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A novel cell-to-cell interaction between mast cells and other cell types
Experimental Cell Research 147 (1983) 1-13
Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any fom reserved CO14-4827/83$3.C0
A Novel Cell-to-Cell Interaction Between Mast Cells and other Cell Types GARY GREENBERG*
and GEOFFREY
BURNSTOCK
University College London, Department of Anatomy and Embryology, London, UK
SUMMARY Time-lapse cinephotomicrography and transmission electron microscopy (TEM) have been used to study the interactions between rat mast cells and different cell monolayers in culture (tibroblasts, vascular endothelial cells and cardiac muscle cells). This report documents a novel form of behavior between mast cells and certain -other cell types. We have tentatively termed this cellular behavior ‘transgranulation’, which involves sequential changes not seen in control cells, including: (1) formation of a granule-containing mast cell pseudopod that becomes closely applied to an adjacent cell; (2) development of specialized plasma membrane interrelationships between apposing cells; (3) alteration of granules and perigranular membranes within the mast cell pseudopod; (4) occasional transfer of exocytosed mast cell granules to the cytoplasm of the adjacent cell; (5) presence of a specialized inclusion body in the mast cell; and finally, (6) either withdrawal of the pseudopod by the mast cell, or casting-off of the pseudopod from the mast cell, leaving it on the surface of the adjacent cell (pseudopod translocation). These mast cell interactions occur specifically with tibroblasts and endothelial cells in vitro and are never observed with cardiac muscle cells or non-cellular substrates. Our investigations of rat mesenteries in situ confirm that these cell-cell interactions also occur in vivo. We suggest it represents a form of cell-to-cell communication involving secretion from a mast cell pseudopod to another cell type. The significance of specialized contacts between mast cells and other cell types in vivo is discussed.
Mast cells are widely distributed throughout the connective tissues of vertebrates, particularly around small blood vessels, nerves and glandular ducts as well as mucosal, serosal and cutaneous surfaces [l]. They respond to mechanical, chemical and immunological stimuli [2] by secreting a large number of biologically active substances, including histamine, serotonin (in rodents), heparin, proteolytic enzymes, platelet-activating factor, neutrophil chemotactic factors, eosinophi1 chemotactic factors, leukotrienes and prostaglandins [3, 41. Mast cells have been shown to interact with a number of different cell types, both in vitro and in vivo, including tibroblasts [5], vascular endothelial cells [6, 71, epithelial cells [8, 91, lymphocytes [lO-121, macrophages [131, neutrophils [14], eosinophils [15, 161,nerve cells [17, 181and cancer cells [19]; however, the mechanisms that underlie these cell-cell interactions are not well understood. Although degranulation (exocytosis of granules) is associated with mast cell secretion during pathological conditions [203,it is now thought that there may be several release mechanisms in mast cells that do not involve the degranulation process [21-241. It has been shown that during secretion, extracellular fluid is allowed access to the granules through a labyrinth of cavities, formed by the fusion of perigranular and plasma membranes and fusion of adjacent perigranular membranes; in this way, soluble mast cell products are released from the granules while they remain within the confines of the cell. * Present address: Laboratory for Developmental Biology, Program in Craniofacial Biology, University of Southern California, University Park-MC-0191, Los Angeles, CA 90089-0191, USA.
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The purpose of this study was to investigate mechanisms of communication between mast cells and other cell types by examining the behavior and ultrastructure of individual mast cells as they interact with different cellular monolayers in short-term cell culture. Our data suggest that mast cells interact specifically with other cell types through the formation of specialized cell-cell contacts.
MATERIALS
AND METHODS
Mast cells Mast cells were obtained from peritoneal washings of adult Wistar rats (approx. 200 g) following 6-ml intraperitoneal injections of saline solution. Separation of mast cells from other peritoneal elements was achieved by density centrifugation. Peritoneal fluid washings were gently layered onto sterile discontinuous density gradients consisting of 1 ml layers of 30 % and then 20 % human serum albumin (Calbiochem, San Diego, Calif.) in a glass centrifuge tube and spun at 900 rpm (6-inch radius) for 8 min. The pellet of mast cells was washed and then resuspended in medium 199 (Gibco, Grand Island, N.Y .) This procedure yielded a 90 % pure population of viable mast cells.
Cellular
monolayers
Primary cultures of fibroblasts, endothelial cells and cardiac muscle cells were obtained from dissections of newborn rat hearts. The hearts were chopped into 1 mm cubes, rinsed in culture medium and then placed in trypsin (from hog pancreas, Nutritional Biochemical Corp., Cleveland, Ohio; 0.0125 g/ml), for 5 min at 37°C. The tissue was agitated with a pipette and the solution was mixed with an equal amount of fetal calf serum (FCS) to neutralize the trypsin. The solution was then spun at 900 rpm for 5 min (the pellet contained mostly fibroblasts). The 1 mm cubes of tissue were trypsinized two more times by the previous method and the solution was then spun at 900 rpm for 5 min (the pellet from the second and third trypsinization contained a larger population of cardiac muscle cells and endothelial cells than the first trypsinization). The cells were resuspended in Eagle’s medium with 10% FCS, then plated into modified Rose chambers and incubated at 37°C. After 3w5 min, the fibroblasts could be seen adhering to the collagen-coated substrate (dialysed rat tail collagen) and the chambers were then inverted so that the cardiac muscle cells and endothelial cells would fall to the opposite coverslip and attach there.
Mast cellicell
monolayer
co-cultures
The primary cultures were grown to confluence, at which time they contained approx. 250000 cells. Approx. 250000 mast cells were then suspended in 2 ml of culture medium and injected into each culture chamber containing the cellular monolayers. The mast cells were allowed to settle on the monolayers for 1 h at 37°C and the chambers were then inverted so that the non-adherent cells and debris would fall to the opposite coverslip. The medium (medium 199 in Hanks salts with L-glutamine and 10% FCS) was then changed and the chambers were maintained in a cell-side-up position at 37°C for the remainder of the experiment. A total of 77 mast cell/cell monolayer co-cultures were prepared and divided into two treatment groups: (1) for light microscope observations over a 12-h period in vitro (using 16 mm time-lapse and 35 mm stills); and (2) for electron microscope observations after 3,6 and 12 h in vitro. In addition, 12 control cultures were prepared which contained peritoneal mast cells plated onto non-cellular substrates (plastic, glass and collagen-coated glass).
