Requirement for vasoactive amines for production of delayed-type hypersensitvity skin reactions

The skin sites of the mouse where delayed-type hypersensitivity (DTH) reactions are most easily elicited (foot pads and ears) are particularly rich in 5-hydroxytryptamine (5-HT)-containing mast cells. Since mice are deficient in circulating basophils, which play a role in at least some DTH reactions, we investigated the possibility that the mast cells were playing an important role in the evolution of the skin reactions of DTH in mice. We found that reserpine, a drug which depletes mast cells of 5-HT, abolished the ability of the mouse to make DTH reactions in the skin. The suppressive effect of reserpine could be partially blocked by monoamine oxidase inhibitors which prevent the degradation of 5-HT in the cytosol of the mast cell. Spleen cells of immune, reserpine-treated mice transferred DTH reactions to nonimmune mice normally, indicating that the reserpine treatment did not affect immune T cells. DTH reactions could not be transferred into reserpine-treated mice. We suggest that T cells are continually emigrating from the blood, through postcapillary venule endothelium, by a mechanism which does not depend on vasoactive amines. If they are appropriately immune and meet the homologous antigen in the tissue, they induce mast cells to release vasoactive amines which cause postcapillary venule endothelial cells to separate, allowing the egress from the blood of cells which ordinarily do not recirculate. The secondarily arriving vasoactive amine-dependent cells are responsible for the micro- and macroscopic lesions of DTH reactions. Chemotactic factors may also be involved in bringing cells to the DTH reaction sites but we propose that T-cell regulation of vasoactive amine-containing cells allows the effector cells to pass through the endothelial gates after they are called.     

Reevaluation of reserpine-induced suppression of contact sensitivity. Evidence that reserpine interferes with T lymphocyte function independently of an effect on mast cells

It has been suggested that reserpine blocks expression of delayed hypersensitivity (DH) by depleting tissue mast cells of serotonin (5-HT), thereby preventing a T cell-dependent release of mast cell 5-HT necessary to localize and to amplify the DH response. However, reserpine blocks expression of DH in mast cell-deficient mice. We therefore decided to reevaluate the mechanism by which reserpine abrogates expression of cellular immunity, and investigated whether the drug might interfere with T cell activity in vitro or in vivo. At concentrations as low as 4 microM, reserpine profoundly suppressed baseline or antigen-augmented levels of [3H]thymidine incorporation by immune lymph node cells obtained from mice sensitized to the contactant oxazolone [I-LNC(Ox)]. This effect was observed both with I-LNC derived from normal mice and with I-LNC derived from congenitally mast cell-deficient W/Wv mice, cell preparations that lacked detectable mast cells, histamine, and 5-HT. Furthermore, treatment of I-LNC with reserpine (20 microM) for 1 h in vitro virtually abolished the ability of these cells to transfer CS to naive mice. This was not a cytolytic effect, as the viability of the I-LNC treated with reserpine was not affected, and washing of the reserpine-treated I-LNC before transfer fully restored their ability to orchestrate a CS response. The action of the drug was not mediated by an effect on mast cells, since the experiment could be performed using mast cell-deficient W/Wv mice as both donors and recipients of I-LNC. In addition, the effect was specific for the treated cells: mice that received reserpine-treated I-LNC(Ox) intravenously together with untreated I-LNC(DNFB) did not develop CS to Ox but responded normally to DNFB; and local intradermal injection of reserpine-treated I-LNC(Ox) which failed to transfer reactivity to Ox, did not interfere with the development of CS to DNFB at the same site. Finally, cotransfer experiments indicated that the effect of reserpine on the transfer of CS was not due to activation of suppressor cells. Our findings strongly suggest that whatever effects reserpine might have on immunologically nonspecific host cells, the drug's effects on sensitized T cells are sufficient to explain its ability to block cell-mediated immune responses in vivo.     

Hyperalgesia due to nerve damage: role of nerve growth factor.

