Microdomains of high calcium are not required for exocytosis in RBL-2H3 mucosal mast cells

We have previously shown that store-associated microdomains of high Ca(2+) are not essential for exocytosis in RBL-2H3 mucosal mast cells. We have now examined whether Ca(2+) microdomains near the plasma membrane are required, by comparing the secretory responses seen when Ca(2+) influx was elicited by two very different mechanisms. In the first, antigen was used to activate the Ca(2+) release-activated Ca(2+) (CRAC) current (I(CRAC)) through CRAC channels. In the second, a Ca(2+) ionophore was used to transport Ca(2+) randomly across the plasma membrane. Since store depletion by Ca(2+) ionophore will also activate I(CRAC), different means of inhibiting I(CRAC) before ionophore addition were used. Ca(2+) responses and secretion in individual cells were compared using simultaneous indo-1 microfluorometry and constant potential amperometry. Secretion still takes place when the increase in intracellular Ca(2+) occurs diffusely via the Ca(2+) ionophore, and at an average intracellular Ca(2)+ concentration that is no greater than that observed when Ca(2+) entry via CRAC channels triggers secretion. Our results suggest that microdomains of high Ca(2+) near the plasma membrane, or associated with mitochondria or Ca(2+) stores, are not required for secretion. Therefore, we conclude that modest global increases in intracellular Ca(2+) are sufficient for exocytosis in these nonexcitable cells.     

ATP inhibits onset of exocytosis in permeabilised mast cells

ATP is not required for exocytosis from permeabilised mast cells, and therefore there is no direct role for protein phosphorylation in the late stages of the activation pathway. We have measured the timecourse of exocytosis from permeabilised cells triggered to release hexosaminidase following addition of Ca²⁺ to cells equilibrated for 2 min with GTP--S. If ATP is included at the time of permeabilisation, then exocytosis commences after a delay, the duration of which depends on the square root of the product [Ca²⁺][GTP--S], and which may extend to beyond 3 min. When ATP is excluded then the maximal rate of exocytosis is established within 3 secs of completing the effector combination. These results suggest that the achievement of a new steady-state, induced by Ca²⁺ and GTP--S, and required for exocytosis is inhibited by ATP. From this we conclude that dephosphorylation of an unknown regulator protein may comprise a step in the exocytotic pathway.     

The dual effector system for exocytosis in mast cells: Obligatory requirement for both Ca2+ and GTP

The secretory process is a coordinated cellular response, initiated by occupation of surface receptors and comprising an ordered sequence of biochemical steps subject to multiple controls. Conceptually we can divide the sequence into two main sections comprising early, receptor-mediated events leading to generation of intracellular second messengers, and later events leading to membrane fusion and exocytosis. With the discovery that occupation of Ca²⁺ mobilising receptors leads to activation of polyphosphoinositide phosphodiesterase (PPI-pde) through the mediation of a G-protein (Gp), all the early events can be ascribed to the plasma membrane. Investigation of the exocytotic stage of secretion has been simplified by the use of permeabilised cells in which the composition of the cytosol can be precisely controlled. We have used streptolysin-O, a bacterial cytolysin which generates protein-sized pores in the plasma membrane, to investigate the exocytotic mechanism of rat mast cells. We find that in addition to the activation of PPI-dpe, GTP also acts in concert with Ca²⁺ at, or close to, the exocytotic site. Exocytosis can occur after substantial depletion of cytosol lactate dehydrogenase and 3-phosphoglycerate kinase indicating that soluble cytosol proteins are unlikely to play any role. There is no absolute requirement for ATP or phosphorylating nucleotide in exocytosis though when present the effective affinities of the two obligatory effectors (i.e. Ca²⁺ and GTP) are substantially enhanced.     

