Complement peptides C3a- and C5a-induced mediator release from dissociated human skin mast cells.

The complement peptides C3a and C5a have been shown previously to release histamine from human basophils but not human lung mast cells. As skin mast cells differ from those of the lung in both immunocytochemical and functional properties, we examined the ability of these anaphylatoxins to release preformed and newly generated mediators from human dispersed skin mast cells. In concentration-response studies, both C3a and C5a released histamine in a concentration related manner with C5a being 40-50 times more potent. However, the extent of histamine, 15-20%, was considerably less than that released from basophils. This was not due to catabolism of the peptides by mast cell proteases, mast cell supernatants that contained C5a being effective in releasing basophil histamine. Removal of the C-terminal arginine from C3a and C5a abolished their activity on skin mast cells. In time-course studies, histamine release induced by C3a and C5a was complete within 15 seconds. Complement-induced histamine release is a non-cytotoxic process as evidenced by 2-deoxy-D-glucose and antimycin A, inhibitors of glycolysis and oxidative phosphorylation, respectively. In contrast to IgE-dependent stimulation, anaphylatoxin-induced histamine release from human skin mast cells is independent of extracellular calcium. Both C3a and C5a at concentrations that induced 10-16% net histamine release caused a negligible release of the newly generated mediator, PGD2. The results suggest that C3a and C5a stimulate human skin mast cells in a manner similar to substance P and related basic secretagogues. However, the activation site for C3a and C5a appears to be different to that for substance P as the substance P antagonist (D-Pro4, D-Trp7,9,10) SP4-11 inhibited histamine release stimulated by substance P but not that induced by C3a and C5a.

Eosinophil granule proteins inhibit substance P-induced histamine release from human skin mast cells.

We have investigated the activity of the four principal cationic proteins of the eosinophil granules, major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil-derived neurotoxin, and eosinophil cationic protein on histamine release from human skin mast cells. These four cationic proteins, over the concentration range of 10 to 200 micrograms/ml, did not induce significant histamine release, nor did they prime anti-IgE-induced histamine release from human skin mast cells significantly. However, a brief incubation (15 minutes) of two of the four principal eosinophil granule proteins, MBP and EPO, at concentrations of 50 to 200 micrograms/ml, caused a significant concentration-related inhibition of histamine release induced by 30 mumol/L substance P. The concentrations producing 50% inhibition for MBP and EPO on substance P-induced histamine release were 30 micrograms/ml and 100 micrograms/ml, respectively. This inhibitory effect appears to be a direct effect of these proteins on skin mast cells because purified (78% to 85%) skin mast cells displayed a similar response to MBP and EPO (n = 4). Also, when skin mast cells were incubated with 100 micrograms/ml MBP and EPO for 15 minutes and washed twice before activation by substance P, the inhibitory effect was not altered. These two proteins also inhibited histamine release induced by 10 micrograms/ml compound 48/80. These results suggest that MBP and EPO affect the same binding site(s) on skin mast cells as those of substance.

Biological properties of human skin mast cells.

Mast cells and basophils, although sharing many constitutive properties, are quite distinct in their development, functions and biological properties. Mast cell granules are composed of a macromolecular matrix of proteoglycan and neutral protease of which heparin and tryptase, respectively, are predominant. The distribution of the other major neutral protease, chymase, allows human mast cell subpopulations to be subdivided immunocytochemically. All human mast cells respond to IgE-dependent stimulation with the secretion of the preformed mediator, histamine, and the newly generated lipid-derived eicosanoids PGD2 and LTC4. Although amounts of these products vary between mast cells dispersed from different tissues, it is uncertain whether this reflects true heterogeneity. Mast cells of the human skin, but not those of other tissues, are sensitive to stimulation by substance P, compound 48/80 and other basic non-immunological stimuli. The mechanism of mediator secretion induced by these agents is distinct from that induced by IgE-dependent stimulation. However, the morphological characteristics of degranulation are similar, suggesting that the distinct biochemical pathways merge into a common pathway before effecting degranulation.

Neuropeptide-induced secretion from human skin mast cells.

Unlike human mast cells associated with mucosal surfaces such as lung, adenoids, tonsils and intestine, skin mast cells may be stimulated to release histamine by the neuropeptides substance P, vaso-active intestinal polypeptide and somatostatin or by other basic secretagogues such as morphine and compound 48/80. Release of histamine by neuropeptides is rapid and accompanied by minimal generation of the eicosanoids prostaglandin D2 and leukotriene C4. Transient elevations of intracellular calcium are associated with mediator secretion induced by both immunological and non-immunological stimulation, that induced by anti-IgE being derived from extracellular sources through channels in the plasma membrane while that stimulated by neuropeptides is mobilized intracellularly. Similarly, elevations of intracellular cyclic AMP induced by anti-IgE occur only in the presence of extracellular calcium, whereas with substance P elevations are apparent even in the absence of extracellular calcium. With the latter stimulus, histamine release is complete before the peak cyclic AMP is achieved. Despite these biochemical and temporal differences, degranulation induced by both secretagogues proceeds by compound exocytosis which is indistinguishable under the electron microscope. From these results we suggest that IgE-dependent and neuropeptide stimulation of human skin mast cells proceed by distinct biochemical pathways which eventually merge to produce exocytosis of their preformed granule-associated mediators.

