senescent cells, since they were part of the hematopoietic popuilation. The present evidence for phagocytosis of reticuilocytes fturther attests to the ingestion of yolung erythroid cells by mast cells of rats. Endocytosis of early erythrocytes couild occtur as a conseqtuence of excessive hematopoiesis. This possibility derives stupport from observations indicating excessive hematopoiesis and disposal of tip to 50% of the cells formed in normal marrow.22 Such compensatory erythrophagocytosis by mast cells seems uinlikely, however, becauise the prevalence of mast cell erythrophagocytosis was not high enough to account for appreciable disposal of red cells in the involved animals, and this process was not encountered in most of the rats examined. Perhaps, then, the phagocytosis of erythrocytes by mast cells results from an abnormality of the erythrocytes in the few animals in which the process was observed. Infection by microorganisms such as Haemobartonella, Eperythrozoon or Grahamella that are known to infest rodent erythrocytes cotuld alter the red blood cells and render them susceptible to phagocytosis by mast cells. Fuirther stuidies wouild be required to relate this process to a specific pathologic process. A nuimber of mast cells disclosed a different alteration that apparently consisted of fusion of the membrane delimiting several granuiles, with consequent sequiestration of these granuiles in one or more irreguilar vacuiolar
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spaces. Since these cells often lacked evidence of endocvtosed material, perhaps this change was unrelated to the erythrophagocvtic activity of neighboring mast cells. However, animals showing the apparent granuile coalescence within common vacuoles were those most clearly involved in phagocytosis of ervthrocvtes; therefore, the two processes appear in some way related. Extracellular attack on erythroid cells by exocytosed mast cell granules could account for their apparent extracelluilar fragmentation and play a role in the degradation of red cells. The nature of the vacuoles arising from coalescence of cytoplasmic granules remains a question. Their continuiity with the extracelluilar space affirms the interpretation previously derived from observations of granuiles lying free in surface-connected spaces2 that the granuiles are uindergoing exocvtosis. The vacuoles, on the other hand, couild contain extrinsic substance and constitute secondary Ivsosomes of a heterophagic natuire. The most central vacuoles lacking apparent connections with the suirface and containing largelv degenerated granuiles-and floccuilent material probably derived there from (Figure 23)-appear unconnected with ouitside space and more likely concerned with endocytosis than exocvtosis. Conceivably, the fusion of granules into commuinal vacuioles and suibsequent digestion of granule material in the vacuiole occuirs in response to microendocytic activity and entails digestion of material not visuialized in the cells because of its small size or minimal electron opacitv. The granules and the other content in the commuinal vacuioles in some cells appeared to undergo dissolution and become a floccuilent mass. The residue from coalescence of granules in a commuinal vacuiole cannot be clearly differentiated by morphologic appearance from that resuilting from digestion of endocytosed material. However, the similaritv of floccuilent material in a portion of some vacuioles to deteriorated granuiles in the same or other vacuoles perhaps identifies the uiniformly loose, floccuilent matter, often present in vacuoles, as a residuulm-in part, at least-of coalesced granules. Such aggregations possibly shrank with continuied coalescence and digestion into the small vacuoles of indeterminate origin seen in a few mast cells.
Referweces 1. Padawer J: The reaction of rat mast cells to polylysine. J Cell Biol 47:352-372, 1970 2. Lagunoff D: Membrane fusion during mast cell secretion. J Cell Biol 57:252-259, 1973 3. Moriyasu S, Yamura T: Electron microscopic studies of mast cell degranulation. Acta Derm Venereol [Suppl 73] (Stockh) 53:149-156, 1973
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4. Tasaka K, Yamasaki H: Local degranulation and histamine release from a single rat mast cell by microelectrophoretic application of-basic histamine releasers and antigen. Acta Derm Venereol [Suppl 73] (Stockh) 53:167-174, 1973 5. Uvntis B: Histamine storage and release. Fed Proc 33:2172-2176, 1974 6. Colvin RB, Dvorak AM, Dvorak HF: Mast cells in the cortical tubular epithelium and interstitium in human renal disease. Hum Path 5:315-326, 1974 7. Padawer J: Ingestion of colloidal gold by mast cells. Proc Soc Exp Biol Med 129:905-907, 1968 8. Padawer J: Phagocytosis of particulate substances by mast cells. Lab Invest 25:320-330, 1971 9. Simson JV, Spicer SS: Ferritin particles in macrophages and in associated mast cells. J Cell Biol 52:536-541, 1972 10. Ishizaka K, Ishizaka T: Reversed type allergic skin reactions by anti-gamma-E globulin antibodies in humans and monkeys. J Immunol 100:554-562, 1968 11. Sheard P, Killingback PG, Blair AMJN:- Antigen induiced release of histamine and SRS-A from human lung passively sensitized with reaginic seruim. Natulre (Lond) 216:283-284, 1967 12. Parish WE: Release of histamine and slow reacting substance with mast cell changes after challenge of huiman lung sensitized passively with reagin in vitro. Nature (Lond) 215:738-739, 1967 13. Orange RP, Austen KF: Immunological release of the chemical mediators of anaphylaxis. Identification of Asthma, Ciba Fouindation Stuidy Grouip No. 38. Edited by R Porter, J Birch. Edinburgh, Churchill Livingstone, 1971 14. Greene WB, Spicer SS: Variability in mast cells of rat bone marrow. J Cell Biol 43:47a, 1969 (Abstr) 15. Karnovsky MJ: A formaldehyde-glutaraldehyde fixative of high osmolality for ulse in electron microscopy. J Cell Biol 27:137a-138a, 1965 (Abstr) 16. Graham RC Jr, Karnovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubuiles of mouise kidney: Ultrastrulctulral cytochemistry by a new technique. J Histochem Cytochem 14:291-302, 1966 17. Spicer SS: Siderosis associated with increased lipofuscins and mast cells in aging mice. Am J Pathol 37:457475, 1960 18. Spicer SS: Histochemical properties of mucopolysaccharide and basic protein in mast cells. Ann NY Acad Sci 103:322-333, 1963 19. Lagunoff D, Benditt EP: Proteolytic enzymes of mast cells. Ann NY Acad'Sci 103:185-198, 1963 20. Komiyama A, Spicer SS: Acid phosphatase demonstrated tultrastruictulrally in mast cell grantules altered by pinocytosis. Lab Invest 32:485491, 1975 21. Essner E: An electron microscopic study of erythrophagocytosis. J Biophys Biochim Cytol 7:329-334, 1960 22. Maloney MA, Patt HM, Lund JE: Granuilocyte dynamics and the quiestion of ineffective granulopoiesis. Cell Tiss Kinet 4:201-209, 1971
Acknowledgments The authors are grateful for the skilled technical and secretarial assistance of W. B. Greene and D. L. Smith.
