Morphological and biochemical aspects of degranulation of human skin mast cells
Morphological and biochemical aspects of degranulation of human skin mast cells
Human skin mast cells release histamine in response to a wide variety of stimuli. Indirect evidence suggests that the complement peptides C3a and C5a may also induce histamine release. The ability of human skin mast cells to respond to anti-IgE and substance P gives a unique opportunity to study the degranulation process, both in terms of ultrastructural and biochemical changes. In concentration-response studies, both C3a and C5a release histamine in a concentration-related manner with C5a being 50 times more potent. However, the extent of histamine release is considerably less than that released from basophils. This is not due to catabolism of the peptides by mast cell proteases. Removal of the C-terminal arginine from C3a and C5a abolishes their activity on skin mast cells. C3a and C5a induced histamine release requires 15 seconds for completion, is independent of extracellular calcium and causes negligible release of prostaglandin D2 (PGD2). These results suggest that C3a and C5a stimulate human mast cells in a manner similar to substance P. However, the actviation site for C3a and C5a appears to be different to that for substance P. The secretory granules in resting mast cells comprised 47.5% of the extranuclear cell volume and contain crystalline structures, namely, scrolls, gratings, and lattices. After anti-IgE and substance P stimulation, about 75% of the cells undergo compound exocytosis, with fusion of the granule membranes with one another and with the plasma membrane to produce degranulation channels. There is no overall decrease in the volume density of lipid droplets at the time when the granules are being discharged. Biochemical studies form the final basis for differentiating anti-IgE and substance P. Anti-IgE- and substance P-mediated release is preceded and accompanied by elevation in cytosolic calcium. Substance P which releases histamine even in a calcium-free medium, stimulates human skin mast cells to mobilize the calcium necessary to activation-secretion coupling from intracellular stores. However, influx of calcium by transient opening of calcium channels in the plasma membrane is a requirement for anti-IgE-mediated histamine release. The role of protein kinase C (PKC) in anti-IgE- and substance P-mediated secretion is investigated by two complementary pharmacological approaches. Inhibition of histamine release in a concentration related manner by staurosporine, Ro-31-7549 and Rop-31-8220, which bind to the catalytic domain of the enzyme, and by calphostin C which binds to the regulatory domain, suggests that PKV plays a positive role in anti-IgE- and substance P-mediated activation-secretion coupling. Down-regulation of PKC by phorbol-12-myristate-13-acetate (PMA) does not completely abolish the response to both stimuli. This suggests that other second messengers, besides PKC, are involved in anti-IgE- and substance P-mediated signalling. Signal transduction generated in human skin mast cells by anti-IgE and substance P has also been expanded to include the measurement of cyclic AMP. With anti-IgE, the elevation of cyclic AMP occurs only in the presence of extracellular calcium and decays before a significant release of histamine occurs. This may suggest a facilatory role of cyclic AMP in the early stage of mast cell activation. In contrast, with substance P, elevations in cyclic AMP are apparent even in the absence of extracellular calcium and occur after histamine release is complete. In contrast, pertussis toxin, which inhibits the activity of a specific G-protein, reduces the response to substance P and has no effect on anti-IgE-mediated release. This indicates that substance P-mediated release is initiated by a pertussis toxin-sensitive G-protein, whereas if a G-protein regulate IgE-mediated release then it is not pertussis toxin-sensitive. In conclusion, IgE- and non-IgE-dependent stimulation of human dispersed skin mast cells proceed by distinct biochemical pathways which eventually merge to produce exocytosis of their preformed granule-associated mediators.
University of Southampton
1992
El-Lati, Suhad George
(1992)
Morphological and biochemical aspects of degranulation of human skin mast cells.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Human skin mast cells release histamine in response to a wide variety of stimuli. Indirect evidence suggests that the complement peptides C3a and C5a may also induce histamine release. The ability of human skin mast cells to respond to anti-IgE and substance P gives a unique opportunity to study the degranulation process, both in terms of ultrastructural and biochemical changes. In concentration-response studies, both C3a and C5a release histamine in a concentration-related manner with C5a being 50 times more potent. However, the extent of histamine release is considerably less than that released from basophils. This is not due to catabolism of the peptides by mast cell proteases. Removal of the C-terminal arginine from C3a and C5a abolishes their activity on skin mast cells. C3a and C5a induced histamine release requires 15 seconds for completion, is independent of extracellular calcium and causes negligible release of prostaglandin D2 (PGD2). These results suggest that C3a and C5a stimulate human mast cells in a manner similar to substance P. However, the actviation site for C3a and C5a appears to be different to that for substance P. The secretory granules in resting mast cells comprised 47.5% of the extranuclear cell volume and contain crystalline structures, namely, scrolls, gratings, and lattices. After anti-IgE and substance P stimulation, about 75% of the cells undergo compound exocytosis, with fusion of the granule membranes with one another and with the plasma membrane to produce degranulation channels. There is no overall decrease in the volume density of lipid droplets at the time when the granules are being discharged. Biochemical studies form the final basis for differentiating anti-IgE and substance P. Anti-IgE- and substance P-mediated release is preceded and accompanied by elevation in cytosolic calcium. Substance P which releases histamine even in a calcium-free medium, stimulates human skin mast cells to mobilize the calcium necessary to activation-secretion coupling from intracellular stores. However, influx of calcium by transient opening of calcium channels in the plasma membrane is a requirement for anti-IgE-mediated histamine release. The role of protein kinase C (PKC) in anti-IgE- and substance P-mediated secretion is investigated by two complementary pharmacological approaches. Inhibition of histamine release in a concentration related manner by staurosporine, Ro-31-7549 and Rop-31-8220, which bind to the catalytic domain of the enzyme, and by calphostin C which binds to the regulatory domain, suggests that PKV plays a positive role in anti-IgE- and substance P-mediated activation-secretion coupling. Down-regulation of PKC by phorbol-12-myristate-13-acetate (PMA) does not completely abolish the response to both stimuli. This suggests that other second messengers, besides PKC, are involved in anti-IgE- and substance P-mediated signalling. Signal transduction generated in human skin mast cells by anti-IgE and substance P has also been expanded to include the measurement of cyclic AMP. With anti-IgE, the elevation of cyclic AMP occurs only in the presence of extracellular calcium and decays before a significant release of histamine occurs. This may suggest a facilatory role of cyclic AMP in the early stage of mast cell activation. In contrast, with substance P, elevations in cyclic AMP are apparent even in the absence of extracellular calcium and occur after histamine release is complete. In contrast, pertussis toxin, which inhibits the activity of a specific G-protein, reduces the response to substance P and has no effect on anti-IgE-mediated release. This indicates that substance P-mediated release is initiated by a pertussis toxin-sensitive G-protein, whereas if a G-protein regulate IgE-mediated release then it is not pertussis toxin-sensitive. In conclusion, IgE- and non-IgE-dependent stimulation of human dispersed skin mast cells proceed by distinct biochemical pathways which eventually merge to produce exocytosis of their preformed granule-associated mediators.
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Published date: 1992
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Local EPrints ID: 461277
URI: http://eprints.soton.ac.uk/id/eprint/461277
PURE UUID: 567c9ff5-26c6-4959-8f37-5d9222c1a1fc
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Date deposited: 04 Jul 2022 18:42
Last modified: 04 Jul 2022 18:42
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Author:
Suhad George El-Lati
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