The Lyme disease spirochete, Borrelia burgdorferi, is introduced into human hosts via tick bites. Among the cell types present in the skin which may initially contact spirochetes are mast cells. Since spirochetes are known to activate a variety of cell types in vitro, we tested whether B. burgdorferi spirochetes could activate mast cells. We report here that freshly isolated rat peritoneal mast cells or mouse MC/9 mast cells cultured in vitro with live or freeze-thawed B. burgdorferi spirochetes undergo low but detectable degranulation, as measured by [5-3H] hydroxytryptamine release, and they synthesize and secrete the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). In contrast to findings in previous studies, where B. burgdorferi-associated activity was shown to be dependent upon protein lipidation, mast cell TNF-alpha release was not induced by either lipidated or unlipidated recombinant OspA. This activity was additionally shown to be protease sensitive and surface expressed. Finally, comparisons of TNF-alpha-inducing activity in known low-, intermediate-, and high-passage B. burgdorferi B31 isolates demonstrated passage-dependent loss of activity, indicating that the activity is probably plasmid encoded. These findings document the presence in low-passage B. burgdorferi spirochetes of a novel lipidation-independent activity capable of inducing cytokine release from host cells. (+info)
Potent mast cell degranulation and vascular permeability triggered by urocortin through activation of corticotropin-releasing hormone receptors.
(2/1340)
Urocortin (Ucn) is related to corticotropin-releasing hormone (CRH), and both are released in the brain under stress where they stimulate CRH 1 and 2 receptors (CRHR). Outside the brain, they may have proinflammatory actions through activation of mast cells, which are located perivascularly close to nerve endings and degranulate in response to acute psychological stress. Here, we report that a concentration of intradermal Ucn as low as 10 nM induced dose-dependent rat skin mast cell degranulation and increased vascular permeability. This effect appeared to be equipotent to that of calcitonin gene-related peptide and neurotensin. Ucn-induced skin vasodilation was inhibited by pretreatment with the mast cell stabilizer disodium cromoglycate (cromolyn) and was absent in the mast cell-deficient W/Wv mice. The selective nonpeptide CRH receptor 1 antagonist, antalarmin and the nonselective peptide antagonist astressin both reduced vascular permeability triggered by Ucn but not that by Substance P or histamine. In contrast, the peptide antagonist alpha-helical CRH-(9-41) reduced the effect of all three. The vasodilatory effect of Ucn was largely inhibited by pretreatment with H1 receptor antagonists, suggesting that histamine is the major mediator involved in vitro. Neuropeptide depletion of sensory neurons, treatment with the ganglionic blocker hexamethonium, or in situ skin infiltration with the local anesthetic lidocaine did not affect Ucn-induced vascular permeability, indicating that its in situ effect was not mediated through the peripheral nervous system. These results indicate that Ucn is one of the most potent triggers of rat mast cell degranulation and skin vascular permeability. This effect of Ucn may explain stress-induced disorders, such as atopic dermatitis or psoriasis, and may lead to new forms of treatment. (+info)
A functional granulocyte colony-stimulating factor receptor is required for normal chemoattractant-induced neutrophil activation.
(3/1340)
Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that is widely used to treat neutropenia. In addition to stimulating polymorphonuclear neutrophil (PMN) production, G-CSF may have significant effects on PMN function. Because G-CSF receptor (G-CSFR)-deficient mice do not have the expected neutrophilia after administration of human interleukin-8 (IL-8), we examined the effect of the loss of G-CSFR on IL-8-stimulated PMN function. Compared with wild-type PMNs, PMNs isolated from G-CSFR-deficient mice demonstrated markedly decreased chemotaxis to IL-8. PMN emigration into the skin of G-CSFR-deficient mice in response to IL-8 was also impaired. Significant chemotaxis defects were also seen in response to N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, or macrophage inflammatory protein-2. The defective chemotactic response to IL-8 does not appear to be due to impaired chemoattractant receptor function, as the number of IL-8 receptors and chemoattractant-induced calcium influx, actin polymerization, and release of gelatinase B were comparable to those of wild-type PMNs. Chemoattractant-induced adhesion of G-CSFR-deficient PMNs was significantly impaired, suggesting a defect in beta2-integrin activation. Collectively, these data demonstrate that selective defects in PMN activation are present in G-CSFR-deficient mice and indicate that G-CSF plays an important role in regulating PMN chemokine responsiveness. (+info)
Neurotensin is a proinflammatory neuropeptide in colonic inflammation.
(4/1340)
The neuropeptide neurotensin mediates several intestinal functions, including chloride secretion, motility, and cellular growth. However, whether this peptide participates in intestinal inflammation is not known. Toxin A, an enterotoxin from Clostridium difficile, mediates pseudomembranous colitis in humans. In animal models, toxin A causes an acute inflammatory response characterized by activation of sensory neurons and intestinal nerves and immune cells of the lamina propria. Here we show that neurotensin and its receptor are elevated in the rat colonic mucosa following toxin A administration. Pretreatment of rats with the neurotensin receptor antagonist SR-48, 692 inhibits toxin A-induced changes in colonic secretion, mucosal permeability, and histologic damage. Exposure of colonic explants to toxin A or neurotensin causes mast cell degranulation, which is inhibited by SR-48,692. Because substance P was previously shown to mediate mast cell activation, we examined whether substance P is involved in neurotensin-induced mast cell degranulation. Our results show that neurotensin-induced mast cell degranulation in colonic explants is inhibited by the substance P (neurokinin-1) receptor antagonist CP-96,345, indicating that colonic mast activation in response to neurotensin involves release of substance P. We conclude that neurotensin plays a key role in the pathogenesis of C. difficile-induced colonic inflammation and mast cell activation. (+info)
Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence.