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Legends for Figures All micrographs illustrate mast cells from bone marrow except Figure 21, which depicts a marrow macrophage, and Figures 7 and 28, which show mast cells from lymph nodes. The specimens illustrated were processed routinely for morphologic examination, except those for Figures 15 to 18, which were incubated for localization of peroxidase. The thin sections were stained with uranyl acetate following lead citrate.
Figure 1-Rounded to slightly irregular profiles of very dense granules fill the cytoplasm of this normal-appearing mast cell. The grains appear fairly uniform in structure. (Rat 6, x 11,700) Figure 2-Small Golgi-associated vesicles contain spherules (arrow) of a density and texture comparable to that of the matrix of nearby mast cell grains (x 8700)
Figure 3-Dense material extends from an erythrocyte toward a mast cell at an area of close contact between these cells enlarged in the inset. This blurred junction and the unique low density of the cytoplasmic grain at this area of contact indicate interaction between the cells. (Rat 2, x 7700; Inset, x approximately 10,700) Figure 4-A misshapen erythroid cell with long processes spreads over the surface of a mast cell and is molded to the surface of a neighboring erythrocyte (E). The cell applied to the mast cell appears less dense than its neighbors. (Rat 2, x 7800) Figure 5-A reticulocyte containing mitochondria, a centriole (arrow), ribosomes, and small vesicles protrudes into a mast cell. The cytoplasmic granules vary considerably in size and densitv. (Rat 2, x 9700) Figure 6-An irregular process of a reticulocyte protrudes into a mast cell in which the granules vary in density and size. Some indication of discharge of a granule is apparent in the region of contact (arrow). (Rat 1, x 12,500) Figure 7-The plasmalemma of this mast cell from a cervical lymph node extends around a somewhat irregular erythrocyte profile, apparently engulfing it A peripheral area from this cell (inset) reveals altered granules apparently in the process of exocytosis. (Rat 4, x 13,700; Inst, x 9400)
Figure 8-A large heterophagic vacuole in a mast cell contains material similar in texture, den-
sity, and homogeneity to neighboring erythrocytes. Some cytoplasmic granules bordering the inclusion (arrow) and a few elsewhere in the cytoplasm differ from the majority of the grains in their less dense, fine grained content (Rat 3, x 9800)
Figure 9-A heterophagic vacuole in a mast cell contains a somewhat irregular body laced with denser material. A few irregular small granules (arrows) distinctly different from the specific cytoplasmic granules of the mast cell border this inclusion. (Rat 1, x 12,200) Fgure 10-A portion of an inclusion like that of Figure 9, but from a neighboring mast cell, is also closely bordered by pleomorphic, small, moderately dense bodies (arrows) (x 16,200).
Figure 11-An inclusion consisting of uniform, moderately dense material lies near the plasmalemma in a cell which contains a similar inclusion close to the nucleus. (Rat 4, x 17,800)
Figure 12-The nucleus of this mast cell nearly encircles a heterophagic vacuole containing dense material. The paranuclear inclusions resemble that of the macrophage in Figure 20. (Rat 1, x 4900)
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Figure 13-Pleomorphic small structures reminiscent of those in macrophage vacuoles containing phagocytized leukocytes fill this mast cell heterophagic vacuole. (Rat 4, x 6600)
Figure 14-This mast cell contains three heterophagic inclusions, the largest and most ir-
regular of which consists mostly of amorphous, moderately dense material suggestive of erythroid stroma. A periphery of denser particles and membrane-limited inclusions, shown enlarged in the inset, could derive from ribosomes and mitochondria of a phagocytized reticulocyte. Two denser inclusions with uniform opacity slightly indent the larger inclusion. Such inclusions resemble somewhat those characteristically observed in bone marrow macrophages and, as in the latter, could arise from endocytosed erythroblast nuclei (compare with Figure 20). The cytoplasmic granules have unusually rounded profiles and lucent rims and, in some instances, appear enlarged. (x 9600; Inset, x 21,900)
Figure 15-Numerous small heterophagic vacuoles which enclose bodies resembling nearby extracellular bodies (arrows) occupy much of the mast cell's cytoplasm. The vacuolar and extracellular bodies resemble neighboring erythrocytes (E) in density and homogeneity and apparently constitute fragments of erythrocytes. The erythrocytes fail to evidence their intrinsic peroxidase, possibly because of inactivation by fixative or nonpenetration of substrate. The inclusions differ from mast cell granules in size and density. Whether the high density in the granules demonstrates peroxidase activity in these organelles remains uncertain because of the great density often encountered in the granules in control specimens incubated in substrate free medium. (Rat 2, x 7500) Figure 16-Small heterophagic vacuoles containing homogeneous bodies (arrows) lie within this mast cell. Morphologically similar bodies lie near but external to the mast cell at about 4, 6, and 11 o'clock. The density of the bodies inside and outside the mast cell resembles that of a neighboring erythrocyte (E). The density of the cytoplasmic granules, all of which appear normal morphologically (compare with Figure 1), greatly exceeds that of these bodies. The membrane delimiting the vacuole that contains two bodies shows continuity with the plasmalemma as evidenced in the inset. (Rat 2, x 7500; Inset, X 10,700) Figure 17-A mast cell displays small heterophagic vacuoles that contain material of moderate, uniform density comparable to that of erythroid cells. The inclusions measure two to three times larger than the mast cell granules which appear unaltered in structure (compare with Figure 1). (Rat 2, X 8400) Figure 18-In one of three similar peripheral areas of another mast cell, bodies of presumed erythroid origin and similar to those in Figure 16 largely fill an invagination of the plasmalemma and resemble a nearby extracellular body while differing markedly from the mast cell granules. (Rat 2, x 11,900)
Figure 19-A mast cell encloses several round homogeneous dense bodies (arrows) and a
larger nodular body. The bodies exceed the specific granules in size but resemble them in density in this routinely processed specimen; they cannot be identified with certainty as of endocytic origin. (Rat 4, x 11,200)
Figure 20-A marrow macrophage contains small dense bodies and irregular heterophagic vacuoles (arrows) together with a body that has a round profile and nearly uniform high density. This latter heterophagic inclusion, probably derived from an erythroblast nucleus, indents the macrophage nucleus as does a similar body in the mast cells of Figure 12. (Rat 1, x 7200) Figure 21-This one of two similar peripheral areas in a mast cell reveals several moderately dense bodies partially enveloped by processes from the mast cell. The bodies differ from