The hypothesis that nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) contribute to hyperalgesia resulting from nerve damage was tested in rats in which the sciatic nerve was partially transected on one side. Administration of antisera raised against NGF and BDNF relieved mechanical and thermal hyperalgesia in these animals. It has been suggested that NGF may elicit hyperalgesia by inducing mast cells to release algesic agents such as serotonin (5-HT). We found that degranulation of mast cells with compound 48/80 relieved mechanical and thermal hyperalgesia produced by nerve damage. We also found that local injection of the 5-HT2A and 5-HT3 receptor antagonists ketanserin and ICS 205-930 into the affected hind paw relieved mechanical hyperalgesia in a dose-dependent fashion. These findings support the idea that in this rat model of hyperalgesia due to peripheral nerve damage, NGF acts on mast cells to induce release of 5-HT, which sensitizes nociceptors. Hyperalgesia due to nerve injury and hyperalgesia due to inflammation may share some common features.     

Mechanical stimulation activates Galphaq signaling pathways and 5-hydroxytryptamine release from human carcinoid BON cells

5-Hydroxytryptamine (5-HT) released from enterochromaffin cells activates secretory and peristaltic reflexes necessary for lubrication and propulsion of intestinal luminal contents. The aim of this study was to identify mechanosensitive intracellular signaling pathways that regulate 5-HT release. Human carcinoid BON cells displayed 5-HT immunoreactivity associated with granules dispersed throughout the cells or at the borders. Mechanical stimulation by rotational shaking released 5-HT from BON cells or from guinea pig jejunum during neural blockade with tetrodotoxin. In streptolysin O-permeabilized cells, guanosine 5'-O- (2-thiodiphosphate) (GDP-beta-S) and a synthetic peptide derived from the COOH terminus of Galphaq abolished mechanically evoked 5-HT release, while the NH(2)-terminal peptide did not. An antisense phosphorothioated oligonucleotide targeted to a unique sequence of Galphaq abolished mechanically evoked 5-HT release and reduced Galphaq protein levels without affecting the expression of Galpha(11). Depletion and chelation of extracellular calcium did not alter mechanically evoked 5-HT release, whereas depletion of intracellular calcium stores by thapsigargin and chelation of intracellular calcium by 1,2-bis (o-Aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM) reduced 5-HT release. Mechanically evoked 5-HT release was inhibited by somatostatin-14 in a concentration-dependent manner. The results suggest that mechanical stimulation of enterochromaffin-derived BON cells directly or indirectly stimulates a G protein-coupled receptor that activates Galphaq, mobilizes intracellular calcium, and causes 5-HT release.     

Release of histamine from dural mast cells by substance P and calcitonin gene-related peptide

The aim of the present study was to examine if the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) can stimulate histamine release from mast cells in the dura mater and thereby play a role in cranial vasoregulation and local neurogenic inflammation. Dura mater mast cells were compared with peritoneal mast cells in the rat. Histamine was released from dura mater mast cells by compound 48/80, SP and CGRP but from peritoneal mast cells only by compound 8/80 and SP. NPY and VIP released quite small amounts of histamine from dural mast cells. The release on SP and CGRP from rat dura mater mast cells was blocked by the receptor antagonists FK888 and CGRP_8–37 respectively, suggesting receptor mediated release mechanisms. None of the stimuli released histamine from human or porcine dural mast cells, possibly because the sampling procedure injures and incapacitates the cells.     