Light microscopy A Zeiss Photomicroscope with phase contrast and Nomarski optics was used with planapo lenses (x63, x40 and x25). The microscope stage was maintained at 37°C with an air curtain incubator. The behavior of individual mast cells was recorded using 16 mm time-lapse techniques. Equipment included a Bolex H16 SBM tine camera, a Wild 50 mm lens with focusing telescope and light meter, a Paillard-Wild MBF-A motor, MBF-B variometer control unit and MBF-C variometer timer. An 0.2set shutter speed was used with a film speed of one frame every 8 sec. The light source was covered by an automatic shutter between each exposure. Kodak Ektachrome infrared film IE 449 was used (specially ordered from Eastman Kodak Co., N.Y .) and was processed in ME-4 developer.
Mast cell interactions
EXD Cell Res 147 (1983)
Transmission
electron microscopy
(TEM)
The coverslips containing the cells were fixed in 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer with 5 % sucrose for 30 min, post-fixed in 1% osmium tetroxide for 30 min, block-stained in 1% aqueous uranyl acetate for 30 min and dehydrated in ethanol. The preparations were embedded in Araldite, and after curing, the glass coverslips were removed from the embedded cells using concentrated hydrofluoric acid. Preselected fields of view were then drawn to scale with a microscope drawing attachment. These drawings were used as maps for locating and orienting the selected cells when tine-trimming the blocks in the microtome. Serial sections (90 nm thick) of selected cells were cut with a glass knife on a Reichert Microtome. The sections were placed on 200-hole copper grids and stained with Reynolds lead citrate for 15 min. Specimens were observed using a Philips 301 electron microscope with tilting goniometer stage.
Examination
of mast cells in vivo
Adult Wistrar rats (200 g) were sacrificed by cervical dislocation. The samll intestines were dissected out and fixed in 2.5% glutaraldehyde for 1 h. The mesenteries were then trimmed away from the intestines and mesenteric spreads were prepared for both light and electron microscopy in order to determine if transgranulation occurs in vivo.
RESULTS In addition to occasional mast cell degranulation and subsequent phagocytosis of the discharged granules by monolayer cells and macrophages, another form of interaction was observed between mast cells and cellular monolayers which we have tentatively termed ‘transgranulation’. In all 77 experiments with mast cell/cell monolayer co-cultures, it was estimated that transgranulation occurred in l-3 % of the mast cells present in each culture after 6 h in vitro. These mast cell interactions occurred specifically with fibroblasts and vascular endothelial cells, and were never observed with cardiac muscle cells or in control cultures that contained mast cells plated onto non-cellular substrates (plastic, glass and collagen-coated glass). The following report describes these specific, heterotypic cell-cell interactions. Pseudopod formation
and granule extrusion
into the pseudopod
The interactions began with the formation of a mast cell pseudopod that flattened against the ventral surface of an adjacent cell (i.e., between the monolayer and
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Fig. 2. Photomicrographs of transgranulation in vitro (Nomarski optics). (a) A mast cell (bright,
round cell) extrudes granules into its specialized pseudopod (arrow) which remains flattened against the surface of an adjacent fibroblast. (b) The same cells 2 h later. The mast cell has cast off its flattened pseudopod and left it on the surface of the adjacent tibroblast as a membrane-bound packet of granules (arrow). x650. Fig. 3. Electron micrographs of transgranulation in vitro. (a) A granule-filled mast cell pseudopod forms along the ventral surface of an adjacent fibroblast (i.e., between the fibroblast and the collagen substrate). N, Fibroblast nucleus. (b) Higher magnification shows a series of intercellular contacts between the apposing cell surfaces (arrows). These intermediate type intercellular junctions are associated with a build-up of cytoplasmic microfilament bundles. (a) ~3 500; (b) x 18500.
Mast cell interactions
Exp Cell Res 147 (1983)
the substrate) (fig. 1). Initially, this pseudopod was devoid of granules and grew to a length of IO-20 urn. This was followed by extrusion of mast cell granules into the flattened pseudopod, which remained closely applied to the adjacent cell surface (figs 2 a, 4, 5). The granule-filled pseudopod grew to a length of up to 40 urn. Time-lapse 16 mm data indicated that the rate of pseudopod formation and granule extrusion occurred over a period of l-3 h. Specialized plasma membrane interrelationships
Specialized intercellular contacts were demonstrated between the mast cell pseudopod and the surface of the adjacent cell. These contacts were characterized by a close apposition of plasma membranes (10-U nm) and were associated with a build-up of cytoplasmic microfilament bundles in both cells (fig. 3). The contacts were most consistently seen where the apical tip of the fibroblast met the mast cell. Specialized contacts at other points between the mast cell pseudopod and the ventral surface of the adjacent monolayer cell appeared to occur at discrete spots, separated by spaces with no specialized membrane appositions. However, an examination of serial sections indicated that the spots of contact seen in thin sections consisted of converging and diverging lines of contact, forming a network of specialized membrane appositions between the two adjacent cell surfaces. Granule alterations
and perigranular
membrane changes
Time-lapse tine observations revealed the occasional, sudden swelling (alteration) of individual granules, which remained within the mast cell pseudopod (fig. 4), whereas the majority of granules within the pseudopod appeared unaltered (with the light microscope, the altered granules appeared large and pale, whereas the unaltered granules appeared small and dark). However, as viewed with the electron microscope, the ‘unaltered’ granules within the pseudopod appeared to undergo progressive, partial alteration: the granule matrix changed from being dense and homogenous to becoming less dense and more particulate. This was accompanied by vacuolation and separation of the perigranular membranes from the granules, and was often followed by fusion of adjacent perigranular membranes (figs 3, 6). Transfer of granules
Time-lapse 16 mm data showed that some of the altered granules within the mast cell pseudopod were finally exocytosed. However, rather than being discharged into the culture medium, these granules were immediately endocytosed by the adjacent cell (fig. 5, arrows). Granule exocytosis and subsequent endocytosis occurred quickly, from 1-2 sec. Electron microscopic data indicated that the granules were transferred to the cytoplasm of the adjacent cell where they were individually enclosed by an endocytotic membrane (fig. 8 a). Specialized
mast cell inclusion
body
Observations with the electron microscope revealed a unique inclusion body, unlike anything previously described in rat mast cells. It was composed of a tine
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Exp Cell Res 147(1983)
Fig. 4. Photomicrograph (Nomarski optics) of granule alteration in vitro. A single granule within the mast cell pseudopod appears swollen (arrow); 16 mm cind observations show that this type of granule alteration occurs suddenly. x 1400.
