Release Of Mast Cell Tryptase Research Articles

Mast-Cell Tryptase Release Contributes to Disease Progression in Lymphangioleiomyomatosis.

Rationale: Lymphangioleiomyomatosis (LAM) is a multisystem disease that causes lung cysts and respiratory failure. Loss of TSC (tuberous sclerosis complex) gene function results in a clone of "LAM cells" with dysregulated mTOR (mechanistic target of rapamycin) activity. LAM cells and fibroblasts form lung nodules that also contain mast cells, although their significance is unknown. Objectives: To understand the mechanism of mast-cell accumulation and the role of mast cells in the pathogenesis of LAM. Methods: Gene expression was examined using transcriptional profiling and qRT-PCR. Mast cell/LAM nodule interactions were examined in vitro using spheroid TSC2-null cell/fibroblast cocultures and in vivo using an immunocompetent Tsc2-null murine homograft model. Measurements and Main Results: LAM-derived cell/fibroblast cocultures induced multiple CXC chemokines in fibroblasts. LAM lungs had increased tryptase-positive mast cells expressing CXCRs (CXC chemokine receptors) (P < 0.05). Mast cells located around the periphery of LAM nodules were positively associated with the rate of lung function loss (P = 0.016). LAM spheroids attracted mast cells, and this process was inhibited by pharmacologic and CRISPR/cas9 inhibition of CXCR1 and CXCR2. LAM spheroids caused mast-cell tryptase release, which induced fibroblast proliferation and increased LAM-spheroid size (1.36 ± 0.24-fold; P = 0.0019). The tryptase inhibitor APC366 and sodium cromoglycate (SCG) inhibited mast cell-induced spheroid growth. In vivo, SCG reduced mast-cell activation and Tsc2-null lung tumor burden (vehicle: 32.5.3% ± 23.6%; SCG: 5.5% ± 4.3%; P = 0.0035). Conclusions: LAM-cell/fibroblast interactions attract mast cells where tryptase release contributes to disease progression. Repurposing SCG for use in LAM should be studied as an alternative or adjunct to mTOR inhibitor therapy.

Birch pollen-specific subcutaneous immunotherapy reduces ILC2 frequency but does not suppress IL-33 in mice.

The underlying mechanism of allergen-specific subcutaneous immunotherapy (SCIT) is not yet fully understood, but suppression of allergen-specific Th2 cells and production of allergen-specific IgG4 antibodies are two hallmarks. The impact on the innate arm of the immune system is far less clear. The aim of this study was to investigate the effect of birch pollen (BP) SCIT on the innate immune response in a BP SCIT mouse model. Mice with birch pollen-induced allergic airway inflammation received weekly subcutaneous immunotherapy injections with birch pollen extract (BPE) adsorbed to alum. The effect of the BP SCIT on innate cytokine levels in lung, the number and the functionality of ILC2s and the airway inflammation was determined. Mice with BP allergy had an increased level of the innate cytokines IL-33, IL-25, GM-CSF and IL-5+ ILC2s in the lungs. BP SCIT suppressed the number of IL-5+ ILC2s, mast cell tryptase release, Th2 cytokine production, eosinophil recruitment and peribronchial inflammatory infiltrates. In contrast, innate cytokine production and collagen deposition in the airways were not affected. BP SCIT is able to suppress the adaptive and part of the innate immune response, but this is not sufficient to inhibit collagen deposition and the IL-33 expression in the airways in mice.

Early weaning stress induces chronic functional diarrhea, intestinal barrier defects, and increased mast cell activity in a porcine model of early life adversity.