(5/1340)
The secretion process of the mucosal mast cell line RBL-2H3 was imaged using infrared three photon excitation (3PE) of serotonin (5-hydroxytryptamine, 5-HT) autofluorescence, a measurement previously difficult because of the technical intractability of deep UV optics. Images of prestimulation 5-HT distributions were analyzed in loaded cell populations (those incubated in a 5-HT-rich medium overnight) and in unloaded populations and were found to be strictly quantifiable by comparison with bulk population high-performance liquid chromatography measurements. Antigenically stimulated cells were observed to characteristically ruffle and spread as granular 5-HT disappeared with no detectable granule movement. Individual cells exhibited highly heterogeneous release kinetics, often with quasi-periodic bursts. Neighboring granule disappearances were correlated, indicative of either spatially localized signaling or granule-granule interactions. In one-half of the granule release events, weak residual fluorescence was visible suggestive of leftover 5-HT still bound to the granule matrix. The terminal stages of secretion (>300 s) consisted primarily of unresolved granules and remainder 5-HT leakage from already released granules. (+info)
Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy.
(6/1340)
We have observed secretory granules beneath the plasma membrane of chromaffin cells. Using evanescent-field excitation by epiillumination, we have illuminated a thin layer of cytosol where cells adhere to glass coverslips. Up to 600 frames could be recorded at diffraction-limited resolution without appreciable photodynamic damage. We localized single granules with an uncertainty of approximately 30 nm and tracked their motion in three dimensions. Granules in resting cells wander randomly as if imprisoned in a cage that leaves approximately 70 nm space around a granule. The "cage" itself moves only slowly (D = 2 x 10(-12) cm2/s). Rarely do granules arrive at or depart from the plasma membrane of resting cells. Stimulation increases lateral motion only slightly. After the plasma membrane has been depleted of granules by exocytosis, fresh granules can be seen to approach it at an angle. The method will be useful for exploring the molecular steps preceding exocytosis at the level of single granules. (+info)
LXA4, aspirin-triggered 15-epi-LXA4, and their analogs selectively downregulate PMN azurophilic degranulation.
(7/1340)
The eicosanoid lipoxin A4 (LXA4) is biosynthesized in vivo by cells present at inflammatory sites and appears to be an endogenous anti-inflammatory mediator. Further, in the presence of aspirin, the 15-epimer of LXA4 (15-epi-LXA4) is biosynthesized and may mediate some of aspirin's desirable bioactions. LXA4, 15-epi-LXA4, and their stable analogs inhibit inflammation in established animal models, indicating that these compounds may be useful for treating inflammatory disease states. To investigate the cellular mechanisms by which these lipid mediators downregulate inflammation, we investigated whether these eicosanoids could influence receptor-mediated degranulation of human neutrophils, an event thought to play a major causative role in several inflammatory disease states. LXA4, 15-epi-LXA4, and their stable analogs potently (IC50 < 1 nM) and selectively downregulated neutrophil release of azurophilic granule contents but did not affect other neutrophil secretory functions. Thus the cellular basis of action of these natural off-switches to inflammation appears to involve downregulation of neutrophil azurophilic granule release. (+info)
Nerve growth factor modifies the expression of inflammatory cytokines by mast cells via a prostanoid-dependent mechanism.
(8/1340)
Nerve growth factor (NGF) is well recognized to have a number of potent effects on mast cells, including increasing mast cell numbers in vivo and inducing mast cell degranulation in vitro. More recently, NGF has been demonstrated to induce PGD2 production by mast cells through the induction of mast cell cyclooxygenase expression. We have observed that NGF at doses as low as 10 ng/ml will induce IL-6 production and inhibit TNF-alpha release from rat peritoneal mast cells in the presence of lysophosphatidylserine as a cofactor. NGF synergizes with LPS treatment of peritoneal mast cells (PMC) for the induction of IL-6. Examination of the mechanism of this phenomenon has revealed that NGF can induce both rat PMC and mouse bone marrow-derived cultured mast cells to produce substantial levels of PGE2. This response is maximal at later time points 18-24 h after NGF activation. The ability of NGF to induce PGE2 is not dependent on mast cell degranulation. Other stimuli capable of inducing IL-6, such as LPS, do not induce production of this prostanoid. Inhibition of cyclooxygenase activity by PMC using either flurbiprofen or indomethacin inhibited both the NGF-induced PGE2 synthesis and the NGF-induced alterations in TNF-alpha and IL-6 production. These results suggest a role for mast cell-derived prostanoids in the regulation of local inflammatory responses and neuronal degeneration after tissue injury involving induction of NGF production. (+info)