Morphological and secretory properties of bronchoalveolar lavage mast cells in respiratory diseases

We have studied various functional and morphological characteristics of mast cells obtained in bronchoalveolar lavage from fifty-two patients with several lung diseases. The percentage of mast cells ranged from 0.04 to 0.6% (bronchial carcinoma), 0.05-0.3% (sarcoidosis), 0.06-0.25% (asthma), 0.04-1.8% (miscellaneous) and 0.02-0.04% (normals). There were no significant differences in the mast cell counts between the disease groups. Lung mast cells exhibited heterogeneity of size, shape and intensity of staining. Cells from thirty-seven subjects were further studied for total histamine content and histamine release using various secretagogues. There was a significant correlation (P less than 0.001) between the histamine content of the total lavage cell population and mast cell counts. The calculated mean histamine content per mast cell was 6.35 pg. Histamine was released in a dose-dependent fashion after stimulation with anti-IgE, calcium ionophore and phorbol myristate acetate with a time course of histamine release characteristic of the mast cell. Unlike peripheral blood basophils, no release was observed following incubation with f-met-leu-phe (10(-6)-10(-8) M) and neither cell type released histamine following incubation with 48/80 (10 micrograms/ml). Inhibition of anti-IgE-induced histamine release was obtained following pre-incubation with salbutamol (10(-4)-10(-6) M). These studies indicate that bronchoalveolar lavage is a suitable model for the study of human lung mast cells.     

Pharmacological aspects of anticancer drug-induced emesis with emphasis on serotonin release and vagal nerve activity

Cytotoxic drug-induced nausea and vomiting are the side effects most feared by cancer patients. Emesis is an instinctive defense reaction caused by the somatoautonomic nerve reflex, which is integrated in the medulla oblongata. Emesis caused by cytotoxic drugs such as cisplatin is associated with an increase in the concentration of 5-hydroxytryptamine (5-HT) in the intestine and the brainstem. It is proposed that cytotoxic drugs evoke 5-HT release from the enterochromaffin (EC) cells in the intestinal mucosa and that the released 5-HT stimulates the 5-HT receptors on the adjacent vagal afferent nerves. The depolarization of the vagal afferent nerves stimulates the vomiting center in the brainstem and eventually induces a vomiting reflex. 5-HT released from EC cells seems to mediate the cisplatin-induced emesis sensitive to 5-HT(3) receptor antagonists. The release of 5-HT from the EC cells, however, is regulated by polymodal mechanisms on autoreceptors or heteroreceptors. The precise role of 5-HT on the occurrence of vomiting has not been fully elucidated. The present review aims to describe the role of 5-HT in anticancer drug-induced emesis from the viewpoint of 5-HT release and afferent vagus nerve activity. Various methods for predicting emesis are also evaluated.     

Sensitization and activation of intracranial meningeal nociceptors by mast cell mediators

Intracranial headaches such as migraine are thought to result from activation of sensory trigeminal pain neurons that supply intracranial blood vessels and the meninges, also known as meningeal nociceptors. Although the mechanism underlying the triggering of such activation is not completely understood, our previous work indicates that the local activation of the inflammatory dural mast cells can provoke a persistent sensitization of meningeal nociceptors. Given the potential importance of mast cells to the pain of migraine it is important to understand which mast cell-derived mediators interact with meningeal nociceptors to promote their activation and sensitization. In the present study, we have used in vivo electrophysiological single-unit recording of meningeal nociceptors in the trigeminal ganglion of anesthetized rats to examine the effect of a number of mast cell mediators on the activity level and mechanosensitivity of meningeal nociceptors. We have found that that serotonin (5-HT), prostaglandin I(2) (PGI(2)), and to a lesser extent histamine can promote a robust sensitization and activation of meningeal nociceptors, whereas the inflammatory eicosanoids PGD(2) and leukotriene C(4) are largely ineffective. We propose that dural mast cells could promote headache by releasing 5-HT, PGI(2), and histamine.     

Effects of relaxin on mast cells. In vitro and in vivo studies in rats and guinea pigs.

