Diverse effects of bacterial cell wall components on mast cell degranulation,cysteinyl leukotriene generation and migration

Nowadays there is more and more evidence that mast cells take part in antibacterial defence. Mast cells have the ability to kill bacteria via phagocytose‐dependent or phagocytose‐independent ways and express antimicrobial peptides that can directly kill pathogens at their site of entry. What is more, mast cells are capable of processing bacterial antigens for presentation through class I and II MHC molecules. Some data indicate that these cells can release various proinflammatory mediators in response to activation with bacteria and/or their products, however this information is still far from complete. Therefore, in this study we examined the ability of PGN from Staphylococcus aureus, LPS from Eschericha coli and LAM from Mycobacterium smegmatis to stimulate mature rat mast cell degranulation as well as cysteinyl LT generation. We also studied the influence of these bacterial components on mast cell migration. We found that PGN, LPS and LAM all failed to induce mast cell degranulation and histamine release. At the same time, activation of mast cells with these bacterial antigens resulted in generation and release of significant amounts of LT. Moreover, we documented that, even in the presence of laminin, none of the bacterial antigens used stimulated mast cell migration. However, PGN did induce migration of RANTES‐primed mast cells, and LPS did stimulate mast cell migratory response after priming with IL‐6. Our results show that PGN, LPS and LAM might be among the important bacterial antigens involved in mast cell activation during bacterial infection.     

The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.

The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of colchicine on histamine secretion and calcium uptake in mast cells

The function of contractile system of microtubules on the mechanism of mast cell exocytosis by using colchicine, a depolymerizing alkaloid of the microtubular system, has been studied. The response of histamine release and 45Ca-uptake in isolated rat mast cells treated with colchicine has been determined. The incubation of mast cells in the presence of 10(-8)-10(-3) M colchicine slightly inhibits histamine secretion induced by the stimulant concentration 50 micrograms/ml of compound 48/80 (35 +/- 5%). Similarly colchicine does not significantly affect histamine values spontaneously elicited in unstimulated mast cells; the percentages of secretion are never greater than 10%. However, high doses of this alkaloid are found to markedly inhibit entry of calcium ions into the cell. These results suggest that microtubules do not participate in the secretory process of mast cells, although they significantly decrease calcium uptake. The microtubules might be connected to the membrane, so that the depolymerization of this contractile system could damage the membrane structures through which Ca2+ is transported.     

Effect of serine phospholipid structure on the enhancement of concanavalin A-induced degranulation in rat mast cells

The correlations among the potentiating activity of various PS analogs on concanavalin A (Con A)-induced rat mast cell degranulation, the hemolytic activity and the incorporation into the mast cell membrane were studied. The following results were obtained. Lysophosphatidylserine (LysoPS) caused rat mast cell activation (degranulation) in the presence of Con A. The order of the activity was as follows: 1-stearoyl lysoPS = 1-palmitoyl lysoPS greater than 1-myristoyl lysoPS greater than 1-lauroyl lysoPS. The relative hemolytic activity of these compounds was similar to that observed in the mast cell activation. Dilauroyl PS, which shows similar hemolytic activity to 1-myristoyl lysoPS, did not activate mast cells appreciably. The relative activity of these phospholipids in the binding to mast cells was 1-stearoyl lysoPS greater than dilauroyl PS greater than 1-lauroyl lysoPS. Hemolytic activity, as well as activity on mast cells, of lysoPS analogs was well correlated to mast cell membrane incorporation, whereas such a correlation was not found with PS analogs. Dilauroyl PS could be accumulated in the mast cell membrane and showed hemolytic activity, but did not activate histamine secretion.     

Different potency of bacterial antigens TLR2 and TLR4 ligands in stimulating mature mast cells to cysteinyl leukotriene synthesis