Early life adversity (ELA) is a risk factor for development of gastrointestinal disorders later in life. The underlying mechanisms through which ELA and sex interact to influence disease susceptibility remains poorly understood. Utilizing a porcine early weaning stress (EWS) model to mimic ELA, we investigated the long-term effects of EWS on functional diarrhea, ileal permeability, mast cell activity and mast cell relationship with enteric ganglia. Juvenile and adult EWS pigs exhibited chronic, functional diarrhea (EWS 43.6% vs late wean control(LWC) 4.8%, P<.0001), increased intestinal permeability (2 fold increase EWS vs LWC, P<.0001), and mast cell numbers (at 7weeks and 20weeks ~1.6 fold increase EWS vs LWC, P<.05). Compared with EWS male castrates (Male-C), females EWS pigs exhibited more frequent diarrhea (58.8% vs 29.9%, P=.0016), and increased intestinal permeability (1-2 fold higher in EWS females, P<.001). Increased mast cell numbers and their enhanced co-localization with neuronal ganglia were observed in both Male-C and female EWS pigs; however, female pigs exhibited greater release of mast cell tryptase upon activation with c48/80 (~1.5 fold increase, P<.05), compared with Male-C pigs. These data demonstrate that pigs exposed to ELA exhibit increased vulnerability to functional diarrhea, intestinal permeability and mast cell activity. Further, these studies also showed that EWS female and Male-C pigs exhibited dimorphic responses to EWS with female piglets exhibited greater susceptibility and severity of diarrhea, intestinal permeability and mast cell tryptase release. Together, these findings mimic some of the key pathophysiologic findings in human functional GI disorders functional gastrointestinal disorders (FGIDs) suggesting that the EWS porcine model could be a valuable preclinical translational model for FGID research associated with ELA.

Association of Mu Opioid Receptor with Neurogenic Inflammation in Sickle Mic

Abstract 895Sickle cell disease (SCD) is accompanied by chronic inflammation and severe pain for which available therapies are unsatisfactory. We found that pain was accompanied by increased peripheral expression of pro-inflammatory and vasoactive neuropeptides, substance P (SP) and calcitonin gene related peptide (CGRP), and decreased expression of mu opioid receptor (MOR) in the skin of BERK mice expressing sickle hemoglobin (HbSS) compared to control HbA-BERK mice expressing normal human hemoglobin (Kohli et al., Blood 2010). Neuropeptide modulations are key features of neurogenic inflammation in chronic pain states. Tryptase released from mast cells is known to activate the release of neuropeptides from peripheral nerve endings, which in turn act on target cells including mast cells, inflammatory cells, endothelial cells and smooth muscle cells, resulting in increased vascular flow, inflammation and plasma extravasation collectively termed neurogenic inflammation. MOR influences neuropeptide release and perhaps modulates neurogenic inflammation. We hypothesize that neurogenic inflammation contributes to chronic pain and inflammation in SCD. We first examined the quantitative expression of MOR-, SP-, CGRP- and tryptase-immunoreactivity (ir) using laser scanning confocal microscopy (LSCM) in thick skin sections of BERK sickle and HbA-BERK control mice. Significantly decreased MOR-ir (p<0.001), but increased SP-, CGRP- (p<0.01 for both) and tryptase-ir (p<0.001) was observed in the skin of BERK as compared to HbA-BERK. Decreased MOR-ir co-localized with epidermal and dermal layers in the skin of BERK, but blood vessels showed robust MOR-ir in BERK similar to HbA-BERK. Tryptase-ir was more pronounced in the epidermal layer, indicative of its proximity to peripheral nerve endings. Increased tryptase co-localized with CD117/c-kit, confirming the mast cell origin. Next, we analyzed if MOR downregulation contributes to these alterations, by using mice with deletion of mu-, delta- and kappa-opioid receptors (MOR, DOR and KOR, respectively) and wild type (WT), 129S6 mice. SP-, CGRP- and tryptase-ir were increased about 2-fold in the skin of MOR, DOR and KOR knockout as compared to WT mice, suggesting that downregulation of MOR in sickle mice may contribute to increased neuropeptide and tryptase expression. Consistent with an increase in cutaneous neuropeptides, BERK and MOR knockout mice showed ∼2.5- and ∼1.5-fold increase in dorsal skin blood flow using Laser Doppler blood perfusion monitor, as compared to HBA-BERK and WT control mice, respectively. Thermography measurements revealed a correlative increase in temperature in dorsal skin of BERK as compared to HbA-BERK (p<0.05), indicative of increased inflammation. These neurochemical and physiological alterations in the skin were accompanied by activation of dorsal root ganglion (DRG) neuron and glial cells. LSCM of whole DRG revealed increased-ir for activating transcription factor-3 (ATF3), a marker of neuropathic and inflammatory insult; and increased glial fibrillary acidic protein (GFAP)-ir, a marker of inflammation, in BERK relative to HbA-BERK mice. It is likely that chronic pain and ischemia-reperfusion injury in SCD activates ATF3 in DRG leading to the release of mast cell tryptase followed by release of neuropeptides in the DRG and their ‘antidromic’ release from the nociceptive C-fibers in the skin. In addition, recurrent/chronic pain and inflammation in SCD may cause peripheral nociceptor sensitization mediated by mast cell-tryptase, and neuropeptide release from nerve endings. Nociceptor sensitization together with decrease in MOR, in turn may activate mast cell tryptase, promoting a vicious cycle between components of central nervous system and inflammation that may exacerbate neurogenic inflammation in SCD. It is likely that the pro-inflammatory neuropeptide-enhanced microenvironment has a compounding effect on inflammation and pain in SCD. We speculate that therapies targeting neuropeptides and their release mechanism(s) may potentiate the effect of analgesics and anti-inflammatory therapies for treating pain in SCD. Disclosures:No relevant conflicts of interest to declare.