The results of the current study demonstrate that relaxin inhibits histamine release by mast cells. This effect is related to the peptide concentrations, and could be observed in both isolated rat serosal mast cells stimulated with compound 48/80 or calcium ionophore A 23187, and in serosal mast cells isolated from sensitized guinea pigs and challenged with the antigen. The morphological findings agree with the functional data, revealing that relaxin attenuates calcium ionophore-induced granule exocytosis by isolated rat serosal mast cells. Similar effects of relaxin have also been recognized in vivo by light microscopic and densitometric analysis of the mesenteric mast cells of rats which received the hormone intraperitoneally 20 min before local treatment of the mesentery with calcium ionophore. Moreover, evidence is provided that relaxin stimulates endogenous production of nitric oxide and attenuates the rise of intracellular Ca2+ concentration induced by calcium ionophore. The experiments with drugs capable of influencing nitric oxide production also provide indirect evidence that the inhibiting effect of relaxin on mast cell histamine release is related to an increased generation of nitric oxide. It is suggested that relaxin may have a physiological role in modulating mast cell function through the L-arginine-nitric oxide pathway.     

Mast cell activation enhances airway responsiveness to methacholine in the mouse.

Mast cell-deficient mutant mice and their normal littermates were used to determine whether activation of mast cells by anti-IgE enhances airway responsiveness to bronchoactive agonists in vivo. Pulmonary conductance was used as an index of airway response as the mice were challenged with increasing intravenous doses of methacholine (Mch) or 5-hydroxytryptamine (5-HT). Mast cell activation with anti-IgE enhanced pulmonary responsiveness to Mch in both types of normal mice (P < 0.0001 by analysis of variance) but not in either genotype of mast cell-deficient mouse. Additionally, anti-IgE pretreatment of genetically mast cell-deficient W/Wv mice whose mast cell deficiency had been repaired by infusion of freshly obtained bone marrow cells or bone marrow-derived cultured mast cells from congenic normal mice led to significant (P < 0.0001) enhancement of Mch responsiveness. 5-HT responsiveness was not significantly influenced by anti-IgE pretreatment in any of the mice studied. The data support the hypothesis that IgE-mediated activation of mast cells enhances pulmonary responsiveness to cholinergic stimulation.     

Evidence for 5-HT1B/1D receptors mediating the antimigraine effect of sumatriptan and dihydroergotamine

Neurogenic plasma extravasation, endothelial cell activation (increase in vesicle number and vacuole formation), platelet aggregation and adhesion, and mast cell degranulation occur selectively in post-capillary venules of the dura mater following electrical trigeminal ganglion stimulation, and are mediated by release of neuropeptides from perivascular unmyelinated C fibres. Pre-treatment with the antimigraine drugs dihydroergotamine and sumatriptan, two drugs that bind with high affinity to 5-HT1B/1D receptors, markedly attenuated plasma protein extravasation induced by electrical trigeminal ganglion stimulation. Trigeminal stimulation increased plasma calcitonin gene-related peptide levels in rat superior sagittal sinus. Pre-treatment with dihydroergotamine and, to a lesser extent, sumatriptan, attenuated this increase. Both drugs reduced morphological changes in post-capillary venules and mast cells within dura mater following electrical trigeminal ganglion stimulation. Plasma protein extravasation was selectively blocked in dura mater (but not in extracranial tissues) by pre-treatment with those receptor agonists showing a rank order of potency suggesting a 3-HT1B/1D interaction (5-CT greater than 5-BT greater than DHE greater than sumatriptan greater than 8-OH-DPAT). Pre-treatment with 5-HT2 and 5-HT3 antagonists was not effective. Taken together, these data are consistent with the interpretation that putative 5-HT-1B/1D receptors located on sensory fibres are coupled to inhibition of peptide release and blockade of neurogenic inflammation. An important therapeutic action of ergot alkaloids and sumatriptan in migraine headaches is so defined.     

Differential effect of histamine 3 receptor-active agents on brain, but not peritoneal, mast cell activation.