Mast cell interactions
EXD Cell Res 147 (1983)
tubular material, sparsely embedded within an amorphous granular matrix, approx. 1.5 urn in diameter and devoid of a limiting perigranular membrane (fig. 6). It was located adjacent to a well developed Golgi apparatus and numerous mitochondria. This specialized inclusion body was observed in 90% of mast cells during transgranulation (18 out of 20 cells from electron microscopic serial sections). In contrast, it was found in only 3 out of 100 control mast cells. Pseudopod
translocation
After several hours, the granule-filled pseudopod was either withdrawn by the mast cell, or it was cast-off from the mast cell and left on the surface of the adjacent cell as a membrane-bound packet of granules (fig. 2 b). These packets contained a variable number of mast cell granules, from just a few to over a hundred (fig. 7). The cast-off packets remained flattened against the adjacent cell surface, usually just one granule-layer thick. Light and electron microscopy showed that the granules within these packets became increasingly disrupted (partially altered), and separation and fusion of the perigranular membranes became more pronounced as time progressed. Following pseudopod translocation, the mast cell remained viable and assumed its formerly spherical shape; furthermore, it often migrated away from its shed packet of granules (see fig. 7a). Morphological
changes in adjacent cell
Electron micrographs revealed that the adjacent cell often contained endocytotic vacuoles and vesicles, particularly on the side of the cell adjacent to the interacting mast cell (figs 3,6, 8 b). At the light microscope level, usually no conspicuous changes occurred in the adjacent cell. Occasionally, however, the adjacent fibroblast or endothelial cell retracted suddenly away from the mast cell. Following these occurrences, the mast cell withdrew its granule-filled pseudopod and resumed a spherical shape. Transgranulation
in vivo
In order to determine if transgranulation occurs in vivo, we investigated the mast cells in mesenteric spreads. Light microscope observations of rat intestinal mesenteries revealed numerous mast cells in situ with long, granule-filled pseudopodia, flattened against the surface of adjacent connective tissue cells. Electron micrographs of these interacting cells in vivo revealed the same characteristic features found during transgranulation in vitro, including specialized membrane contacts between the apposing cell surfaces; the presence of the specialized mast cell inclusion body; partial alteration of granules; and occasionally, the presence of individual mast cell granules within the cytoplasm of the adjacent cell (fig. 8 a), as well as endocytotic vesicles within the adjacent cell (fig. 8 b).
Fig. 5. Photomicrograph of the transfer of individual granules from specialized mast cell pseudopodia
to adjacent tibroblasts in vitro. Cine observations show that swollen granules are exocytosed from mast cell pseudopodia and immediately endocytosed by the adjacent cell. (arrows). (a) Phase contrast optics. (b) In contrast to transgranulation (arrows), a degranulating mast cell (open arrow) sheds its exocytosed granules into the culture medium (Normarski optics) (a) x800; (b) x650.
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Fig. 6. Electron micrograph of a unique mast cell inclusion body during transgranulation in vitro. The
specialized inclusion (see *) consists of a tine tubular material embedded within an amorphous granular matrix, approx. 1.5 urn in diameter and is devoid of a limiting perigranular membrane. N, Mast cell nucleus; M, mitochondria; G, Golgi apparatus; F, adjacent tibroblast. ~25 500. Fig. 7. Photomicrographs in vitro show that cast-off packets of mast cell granules form plaques on the surface of adjacent cells. (a) A mast cell leaves a small membrane-bound packet of granules (open arrow) on an adjacent fibroblast. A retraction fiber (arrow) traces the migration of the mast cell, following pseudopod translocation (Nomarski optics). (b) A mast cell leaves a large packet of granuels on the surface of an adjacent fibroblast. Note that some of the swollen granules have been endocytosed by the tibroblast (arrows) (phase contrast optics). (a) x650; (b) x 1000.
(1983)
Exp Cell
Res 147(1983)
Mast cell interactions
Fig. 8. Electron micrographs of transgranulation in vivo in rat mesentery. (a) Mast cell granules can be seen within the cytoplasm of an adjacent connective tissue cell (nrrows), whereas serial sections of the same cells show no evidence that granules had been shed into the extracellular space. Open arrow, Mast cell; N, connective tissue cell nucleus. (b) Endocytotic vesicles (arrows) occur in a connective tissue cell, adjacent to an interacting mast cell. (a) x 11500; (6) X21 500.