Washout phenomena in dialyzed mast cells allow discrimination of different steps in stimulus-secretion coupling

Transient increases of intracellular calcium and exocytotic activity of rat peritoneal mast cells following stimulation with compound 48/80 were monitored using the Ca-indicator dye fura-2 and the capacitance measurement technique. It is known that mast cells very rapidly lose their secretory response towards antigenic or compound 48/80-induced stimulation in the whole-cell recording configuration of the patch-clamp technique due to washout of signal mediators. In contrast, we found that calcium transients remained unaffected by intracellular dialysis for as long as 10 min.The fast washout phenomenon of exocytosis could be overcome by supplementing the pipette filling solution with guanosinetriphosphate (GTP) indicating a major role for GTP-binding proteins in secretion. The restoration of exocytosis was transient and decayed within three minutes, suggesting diffusional escape of one or several other cytoplasmic substances involved in stimulus-secretion coupling. Quantitative aspects of this process and the implications of its differential effects on Ca-transients versus secretion are discussed.     

Sympathetic nerve contact causes maturation of mast cells in vitro

Using a tissue culture model developed to study interactions between peripheral neurons and mast cells (MC), time-lapse microscopy showed that RBL- 2H3 cells (a model of the mucosal MC) formed attachments with sympathetic neurons, ceased to divide, and moved along neurites toward the cell bodies. Electron microscopy showed significant increase in granules compared to intrinsic controls (RBL cells in coculture but lacking neurite contact). In studies using cohort cultures of 12- to 14-day-old sympathetic neurons, RBL cells adhered more rapidly to neurons than did control YB2/0 cells (a neutral target cell), and were inhibited in growth compared with RBL cells cultured in parallel without neurons. RBL cells cocultured with neurons for 24–48 h took up significantly more ³H-5HT and released a significantly larger percentage of ³H-5HT in response to the calcium ionophore A23187 than RBL cells in parallel pure cultures. Since no change in MC phenotype was seen, we conclude that contact with nerve membrane may be a developmental cue leading to maturation of MC. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 173–182, 1998     

The effect of 2450 MHz microwave radiation on histamine secretion by rat peritoneal mast cells

Isolated rat peritoneal mast cells actively secrete histamine in response to reaginic or chemical stimulation. Mast cells were irradiated in a waveguide microwave exposure chamber at 2450 MHz with power absorptions of 8.2 and 41.0 mW/g for periods up to 3 h. These levels of microwave absorption caused no change in the morphological characteristics or viability of the cells. Irradiated mast cells were stimulated with compound 48/80, a potent, noncytotoxic histamine releasing agent. The dose response curves showed that neither prior nor simultaneous irradiation of mast cells at 37°C affected 48/80-induced secretion. However, microwave power absorptions of 41.0 mW/g inhibited secretion at 44.0°C. Precise measurements of the effect of heat on secretion indicated that this level of inhibition could have been produced by a radiation induced increase in cell temperature between 0.4 and 0.9°C above ambient levels. Alternatively, the heat stress produced at 44°C may have sensitized the cells to the electromagnetic effects of the microwave radiation. Rat peritoneal mast cells can therefore be useful as a model for the study of functioning secretory cells during microwave irradiation and can also be used to monitor the synergistic effects of cell heating during in vitro exposure.     

Studies on choline permeation through the plasma membrane and its incorporation into phosphatidyl choline of Ehrlich-Lettré-ascites tumor cells in vitro.