nearby erythrocytes (E) and cytoplasmic granules, their fuzzy border and variable content resembling that in granules of communal vacuoles (compare with Figure 22). The absence of heterophagic inclusions from the cell elsewhere favor interpreting them as granules in the process of exocytosis. (Rat 2, x 16,900)
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Figure 22-Cytoplasmic granules in various stages of disruption and loose flocculent material occupy communal vacuoles in a mast cell. The vacuole at the lower left opens to extracellular space. Cytoplasmic granules retaining their limiting membrane possess the usual uniform high density. A body resembling an unaltered cytoplasmic granule lies beside the cell at the bottom (arrow), suggesting that admission to a communal vacuole alters the granule structure more than exocytosis. (Rat 2, x 11,200)
Figure 23-At higher magnification, structures interpreted as disrupted granules lie in communal vacuoles in a mast cell. (Rat 5, x 25,000)
Figure 24-Communal vacuoles in the bottom half of this mast cell enclose profiles consistent with granules in stages of disruption as well as dispersed finely particulate matter. The remaining cytoplasmic granules vary markedly in size and density. Two enlarged profiles, probably representing altered granules, occupy a single space (arrow) and another similarly enlarged profile with less compact content (arrowhead) borders the large space (Rat 1, x 5300)
Figure 25-A vacuole near the center of an otherwise normal-appearing mast cell contains profiles consistent with altered cytoplasmic granules and a mass of similar flocculent material apparently comprised of coalesced granules. (Rat 1, x 8400)
Figure 26-This membrane-limited inclusion resembles that in Figure 25 but contains only uniform, finely particulate material. Such an indeterminate vacuole could arise through coalescence of disrupted granules or degradation of endocytosed matter (compare with Figure 21). Specific granules in the cytoplasm appear unaltered. (Rat 1, x 7800) Figure 27-These two moderately large, electron-lucent structures (arrows) cannot be identified as heterophagic bodies or altered or coalesced granules and are classed as inclusions of indeterminate type. (Rat 2, x 8800) Figure 28-A mast cell from a cervical lymph node contains an indeterminate vacuole filled with fine-grained, lucent matter. (Rat 6, x 15,000)
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The prevalence of mast cells infiltrating bone marrow of different rats varied widely. as did the staining properties and size of their cytoplasmic granules. Bone marrow mast cells from several rats revealed large membrane-limited inclusions which stained metachromatically or orthochromatically and resembled inclusions in some macrophages. Ultrastructurally, mast cells varied widely in content of uniform dense granules or enlarged granules with less dense. fine grained content. Some of the large inclusions observed ultrastructurally in mast cells were heterophagic vacuoles which contained ervthrocvtes or reticulocytes, or remnants from other phagocytized cells, possibly neutrophils or unidentified homogeneous material. Smaller bodies. interpreted as fragments of ervthrocytes, lay extracellularly near mast cells and occupied small, membrane-limited, heterophagic vacuoles in some mast cells. In other mast cells. communal vacuoles enclosed several specific cytoplasmic granules in various stages of disruption. The communal vacuoles occasionally opened to the extracellular space. A few large indeterminate vacuoles in mast cells contained amorphous flocculent matter which apparently derived either from coalescence of cvtoplasmic granules through fusion of granule membranes or from endocytosis. (Am J Pathol 80:481-498, 1975)
EVIDEN-CE HAS ACCU-NIULATED that. in addition to secreting their granules or the contents thereof.1 6 mast cells may also endocytose extrinsic material.7- Antigen binding to immuinoglobulin E (IgE) on the surface of mast cells 10 initiates release of vasoactive amines that mediate inflammatorv responses."-13 Mast cells have been shown to incorporate a varietv of particulates into their cvtoplasmic grantules. converting them to heterophagosomes.78 The presence of ferritin particles in rat mast cells closely adjacent to hemosiderin-laden phagocytes supports the interpretation that the cells are endocytic.9 In the present studvy mast cells in rat bone marrow and lymph nodes were examined tultrastnrcturallv for evidence of endocvtic activitv. The material examined provided several examples of phagocytosis of ervthroid cells as well as of other unidentified bodies by mast cells in some rats. A preliminary account of this studv has appeared." Materials and Methods Twentx- one adult albino rats of the Sprague-Dawlev. Fischer, or Wistar strains were obtained from commercial souirces and houised for several w-eeks to months in the local From the Department of Pathologv. Medical University of Souith Carolina. Charleston. Sou,th Carolina Supported by Grants AM-10956 and ANI-11028 from the National Institutes of Health and Training Grant TR-1681 from the Veterans Administration, Accepted for puiblication Mav 20. 19753 Address reprint requests to Dr S S Spicer. Department of Pathologv. Medical UnisersitN- of Souith Carolina. 80 Barre Street. Charleston. SC 29401 481
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animal facility. Rats 1 to 8 and 16 to 21 were maintained on a standard pellet diet. Rats 9 to 12 and 13 to 15 were Fischer rats maintained in a separate experiment on a Remington or Evans low iodine diet, respectively. The rats were sacrificed uinder anesthesia. Femoral marrow from Rats 1 to 15 was minced promptly in 2 to 6% gluitaraldehyde, buiffered with 0.1 M cacodylate at pH 7.2, and fixed for 90 minuites at 4 C in this soluition. Cervical lymph nodes from Rats 4 and 16 to 21 were fixed similarly. A portion of the bone marrow from Rat 2 was also fixed for 90 minutes in a 4% formaldehyde 2.5% glutaraldehyde mixture,15 and rinsed in buffered sucrose. The latter specimen was incubated for 30 minutes at 25 C in 3,3'-diaminobenzidine-H202 substrate medium for demonstration of peroxidase16 as a possible means of identifying erythroid cells by their content of peroxidase. Control tissues were incubated in suibstrate medium lacking the diaminobenzidine. All of the specimens were then postfixed for 1 hour at 24 C in 2% osmium tetroxide, buffered at pH 7.2 with 0.1 M cacodylate. This was followed by brief rinsing with water, routine dehydration through alcohols, and embedment in Epon. Thick epoxy sections of several blocks of each specimen were stained with alkaline ethanolic toluidine blue for light microscopic examination. Thin sections were prepared of bone marrow from Rats 1 to 6 and of lymph nodes from Rats 16 to 21. The sections of specimens processed for morphologic examination were stained with a uranyl acetate-lead citrate sequence and examined in an AEI-6B electron microscope. Thin sections of the portion of the specimen from Rat 2, incubated for peroxidase, were examined with and without uranyl acetate-lead citrate staining.