Shedding of the coronary endothelial glycocalyx: effects of hypoxia/reoxygenation vs ischaemia/reperfusion

Vascular endothelium is covered by a glycocalyx. Damage to the glycocalyx after systemic inflammation or ischaemia/reperfusion contributes to increased vascular permeability and leucocyte adhesion. The underlying mechanisms leading to ischaemia/reperfusion-induced glycocalyx shedding are incompletely understood, in terms of lack of oxygen, absence of flow, or return of oxygen. Isolated guinea pig hearts perfused with Krebs-Henseleit buffer at 37°C underwent 20 min of either stopped-flow ischaemia or hypoxic perfusion with subsequent reperfusion/reoxygenation (n = 6 each). Hearts perfused with normoxic buffer served as time controls. Epicardial transudate was collected to assess coronary net fluid filtration, colloid extravasation, and histamine release by mast cells. Syndecan-1 and heparan sulphate were measured in coronary effluent, together with lactate, purines, and the release of mast-cell tryptase β. Additional hearts were perfusion-fixed to visualize the glycocalyx. Both ischaemia and hypoxia with reperfusion/reoxygenation resulted in significant increases in net fluid filtration (P < 0.05) and release of syndecan-1 and heparan sulphate in coronary effluent. These effects were already seen with the onset of hypoxic perfusion. Histamine was released during hypoxia and reoxygenation and also reperfusion, as was tryptase β, and high concentrations of adenosine (>1 µmol litre⁻¹, hypoxia group) and inosine (> 7 µmol litre⁻¹, ischaemia group) were measured in effluent (P < 0.05). Damage to the coronary glycocalyx was evident upon electron microscopy. Both ischaemic and hypoxic hypoxia initiate glycocalyx degradation, promoting an increase in permeability. A contributing mechanism could be purine-mediated degranulation of resident mast cells, with liberated tryptase β acting as potential 'sheddase'.

Release of Mast Cell Tryptase into Saliva: A Tool to Diagnose Food Allergy by a Mucosal Challenge Test?