The activation of presynaptic histamine 3 (H(3)) receptors inhibits the release of histamine and other neurotransmitters from central nervous system neurons. Rat brain mast cells (MCs) release histamine and 5-hydroxytryptamine (5-HT) in response to neuropeptides and neurotransmitters secreted from adjacent neurons. Dura MCs also degranulate in response to antidromic trigeminal nerve stimulation and with acute psychological stress. Such findings have implicated brain MCs in certain neuroinflammatory disorders, such as migraines. We investigated the ultrastructural appearance of control and stimulated thalamic/hypothalamic (brain) MCs before and after treatment with the H(3) receptor agonist N(alpha)-methylhistamine (N(alpha)-mH) and the H(3) receptor antagonist thioperamide (Th). Ultrastructural investigation of brain MCs stimulated with compound 48/80 revealed extensive intragranular changes that paralleled 5-HT secretion but without degranulation by exocytosis typical of connective tissue MCs. N(alpha)-mH significantly reduced these morphological changes, as well as 5-HT release from brain MCs and neurons stimulated with KCl; conversely, Th augmented both histamine and 5-HT release from brain neurons and MCs. Neither N(alpha)-mH nor Th had any effect on peritoneal MCs. Simultaneous addition of both drugs largely antagonized each other's effects on brain MC activation and 5-HT secretion. Ultrastructural observations and lack of lactic dehydrogenase release in the perfusate excluded any cytotoxic effect. The ability of H(3) agonists to inhibit brain MC activation, as well as secretion of 5-HT from both brain MCs and neurons, may be useful in the management of migraines.     

Mast Cells Can Secrete Vascular Permeability Factor/ Vascular Endothelial Cell Growth Factor and Exhibit Enhanced Release after Immunoglobulin E–dependent Upregulation of Fcε Receptor I Expression

Vascular permeability factor/vascular endothelial cell growth factor (VPF/VEGF) can both potently enhance vascular permeability and induce proliferation of vascular endothelial cells. We report here that mouse or human mast cells can produce and secrete VPF/VEGF. Mouse mast cells release VPF/VEGF upon stimulation through Fcε receptor I (FcεRI) or c-kit, or after challenge with the protein kinase C activator, phorbol myristate acetate, or the calcium ionophore, A23187; such mast cells can rapidly release VPF/VEGF, apparently from a preformed pool, and can then sustain release by secreting newly synthesized protein. Notably, the FcεRI-dependent secretion of VPF/VEGF by either mouse or human mast cells can be significantly increased in cells which have undergone upregulation of FcεRI surface expression by a 4-d preincubation with immunoglobulin E. These findings establish that at least one cell type, the mast cell, can be stimulated to secrete VPF/VEGF upon immunologically specific activation via a member of the multichain immune recognition receptor family. Our observations also identify a new mechanism by which mast cells can contribute to enhanced vascular permeability and/or angiogenesis, in both allergic diseases and other settings.     

Serotonin-stimulated release of [3H]dopamine via reversal of the dopamine transporter in rat striatum and nucleus accumbens: a comparison with release elicited by potassium, N-methyl-D-aspartic acid, glutamic acid and D-amphetamine.

Release of preloaded radiolabeled dopamine ([3H]DA) elicited by several agents from terminal fields of mesolimbic and nigrostriatal projections in rats was compared. Several similarities between the two areas were observed. For example, potassium, which stimulates release both directly, through altering the potential across the membrane of the dopaminergic neuron, as well as indirectly, presumably by releasing endogenous excitatory neurotransmitters, exhibited some similarities to release stimulated by L-glutamate and N-methyl-D-aspartic acid. These included sensitivity to tetrodotoxin (TTX), Mg++ and Ca++. In contrast, release of [3H]DA stimulated by serotonin (5-HT), like that stimulated by D-amphetamine, depended upon a functional dopamine transport system and was less sensitive to TTX, Mg++ and Ca++. 5-HT-stimulated [3H]DA release in striatum (STR) and nucleus accumbens (NACC) was not modified by antagonists at 5-HT2 or 5-HT3 receptors. Differences were observed in release of [3H]DA from STR and NACC. Elevated potassium (20 mM) released about twice as much [3H]DA from NACC as it did from STR. 5-HT was also able to release more [3H]DA from NACC than from STR. Conversely, D-amphetamine released more [3H]DA from STR than from NACC. TTX increased release stimulated by potassium in STR, but decreased release stimulated by potassium in NACC. These observations suggest that receptor- and non-receptor-mediated mechanisms may contribute to regulation of [3H]DA release in mesolimbic and nigrostriatal areas of the brain. It is possible that endogenous 5-HT in STR or NACC acts as a local regulator of DA release acting via a transport-dependent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of leukotrienes by purified human lung mast cells.