The aim of study was to compare the potency of different bacterial antigens to induce rat mature mast cell to cysteinyl leukotriene (cysLT) generation. We examined Toll-like receptor (TLR)2 agonists, i.e. lipoteichoic acid (LTA) Staphylococcus faecalis, Streptococcus pyogenes, Bacillus subtilis and Staphylococcus aureus, lipoarabinomannan (LAM) Mycobacterium smegmatis, peptydoglican (PGN) Staphylococcus aureus, as well as TLR4 agonists, i.e. lipopolysaccharide (LPS) Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis, Pophyromonas gingivalis and Escherichia coli. We also estimated the effect of tumor necrosis factor (TNF)-, interleukin (IL)-6-, CCL5-, and IL-10-priming on mast cell cysLT synthesis following bacterial antigen activation. We found that all bacterial antigens activated mast cells to cysLT generation; however, the extent of cysLT release in response to stimulation varied. Out of the examined antigens LPS P. gingivalis exhibited the highest potency, as it induced cysLT generation acting at a very low concentration (10(-4) ng/mL). Other LPSs affected mast cells at higher (up to 10(5) -fold) concentrations. LTAs were the most effective at concentrations of 5 × 10(2) ng/mL, while LAM and PGN stimulated mast cells to maximal cysLT generation at concentrations as high as 10(5) ng/mL. Anti-TLR2 and anti-TLR4 antibodies, as well as nuclear factor κB (NF-κB) inhibitor significantly diminished cysLT generation in response to bacterial antigen stimulation. Priming with TNF, IL-6 and CCL5 did not affect bacterial antigen-induced cysLT generation, while IL-10-pretreatment caused significant decrease in cysLT synthesis by mast cells. These observations might have a great pathophysiological importance; inasmuch cysLTs strongly influence the development and intensity of inflammation during bacterial infection.     

Histamine-containing cells obtained from the nose hours after antigen challenge have functional and phenotypic characteristics of basophils.

The pattern of mediators and appearance of cells that stain with alcian blue during human experimental early and late phase allergic reactions suggest that basophils accumulate in nasal secretions within hours of local Ag stimulation. To further explore whether the histamine containing cells that enter the nose after Ag challenge are mast cells or basophils, we studied their functional and phenotypic characteristics. Approximately 24 h after intranasal Ag provocation of subjects with allergic rhinitis, nasal lavage was performed, and the cells were isolated for degranulation studies, analysis of surface Ag, and viability. The average histamine content per alcian blue staining cell was 0.78 +/- 0.2 pg (n = 7), similar to that reported for peripheral blood basophils. Nasal cells were challenged in vitro with anti-IgE, ragweed Amb a I, and FMLP and their responses were compared to those of peripheral blood basophils isolated simultaneously from the same donors. Nasal leukocytes released histamine maximally at 0.1 micrograms/ml of anti-IgE (35.8 +/- 7.8%, n = 7) and responded to FMLP (25.4 +/- 9.9%, n = 7). The response of the cells to ragweed Amb a I and anti-IgE was attenuated compared to peripheral blood basophils. Anti-IgE-induced histamine release was calcium and temperature dependent. Dual color immunofluorescence and flow cytometric analysis of the recovered nasal cells coexpressed CD18, a leukocyte marker not expressed by mast cells. The nasal cells consistently had high levels of spontaneous histamine release (19.5 +/- 2.0%, n = 22). The viability of all cells, assessed by erythrosin B dye exclusion, was 70 +/- 2% (n = 15). However, the viability of IgE-bearing cells was only 28.3 +/- 5.7% (n = 4). The characteristics of histamine release and the nature of the cellular surface markers provide functional proof that the histamine-containing cells accumulating after nasal Ag challenge are basophils and not mast cells.     

Polymyxin B inadequately quenches the effects of contaminating lipopolysaccharide on murine dendritic cells

Dendritic cell (DC) activation is commonly used as a measure of the immunomodulatory potential of candidate exogenous and endogenous molecules. Residual lipopolysaccharide (LPS) contamination is a recurring theme and the potency of LPS is not always fully appreciated. To address this, polymyxin B (PmB) is often used to neutralise contaminating LPS. However, the limited capacity of this antibiotic to successfully block these effects is neglected. Therefore, this study aimed to determine the minimum LPS concentration required to induce murine bone marrow-derived dendritic cell (BMDC) maturation and cytokine secretion and to assess the ability of PmB to inhibit these processes. LPS concentrations as low as 10 pg/ml and 20 pg/ml induced secretion of interleukin (IL)-6 and tumor necrosis factor (TNF)-α respectively, while a concentration of 50 pg/ml promoted secretion of IL-12p40. A much higher threshold exists for IL-12p70 as an LPS concentration of 500 pg/ml was required to induce secretion of this cytokine. The efficacy of PmB varied substantially for different cytokines but this antibiotic was particularly limited in its ability to inhibit LPS-induced secretion of IL-6 and TNF-α. Furthermore, an LPS concentration of 50 pg/ml was sufficient to promote DC expression of costimulatory molecules and PmB was limited in its capacity to reverse this process when LPS concentrations of greater than 20 ng/ml were used. There is a common perception that LPS is heat resistant. However, heat treatment attenuated the ability of low concentrations of LPS to induce secretion of IL-6 and IL-12p40 by BMDCs, thus suggesting that heat-inactivation of protein preparations is also an ineffective control for discounting potential LPS contamination. Finally, LPS concentrations of less than 10 pg/ml were incapable of promoting secretion of IL-6 independently but could synergise with heat-labile enterotoxin (LT) to promote IL-6, indicating that reducing contaminating endotoxin concentrations to low pg/ml concentrations is essential to avoid misleading conclusions regarding candidate immunomodulators.     