Background: Our aim was to examine whether measurement of the saliva mast cell tryptase (MCT) concentrations before and after a mucosal challenge test with the offending food would be helpful in diagnosing food allergy. Methods: We performed a retrospective analysis of 44 food challenge tests performed in 38 patients between 2006 and 2009. Patients with a suspected history of food allergy chewed the food until they developed symptoms or until the amount of time known from the patients’ history to usually be required for the provocation of symptoms had passed. In 5 patients, saliva samples for the measurement of MCT were collected at minutes 0, 1, 4, 8, 11, and 16 after the first onset of symptoms. The remainder of the patients only had samples taken before chewing and 4 min after the end of the test period. Results: During repeated measurements, MCT peaked about 4 min after the onset of symptoms (p = 0.028). During 33 of the 44 tests (75.0%), we observed oral symptoms during testing; after 25 of the 33 (75.8%) tests evoking symptoms, the saliva MCT concentration increased. The MCT increase was negative in all other tests where no oral symptoms could be provoked. Conclusions: The measurement of saliva MCT 4 min after the onset of symptoms may be helpful to diagnose food allergy. Because of numerous confounding variables, however, a negative saliva MCT increase does not exclude food allergy.

293 Increased Intraepithelial Lymphocytes and Mast Cells with Depressed Serotonin Transporter in Duodenal Biopsies and Platelets of Patients with IBS and Diarrhoea (IBS-D): Correlation with Bowel Frequency

INTRODUCTION: Several groups report increased mucosal inflammation and increased circulating cytokines, which both cell and animal studies show to depress SERT, in IBS. We therefore assessed mucosal and platelet SERT function together with 5HT & Mast Cell tryptase release (MCT) from duodenal biopsies which were also assessed for mast cells (MC), intra-epithelial lymphocytes (IEL) & 5HT-containing enterochromaffin cells (EC) compared to coeliac patients who are known to have mucosal inflammation with depressed SERT mRNA. AIMS & METHODS: 28 healthy volunteers, 20 patients with IBS-D & 20 with untreated coeliac disease (CD) completed bowel symptom questionnaires and underwent duodenal biopsy. 5HT & MCT release was assayed after 6 hour incubation. MC, IELs & ECs were identified by immuno-histochemistry. SERT and cytokeratin 20(CK20) mRNA were assessed by Taqman qRT-PCR. Platelet SERT function was assayed by H3-5HT uptake. Project supported by Novartis Pharma AG. RESULTS: IBS-D showed increase IELs, mast cell numbers and increased mast cell tryptase release (see Table). DIBS also showed depressed SERT/CK20(arbitrary units) at 0.19(0.1-0.43) versus 0.96(0.14-1.40) for HV, p<0.02. Platelet uptake of H3-5HT was also depressed at 12.8(8.3-23.2) versus 45.2(27.7-69.3)pmol/min/ mg protein for HV, p<0.01. CD had intermediate values, difference NS v HV. Mast cell numbers correlated (r=0.56, p=0.02) with stool frequency in IBS patients but not in coeliacs nor HV. IELs, in HV and IBS, correlated positively with mast cell numbers r=0.45 , p=0.001 and with EC count r=0.39, p=0.006 but negatively with SERT r=-0.37 ,p=0.01 and platelet 5HT uptake -0.53, p=0.001. CONCLUSIONS: IBS-D is characterised by increased duodenal IELs and mast cells associated with increased mast cell tryptase release. The depressed SERT in mucosa and platelets correlates with mucosal IELs and may be secondary to increased inflammatory mediators. Depressed SERT may contribute to increased 5HT availability and diarrhoeal symptoms. Mast cell numbers correlate with stool frequency suggesting mast cell products also play a part in symptoms.