Although mediator release from mast cells and basophils plays a central role in the pathogenesis of human allergic disease, biochemical studies have been restricted to rat peritoneal mast cells and basophilic leukemia cells because they could be easily purified. We have used two new techniques of cell separation to purify human lung mast cells to 98% homogeneity. Lung cell suspensions were obtained by dispersion of chopped lung tissue with proteolytic enzymes. Mast cells were then purified from the suspensions by countercurrent centrifugal elutriation and affinity chromatography. The purified mast cells released both histamine and slow-reacting substance of anaphylaxis (SRS-A) (leukotriene C and D) during stimulation with goat anti-human IgE antibody. Moreover, these preparations were able to generate significant quantities of SRS-A (32 +/- 7 x 10(-17) LTD mole-equivalents/mast cell) at all stages of purification, indicating that a secondary cell is not necessary for the antigen-induced release of SRS.     

The site of action of the histamine releaser compound 45/80 in causing mast cell degranulation.

A primary amine analog of compound 48/80 (nor-48/80) was purified and attached to Sepharose beads through an albumin link. Suspensions of rat peritoneal cells were passed over the beads in an affinity chromatography column, and the proportion of the mast cells that was retained by the beads was determined. Sixty-seven percent of the mast cells were found to be retained by the column, indicating the existence of a binding site for nor-48/80 on the exterior of mast cells. The beads were larger than the cells, and hence precluded the entry of the nor-48/80 into the cells. Neither the Sepharose beads nor the albumin link was responsible for this amount of binding because control beads without nor-48-80 retained only 22% of the mast cells. Mast cells incubated with large quantities of the beads in a batch procedure released up to 53% of the mast cell histamine, whereas control beads without the polymer released only 18%. This release could not be attributable to soluble nor-48/80 because only trace amounts of radioactive nor-48/80 were released from beads soaked overnight, and these supernatants released insignificant amounts of histamine when incubated with mast cells. These studies indicate the presence of a receptor for 48/80 on the mast cell membrane which can trigger histamine release.     

Immunologic and Nonimmunologic Generation of Superoxide from Mast Cells and Basophils

Mediator release from rat peritoneal and human lung mast cells as well as human leukemic basophils was examined to determine whether super-oxide (O(-) (2)) was concomitantly generated. Immunologic or nonimmunologic stimulation of each preparation induced parallel release of histamine and O(-) (2) within 2 min. O(-) (2) production was quantitated by superoxide dismutase (SOD)-inhibitable chemiluminescence and cytochrome c reduction. SOD was detected in basophil and mast cell lysates and was also released by rat mast cells stimulated by anti-IgE. Secretory granules isolated from purified rat mast cells released histamine, O(-) (2), and SOD upon exposure to cations. Thus, both superoxide radicals and SOD may play a role in host defenses involved in immediate hypersensitivity reactions.     

Mast Cell Accumulation and Degranulation in Rat Bladder with Partial Outlet Obstruction

Introduction

Benign prostatic hyperplasia causes partial bladder outlet obstruction (pBOO), and many patients with pBOO are affected by not only voiding symptoms but also storage symptoms. We previously suggested that enhancement of 5-hydroxytryptamine (5-HT)-induced bladder contraction in the pBOO bladder may be one cause of storage symptoms. However, little is known about the presence of 5-HT in rat bladders. In this study, we hypothesized that mast cells are a source of 5-HT and investigated the distribution of mast cells and 5-HT in the bladders of rats with pBOO.