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DISCUSSION This report documents a novel sequence of specialized interactions between mast cells and other cell types, which we tentatively term ‘transgranulation’. Behavioral and ultrastructural data from in vitro experiments using mast cell/cell monolayer co-cultures demonstrate that mast cells specifically interact with fibroblasts and vascular endothelial cells, whereas this behavior does not occur with cardiac muscle cells or in control cultures containing mast cells plated onto non-cellular substrates. In addition, investigations of rat mesenteries confirm that this behavior, which results in specialized cell-cell contacts between mast cells and other cell types, occurs in vivo (fig. 8) as well as in vitro. Furthermore, the conspicuous morphology, characteristic of transgranulation, has been reported in previous in vivo studies of mast cells. Shed packets of mast cell granules have been observed at the light microscope level in rat calvarial periosteum [25] and in rat mesenteric spreads in response to experimental lipemia [26]; and at the ultrastructural level, close contacts between mast cell processes and fibroblasts have been observed in human nasopharyngeal fibromas [27] and between mast cell processes and nerve tibres in human solitary glomus tumors [ 18, 281. We propose that the interactions described in this report represent a specialized form of cell-to-cell communication involving secretion. First, the formation of a specialized mast cell pseudopod increases the surface area contact between the interacting cell types, bringing a large number of mast cell granules into close association with the adjacent cell (figs 2-5), which otherwise would not occur, since mast cells are usually spherical in shape. Ultrastructural evidence of specialized contacts between the adjacent cell membranes during transgranulation (fig. 3) supports the view that a specific cell-to-cell interaction is taking place. Electron microscopic serial sections show that these intermediate type intercellular junctions form converging and diverging lines of close contact between the adjacent cells. They are distinct from other types of intercellular junctions such as tight junctions, gap junctions and desmosomes [29]. The membrane contacts formed during transgranulation resemble intercellular junctions described between aggregating cells in reaggregation experiments with heterotypic cell suspensions [30], between adjacent tibroblasts during the early stages of contact inhibition of movement [31], and between lymphocytes and target cells during the initial stages of cell-mediated cytotoxicity [32]. By analogy, the ultrastructural similarities observed during the mast cell interactions suggest that the specialized contacts mediate adhesion between adjacent cells and function during pseudopod formation, and in addition, they create a common microenvironment between the interacting cells. The morphological data presented in this report support the notion that mast cell secretion occurs in the specialized pseudopod. The sudden swelling (alteration) of individual granules within the pseudopod (fig. 4) suggests the focal release of mast cell amines around the area of contact with the adjacent cell. Although the granule remains within the pseudopod it is known that histamine secretion can occur in mast cells, without degranulation, through small pores formed by fusion of the plasma membrane with the perigranular membrane [22,
Exp Cell Res 147 (1983)
Mast cell interactions
241. In addition, focal secretion from mast cells has been demonstrated in vitro by other investigators [33]. Furthermore, the progressive, partial alteration of granules and formation of perigranular vacuoles within the pseudopod, seen with electron microscopy (figs 3 b, 6) is also suggestive of mast cell secretion during transgranulation. Electron microscope autoradiographic studies using [3H]histamine have shown that partially altered granules contain a moderate amount of histamine in comparison to fully altered granules which are nearly devoid of histamine [34]. In addition, the ultrastructure of the partially altered granules is morphologically similar to granules that have been exposed to a hypertonic salt solution, which extracts proteins from the granule matrix [35]. Thus, it is likely that a moderate amount of histamine, as well as a portion of the granule protein, is being released from the granules during transgranulation, possibly via perigranular vacuoles. Ultrastructural data plus time-lapse tine observations indicate that the granule constituents continue to dissolve after the pseudopod has been cast-off by the mast cell and left on the surface of the adjacent cell (figs 2 b, 7). This suggests a prolonged form of secretion, somewhat analogous to a ‘timed-release capsule’. Time-lapse tine observations demonstrate that some of the granules are exocytosed from the mast cell pseudopod and immediately endocytosed by the adjacent cell (fig. 5). Furthermore, electron microscopic data confirm that the granules are transferred to the cytoplasm of the adjacent cell, where they are individually surrounded by an endocytotic membrane (fig. 8a). In addition to the transfer of granules, ultrastructural evidence of endocytotic vacuoles and vesicles in the adjacent cell suggests that the interacting cells actively take-up soluble mast cell substances by pinocytosis (figs 3 b, 8 b). The function of the unique mast cell inclusion body (fig. 6) described in this report, is not as yet known; however, it is tempting to speculate that this cellular inclusion is involved in the synthesis of specialized secretory substances (several potent products are generated de novo by mast cells and are not associated with the storage granules) [3, 41. The proximity of the inclusion body to mitochondria and a well developed Golgi apparatus is consistent with this possibility. The dramatic increase in percentage of mast cells containing the specialized inclusion body during cell-to-cell interactions in vitro, as compared to controls, suggests a process that may involve the selection of a pre-existing subpopulation of mast cells, distinguished by the presence of this inclusion body. In conclusion, we propose that as the mast cell pseudopod moves across the adjacent cell surface, the specialized intercellular contacts hold the cells firmly together, while the network of close plasma membrane appositions create a common microenvironment between the cells. The focal secretion of substances into this common microenvironment might function to protect them from rapid diffusion and/or degradation by extracellular enzymes, thus facilitating the adjacent cell to take-up the substances more effectively. Substances secreted into this common microenvironment might mediate communication by binding to specific receptors on the apposing cell surface and/or by cleavage of adjacent plasma membrane proteins by mast cell proteolytic enzymes. It is known that several different cell types have histamine receptors (termed H1 and H2 receptors) which
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Exp Cell Res 147(1983)
mediate a wide variety of physiological and pathological responses [lo, 36, 371. Since the local concentration of histamine is critical in determining the functional responses of these histamine-receptor-bearing cells, the discrete release of histamine from mast cells during contact with other cell types may serve to mediate specific cell-to-cell communication, without resulting in a substantial release of histamine into the extracellular milieu, which would lead to an inflammatory response. Mast cells reside adjacent to many other cell types in vivo and are known to release substances that affect their behavior. Therefore, the selection of which cell types make intimate contact with mast cells under different physiological and pathological conditions is fundamental towards understanding mast cell functions. Thus, the factors that control adhesion between mast cells and other cell types may be essential in the modulation of mast cell functions in vivo. We thank George Barrett for his excellent technical assistance, and Dr J. Mongar and Dr M. Rosendaal for their advise and constructive criticism.
REFERENCES 1. 2. 3. 4.
Selye, H, The mast cells. Butterworth, Washington DC (1965). Kazimierczak, W & Diamont, B, Progr allergy 24 (1978) 295. Lewis, R & Austen, K, Nature 293 (1981) 103. Wasserman, S, The mast cell: its role in health and disease (ed J Pepys & A Edwards) p. 9. Pitman Med. Publ. Co, London (1979). 5. Norrby, K, Virchows arch B cell path01 38 (1981) 57. 6. Majno, G & Palade, G, J biophys biochem cytol 11 (1961) 571. 7. Azizkhan, R, Azizkhan, J, Zetter, B & Folkman, J, J exp med 152 (1980) 931. 8. Bamett, M, J ultrastruct res 43 (1973) 247. 9. Murray, M, Miller, H R P & Jarret, W F H, Lab invest 19 (1968) 222. 10. Askenase, P, Progr allergy 23 (1977) 199. 11. Rocklin, R, J clin invest 57 (1976) 1051. 12. Roszkowski, W, Plaut, M & Lichtenstein, L, Science 195 (1977) 683. 13. Lindah, U, Pertoft, H & Seljelid, R, Biochem j 182 (1979) 189. 14. Wasserman, S, Soter, N, Center, D & Austen, K, J clin invest 60 (1977) 189. 15. Baggiolini, M, Horisberger, U & Martin, U, Int arch allergy appl immunol 67 (1982) 219. 16. Boswell, R, Austen, K & Goetzl, E, J immunol 120 (1978) 15. 17. Kieman, J, Quart j exp physiol60 (1975) 123. 18. Wiesner-Menzel, L, Schulz, B, Vakilzadeh, F & Czametski, B, Acta dermatovener 61 (1981) 465. 19. Alexander, P, The mast cell: its role in health and disease (ed J Pepys & A Edwards) pp. 137. Pitman Med. Publ. Co, London (1979). 20. Asboe-Hansen, G, Acta dermatol venereol, suppl. 73 (1973) 139. 21. Padawer, J, The mast cell: its role in health and disease (ed J Pepys & A Edwards) p. 1. Pitman Med. Publ. Co., London (1979). 22. Lagunoff, D, Biochem pharmachol21 (1972) 1889. 23. Ginsburg, H, Nir, I, Hammell, I, Eren, R, Weissman, B & Naot, Y, Immunology 35 (1978) 485. 24. Rohlich, P, Anderson, P & Uvnas, B, J cell biol 51 (1971) 465. 25. Selye, H, Gabbiani, G & Nielsen, K, Proc sot exp biol 112 (1963) 460. 26. Hill, M, Nature 180 (1957) 654. 27. Arnold, W & Huth, F, Virchows Arch B cell path01 379 (1978) 285. 28. Kimura, S, Acta dermatovener 59 (1979) 415. 29. Staehelin, L, Int rev cytol 39 (1974) 191. 30. Armstrong, P, J cell bio147 (1970) 197. 31. Heaysman, J & Pegrum, S, Exp cell res 78 (1973) 71. 32. Matter, A, Immunology 36 (1979) 179. 33. Lawson, D, Fewtrell, C & Raff, M, J cell bio179 (1978) 394.