Results Light Microscopy
The abuindance of mast cells varied widely in the different bone marranging from 0.3 to 12.3 cells per oil immersion field (Table 1).The staining and morphologic characteristics of the granuiles differed markedly in the individual mast cells of some specimens and varied from one rows
Table 1-The Variable Prevalence and Structure of Mast Cells in Rat Bone Marrow Percent of mast cells With With over With over No. of mast 20% enlarged inclusions Mast cells 80% small cells/oil >2 u granules Rat granules observed immersion field 97 6.6 3 1 30 0.5 6.3 55 45 2 251 1.5 14 3.6 3.7 86 111 3 21.3 11 89 4 61 0.3 1 4.8 6.2 99 1335 5 3.8 17 83 4.2 264 6 17.6 75 17 25 7 0.3 62 38 6.9 0.5 29 8 1 19.0 99 12.3 530 9 14.4 1 99 5.1 382 10 17.5 33 171 5.3 66 11 5.3 18 82 10.4 12 785 8.3 20 80 528 8.6 13 6.5 94 6 9.9 14 596 5.2 34 66 4.9 228 15
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specimen to another. Many cells contained excluisivelv or predominantlv a population of abundant, fairlv uniform, rouind granuiles which resembled granules in mast cells of rat connective tissuies in that they were uiniform in size and stained dark purple with toluidine bluje. Nuimerouis cells possessed a variable proportion of granules which were moderately enlarged, stained relatively lightly, and were highly metachromatic. There was no obviouis correlation between the abuindance of mast cells in bone marrow and the extent to which mast cells in a given sample contained enlarged granules (Table 1). In addition to classic granuiles, the bone marrow mast cells occasionally disclosed incluisions (Table 1) which were rouighly two to five times the size of normal granules and either stained gray-bluie like neighboring ervthrocvtes or, more often, exhibited a metachromatic pink coloration. Occasional macrophages in these specimens enclosed gray-bluie inclusions like some in mast cells. Electron Microscopy
Many of the mast cells in the specimens examined uiltrastnrctuirallv contained predominantly uniform granuiles which were very dense, roughly spherical, and crowded together in the cvtoplasm (Figuire 1). Other mast cells contained such granules admixed with a variable proportion of enlarged granules whose content was often less dense and finelv grained. The latter granules apparently corresponded with the lighter staining, more metachromatic granules in toluiidine-bluie-stained thick sections. The proportion of cells with mostly small, dense granuiles varied in different specimens. Small vesicles containing sphenrles of densitv similar to that of the cytoplasmic granules often lay near Golgi zones in the latter cells (Figure 2). Some of the mast cells from each rat showed one or more unusual ultrastructural features (Table 2). In Rats 1 and 2, mast cells of the bone marrow appeared to lie in frequent and close approximation to ervthroblasts, reticulocytes, or erythrocytes over a variable distance (Figures 3 and 4). Erythroid cells in extensive contact with these mast cells often had an irregular profile characterized by long curved processes which conformed to the surface of the mast cell (Figure 4). Processes from suich irreguilar erythroid cells on occasion deeply indented the surface of some mast cells (Figures 5 and 6). Dense or filaimentous material occasionally extended inward from the blurred plasmalemma of erythroid cells where they contacted mast cells (Figure 3, Inset). The cytoplasmic granules in such cells varied widely in densitv. A profile of a mast cell from a lymph node of Animal 4 disclosed an irregular erythrocyte within a vacuole-like space which was delimited peripherally
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Table 2-Inclusions Observed Ultrastructurally in Mast Cells of Rat Bone Marrow and Lymph Nodes
Small
Rat
multiple Large heteorphagic heterophagic vacuoles vacuoles Homogeneous content consistent with Hetero- Of probable erythroid Communal geneous erythroid vacuoles content origin cells ++ + +++
Indeterminate vacuoles
Tissue 1 Bone marrow ++ ++ + ++ 2 Bone marrow + 3 Bone marrow 4 + + Bone marrow + Lymph node 5 Bone marrow 6 Bone marrow Lymph 16 node + to +++ = Estimated relative prevalence of structure, - = not encountered in terial examined.
+ +
-
+ + +
the ma-
by thin cytoplasmic processes (Figuire 7). Several grantules in the periphery of the latter mast cell were slightly enlarged and decreased in density, and appeared to be in a process of discharge from the cell (Figure 7, Inset). Mast cells from the bone marrow of Rats 1 to 6 and lymph nodes of Rats 4 and 16 occasionally contained one or more membrane-limited, rolundto-somewhat-irreguilar vactuoles measuiring 2 to 6 ,u in diameter. Suich vacuioles varied in abuindance, size, and content in different specimens (Table 2) and were interpreted as the uiltrastruictuiral couinterpart of the large metachromatic and orthochromatic inclutsions observed in thick sections. These mast cell incluisions couild be classified into folur basic types: a) large heterophagic (endocytic) vacuioles, b) small, muiltiple, heterophagic vacuoles, c) commuinal vactioles with coalesced grantules, or d) vacuioles of indeterminate natuire.