Mediator secretion from human skin mast cells provoked by immunological and non-immunological stimulation.

Mast cells of the human skin not only release mediators following immunological activation, but may also be stimulated to release histamine by the neuropeptides substance P, vasoactive intestinal polypeptide and somatostatin or by other basic secretagogues such as morphine, poly-L-lysine and compound 48/80. Release of histamine under these conditions is rapid and accompanied by minimal generation of the eicosanoids, prostaglandin (PG)D2 and leukotriene (LT)C4. Transient elevations of intracellular calcium are associated with mediator secretion induced by both stimuli, that induced by anti-IgE being derived from extracellular sources through channels in the plasma membrane while that stimulated by neuropeptides is mobilized intracellularly. Similarly, elevations of intracellular cyclic adenosine monophosphate (AMP) induced by anti-IgE occur only in the presence of extracellular calcium whereas with substance P elevations are apparent even in the absence of extracellular calcium. With the latter stimulus, histamine release is complete before the peak cyclic AMP is achieved. Histamine release stimulated by both secretagogues is unaffected by sodium cromoglycate or nedocromil sodium but is reduced by both salbutamol and isobutylmethylxanthine. Despite these biochemical and temporal differences, degranulation induced by both secretagogues proceeds by compound exocytosis which is indistinguishable under the electron microscope.

Dissociated human foreskin mast cells degranulate in response to anti-IgE and substance P.

Human skin mast cells release histamine in response to both immunologic stimulation mediated by anti-IgE and IgE-independent mechanisms of which substance P is a prototypical secretagogue. We compared the ultrastructural changes produced in dissociated foreskin mast cells by these two stimuli with histamine release. Mast cells were isolated and pooled from the foreskins of 2- to 7-year-old boys in four separate experiments and comprised 25 to 60% of the total dissociated cells. The secretory granules in resting mast cells comprised 47.5% of the extranuclear cell volume and contained crystalline structures, namely, scrolls, gratings, and lattices, in an electron-dense matrix. Stimulation with either anti-IgE or substance P resulted in a net histamine release of 10.2 +/- 1.7% or 21.4 +/- 4.0%, respectively. After either secretagogue, about 75% of the cells underwent compound exocytosis, with fusion of the granule membranes with one another and with the plasma membrane to produce large degranulation channels that opened to the extracellular space. The granules lost their crystalline structure and electron density during secretion but retained the round shape of the original granule as a core that subsequently formed a fibrillar residue. Degranulation channels occupied 30 to 60% of the cytoplasmic volume after substance P stimulation and 10 to 40% after anti-IgE, which compared well with the greater histamine release measured after substance P. The rapid increase in the volume of the degranulation channels after substance P was accompanied by a decrease in cytoplasmic volume, suggesting water moved from the cytoplasm into the granules after stimulation. This study shows that secretion produced in dissociated human foreskin mast cells by two different stimuli, anti-IgE and substance P, which act through different membrane receptors and have distinct secretory characteristics, is similar morphologically.

Changes in the activity of glutamate related enzymes in cerebral cortex, during insulin-induced seizures.

The activity of glutamate related enzymes and the concentration of glutamine, glutamate and gamma-amino n-butyric acid (GABA) were investigated in the cerebral cortex of rats, in different stages of insulin-induced hypoglycemia. Hypoglycemia was produced by intraperitoneal injection of insulin 0.05-100 units per kg body weight. The minimum required dose to produce irreversible severe hypoglycemia was 0.5 units/kg. In 85% of the cases an insulin induced hypoglycemic convulsion, was achieved 130-150 minutes after injection. Blood glucose levels during insulin induced seizures ranged between 8-15 mg%. In the range of 0.5-100 u insulin/kg the degree of hypoglycemia and the onset of convulsions were identical. The concentration of glutamine was significantly reduced during convulsive and postconvulsive stages. Glutamate and GABA concentrations were reduced significantly in all stages of insulin-induced hypoglycemia. The decrease in glutamine concentration was concurrent with an increase in the activity of its degradative enzyme, glutaminase. This was apparent at the preconvulsive, convulsive and postconvulsive stages. The activity of other enzymes related to energy production such as glutamate dehydrogenase (GDH), glutamate transaminase (GPT) and aspartate aminotransferase (AAT) were also increased. The activity of glutamine synthase (GS) was unaffected by hypoglycemia. Insulin induced changes in glutamine, glutamate and their related enzymes could not be attributed to convulsion since a similar pattern of changes was observed in the preconvulsive and postconvulsive stages, and no changes were detected following picrotoxin-induced seizures.