Exr, Cell Res 147 (1983)
34. 35. 36. 37.
Anderson, P & Uvnls, B, Acta physiol Stand 94 (1975) 63. Lagunoff, D, J invest dermatol58 (1972) 296. Beaven, M, New Engl j med 294 (1976) 30 & 320. Hirschowitz, B, Ann rev pharmacol 19 (1979) 203.
Received November 18, 1982 Revised version received March 11, 1983
Printed
in Sweden
Mast cell interactions
13
Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any fom reserved CO14-4827/83$3.C0
A Novel Cell-to-Cell Interaction Between Mast Cells and other Cell Types GARY GREENBERG*
and GEOFFREY
BURNSTOCK
University College London, Department of Anatomy and Embryology, London, UK
SUMMARY Time-lapse cinephotomicrography and transmission electron microscopy (TEM) have been used to study the interactions between rat mast cells and different cell monolayers in culture (tibroblasts, vascular endothelial cells and cardiac muscle cells). This report documents a novel form of behavior between mast cells and certain -other cell types. We have tentatively termed this cellular behavior ‘transgranulation’, which involves sequential changes not seen in control cells, including: (1) formation of a granule-containing mast cell pseudopod that becomes closely applied to an adjacent cell; (2) development of specialized plasma membrane interrelationships between apposing cells; (3) alteration of granules and perigranular membranes within the mast cell pseudopod; (4) occasional transfer of exocytosed mast cell granules to the cytoplasm of the adjacent cell; (5) presence of a specialized inclusion body in the mast cell; and finally, (6) either withdrawal of the pseudopod by the mast cell, or casting-off of the pseudopod from the mast cell, leaving it on the surface of the adjacent cell (pseudopod translocation). These mast cell interactions occur specifically with tibroblasts and endothelial cells in vitro and are never observed with cardiac muscle cells or non-cellular substrates. Our investigations of rat mesenteries in situ confirm that these cell-cell interactions also occur in vivo. We suggest it represents a form of cell-to-cell communication involving secretion from a mast cell pseudopod to another cell type. The significance of specialized contacts between mast cells and other cell types in vivo is discussed.
Mast cells are widely distributed throughout the connective tissues of vertebrates, particularly around small blood vessels, nerves and glandular ducts as well as mucosal, serosal and cutaneous surfaces [l]. They respond to mechanical, chemical and immunological stimuli [2] by secreting a large number of biologically active substances, including histamine, serotonin (in rodents), heparin, proteolytic enzymes, platelet-activating factor, neutrophil chemotactic factors, eosinophi1 chemotactic factors, leukotrienes and prostaglandins [3, 41. Mast cells have been shown to interact with a number of different cell types, both in vitro and in vivo, including tibroblasts [5], vascular endothelial cells [6, 71, epithelial cells [8, 91, lymphocytes [lO-121, macrophages [131, neutrophils [14], eosinophils [15, 161,nerve cells [17, 181and cancer cells [19]; however, the mechanisms that underlie these cell-cell interactions are not well understood. Although degranulation (exocytosis of granules) is associated with mast cell secretion during pathological conditions [203,it is now thought that there may be several release mechanisms in mast cells that do not involve the degranulation process [21-241. It has been shown that during secretion, extracellular fluid is allowed access to the granules through a labyrinth of cavities, formed by the fusion of perigranular and plasma membranes and fusion of adjacent perigranular membranes; in this way, soluble mast cell products are released from the granules while they remain within the confines of the cell. * Present address: Laboratory for Developmental Biology, Program in Craniofacial Biology, University of Southern California, University Park-MC-0191, Los Angeles, CA 90089-0191, USA.
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The purpose of this study was to investigate mechanisms of communication between mast cells and other cell types by examining the behavior and ultrastructure of individual mast cells as they interact with different cellular monolayers in short-term cell culture. Our data suggest that mast cells interact specifically with other cell types through the formation of specialized cell-cell contacts.
MATERIALS
AND METHODS
Mast cells Mast cells were obtained from peritoneal washings of adult Wistar rats (approx. 200 g) following 6-ml intraperitoneal injections of saline solution. Separation of mast cells from other peritoneal elements was achieved by density centrifugation. Peritoneal fluid washings were gently layered onto sterile discontinuous density gradients consisting of 1 ml layers of 30 % and then 20 % human serum albumin (Calbiochem, San Diego, Calif.) in a glass centrifuge tube and spun at 900 rpm (6-inch radius) for 8 min. The pellet of mast cells was washed and then resuspended in medium 199 (Gibco, Grand Island, N.Y .) This procedure yielded a 90 % pure population of viable mast cells.