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Large Heterophagic Vacuoles
The large heterophagic vactuoles measuired tup to 5.0 g or more in diameter and contained material that obviouLslv had been endocvtosed (Figures 8-14). Occasional mast cells of Rats 1, 3, and 4 disclosed one to three of these vacuoles. The material differed in the vactuoles of different or even the same cell, varving in density and degree of heterogeneitv. Some of the vacuoles enclosed a large stnrctuire resembling an ervthrocvte (Figure 8) or smaller bodies consistent with an ervthroid derivation (Figure 11). Other such vacuoles enclosed similar bodies which contained infrequient vesicles and peripheral dense material consistent with degenerated mitochondria and ribosomes of reticuilocvtes (Figures 9, 10, 14). Infrequent vacuoles, generally bordering the nucleuis, possessed fairlv homogeneous material of high density (Figuires 12 and 14). Vacuioles encountered in a few marrow cells of Rats 1, 2, and 4 contained pleomorphic or heterogeneous material (Figure 13) reminiscent of that occasionallv encountered in macrophage vacuoles containing phagocytized leukocytes with their abundant granules. Occasional cells contained lip to three vacuolar profiles which sometimes resembled one another buit often differed in content (Figure 14). A few moderatelv dense, small granuiles measuring abouit 0.1 yI in diameter sometimes bordered the large heterophagic inclusions (Figures 9 and 10). Small Heterophagic Vacuoles
Small heterophagic vacuoles measuired tip to 2.5 u in diameter and contained endocytosed material. These vacuioles occturred in varving numbers in bone marrow mast cells of Rat 2 (Figuires 15-18). Thev generally exceeded neighboring cytoplasmic granuiles in size and were less dense than the granules, especiallv in specimens processed for peroxidase (Figures 15-18). In the latter, the incluisions resembled neighboring erythrocytes in density and textutre. Extracelluilar bodies similar in densitv and texture to both the incluisions and nearby ervthrocytes also bordered these mast cells (Figuires 15, 16, and 18) and occulpied invaginations of the cell periphery, apparently in an incipient stage of endocvtosis (Figuires 16-18). The small bodies in the vicinity of mast cells appeared to be fragments of erythrocytes which, after engulfment, became the muiltiple small mast cell inclusions. Incluisions observed in rouitinelv processed bone marrow from Rat 4 resembled the small heterophagic vactuoles of Rat 2 except that thev were less clearly distinguishable from the equiallv dense buit generallv smaller and more spherical cvtoplasmic granuiles (Figuire 19). Cytoplasmic granules in cells with small heterophagic vacuiloes were small
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and dense and disclosed no evident alteration. Other struictuires occulpying surface-connected spaces in the periphery of rat mast cells appeared rather to be grantules altered following exocytosis (Figuire 21). Inclusions in neighboring macrophages often resembled some of the heterophagic vacuoles of the mast cells. The uiniformly very dense macrophage inclusions, known to arise from ingestion of erythroblast nuclei, displayed striking similarity to some paranuiclear incluisions in mast cells (compare Figures 20 and 12). Communal Vacuoles
Communal vacuoles consisted of membrane-limited spaces which were partially filled by cytoplasmic granuiles in stages of degeneration and apparently resulted from fusion of the limiting membrane of several granules (Figures 22-25). These inclusions were encouintered in marrow cells of Rats 1, 2 and 5 (Table 2). The commuinal vacuioles occuipied extensive areas within otherwise unaltered mast cells. In a few instances, the vacuoles appeared continuouis with the extracelluilar space (Figulre 22). A mast cell profile in the marrow of Rat 1 enclosed a rouind, membranelimited vacuole containing crowded residuies interpreted as decomposing granuiles. At an adjacent level, the content of this vacuiole incluided also an' eccentric area composed of flocculent material like that in the adjoining disintegrated granules (Figuire 25). The floccuilent material in this instance was apparently formed from granule coalescence. It resembled the content in some of the vacuioles in the following category (compare Figuire 25 with Figuires 26-28). Vacuoles of Indeterminate Origin
Vacuoles of indeterminate origin encouintered in occasional mast cells in marrow of Rats 1, 2, and 4 (Figuires 26 and 27) and lymph node of Rat 16 (Figuire 28) afforded little evidence as to their origin. These moderately large, membrane-limited struictures contained finely partictulate, fairly homogenouis material of rather low density. Discussion
Althouigh mast cells have been fouind to incorporate exogenouis particles of widely varying size uinder experimental conditions,4 relatively little evidence has accumuilated for endocytic activity in the abuindant mast cells infiltra.ting variouis normal tisstues. These cells are often associated closely with phagocytes containing lipid and hemosiderin, especially in older animals.17 Such siderophage-associated mast cells have been shown histochemically to possess diminished content of acid muicosuibstance and
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stronglv basic protein."8 The content of ferritin in cytoplasm and granuiles of mast cells intimatelv bordering siderophages described previouislv9 attested to in vivo exchange between macrophage and mast cell and microendocvtic activity in the latter. The present findings demonstrate macroendocvtic activitv (i.e., uiptake of particles > 2000 A in diameter) by mast cells in bone marrow. Observation of stages of mast cell ingestion of ervthroid cells and of large mast cell incluisions composed of a generally uiniform material similar to that of erythroid cells implies that these cells are capable of endocvtosing whole ervthrocvtes. Some inclusions contained dense material at the border and membranouis internal struictures, interpreted as ribosomes and mitochondria of reticulocytes. That reticuilocvtes as well as ervthrocvtes may be engulfed was fuirther indicated by the intimate fuill approximation of reticulocytes to the mast cell surface and protrusion of reticulocyte processes into mast cells. Several inclusions in mast cells resembled some in macrophages in their uniform high density and frequient paranuiclear location. Such inclusion bodies appear similar to those which have been observed frequently in normal bone marrow and have been interpreted as ingested nuclei extruded from erythroblasts. In addition, occasional mast cells contained heterogenous structures, some of which were reminiscent of leukocvte