The initial rate of incorporation of 14C or 3H-labeled choline into Ehrlich-Lettre ascites cells of the glycogen-free strain seven days after inoculation was investigated in vitro. 1. At choline concentrations in the medium between 6 to 30 muM and 100 to 500 muM the choline uptake by the cells followed Michaelis-Menton Kinetics with V values between 31 to 100 and 59 to 500 pmol per minute at a given cell density, and average Q10-values of 2.1 at the high and of 2.4 at the low choline molarity. The K-m-values increased from 27 muM to 58.8 muM at low and from 0.11 mM to 0.22 mM at high choline concentrations over a temperature range between 15 degrees C and 37 degrees C. Arrhenius plot of the V values gave two lines, one with a transition temperature at 25 degrees C at low and one straight line at high choline concentrations, from which the energy of activation for choline uptake was determined to be 16 kcal/mol. 2. It is assumed that two systems exist for the choline uptake by the ascites cells. One, operative at low substrate concentrations, which is saturable and probably is to be classified as a carrier-mediated facilitated diffusion process, can be strongly inhibited by deoxyglucose or 2,4-dinitrophenol and also by substrate analogues such as chlorocholine or benzoylcholine. Ouabain affects this system to a lesser extent. The other system functioning at high choline concentrations may be a simple diffusion process, which is little inhibited by substrate analogues, ouabain and deoxyglucose; however, it is also inhibited by 2,4-dinitrophenol and p-chloromercuribenzoate. 3. Choline incorporation into the acid-insoluble material (lecithin) gave linear Michaelis-Menton kinetics at the low and the high substrate concentration respectively. K-m-values decreased with an increase in temperature at low and increased with rising temperature at high substrate concentrations thus reflecting a close relationship between choline uptake and its metabolism. Labeling of lecithin choline in the various subcellular fractions under the conditions of the functioning of a carrier-mediated process was in the order: mitochondria (50%) greater than plasma membranes (25%) greater nuclei (14%) greater than microsomes (9%) greater than supernatant (1.5%). 4. Treatment of the cells with p-chloromercuribenzoate or heat shock at 50 degrees C markedly reduced the cholinee uptake and concomitantly its conversion into lecithin. Kinetic analysis revealed that the inhibitory effect of p-chloromercuribenzoate was competitive and that of the heat shock non-competitive in nature. Further the choline uptake by the cells was found to be the rate-limiting step, since the rate of choline phosphorylation was determined by the extracellular choline concentration. Pulse chase experiments showed a rapid turnover of the choline moiety with a concomitant increase in activity of the lecithin fraction and little change within the choline phosphate pool.     

Prolonged effect of a single serotonin treatment in adult age on the serotonin and histamine content of white blood cells and mast cells of rats

Hormonal imprinting was provoked by serotonin treatment in adult age. Three weeks after treatment with 100 microg serotonin, the serotonin and histamine content of peritoneal cells (mast cells, lymphocytes and the monocyte-macrophage-granulocyte group), white blood cells (lymphocytes, granulocytes and monocytes) and thymic lymphocytes was studied by flow cytometry. The content of both amines was significantly higher in the mast cells of males and lower in females. Blood lymphocytes contained a higher serotonin and histamine level in males, and a lower serotonin level in females. The peritoneal monocyte-macrophage-granulocyte group contained less serotonin in both males and females. Thymocytes contained higher levels of both amines in females and higher histamine level in males. The experiments demonstrate that a single treatment at adult age can provoke imprinting, which alters-in the present case-the serotonin and histamine content of immune cells durably.     

Potentiation by histamine of synaptically mediated excitotoxicity in cultured hippocampal neurones: a possible role for mast cells

Excessive glutamatergic neurotransmission, particularly when mediated by the N:-methyl-D-aspartate (NMDA) subtype of glutamate receptor, is thought to underlie neuronal death in a number of neurological disorders. Histamine has been reported to potentiate NMDA receptor-mediated events under a variety of conditions. In the present study we have utilized primary hippocampal neurone cultures to investigate the effect of mast cell-derived, as well as exogenously applied, histamine on neurotoxicity evoked by excessive synaptic activity. Exposure of mature cultures for 15 min to an Mg(2+)-free/glycine-containing buffer to trigger synaptic transmission through NMDA receptors, caused a 30-35% neuronal loss over 24 h. When co-cultured with hippocampal neurones, activated mast cells increased excitotoxic injury to 60%, an effect that was abolished in the presence of histaminase. Similarly, addition of histamine during magnesium deprivation produced a concentration-dependent potentiation (+ 60%; EC(50) : 5 microM) of neuronal death which was inhibited by sodium channel blockers and NMDA receptor antagonists, although this effect did not involve known histamine receptors. The histamine effect was further potentiated by acidification of the culture medium. Cultures 'preconditioned' by sublethal (5 min) Mg(2+) deprivation exhibited less neuronal death than controls when exposed to a more severe insult. NMDA receptor activation and the extracellular regulated kinase cascade were required for preconditioning neuroprotection. The finding that histamine potentiates NMDA receptor-mediated excitotoxicity may have important implications for our understanding of conditions where enhanced glutamatergic neurotransmission is observed in conjunction with tissue acidification, such as cerebral ischaemia and epilepsy.     