Cellular
monolayers
Primary cultures of fibroblasts, endothelial cells and cardiac muscle cells were obtained from dissections of newborn rat hearts. The hearts were chopped into 1 mm cubes, rinsed in culture medium and then placed in trypsin (from hog pancreas, Nutritional Biochemical Corp., Cleveland, Ohio; 0.0125 g/ml), for 5 min at 37°C. The tissue was agitated with a pipette and the solution was mixed with an equal amount of fetal calf serum (FCS) to neutralize the trypsin. The solution was then spun at 900 rpm for 5 min (the pellet contained mostly fibroblasts). The 1 mm cubes of tissue were trypsinized two more times by the previous method and the solution was then spun at 900 rpm for 5 min (the pellet from the second and third trypsinization contained a larger population of cardiac muscle cells and endothelial cells than the first trypsinization). The cells were resuspended in Eagle’s medium with 10% FCS, then plated into modified Rose chambers and incubated at 37°C. After 3w5 min, the fibroblasts could be seen adhering to the collagen-coated substrate (dialysed rat tail collagen) and the chambers were then inverted so that the cardiac muscle cells and endothelial cells would fall to the opposite coverslip and attach there.
Mast cellicell
monolayer
co-cultures
The primary cultures were grown to confluence, at which time they contained approx. 250000 cells. Approx. 250000 mast cells were then suspended in 2 ml of culture medium and injected into each culture chamber containing the cellular monolayers. The mast cells were allowed to settle on the monolayers for 1 h at 37°C and the chambers were then inverted so that the non-adherent cells and debris would fall to the opposite coverslip. The medium (medium 199 in Hanks salts with L-glutamine and 10% FCS) was then changed and the chambers were maintained in a cell-side-up position at 37°C for the remainder of the experiment. A total of 77 mast cell/cell monolayer co-cultures were prepared and divided into two treatment groups: (1) for light microscope observations over a 12-h period in vitro (using 16 mm time-lapse and 35 mm stills); and (2) for electron microscope observations after 3,6 and 12 h in vitro. In addition, 12 control cultures were prepared which contained peritoneal mast cells plated onto non-cellular substrates (plastic, glass and collagen-coated glass).
Light microscopy A Zeiss Photomicroscope with phase contrast and Nomarski optics was used with planapo lenses (x63, x40 and x25). The microscope stage was maintained at 37°C with an air curtain incubator. The behavior of individual mast cells was recorded using 16 mm time-lapse techniques. Equipment included a Bolex H16 SBM tine camera, a Wild 50 mm lens with focusing telescope and light meter, a Paillard-Wild MBF-A motor, MBF-B variometer control unit and MBF-C variometer timer. An 0.2set shutter speed was used with a film speed of one frame every 8 sec. The light source was covered by an automatic shutter between each exposure. Kodak Ektachrome infrared film IE 449 was used (specially ordered from Eastman Kodak Co., N.Y .) and was processed in ME-4 developer.
Mast cell interactions
EXD Cell Res 147 (1983)
Transmission
electron microscopy
(TEM)
The coverslips containing the cells were fixed in 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer with 5 % sucrose for 30 min, post-fixed in 1% osmium tetroxide for 30 min, block-stained in 1% aqueous uranyl acetate for 30 min and dehydrated in ethanol. The preparations were embedded in Araldite, and after curing, the glass coverslips were removed from the embedded cells using concentrated hydrofluoric acid. Preselected fields of view were then drawn to scale with a microscope drawing attachment. These drawings were used as maps for locating and orienting the selected cells when tine-trimming the blocks in the microtome. Serial sections (90 nm thick) of selected cells were cut with a glass knife on a Reichert Microtome. The sections were placed on 200-hole copper grids and stained with Reynolds lead citrate for 15 min. Specimens were observed using a Philips 301 electron microscope with tilting goniometer stage.
Examination
of mast cells in vivo
Adult Wistrar rats (200 g) were sacrificed by cervical dislocation. The samll intestines were dissected out and fixed in 2.5% glutaraldehyde for 1 h. The mesenteries were then trimmed away from the intestines and mesenteric spreads were prepared for both light and electron microscopy in order to determine if transgranulation occurs in vivo.
RESULTS In addition to occasional mast cell degranulation and subsequent phagocytosis of the discharged granules by monolayer cells and macrophages, another form of interaction was observed between mast cells and cellular monolayers which we have tentatively termed ‘transgranulation’. In all 77 experiments with mast cell/cell monolayer co-cultures, it was estimated that transgranulation occurred in l-3 % of the mast cells present in each culture after 6 h in vitro. These mast cell interactions occurred specifically with fibroblasts and vascular endothelial cells, and were never observed with cardiac muscle cells or in control cultures that contained mast cells plated onto non-cellular substrates (plastic, glass and collagen-coated glass). The following report describes these specific, heterotypic cell-cell interactions. Pseudopod formation
and granule extrusion
into the pseudopod
The interactions began with the formation of a mast cell pseudopod that flattened against the ventral surface of an adjacent cell (i.e., between the monolayer and
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Fig. 2. Photomicrographs of transgranulation in vitro (Nomarski optics). (a) A mast cell (bright,
round cell) extrudes granules into its specialized pseudopod (arrow) which remains flattened against the surface of an adjacent fibroblast. (b) The same cells 2 h later. The mast cell has cast off its flattened pseudopod and left it on the surface of the adjacent tibroblast as a membrane-bound packet of granules (arrow). x650. Fig. 3. Electron micrographs of transgranulation in vitro. (a) A granule-filled mast cell pseudopod forms along the ventral surface of an adjacent fibroblast (i.e., between the fibroblast and the collagen substrate). N, Fibroblast nucleus. (b) Higher magnification shows a series of intercellular contacts between the apposing cell surfaces (arrows). These intermediate type intercellular junctions are associated with a build-up of cytoplasmic microfilament bundles. (a) ~3 500; (b) x 18500.