granules (Figure 3). These heterophagic vactuoles suiggest that mast cells, on occasion, phagocvtose leuikocytes as well as red blood cells. The varied content of mast cell vacuoles possiblv reflects stages of digestion of ervthrocvtes or other endocvtosed material bv hvdrolases known to exist in mast cells."9 Suich digestion conceivably couild transform the endocvtosed component into material like that in indeterminate vactuoles. However, little evidence was obtained for depletion of the specific granuile population throuigh fuision with the heterophagic vacuioles in the way that neutrophil leuikocvte granuiles fuise with and admit hydrolases into phagocytic vacuioles. The granuiles in cells with muiltiple small inclusions appeared normal in nuimber and stnrctture and those in cells with large inclusions were apparentlv normal in abtindance, althouigh often altered in struieture. Transport of matrix content from cytoplasmic granuiles into phagocvtic vacuioles conceivablv couild occuir here without degranuilation. Loss of granule content withouit secretion or exocvtosis of granules apparently transpires in some hypersensitive states.5 6 However, cell mechanisms have not been described for transport of granuile content to phagocytic vacuioles or extracelluilar space withouit fuision between granuile membrane and the vacuiolar membrane or plasmalemma. The metachromatic staining of some of the large incluisions indicates they contain acid mucosuibstance which they presuimably acquiire bv some means
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from the heparin-rich granules. Alternatively, hydrolases may be transported to the vacuoles by Golgi-derived vesicles or the small dense bodies seen bordering vacuioles (Figures 9-11). Suich a souirce, i.e., primary lysosomes other than specific granuiles, was previouisly considered for the acid phosphatase demonstrable in mast cell granuiles that had been converted to heterophagosomes by tuptake of colloidal gold.20 That the mast cell grantules converted to heterophagosomes may acquiire enzymes which the unaltered specific granules lack was indicated by demonstration of acid phosphatase in the converted buit not in the tunaltered granuiles in endocytosing cells. Some mast cells showed smaller incluisions which differed from mast cell granules. The inclusions resembled extracelltular bodies suiggestive of fragmentated erythrocytic cells. Fragmentation of red blood cells conceivably cotuld occtur as a spontaneouis event restulting from an abnormality of the cells, as a consequence of the release of lysosomal hydrolases into intercelltular space, or as a restult of stepwise cleavage of parts of erythrocytes by mast cells or other endocytes. Fragmentation of red blood cells and ingestion of the fragments by macrophages has been described previously. 21 The erythrocytes observed in bone marrow mast cells in the present sttudy apparently were young rather than senescent cells, since they were part of the hematopoietic popuilation. The present evidence for phagocytosis of reticuilocytes fturther attests to the ingestion of yolung erythroid cells by mast cells of rats. Endocytosis of early erythrocytes couild occtur as a conseqtuence of excessive hematopoiesis. This possibility derives stupport from observations indicating excessive hematopoiesis and disposal of tip to 50% of the cells formed in normal marrow.22 Such compensatory erythrophagocytosis by mast cells seems uinlikely, however, becauise the prevalence of mast cell erythrophagocytosis was not high enough to account for appreciable disposal of red cells in the involved animals, and this process was not encountered in most of the rats examined. Perhaps, then, the phagocytosis of erythrocytes by mast cells results from an abnormality of the erythrocytes in the few animals in which the process was observed. Infection by microorganisms such as Haemobartonella, Eperythrozoon or Grahamella that are known to infest rodent erythrocytes cotuld alter the red blood cells and render them susceptible to phagocytosis by mast cells. Fuirther stuidies wouild be required to relate this process to a specific pathologic process. A nuimber of mast cells disclosed a different alteration that apparently consisted of fusion of the membrane delimiting several granuiles, with consequent sequiestration of these granuiles in one or more irreguilar vacuiolar
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spaces. Since these cells often lacked evidence of endocvtosed material, perhaps this change was unrelated to the erythrophagocvtic activity of neighboring mast cells. However, animals showing the apparent granuile coalescence within common vacuoles were those most clearly involved in phagocytosis of ervthrocvtes; therefore, the two processes appear in some way related. Extracellular attack on erythroid cells by exocytosed mast cell granules could account for their apparent extracelluilar fragmentation and play a role in the degradation of red cells. The nature of the vacuoles arising from coalescence of cytoplasmic granules remains a question. Their continuiity with the extracelluilar space affirms the interpretation previously derived from observations of granuiles lying free in surface-connected spaces2 that the granuiles are uindergoing exocvtosis. The vacuoles, on the other hand, couild contain extrinsic substance and constitute secondary Ivsosomes of a heterophagic natuire. The most central vacuoles lacking apparent connections with the suirface and containing largelv degenerated granuiles-and floccuilent material probably derived there from (Figure 23)-appear unconnected with ouitside space and more likely concerned with endocytosis than exocvtosis. Conceivably, the fusion of granules into commuinal vacuioles and suibsequent digestion of granule material in the vacuiole occuirs in response to microendocytic activity and entails digestion of material not visuialized in the cells because of its small size or minimal electron opacitv. The granules and the other content in the commuinal vacuioles in some cells appeared to undergo dissolution and become a floccuilent mass. The residue from coalescence of granules in a commuinal vacuiole cannot be clearly differentiated by morphologic appearance from that resuilting from digestion of endocytosed material. However, the similaritv of floccuilent material in a portion of some vacuioles to deteriorated granuiles in the same or other vacuoles perhaps identifies the uiniformly loose, floccuilent matter, often present in vacuoles, as a residuulm-in part, at least-of coalesced granules. Such aggregations possibly shrank with continuied coalescence and digestion into the small vacuoles of indeterminate origin seen in a few mast cells.