Augmentation of respiratory mast cell secretion of histamine caused by vagus nerve stimulation during antigen challenge

We studied the effect of vagus nerve stimulation on the mast cell secretion of histamine after intraarterial (i.a.) administration of Ascaris suum antigen (AA) into the bronchial circulation of 10 randomly selected, natively allergic dogs in vivo. Respiratory mast cell response was measured as the arteriovenous difference (AVd) in histamine concentration across the bronchus. Plasma histamine concentration was determined simultaneously from right atrium, right ventricle, and femoral artery 60 and 15 sec before and 15, 30, 45, 60, 75, and 90 sec after i.a. injection of sham (Kreb-Henseleit) diluent, 1:100, and 1:30 concentrations of AA. The mean AVd in plasma histamine for five parasympathetically blocked animals (neural blockade with hexamethonium and beta-adrenergic blockade with propranolol) was 1.28 +/- 0.61 ng/ml (sham), 5.16 +/- 19.7 ng/ml (1:100 AA), and 36.6 +/- 11.1 ng/ml (1:30 AA). Substantial augmentation was obtained when AA was administered during parasympathetic stimulation in five other animals (beta-adrenergic blockade, no neural blockade), which was caused by continuous bilateral electrical stimulation of the vagus nerves. A mean AVd in plasma histamine of 110 +/- 27.6 ng/ml was obtained after 1:100 AA (p less than 0.001 vs parasympathetic blockade) and 166 +/- 32.4 ng/ml for 1:30 AA (p less than 0.001 vs parasympathetic blockade). Parasympathetic stimulation alone did not cause secretion of histamine. In contrast to the AVd response, parasympathetic stimulation did not augment nonrespiratory mast cell secretion after AA challenge. We conclude that vagus nerve stimulation augments secretion of histamine from respiratory mast cells during antigen challenge. We demonstrate that parasympathetic stimulation may potentiate the response to antigen challenge in central airways through augmented mast cell secretion of mediator.     

Receptor-independent effects of natural cannabinoids in rat peritoneal mast cells in vitro

Cannabinoids can activate CB(1) and CB(2) receptors. Since a CB(2) mRNA has been described in rat peritoneal mast cells (RPMC), we investigated a series of cannabinoids and derivatives for their capacity to stimulate RPMC. Effects of natural cannabinoids Delta(9)-tetrahydrocannabinol (Delta(9)-THC), Delta(8)-THC, endocannabinoids (anandamide, palmitoylethanolamide) and related compounds (N-decanoyl-, N-lauroyl-, N-myristoyl-, N-stearoyl- and N-oleoyl-ethanolamines; N-palmitoyl derivatives (-butylamine, -cyclohexylamine, -isopropylamine); and N-palmitoyl, O-palmitoylethanolamine), and synthetic cannabinoids including WIN 55,212-2, SR141716A and SR144528 were assessed for their capacity to induce histamine release or prime RPMC stimulated by compound 48/80. Only Delta(9)-THC and Delta(8)-THC could induce non-lytic, energy- and concentration-dependent histamine releases from RPMC (respective EC(50) values: 23.5+/-1.2; 53.4+/-20.6 microM, and maxima: 71.2+/-5.5; 55.7+/-2.7% of the total RPMC histamine content). These were not blocked by CB(1) (SR141716A) or CB(2) (SR144528) antagonists, but reduced by pertussis toxin (100 ng/ml). Endocannabinoids and analogues did neither induce histamine secretion, nor prime secretion induced by compound 48/80 (0.2 microg/ml). Delta(9)-THC and Delta(8)-THC induced in vitro histamine secretion from RPMC through CB receptor-independent interactions, partly involving G(i/o) protein activation.     

Histochemical heterogeneity of dispersed human lung mast cells.