Mast cell interactions
Exp Cell Res 147 (1983)
the substrate) (fig. 1). Initially, this pseudopod was devoid of granules and grew to a length of IO-20 urn. This was followed by extrusion of mast cell granules into the flattened pseudopod, which remained closely applied to the adjacent cell surface (figs 2 a, 4, 5). The granule-filled pseudopod grew to a length of up to 40 urn. Time-lapse 16 mm data indicated that the rate of pseudopod formation and granule extrusion occurred over a period of l-3 h. Specialized plasma membrane interrelationships
Specialized intercellular contacts were demonstrated between the mast cell pseudopod and the surface of the adjacent cell. These contacts were characterized by a close apposition of plasma membranes (10-U nm) and were associated with a build-up of cytoplasmic microfilament bundles in both cells (fig. 3). The contacts were most consistently seen where the apical tip of the fibroblast met the mast cell. Specialized contacts at other points between the mast cell pseudopod and the ventral surface of the adjacent monolayer cell appeared to occur at discrete spots, separated by spaces with no specialized membrane appositions. However, an examination of serial sections indicated that the spots of contact seen in thin sections consisted of converging and diverging lines of contact, forming a network of specialized membrane appositions between the two adjacent cell surfaces. Granule alterations
and perigranular
membrane changes
Time-lapse tine observations revealed the occasional, sudden swelling (alteration) of individual granules, which remained within the mast cell pseudopod (fig. 4), whereas the majority of granules within the pseudopod appeared unaltered (with the light microscope, the altered granules appeared large and pale, whereas the unaltered granules appeared small and dark). However, as viewed with the electron microscope, the ‘unaltered’ granules within the pseudopod appeared to undergo progressive, partial alteration: the granule matrix changed from being dense and homogenous to becoming less dense and more particulate. This was accompanied by vacuolation and separation of the perigranular membranes from the granules, and was often followed by fusion of adjacent perigranular membranes (figs 3, 6). Transfer of granules
Time-lapse 16 mm data showed that some of the altered granules within the mast cell pseudopod were finally exocytosed. However, rather than being discharged into the culture medium, these granules were immediately endocytosed by the adjacent cell (fig. 5, arrows). Granule exocytosis and subsequent endocytosis occurred quickly, from 1-2 sec. Electron microscopic data indicated that the granules were transferred to the cytoplasm of the adjacent cell where they were individually enclosed by an endocytotic membrane (fig. 8 a). Specialized
mast cell inclusion
body
Observations with the electron microscope revealed a unique inclusion body, unlike anything previously described in rat mast cells. It was composed of a tine
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Fig. 4. Photomicrograph (Nomarski optics) of granule alteration in vitro. A single granule within the mast cell pseudopod appears swollen (arrow); 16 mm cind observations show that this type of granule alteration occurs suddenly. x 1400.
Mast cell interactions
EXD Cell Res 147 (1983)
tubular material, sparsely embedded within an amorphous granular matrix, approx. 1.5 urn in diameter and devoid of a limiting perigranular membrane (fig. 6). It was located adjacent to a well developed Golgi apparatus and numerous mitochondria. This specialized inclusion body was observed in 90% of mast cells during transgranulation (18 out of 20 cells from electron microscopic serial sections). In contrast, it was found in only 3 out of 100 control mast cells. Pseudopod
translocation
After several hours, the granule-filled pseudopod was either withdrawn by the mast cell, or it was cast-off from the mast cell and left on the surface of the adjacent cell as a membrane-bound packet of granules (fig. 2 b). These packets contained a variable number of mast cell granules, from just a few to over a hundred (fig. 7). The cast-off packets remained flattened against the adjacent cell surface, usually just one granule-layer thick. Light and electron microscopy showed that the granules within these packets became increasingly disrupted (partially altered), and separation and fusion of the perigranular membranes became more pronounced as time progressed. Following pseudopod translocation, the mast cell remained viable and assumed its formerly spherical shape; furthermore, it often migrated away from its shed packet of granules (see fig. 7a). Morphological
changes in adjacent cell
Electron micrographs revealed that the adjacent cell often contained endocytotic vacuoles and vesicles, particularly on the side of the cell adjacent to the interacting mast cell (figs 3,6, 8 b). At the light microscope level, usually no conspicuous changes occurred in the adjacent cell. Occasionally, however, the adjacent fibroblast or endothelial cell retracted suddenly away from the mast cell. Following these occurrences, the mast cell withdrew its granule-filled pseudopod and resumed a spherical shape. Transgranulation
in vivo
In order to determine if transgranulation occurs in vivo, we investigated the mast cells in mesenteric spreads. Light microscope observations of rat intestinal mesenteries revealed numerous mast cells in situ with long, granule-filled pseudopodia, flattened against the surface of adjacent connective tissue cells. Electron micrographs of these interacting cells in vivo revealed the same characteristic features found during transgranulation in vitro, including specialized membrane contacts between the apposing cell surfaces; the presence of the specialized mast cell inclusion body; partial alteration of granules; and occasionally, the presence of individual mast cell granules within the cytoplasm of the adjacent cell (fig. 8 a), as well as endocytotic vesicles within the adjacent cell (fig. 8 b).
Fig. 5. Photomicrograph of the transfer of individual granules from specialized mast cell pseudopodia
to adjacent tibroblasts in vitro. Cine observations show that swollen granules are exocytosed from mast cell pseudopodia and immediately endocytosed by the adjacent cell. (arrows). (a) Phase contrast optics. (b) In contrast to transgranulation (arrows), a degranulating mast cell (open arrow) sheds its exocytosed granules into the culture medium (Normarski optics) (a) x800; (b) x650.
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Fig. 6. Electron micrograph of a unique mast cell inclusion body during transgranulation in vitro. The
specialized inclusion (see *) consists of a tine tubular material embedded within an amorphous granular matrix, approx. 1.5 urn in diameter and is devoid of a limiting perigranular membrane. N, Mast cell nucleus; M, mitochondria; G, Golgi apparatus; F, adjacent tibroblast. ~25 500. Fig. 7. Photomicrographs in vitro show that cast-off packets of mast cell granules form plaques on the surface of adjacent cells. (a) A mast cell leaves a small membrane-bound packet of granules (open arrow) on an adjacent fibroblast. A retraction fiber (arrow) traces the migration of the mast cell, following pseudopod translocation (Nomarski optics). (b) A mast cell leaves a large packet of granuels on the surface of an adjacent fibroblast. Note that some of the swollen granules have been endocytosed by the tibroblast (arrows) (phase contrast optics). (a) x650; (b) x 1000.
(1983)
Exp Cell
Res 147(1983)
Mast cell interactions
Fig. 8. Electron micrographs of transgranulation in vivo in rat mesentery. (a) Mast cell granules can be seen within the cytoplasm of an adjacent connective tissue cell (nrrows), whereas serial sections of the same cells show no evidence that granules had been shed into the extracellular space. Open arrow, Mast cell; N, connective tissue cell nucleus. (b) Endocytotic vesicles (arrows) occur in a connective tissue cell, adjacent to an interacting mast cell. (a) x 11500; (6) X21 500.