IL-4 modulates the histamine content of mast cells in a mast cell/fibroblast co-culture through a Stat6 signaling pathway in fibroblasts

IL-4 plays a crucial role in the pathogenesis of allergic diseases, such as the induction of IgE synthesis and the development of mast cells. To further understand the effect of IL-4 on mast cells in skin, we utilized a mast cell/fibroblast co-culture system as an in vitro model of dermal mast cells. IL-4 induced mast cell growth in the culture with fibroblasts. Immunoblot analysis revealed that IL-4 activated Stat6 in both mast cells and fibroblasts. The over-expression of dominant-negative Stat6 in fibroblasts in the presence of IL-4 decreased the histamine content per mast cell, but not the number of mast cells. In contrast, the over-expression of constitutively-active Stat6 in fibroblasts increased the histamine content per mast cell, indicating that the activation of Stat6 in fibroblasts supports the maturation of mast cells co-cultured with fibroblasts.     

Microtubule-dependent transport of secretory vesicles in RBL-2H3 cells

Antigen-mediated activation of mast cells results in Ca²⁺-dependent exocytosis of preformed mediators of the inflammatory response. To investigate the role of secretory vesicle motility in this response, we have performed time-lapse confocal microscopy on RBL-2H3 cells transfected with a green fluorescent protein-Fas ligand fusion protein (GFP-FasL). Green fluorescent protein-labeled vesicles exhibit rapid, bidirectional movement in both resting and activated cells and can be localized adjacent to microtubules. Colchicine treatment inhibits the motility of secretory vesicles as measured by fluorescence recovery after photobleaching (FRAP). Colchicine also inhibits both the extent and the rate of exocytosis triggered by receptor activation or by Ca²⁺ ionophore, demonstrating that microtubule-dependent movement of secretory vesicles plays an important role in the exocytic response .     

Regulation of apoptosis in mast cells

Apoptosis is a physiological process of cell death that occurs in all multicellular organisms. Its dysregulation has been postulated as one of the main causes in the development of diseases such as cancer, AIDS, autoimmune diseases and allergy. Apoptosis has been mainly studied in the inflammatory cells that participate in the late and chronic stages of allergy (eosinophils, neutrophils, lymphocytes and macrophages) as a new way to elucidate the pathogenesis of this disease. Nevertheless, much less it is known about the regulation of apoptosis in the initiators of the allergic process: The Mast Cells. In normal conditions, mast cells are described as long-living cells that keep a constant number of cells in tissues. However, increased numbers of mast cells are observed in the late phase of asthma and in both the inflammatory and in the repair/remodeling stage of various inflammatory/fibrotic disorders. In this report, we discuss the possible mechanisms that regulate the apoptotic process in normal conditions and disease, such as survival factors and death receptors. A link between mast cell activation, during the early stages of the allergic process, and triggering of anti-apoptotic signaling pathways is also suggested as an important contributor to the extended life of mast cells.     

Analysis of the effect of a 60 Hz AC field on histamine release by rat peritoneal mast cells

Reports have indicated effects of electromagnetic fields on inflammatory processes in vivo. To begin a systematic approach toward separating and examining the many components of such responses, we created and tested a temperature-controlled device to develop 5 mT 60 Hz magnetic fields for studies of the effects of fields on mast cells, a key component in acute inflammatory responses. Such fields have been reported to modulate cell activity, including changes in membrane function, in various systems. The magnetic field was generated using a solenoid and calibrated with an induction probe. Tests of mast cell function were determined by histamine release response to stimulation by compound 48/80, using both an “expose then test” and a “test during exposure” protocol. Aliquots not treated with 48/80 were used to evaluate field treatment effects on spontaneous histamine release. Freshly harvested rat peritoneal mast cells were exposed to the magnetic field for periods of 30 min to 2 h at 37 °C. They showed no significant degranulation during treatment, nor did they show reduced sensitivity to the degranulating agent 48/80. These observations are consistent with a model in which such processes are exclusively reflexive by the cells using field-independent membrane systems. This observation is very useful and was needed before examining longer term exposures in which gene expression in the cells might be influenced; this is the first such report of in vitro exposure of purified mast cells under these conditions and will further the study of the effects of electromagnetic fields on cell types active in acute inflammation. Bioelectromagnetics 19:192–198, 1998. © 1998 Wiley-Liss, Inc.     