Cytocentrifuge preparations of enzymatically dispersed human lung parenchymal mast cells were examined by light microscopy after fixation in either Mota's basic lead acetate or 10% neutral buffered formalin followed by toluidine blue staining at pH 0.5. Fixation in Mota's basic lead acetate allowed detection of all mast cells. However, after formalin fixation only 10.8 +/- 1.3%, range 4.7 to 17%, n = 8 remained detectable (i.e., formalin "resistant"). Therefore, the vast majority of human lung mast cells lose their metachromatic staining after formalin fixation (i.e., are formalin "sensitive"). Mast cells were then separated on the basis of diameter by countercurrent elutriation and on the basis of density by discontinuous Percoll gradients. Histochemically distinct populations of mast cell types emerged in all lungs studied. The proportion of formalin-resistant mast cells increased as a function of diameter: less than 5% at diameters of less than or equal to 11 mu and densities less than or equal to 1.063 g/ml, to 30 to 40% in cells of diameters greater than or equal to 16 mu and densities greater than or equal to 1.100 g/ml. Maximum anti-IgE challenge of nearly homogeneous formalin-sensitive mast cells (94.3 +/- 2.1% purity, n = 6) caused the generation of both leukotriene C4 (64.6 +/- 26.4 pg/mast cell) and PGD2 (114.8 +/- 37.5 pg/mast cell). Six- to eight-fold enrichment of formalin-resistant mast cells did not significantly alter the histamine release response or profiles of arachidonate metabolites. Similar results were obtained for the nonimmunologic stimulus ionophore A23187. We conclude that two histochemically distinct subpopulations, of mast cells are present in human lung suspensions. Although formalin-sensitive cells account for almost 90% of lung mast cells, formalin-resistant cells are separable by their large diameters and higher densities. Both subtypes show similar histamine release responses and arachidonate oxidation profiles.     

Group II phospholipase A2 inhibitors suppressed lysophosphatidylserine-dependent degranulation of rat peritoneal mast cells.

Rat peritoneal mast cells were sensitized with IgE and challenged with the specific antigen in the presence of lysophosphatidylserine (lysoPS), an essential co-factor for rodent connective tissue mast cell degranulation, and the effects of phospholipase A2 inhibitors were examined. Mepacrine, a known inhibitor of phospholipase A2, at concentrations below 10(-5) M and anti-rat 14-kDa group II phospholipase A2 antibody inhibited histamine release, while they did not affect the prostaglandin generation. Like histamine release, prostaglandin generation in IgE- and antigen- challenged rat peritoneal mast cells was dependent on the presence of lysoPS. These results indicate that 14-kDa group II phospholipase A2 may play an essential role in IgE-, antigen-, and lysoPS-dependent degranulation process of rat peritoneal mast cells and that the mechanism whereby it participates may not be due to the production of lysoPS from PS in mast cell membranes.     

Abdominal vagotomy decreased the number of ova shed and serum progesterone levels on estrus in the cyclic hamster.

The effects of abdominal vagotomy (AVGT) on ovarian function were studied in cyclic hamsters. AVGT significantly decreased the number of ova shed (AVGT: 10.5 +/- 1.5 ova/hamster, sham: 15.8 +/- 0.7 ova/hamster; P less than 0.05) and serum progesterone levels (AVGT: 2.1 +/- 0.3 ng/ml, sham: 3.9 +/- 0.7 ng/ml; P less than 0.05) on the morning of estrus. However, progesterone concentrations in the corpora lutea and non-luteal ovary on estrus in the AVGT and sham groups were similar. The serum estradiol levels in both groups on proestrus increased from 0900 h (AVGT: 75 +/- 10 pg/ml, sham: 72 +/- 6 pg/ml) to 1500 h (AVGT: 204 +/- 27 pg/ml, sham: 196 +/- 35 pg/ml) but there was no significant difference between the two groups. Partial degranulation of ovarian mast cells was not increased in the AVGT group. Also, vasoactive intestinal peptide (VIP) content in the ovary was not increased by AVGT at 0900 h on proestrus (AVGT: 60.1 +/- 16.8 pg/ovary, sham: 37.2 +/- 14.3 pg/ovary). These results indicated that AVGT interferes with normal ovulation and results in an increase in the number of atretic follicles, but that these effects by AVGT seemed not to be mediated through ovarian mast cells and VIP.     

Neutrophil defensins induce histamine secretion from mast cells: mechanisms of action.