Protease‐activated receptor 2 mediates the proinflammatory effects of synovial mast cells

Mast cells are hypothesized to play a role in the pathogenesis of rheumatoid arthritis (RA) by mechanisms requiring elucidation. Tryptase released from these cells can activate protease-activated receptor 2 (PAR-2), which was recently shown to have proinflammatory actions. The purpose of this study was to examine the relationship between synovial mast cells and PAR-2. Mast cell proximity to PAR-2-expressing cells was investigated in RA synovium. In murine studies, we assessed the capacity of mast cell tryptase to mediate synovial proinflammatory responses via PAR-2 and whether degranulating mast cells induced synovial hyperemia by PAR-2 activation. RA synovial tissue was examined by immunohistochemistry. PAR-2(+/+) and PAR-2(-/-) C57BL/6J mice were used to investigate the PAR-2 dependence of compound 48/80-induced synovial hyperemia, as measured by laser Doppler imaging, and joint swelling and hyperemic responses to recombinant human beta-tryptase. Mast cells and synovial lining cells staining for PAR-2 were colocalized in RA articular tissue. Compound 48/80 administration resulted in vasodilatation in PAR-2(+/+) mice but not in PAR-2(-/-) mice, which showed a vasoconstrictor response. Eliminating the 5-hydroxytryptamine-mediated component of this response with methysergide unveiled an enhanced PAR-2-mediated vasodilatation to compound 48/80 in PAR-2(+/+) mice and ablated the vasoconstrictor response in PAR-2(-/-) mice. Treatment with beta-tryptase resulted in dose-dependent knee joint swelling and synovial vasodilatation in PAR-2(+/+) mice but not PAR-2(-/-) mice. This in vivo study is the first to explore the relationship between synovial mast cells and PAR-2. Our results support the hypothesis that mast cells contribute to the pathogenesis of inflammatory arthritis through PAR-2 activation via release of mast cell tryptase.

Physiology and Pathophysiology of Proteinase-Activated Receptors (PARs): Role of Tryptase/PAR-2 in Vascular Endothelial Barrier Function

Proteinase-activated receptor-2 (PAR-2) plays important roles in a variety of pathophysiological functions, including inflammatory responses and nociception. In this minireview, we describe the role of PAR-2 in acute inflammatory responses in lungs associated with iodinated radiographic contrast medium (RCM). Intravenous injection of RCM to rats induces lung injury characterized by vascular hyperpermeability, edema, and respiratory depression. Nafamostat, which is found to be the most potent and specific tryptase inhibitor, prevents RCM-induced lung injury. In cultured endothelial cells of human pulmonary artery and bovine aorta, RCM, when applied in combination with mast cells, disrupts barrier function evaluated by the permeability of Evans blue through a monolayer of cultured cells, which is blocked by nafamostat and mimicked by tryptase and PAR-2-activating peptide. The tryptase-induced barrier dysfunction is blocked completely by a phospholipase C inhibitor and partially inhibited by a IP3 receptor blocker, protein kinase C inhibitor, or Rho kinase inhibitor. Morphological observations reveal the formation of actin stress fibers and disappearance of the intercellular meshwork structure of vascular endothelial-cadherin after application of RCM or PAR-2 ligands. Therefore, the release of mast cell tryptase and subsequent activation of endothelial PAR-2 are involved in acute lung injury induced by RCM.