Role of mast cells as IL10 producing cells in paracoccidioidomycosis skin lesions

Recent works have demonstrated that mast cells may have an important role in immunologic reactions and inflammation once they synthesize and secrete many cytokines including IL4, IL5, IL6 and TNF-α. We have conducted research in order to verify if mast cells would participate in the local inflammatory immune response against Paracoccidioides brasiliensis in skin lesions characterized by a Th2 pattern of cytokines. Fifty-nine skin biopsies with previous histopathological diagnosis of paracoccidioidomycosis and immunohistochemical characterization of cytokines present in the inflammatory infiltrate were classified in three groups: group 1 (G1), with compact granuloma and a Th1 pattern of cytokines; group 2 (G2), with loose granuloma and a Th2 pattern of cytokines; group 3 (G3), both kind of granuloma in the same lesion, characterized by cytokines from Th1 and Th2 patterns. Ten biopsies from normal skin were used as control group. Mast cells were visualized and quantified by a toluidine blue/HCl staining and a double immunostaining was performed to detect a co-localization of mast cells and IL10. G2 presented an increased number of mast cells when compared to G1, G3 and control group and we frequently could find mast cells expressing IL10 in G2. The data obtained suggest that mast cells participate in the immune response against P. brasiliensis in skin lesions with loose granuloma and a Th2 pattern of cytokines. Considering these results, mast cells could constitute a source of IL10, contributing to a non-effective response against fungal antigens.     

The effect of polymyxin B and some mast-cell constituents on mucosal mast cells in the duodenum of the rat

Summary Mucosal mast cells in the rat duodenum show no morphological signs of exocytosis of granules and do not release histamine after treatment with polymyxin B in doses large enough to cause almost complete degranulation of connective-tissue mast cells of tongue, skin, and mesentery with concomitant release of 60% of the tissue histamine. Administration of polymyxin B in gradually increasing doses over a period of 5ds resulted in a statistically significant increase in mucosal mast cells and a comparable increase in duodenal histamine content, whereas the connective-tissue mast cells in the other tissues examined became fewer in number, the remaining cells showing profound morphological changes, and tissue histamine levels, were reduced to 40% of the controls. A similar increase in mucosal mast cells has been observed after treatment with another mast-cell secretagogue, compound 48/80. This suggests that the increase in mucosal mast cells may be an indirect effect of these compounds, related to their activation of other mast cells and mediated by material(s) secreted by the connective-tissue mast cells. Possible mediators such as heparin, histamine, and 5-hydroxytryptamine injected for 5 ds in doses large enough to account for the amount released from the degranulated mast cells had no effect on the morphology or numbers of mast cells in any of the tissues examined.Supported by grants from the Swedish Medical Research Council, Project no 2235     

Role of mast cells in wound healing process after glass-fiber composite implant in rats

Glass-fiber composites are frequently used in dentistry. In order to evaluate their biocompatibility we tested, in an experimental model "in vivo", their tissue response pointing our attention on presence of mast cells (MCs) and fibrotic process. Sprague Dawley rats were used for the experimental design. The fibers were introduced in a subcutaneous pocket along the middle dorsal line between the two scapulas for 7, 14 or 21 days. At the end of the treatments the skins were excised and then processed for Toluidine Blue, to determine the presence of MCs, and Picrosirius Red staining, to evaluate the presence of fibrotic tissue. Our preliminary results showed and increase of both MC number and deposition of collagen type I, which characterized the fibrotic tissue. So, subsequent aims of our study were to evaluate the role played by MCs in tissue fibrosis and to give a possible explanation regarding the mechanisms that were responsible of biological response observed, through the analyses of some proteins, such as metalloproteinase-2 (MMP-2), its inhibitor (TIMP-2) and transforming growth factor-beta (TGF-beta). Our data confirmed the involvement of TGF-beta, released by MCs, in the disruption of the equilibrium between MMP-2 and TIMP-2 that were implicated in the enhancement of fibrosis. In summary, this study demonstrate that this type of materials induced an inflammatory response at the site of implant and help to clarify what type of mechanism and which proteins are involved in this biological response. Nevertheless, more extensive investigations are in progress to better evaluate the inflammatory process.     