Methods

The bladders of female Sprague–Dawley rats were subjected to pBOO and sham operations for 1 week, were isolated, and were fixed for light or electron microscopy. Mast cells and 5-HT in the bladders were detected by toluidine blue staining and immunohistochemical staining, respectively. The mast cells were counted under a light microscope. Degranulated mast cells were observed under an electron microscope and counted under a light microscope.

Results

Mast cells were present in the mucosa/submucosa region in sham rat bladders. Their number was increased in the detrusor muscle/subserosa/serosa region, especially the subserosal layer, in pBOO rat bladders. The localization of mast cells almost matched that of 5-HT-positive cells in consecutive sections. Degranulated mast cells were present in sham and pBOO rat bladders, but the proportion of degranulated mast cells was significantly increased in every region in pBOO rat bladders compared with that in sham rat bladders.

Conclusion

These results suggest that mast cells contain 5-HT and are more abundant locally in the subserosal layer of pBOO rat bladders. 5-HT released from mast cells could stimulate 5-HT₂ receptors on the detrusor muscle, and this may underlie storage symptoms.

Funding

Asahi Kasei Pharma Corp.

     

Modulation of macrophage superoxide-induced cytochrome c reduction by mast cells

The effect of mast cells and mast cell granules on macrophage O2- release as determined by cytochrome c reduction was studied. In vitro activation of mast cells before macrophage activation caused a decrease in O2- -mediated cytochrome c reduction. This decrease was proportional to mast cell activation and reached 80% to 100% when mast cell mediator release was 40% to 50%. Incubation of isolated mast cell granules with macrophages before activation also inhibited O2- -mediated cytochrome c reduction in a dose-dependent manner. Mast cell granule-mediated inhibition of cytochrome c reduction was not caused by histamine, serotonin, or any other dialyzable components but was found to be caused by the scavenging of O2- by mast cell granule-bound superoxide dismutase. Macrophage uptake of sulfur 35-labeled mast cell granules, electron microscopic localization of mast cell granules in the macrophage phagosomes, and the abrogation of mast cell granule effect when the cells were preincubated at 0 degree C indicate that the effect was associated with the adherence or phagocytosis (or both) of mast cell granules. These results suggest that mast cell granules interact with macrophages and that granule superoxide dismutase scavenges O2- generated by the phagocytes.     

Substance P and vasoactive intestinal peptide degradation by mast cell tryptase and chymase

The peptides substance P (SP) and vasoactive intestinal peptide (VIP) released from peptidergic neurons have potent effects on gland secretion and on smooth muscle tone. Because mast cells release proteases during degranulation, and are located in many of the same tissue microenvironments into which SP and VIP are released, we wished to examine whether mast cell proteases, by cleaving and thus inactivating these peptides, could modulate their effects. We used active site-titrated preparations of the two major neutral proteases of mast cell granules, tryptase and chymase, to determine the sites and rates of cleavage of SP and VIP. The proteases were purified from dog mastocytomas. Tryptase cleaved VIP rapidly at two sites with a kcat/Km of 2.2 X 10(5) sec-1 M-1, but had no effect on SP. Chymase cleaved both SP and VIP at primarily a single site with kcat/Km of 3.9 X 10(4) and 5.4 X 10(4) sec-1 M-1, respectively. Thus, these data show that mast cell proteases degrade SP and VIP. The differences in peptidase activity between tryptase and chymase suggest that the consequences of protease release could vary according to mast cell protease phenotype and location in various tissues and species. Tryptase, by cleaving the bronchodilator VIP but not the bronchoconstrictor SP, might promote bronchial hyper-responsiveness in asthma by decreasing the nonadrenergic neural inhibitory influence mediated by VIP. In skin and other tissues, chymase might interrupt axon reflex-mediated neurogenic inflammation by cleaving SP.