Referweces 1. Padawer J: The reaction of rat mast cells to polylysine. J Cell Biol 47:352-372, 1970 2. Lagunoff D: Membrane fusion during mast cell secretion. J Cell Biol 57:252-259, 1973 3. Moriyasu S, Yamura T: Electron microscopic studies of mast cell degranulation. Acta Derm Venereol [Suppl 73] (Stockh) 53:149-156, 1973
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4. Tasaka K, Yamasaki H: Local degranulation and histamine release from a single rat mast cell by microelectrophoretic application of-basic histamine releasers and antigen. Acta Derm Venereol [Suppl 73] (Stockh) 53:167-174, 1973 5. Uvntis B: Histamine storage and release. Fed Proc 33:2172-2176, 1974 6. Colvin RB, Dvorak AM, Dvorak HF: Mast cells in the cortical tubular epithelium and interstitium in human renal disease. Hum Path 5:315-326, 1974 7. Padawer J: Ingestion of colloidal gold by mast cells. Proc Soc Exp Biol Med 129:905-907, 1968 8. Padawer J: Phagocytosis of particulate substances by mast cells. Lab Invest 25:320-330, 1971 9. Simson JV, Spicer SS: Ferritin particles in macrophages and in associated mast cells. J Cell Biol 52:536-541, 1972 10. Ishizaka K, Ishizaka T: Reversed type allergic skin reactions by anti-gamma-E globulin antibodies in humans and monkeys. J Immunol 100:554-562, 1968 11. Sheard P, Killingback PG, Blair AMJN:- Antigen induiced release of histamine and SRS-A from human lung passively sensitized with reaginic seruim. Natulre (Lond) 216:283-284, 1967 12. Parish WE: Release of histamine and slow reacting substance with mast cell changes after challenge of huiman lung sensitized passively with reagin in vitro. Nature (Lond) 215:738-739, 1967 13. Orange RP, Austen KF: Immunological release of the chemical mediators of anaphylaxis. Identification of Asthma, Ciba Fouindation Stuidy Grouip No. 38. Edited by R Porter, J Birch. Edinburgh, Churchill Livingstone, 1971 14. Greene WB, Spicer SS: Variability in mast cells of rat bone marrow. J Cell Biol 43:47a, 1969 (Abstr) 15. Karnovsky MJ: A formaldehyde-glutaraldehyde fixative of high osmolality for ulse in electron microscopy. J Cell Biol 27:137a-138a, 1965 (Abstr) 16. Graham RC Jr, Karnovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubuiles of mouise kidney: Ultrastrulctulral cytochemistry by a new technique. J Histochem Cytochem 14:291-302, 1966 17. Spicer SS: Siderosis associated with increased lipofuscins and mast cells in aging mice. Am J Pathol 37:457475, 1960 18. Spicer SS: Histochemical properties of mucopolysaccharide and basic protein in mast cells. Ann NY Acad Sci 103:322-333, 1963 19. Lagunoff D, Benditt EP: Proteolytic enzymes of mast cells. Ann NY Acad'Sci 103:185-198, 1963 20. Komiyama A, Spicer SS: Acid phosphatase demonstrated tultrastruictulrally in mast cell grantules altered by pinocytosis. Lab Invest 32:485491, 1975 21. Essner E: An electron microscopic study of erythrophagocytosis. J Biophys Biochim Cytol 7:329-334, 1960 22. Maloney MA, Patt HM, Lund JE: Granuilocyte dynamics and the quiestion of ineffective granulopoiesis. Cell Tiss Kinet 4:201-209, 1971
Acknowledgments The authors are grateful for the skilled technical and secretarial assistance of W. B. Greene and D. L. Smith.
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Legends for Figures All micrographs illustrate mast cells from bone marrow except Figure 21, which depicts a marrow macrophage, and Figures 7 and 28, which show mast cells from lymph nodes. The specimens illustrated were processed routinely for morphologic examination, except those for Figures 15 to 18, which were incubated for localization of peroxidase. The thin sections were stained with uranyl acetate following lead citrate.
Figure 1-Rounded to slightly irregular profiles of very dense granules fill the cytoplasm of this normal-appearing mast cell. The grains appear fairly uniform in structure. (Rat 6, x 11,700) Figure 2-Small Golgi-associated vesicles contain spherules (arrow) of a density and texture comparable to that of the matrix of nearby mast cell grains (x 8700)
Figure 3-Dense material extends from an erythrocyte toward a mast cell at an area of close contact between these cells enlarged in the inset. This blurred junction and the unique low density of the cytoplasmic grain at this area of contact indicate interaction between the cells. (Rat 2, x 7700; Inset, x approximately 10,700) Figure 4-A misshapen erythroid cell with long processes spreads over the surface of a mast cell and is molded to the surface of a neighboring erythrocyte (E). The cell applied to the mast cell appears less dense than its neighbors. (Rat 2, x 7800) Figure 5-A reticulocyte containing mitochondria, a centriole (arrow), ribosomes, and small vesicles protrudes into a mast cell. The cytoplasmic granules vary considerably in size and densitv. (Rat 2, x 9700) Figure 6-An irregular process of a reticulocyte protrudes into a mast cell in which the granules vary in density and size. Some indication of discharge of a granule is apparent in the region of contact (arrow). (Rat 1, x 12,500) Figure 7-The plasmalemma of this mast cell from a cervical lymph node extends around a somewhat irregular erythrocyte profile, apparently engulfing it A peripheral area from this cell (inset) reveals altered granules apparently in the process of exocytosis. (Rat 4, x 13,700; Inst, x 9400)
Figure 8-A large heterophagic vacuole in a mast cell contains material similar in texture, den-
sity, and homogeneity to neighboring erythrocytes. Some cytoplasmic granules bordering the inclusion (arrow) and a few elsewhere in the cytoplasm differ from the majority of the grains in their less dense, fine grained content (Rat 3, x 9800)
Figure 9-A heterophagic vacuole in a mast cell contains a somewhat irregular body laced with denser material. A few irregular small granules (arrows) distinctly different from the specific cytoplasmic granules of the mast cell border this inclusion. (Rat 1, x 12,200) Fgure 10-A portion of an inclusion like that of Figure 9, but from a neighboring mast cell, is also closely bordered by pleomorphic, small, moderately dense bodies (arrows) (x 16,200).