Practical considerations regarding the use of streptolysin-O as a permeabilising agent for cells in the investigation of exocytosis

Streptolysin-O is widely used in cell biological investigations in order to make large (>12 nm) pores in the plasma membrane and so to render the cytosol directly accessible to experimental manipulation. We have compared the effect of streptolysin-O commercially formulated (Murex Diagnostics) as a diagnostic reagent in pathology with two pure reagents (a conventional purified protein, and a recombinant protein generated in E.coli) on exocytotic secretion from mast cells. For mast cells permeabilised by streptolysin obtained from the commercial source, exocytosis (of -D-N-acetylglucosaminidase) is dependent on provision of both Ca²⁺ and a guanine nucleotide. In contrast, for cells permeabilised by either of the two pure proteins, a substantial extent of Ca²⁺-independent exocytosis can be elicited. When the Murex material is subject to dialysis or ultrafiltration, some secretion can be induced in the absence of Ca²⁺, indicating a modulatory function of the low mol wt additives of formulation, mainly phosphate and cysteine. However, Ca²⁺-independent exocytosis is still manifest when the pure proteins are reconstituted with ultrafiltrates from the Murex material. These observations indicate that reagents used to permeabilise cells should be characterised thoroughly and used with great care. Confirmation that the cytolytic activity of the Murex material derives from a cholesterol directed factor was demonstrated by inhibition of exocytosis when red blood cell derived (and hence cholesterol containing) sonicated liposomes were provided.     

Serine and ethanolamine incorporation into different plasmalogen pools: Subcellular analyses of phosphoglyceride synthesis in cultured glioma cells

In cultured glioma cells, plasma membrane (PM) is enriched in phosphatidylserine (PtdSer) and plasmalogens (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Serine can be a precursor of headgroups of both ptdSer and ethanolamine phosphoglycerides (PE) including plasmalogens and non-plasmalogen PE (NP-PE). Synthesis of phospholipids was investigated at the subcellular level using established fractionation procedures and incorporation of [³H(G)]L-serine and [1,2-¹⁴C]ethanolamine. Specific radioactivity of PtdSer from [³H]serine was 2-fold greater in PM than in microsomes, reaching maximum by 2–4 h. Labeled plasmalogen from [³H]serine appeared in PM by 4 h and increased to 48 h, whereas almost no plasmalogen accumulated in microsomes within 12 h. In contrast, labeled plasmalogen from [1,2-¹⁴C]ethanolamine appeared in both PM and microsomes at early incubation times and became enriched in PM beyond 12 h. Thus, in glioma cells: (1) greater and faster accumulation of labeled PtdSer in PM may reflect direct synthesis from serine within PM; (2) PM is a major source of PtdSer for decarboxylation and PE synthesis; (3) NP-PE in both PM and microsome provides headgroup for synthesis of plasmalogen; and, (4) plasmalogen synthesis may involve different intracellular pools depending on headgroup origin.Abbreviations NP-PE nonplasmenylethanolamine phosphoglycerides including both diacyl and alkylacyl species - PE total ethanolamine phosphoglycerides: plasmalogen-plasmenylethanolamine or alkenylacyl ethanolamine phosphoglyceride (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) - PL phospholipid - PM plasma membrane - PtdCho phosphatidylcholine - PtdSer phosphatidylserine     

Ultrastructure of human dermal mast cells in 29 different lysosomal storage diseases

The effect of lysosomal storage diseases on the ultrastructure of human mast cells has not previously been reported. Indeed, there has been little published evidence indicating that mast cells contain typical lysosomes. However, mast cell cytoplasmic granules contain hydrolases similar to those found in lysosomes, but which differ from lysosomal hydrolases in exhibiting optimal activity at higher pH. We therefore examined by transmission electron microscopy the dermal mast cells in 58 biopsies of patients exhibiting 1 of 29 different lysosomal storage diseases. We found mast cells containing abnormal lysosomes in 16 of these disorders. In 6 of these 16 diseases, the mast cells' cytoplasmic granules appeared normal. These observations indicate that human mast cells can contain lysosomes, and provide evidence that the enzymes affected by lysosomal storage diseases are active in mast cells.