Flea Bite Hypersensitivity: New Aspects On The Involvement Of Mast Cells

A study was performed to test the effect of sensitization to flea antigen, followed by exposure to fleas on mast cells (MCs), their subtypes, and IgE+ cells. Biopsies were taken from flea-sensitized dogs ( n=28) and non-sensitized dogs ( n=5) that had been exposed to fleas. Control groups consisted of flea-sensitized ( n=12) and non-sensitized dogs ( n=9) that were not exposed to fleas. Biopsies, taken before, 24 and 72 h after local flea exposure, were stained with haematoxylin and eosin (H&E), toluidine blue, a double labelling technique for MC chymase and tryptase and anti-IgE. An intradermal test for flea antigen was performed and serum titres of allergen-specific IgE and IgG were measured. Significantly higher numbers ( P<0.001) of double labelled MCs compared to toluidine blue stained MCs were detectable in flea-sensitized dogs independent of flea exposure. In contrast, in non-sensitized dogs, the number of toluidine blue stained MCs and the number of double labelled MCs did not differ. In flea-sensitized dogs after flea exposure the percentage of C-MC was significantly increased at day 1 ( P<0.001) and day 3 ( P<0.001), whereas the percentage of TC-MCs decreased significantly at day 1 ( P<0.001) and day 3 ( P<0.05). The percentage of T-MCs decreased ( P<0.05 day 0 versus day 1; P<0.05 day 0 versus day 3). No significant difference was detectable after toluidine blue staining and staining for IgE+ cells between the groups nor between the MC density and the number of IgE+ cells. All flea-sensitized dogs had positive skin tests to flea antigen and high serum titres of flea-specific serum IgE and IgG antibodies. In non-sensitized dogs, these results were negative. Our data provide strong evidence for an upregulation of MC proteases during the process of sensitization and a generalized selective release of mast cell tryptase after exposure to the antigen.

Activation of ion secretion via proteinase-activated receptor-2 in human colon

Proteinase-activated receptor (PAR) type 2 (PAR-2) has been shown to mediate ion secretion in cultured epithelial cells and rat jejunum. With the use of a microUssing chamber, we demonstrate the role of PAR-2 for ion transport in native human colonic mucosa obtained from 30 normal individuals and 11 cystic fibrosis (CF) patients. Trypsin induced Cl(-) secretion when added to the basolateral but not luminal side of normal epithelia. Activation of Cl(-) secretion by trypsin was inhibited by indomethacin and was further increased by cAMP in normal tissues but was not present in CF colon, indicating the requirement of luminal CF transmembrane conductance regulator. Effects of trypsin were largely reduced by low Cl(-), by basolateral bumetanide, and in the presence of barium or clotrimazole, but not by tetrodotoxin. Furthermore, trypsin-induced secretion was inhibited by the Ca(2+)-ATPase inhibitor cyclopiazonic acid and in low-Ca(2+) buffer. The effects of trypsin were almost abolished by trypsin inhibitor. Thrombin, an activator of PAR types 1, 3, and 4, had no effects on equivalent short-circuit currents. The presence of PAR-2 in human colon epithelium was confirmed by RT-PCR and additional experiments with PAR-2-activating peptide. PAR-2-mediated intestinal electrolyte secretion by release of mast cell tryptase and potentiation of PAR-2 expression by tumor necrosis factor-alpha may contribute to the hypersecretion observed in inflammatory processes such as chronic inflammatory bowel disease.

Release of mast cell tryptase from human colorectal mucosa in inflammatory bowel disease.

Histologic detection of mast cells cannot adequately reflect their function and state of activation, since degranulated mast cells may escape from histologic assessment. To better define the role of mast cells in inflammatory bowel disease, the spontaneous secretion of mast cell tryptase, a highly mast cell specific protease, was measured from colorectal samples. After detection of the initial basal tryptase release, gut mucosal samples were incubated in a modified Hanks/RPM1 medium using a mucosa oxygenation system. Spontaneous tryptase secretion from 153 viable samples of 22 controls, 30 patients with Crohn disease (CD) and 19 with ulcerative colitis (UC) was followed over 4 h. Tryptase was measured by radioimmunoassay. The rates of the initial basal tryptase release revealed that mast cell activation occurs during active inflammation in CD and UC. While the time course of tryptase release was similar in all three groups, spontaneous tryptase secretion (over 4 h) was found to be significantly enhanced and prolonged only in UC (P < 0.01 compared to controls), but not in CD. This study provides clear evidence from viable endoscopic colorectal samples that mast cell mediators were secreted during active inflammation in CD and UC. However, the extent of mast cell involvement and activation differs considerably between CD and UC. Significantly increased rates of tryptase secretion were found both in non-inflamed and inflamed tissue of UC, indicating that mast cell involvement is a typical feature of UC.