Figure 11-An inclusion consisting of uniform, moderately dense material lies near the plasmalemma in a cell which contains a similar inclusion close to the nucleus. (Rat 4, x 17,800)
Figure 12-The nucleus of this mast cell nearly encircles a heterophagic vacuole containing dense material. The paranuclear inclusions resemble that of the macrophage in Figure 20. (Rat 1, x 4900)
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Figure 13-Pleomorphic small structures reminiscent of those in macrophage vacuoles containing phagocytized leukocytes fill this mast cell heterophagic vacuole. (Rat 4, x 6600)
Figure 14-This mast cell contains three heterophagic inclusions, the largest and most ir-
regular of which consists mostly of amorphous, moderately dense material suggestive of erythroid stroma. A periphery of denser particles and membrane-limited inclusions, shown enlarged in the inset, could derive from ribosomes and mitochondria of a phagocytized reticulocyte. Two denser inclusions with uniform opacity slightly indent the larger inclusion. Such inclusions resemble somewhat those characteristically observed in bone marrow macrophages and, as in the latter, could arise from endocytosed erythroblast nuclei (compare with Figure 20). The cytoplasmic granules have unusually rounded profiles and lucent rims and, in some instances, appear enlarged. (x 9600; Inset, x 21,900)
Figure 15-Numerous small heterophagic vacuoles which enclose bodies resembling nearby extracellular bodies (arrows) occupy much of the mast cell's cytoplasm. The vacuolar and extracellular bodies resemble neighboring erythrocytes (E) in density and homogeneity and apparently constitute fragments of erythrocytes. The erythrocytes fail to evidence their intrinsic peroxidase, possibly because of inactivation by fixative or nonpenetration of substrate. The inclusions differ from mast cell granules in size and density. Whether the high density in the granules demonstrates peroxidase activity in these organelles remains uncertain because of the great density often encountered in the granules in control specimens incubated in substrate free medium. (Rat 2, x 7500) Figure 16-Small heterophagic vacuoles containing homogeneous bodies (arrows) lie within this mast cell. Morphologically similar bodies lie near but external to the mast cell at about 4, 6, and 11 o'clock. The density of the bodies inside and outside the mast cell resembles that of a neighboring erythrocyte (E). The density of the cytoplasmic granules, all of which appear normal morphologically (compare with Figure 1), greatly exceeds that of these bodies. The membrane delimiting the vacuole that contains two bodies shows continuity with the plasmalemma as evidenced in the inset. (Rat 2, x 7500; Inset, X 10,700) Figure 17-A mast cell displays small heterophagic vacuoles that contain material of moderate, uniform density comparable to that of erythroid cells. The inclusions measure two to three times larger than the mast cell granules which appear unaltered in structure (compare with Figure 1). (Rat 2, X 8400) Figure 18-In one of three similar peripheral areas of another mast cell, bodies of presumed erythroid origin and similar to those in Figure 16 largely fill an invagination of the plasmalemma and resemble a nearby extracellular body while differing markedly from the mast cell granules. (Rat 2, x 11,900)
Figure 19-A mast cell encloses several round homogeneous dense bodies (arrows) and a
larger nodular body. The bodies exceed the specific granules in size but resemble them in density in this routinely processed specimen; they cannot be identified with certainty as of endocytic origin. (Rat 4, x 11,200)
Figure 20-A marrow macrophage contains small dense bodies and irregular heterophagic vacuoles (arrows) together with a body that has a round profile and nearly uniform high density. This latter heterophagic inclusion, probably derived from an erythroblast nucleus, indents the macrophage nucleus as does a similar body in the mast cells of Figure 12. (Rat 1, x 7200) Figure 21-This one of two similar peripheral areas in a mast cell reveals several moderately dense bodies partially enveloped by processes from the mast cell. The bodies differ from
nearby erythrocytes (E) and cytoplasmic granules, their fuzzy border and variable content resembling that in granules of communal vacuoles (compare with Figure 22). The absence of heterophagic inclusions from the cell elsewhere favor interpreting them as granules in the process of exocytosis. (Rat 2, x 16,900)
MAST CELL PHAGOCYTOSIS
Vol. 80, No. 3 September 1975
493
Figure 22-Cytoplasmic granules in various stages of disruption and loose flocculent material occupy communal vacuoles in a mast cell. The vacuole at the lower left opens to extracellular space. Cytoplasmic granules retaining their limiting membrane possess the usual uniform high density. A body resembling an unaltered cytoplasmic granule lies beside the cell at the bottom (arrow), suggesting that admission to a communal vacuole alters the granule structure more than exocytosis. (Rat 2, x 11,200)
Figure 23-At higher magnification, structures interpreted as disrupted granules lie in communal vacuoles in a mast cell. (Rat 5, x 25,000)
Figure 24-Communal vacuoles in the bottom half of this mast cell enclose profiles consistent with granules in stages of disruption as well as dispersed finely particulate matter. The remaining cytoplasmic granules vary markedly in size and density. Two enlarged profiles, probably representing altered granules, occupy a single space (arrow) and another similarly enlarged profile with less compact content (arrowhead) borders the large space (Rat 1, x 5300)
Figure 25-A vacuole near the center of an otherwise normal-appearing mast cell contains profiles consistent with altered cytoplasmic granules and a mass of similar flocculent material apparently comprised of coalesced granules. (Rat 1, x 8400)
Figure 26-This membrane-limited inclusion resembles that in Figure 25 but contains only uniform, finely particulate material. Such an indeterminate vacuole could arise through coalescence of disrupted granules or degradation of endocytosed matter (compare with Figure 21). Specific granules in the cytoplasm appear unaltered. (Rat 1, x 7800) Figure 27-These two moderately large, electron-lucent structures (arrows) cannot be identified as heterophagic bodies or altered or coalesced granules and are classed as inclusions of indeterminate type. (Rat 2, x 8800) Figure 28-A mast cell from a cervical lymph node contains an indeterminate vacuole filled with fine-grained, lucent matter. (Rat 6, x 15,000)
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