Defensins are endogenous antimicrobial peptides stored in neutrophil granules. Here we report that a panel of defensins from human, rat, guinea pig, and rabbit neutrophils all have histamine-releasing activity, degranulating rat peritoneal mast cells with EC50 ranging from 70 to 2500 nM, and between 45 and 60% of the total histamine released. The EC50 for defensin-induced histamine secretion correlates with their net basic charge at neutral pH. There is no correlation between histamine release and antimicrobial potency. Degranulation induced by defensins has characteristics similar to those of activation by substance P. The maximum percent histamine release is achieved in <10 s, and it can be markedly inhibited by pertussis toxin (100 ng/ml) and by pretreatment of mast cells with neuraminidase. These properties differ from those for degranulation induced by IgE-dependent Ag stimulation and by the calcium ionophore A23187. GTPase activity, a measure of G protein activation, was induced in a membrane fraction from mast cells following treatment with defensin. Thus, neutrophil defensins are potent mast cell secretagogues that act in a manner similar to substance P and 48/80, through a rapid G protein-dependent response that is mechanistically distinct from Ag/IgE-dependent mast cell activation. Defensins may provide important pathways for communication between neutrophils and mast cells in defenses against microbial agents and in acute inflammatory responses.     

The influence of anaerobic bacteria and mycoplasma isolated from the genital tract on mast cell activation

Nowadays, it is known that mast cells, numerously appearing in all organs and being a source of a wide range of mediators and cytokines, are involved both in physiological and pathological processes. The aim of our study was to examine whether vaginal bacteria, especially those participating in Bacterial vaginosis, are able to activate mast cells to mediators secretion. The study was done on rat peritoneal mast cells. The mast cells were incubated in vitro with suspensions of Bacteroides capillosus, Actinomyces naeslundii (2 strains), Peptostreptococcus spp., Lactobacillus fermentum (2 strains), Mycoplasma hominis or Ureaplasma urealyticum killed by temperature. Activation of mast cells was estimated on the basis of histamine release. It was established that M. hominis, U. urealyticum and B. capillosus strongly stimulated rat mast cells to histamine secretion (histamine release 53.0%, 17.4% and 10.0%, respectively). Histamine release induced by Peptostreptococcus spp., A. naeslundii and L. fermentum was lower (at a range of 2.4%-8.2%). The obtained results can suggest that presumably interactions between vaginal bacteria and placental mast cells could influence the course of pregnancy.     

Endothelin-1 mediates cardiac mast cell degranulation, matrix metalloproteinase activation, and myocardial remodeling in rats

The objective of this study was to determine whether elevated circulating levels of endothelin (ET)-1 are capable of mediating left ventricular (LV) mast cell degranulation and thereby induce matrix metalloproteinase (MMP) activation. After the administration of 20 pg/ml ET-1 to blood-perfused isolated rat hearts, LV tissue was analyzed for signs of mast cell degranulation and MMP activation. Relative to control, ET-1 produced extensive mast cell degranulation as well as a significant increase in myocardial water content (78.8 +/- 1.5% vs. 74.2 +/- 2.2%, P <0.01), a marked 107% increase in MMP-2 activity (P <0.05), and a substantial decrease in collagen volume fraction (0.69 +/- 0.09% vs. 0.99 +/- 0.04%, P <0.001). Although the myocardial edema would be expected to increase ventricular stiffness, compliance was not altered, and moderate ventricular dilatation was observed (end-diastolic volume at end-diastolic pressure of 0 mmHg of 330.2 +/- 22.1 vs. 298.9 +/- 17.4 microl in ET-1 treated vs. control, respectively, P=0.07). Additionally, pretreatment with the mast cell stabilizer nedocromil prevented ET-1-induced changes in MMP-2 activity, myocardial water content, collagen volume fraction, and end-diastolic volume. These findings demonstrate that ET-1 is a potent cardiac mast cell secretogogue and further indicate that ET-1-mediated mast cell degranulation is a potential mechanism responsible for myocardial remodeling.     

The relationship between adrenal vascular events and steroid secretion: the role of mast cells and endothelin.