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DISCUSSION This report documents a novel sequence of specialized interactions between mast cells and other cell types, which we tentatively term ‘transgranulation’. Behavioral and ultrastructural data from in vitro experiments using mast cell/cell monolayer co-cultures demonstrate that mast cells specifically interact with fibroblasts and vascular endothelial cells, whereas this behavior does not occur with cardiac muscle cells or in control cultures containing mast cells plated onto non-cellular substrates. In addition, investigations of rat mesenteries confirm that this behavior, which results in specialized cell-cell contacts between mast cells and other cell types, occurs in vivo (fig. 8) as well as in vitro. Furthermore, the conspicuous morphology, characteristic of transgranulation, has been reported in previous in vivo studies of mast cells. Shed packets of mast cell granules have been observed at the light microscope level in rat calvarial periosteum [25] and in rat mesenteric spreads in response to experimental lipemia [26]; and at the ultrastructural level, close contacts between mast cell processes and fibroblasts have been observed in human nasopharyngeal fibromas [27] and between mast cell processes and nerve tibres in human solitary glomus tumors [ 18, 281. We propose that the interactions described in this report represent a specialized form of cell-to-cell communication involving secretion. First, the formation of a specialized mast cell pseudopod increases the surface area contact between the interacting cell types, bringing a large number of mast cell granules into close association with the adjacent cell (figs 2-5), which otherwise would not occur, since mast cells are usually spherical in shape. Ultrastructural evidence of specialized contacts between the adjacent cell membranes during transgranulation (fig. 3) supports the view that a specific cell-to-cell interaction is taking place. Electron microscopic serial sections show that these intermediate type intercellular junctions form converging and diverging lines of close contact between the adjacent cells. They are distinct from other types of intercellular junctions such as tight junctions, gap junctions and desmosomes [29]. The membrane contacts formed during transgranulation resemble intercellular junctions described between aggregating cells in reaggregation experiments with heterotypic cell suspensions [30], between adjacent tibroblasts during the early stages of contact inhibition of movement [31], and between lymphocytes and target cells during the initial stages of cell-mediated cytotoxicity [32]. By analogy, the ultrastructural similarities observed during the mast cell interactions suggest that the specialized contacts mediate adhesion between adjacent cells and function during pseudopod formation, and in addition, they create a common microenvironment between the interacting cells. The morphological data presented in this report support the notion that mast cell secretion occurs in the specialized pseudopod. The sudden swelling (alteration) of individual granules within the pseudopod (fig. 4) suggests the focal release of mast cell amines around the area of contact with the adjacent cell. Although the granule remains within the pseudopod it is known that histamine secretion can occur in mast cells, without degranulation, through small pores formed by fusion of the plasma membrane with the perigranular membrane [22,
Exp Cell Res 147 (1983)
Mast cell interactions
241. In addition, focal secretion from mast cells has been demonstrated in vitro by other investigators [33]. Furthermore, the progressive, partial alteration of granules and formation of perigranular vacuoles within the pseudopod, seen with electron microscopy (figs 3 b, 6) is also suggestive of mast cell secretion during transgranulation. Electron microscope autoradiographic studies using [3H]histamine have shown that partially altered granules contain a moderate amount of histamine in comparison to fully altered granules which are nearly devoid of histamine [34]. In addition, the ultrastructure of the partially altered granules is morphologically similar to granules that have been exposed to a hypertonic salt solution, which extracts proteins from the granule matrix [35]. Thus, it is likely that a moderate amount of histamine, as well as a portion of the granule protein, is being released from the granules during transgranulation, possibly via perigranular vacuoles. Ultrastructural data plus time-lapse tine observations indicate that the granule constituents continue to dissolve after the pseudopod has been cast-off by the mast cell and left on the surface of the adjacent cell (figs 2 b, 7). This suggests a prolonged form of secretion, somewhat analogous to a ‘timed-release capsule’. Time-lapse tine observations demonstrate that some of the granules are exocytosed from the mast cell pseudopod and immediately endocytosed by the adjacent cell (fig. 5). Furthermore, electron microscopic data confirm that the granules are transferred to the cytoplasm of the adjacent cell, where they are individually surrounded by an endocytotic membrane (fig. 8a). In addition to the transfer of granules, ultrastructural evidence of endocytotic vacuoles and vesicles in the adjacent cell suggests that the interacting cells actively take-up soluble mast cell substances by pinocytosis (figs 3 b, 8 b). The function of the unique mast cell inclusion body (fig. 6) described in this report, is not as yet known; however, it is tempting to speculate that this cellular inclusion is involved in the synthesis of specialized secretory substances (several potent products are generated de novo by mast cells and are not associated with the storage granules) [3, 41. The proximity of the inclusion body to mitochondria and a well developed Golgi apparatus is consistent with this possibility. The dramatic increase in percentage of mast cells containing the specialized inclusion body during cell-to-cell interactions in vitro, as compared to controls, suggests a process that may involve the selection of a pre-existing subpopulation of mast cells, distinguished by the presence of this inclusion body. In conclusion, we propose that as the mast cell pseudopod moves across the adjacent cell surface, the specialized intercellular contacts hold the cells firmly together, while the network of close plasma membrane appositions create a common microenvironment between the cells. The focal secretion of substances into this common microenvironment might function to protect them from rapid diffusion and/or degradation by extracellular enzymes, thus facilitating the adjacent cell to take-up the substances more effectively. Substances secreted into this common microenvironment might mediate communication by binding to specific receptors on the apposing cell surface and/or by cleavage of adjacent plasma membrane proteins by mast cell proteolytic enzymes. It is known that several different cell types have histamine receptors (termed H1 and H2 receptors) which
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mediate a wide variety of physiological and pathological responses [lo, 36, 371. Since the local concentration of histamine is critical in determining the functional responses of these histamine-receptor-bearing cells, the discrete release of histamine from mast cells during contact with other cell types may serve to mediate specific cell-to-cell communication, without resulting in a substantial release of histamine into the extracellular milieu, which would lead to an inflammatory response. Mast cells reside adjacent to many other cell types in vivo and are known to release substances that affect their behavior. Therefore, the selection of which cell types make intimate contact with mast cells under different physiological and pathological conditions is fundamental towards understanding mast cell functions. Thus, the factors that control adhesion between mast cells and other cell types may be essential in the modulation of mast cell functions in vivo. We thank George Barrett for his excellent technical assistance, and Dr J. Mongar and Dr M. Rosendaal for their advise and constructive criticism.
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Received November 18, 1982 Revised version received March 11, 1983
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