The actions of ACTH on the adrenal cortex are known to be 2-fold. In addition to increased steroidogenesis, ACTH also causes marked vasodilation, reflected by an increased rate of blood flow through the gland. Our studies, using the in situ isolated perfused rat adrenal preparation, have shown that zona fasciculata function and corticosterone secretion are closely related to vascular events, with an increase in perfusion medium flow rate causing an increase in corticosterone secretion, in the absence of any known stimulant. These observations give rise to two important questions: how does ACTH stimulate blood flow; and how does increased blood (or perfusion medium) flow stimulate steroidogenesis? Addressing the first question, we have recently identified mast cells in the adrenal capsule, and shown that Compound 48/80, a mast cell degranulator, mimics the actions of ACTH on adrenal blood flow and corticosterone secretion. We have also demonstrated an inhibition of the adrenal vascular response to ACTH in the presence of disodium cromoglycate, which prevents mast cell degranulation. We conclude, therefore, that ACTH stimulates adrenal blood flow by its actions on mast cells in the adrenal capsule. Addressing the second question, we looked at the role of endothelin in the rat adrenal cortex. Endothelin 1, 2 and 3 caused significant stimulation of steroid secretion by collagenase dispersed cells from both the zona glomerulosa and the zona fasciculata. A sensitive response was seen, with significant stimulation at an endothelin concentration of 10(-13) mol/l or lower. Endothelin secretion by the in situ isolated perfused rat adrenal gland was measured using the Amersham assay kit. Administration of ACTH (300 fmol) caused an increase in the rate of immunoreactive endothelin secretion, from an average of 28.7 +/- 2.6 to 52.6 +/- 6 fmol/10 min (P less than 0.01, n = 5). An increase in immunoreactive endothelin secretion was also seen in response to histamine, an adrenal vasodilator, which stimulates corticosterone secretion in the intact gland, but has no effect on collagenase-dispersed cells. From these data we conclude that endothelin may mediate the effects of vasodilation on corticosterone secretion, and this mechanism may explain some of the differences in response characteristics between the intact gland and dispersed cells.     

Cutting edge: distinct Toll-like receptor 2 activators selectively induce different classes of mediator production from human mast cells

Mast cells play a critical role in host defense against bacterial infection. Murine mast cells produce cytokines in response to bacterial peptidoglycan and LPS via Toll-like receptor (TLR) TLR2- and TLR4-dependent mechanisms. The expression of TLRs by human mast cells and responses to known TLR activators was examined. Human mast cells expressed mRNA for TLR1, TLR2, and TLR6 but not TLR4. Bacterial peptidoglycan and yeast zymosan were potent inducers of GM-CSF and IL-1beta and also induced substantial short-term cysteinyl leukotriene generation. In contrast, a synthetic triacylated lipopeptide induced short-term degranulation but failed to induce cysteinyl leukotriene production. The TLR4 activator Escherichia coli LPS did not induce a GM-CSF, IL-1beta leukotriene, or degranulation response. These data demonstrate highly selective production of different classes of mast cell mediators in response to distinct TLR activators of potential importance to the host response to bacterial or fungal pathogens.     

Carnosine attenuates mast cell degranulation and histamine release induced by oxygen-glucose deprivation

Carnosine (beta-alanyl-histidine) is a naturally occurring dipeptide that has been characterized as a putative hydrophilic antioxidant. The protective function of carnosine has been demonstrated in neuronal cells under ischemic injury. The purpose of this study was to investigate the effects of carnosine on oxygen-glucose deprivation (OGD)-induced degranulation and histamine release from mast cells. Cultured mast cells were exposed to OGD for 4 h, and then the degranulation was observed immediately by microscopy. Histamine release was analyzed by high-performance liquid chromatography (HPLC). OGD caused degranulation of mast cells, and increased histamine and lactate dehydrogenase (LDH) release. Carnosine (at a concentration of 5 mM) alone did not produce any appreciable effect on degranulation, histamine, and LDH release from mast cells under normal condition, but significantly inhibited the degranulation, histamine, and LDH release of mast cells induced by OGD. These results indicate that carnosine can protect mast cells from degranulation and histamine release and it may be an endogenous mast cell stabilizer in the pathological processes induced by ischemia.     

Influence of ethanolic extract of Tephrosia purpurea Linn. on mast cells and erythrocytes membrane integrity

The ethanolic extract of T. purpurea Linn. was studied for its in vitro effect on rat mast cell degranulation and erythrocyte membrane integrity in vitro. The extract in concentration of 25-200 microg/ml showed a dose-dependant inhibition of rat mast cell degranulation induded by compound 48/80 and egg albumin. T. purpurea extract was found to inhibit haemolysis of erythrocytes induced by hypotonic solution but accelerated haemolysis induced by heat at a concentration of 100 microg/ml. The studies reveal that the ethanolic extract of T. purpurea may inhibit degranulation of mast cells by a mechanism other than membrane stabilization.