Release of Mast Cell Tryptase from Human Colorectal Mucosa in Inflammatory Bowel Disease

Background: Histologic detection of mast cells cannot adequately reflect their function and state of activation, since degranulated mast cells may escape from histologic assessment. To better define the role of mast cells in inflammatory bowel disease, the spontaneous secretion of mast cell tryptase, a highly mast cell specific protease, was measured from colorectal samples. Methods: After detection of the initial basal tryptase release, gut mucosal samples were incubated in a modified Hanks/RPMI medium using a mucosa oxygenation system. Spontaneous tryptase secretion from 153 viable samples of 22 controls, 30 patients with Crohn disease (CD) and 19 with ulcerative colitis (UC) was followed over 4 h. Tryptase was measured by radioimmunoassay. Results: The rates of the initial basal tryptase release revealed that mast cell activation occurs during active inflammation in CD and UC. While the time course of tryptase release was similar in all three groups, spontaneous tryptase secretion (over 4 h) was found to be significantly enhanced and prolonged only in UC (P < 0.01 compared to controls), but not in CD. Conclusions: This study provides clear evidence from viable endoscopic colorectal samples that mast cell mediators were secreted during active inflammation in CD and UC. However, the extent of mast cell involvement and activation differs considerably between CD and UC. Significantly increased rates of tryptase secretion were found both in non-inflamed and inflamed tissue of UC, indicating that mast cell involvement is a typical feature of UC.

Mast cell tryptase release and asthmatic responses to allergen increase with regular use of salbutamol

Background: Increased asthmatic responses to allergen, both early and late, have been demonstrated after regular use of β2-agonists in as few as 7 days. Desensitization of β2-adrenergic receptors on airway mast cells may contribute to this effect by allowing greater release of mast cell mediator on allergen-induced degranulation. Tryptase released from lung mast cells can be measured in serum 1 hour after allergen challenge and serves as a marker of mast cell degranulation. Objective: To examine the effect of regular treatment with salbutamol, a β2-agonist, on mast cell mediator release after allergen challenge and its influence on the early asthmatic response (EAR) and the late allergic response, we measured the EAR, serum tryptase levels, the 7-hour FEV1, and sputum tryptase levels and cell profiles. Methods: We conducted a placebo-controlled, double-blind, randomized cross-over comparison of treatments for 10 days with either a salbutamol metered-dose inhaler (100 μg, 2 puffs 4 times daily) or a matched placebo inhaler with at least a 7-day washout between treatments. Atopic subjects (n = 14) with mild-to-moderate asthma performed same-dose allergen inhalation tests after both treatments 12 to 15 hours after the last dose of study inhaler. Baseline and 7-hour FEV1 and the EAR to allergen were measured by using spirometry; venous blood was drawn at 1 hour for analysis of serum tryptase; and sputum was induced and collected at 1 and 7 hours. Results: Salbutamol treatment resulted in a significantly greater EAR (20% ± 1.6% [SEM] vs 15% ± 2.1%; P = .047); increased 1-hour serum tryptase levels (9.09 ± 1.57 vs 7.52 ± 1.12 μg/L; P = .011); increased proportions of eosinophils in the 7-hour sputum sample (39.1% ± 5.1% vs 28.4% ± 4.4%; P < .05); increased proportion of metachromatic cells in the 7-hour sputum sample (4.4% ± 1.1% vs 2.2% ± 0.6%; P = .032); and lower 7-hour FEV1 (2.77 ± 0.18 vs 2.97 ± 0.20 L; P = .014). Baseline FEV1 was not significantly different after salbutamol treatment compared with values after placebo treatment (2.90 ± 0.20 vs 3.00 ± 0.19 L; P = .11). Conclusion: Regular 10-day treatment with salbutamol increases the allergen-induced release of mediator from airway mast cells, and this is reflected in an increased EAR to allergen. Late-phase responses to allergen were also enhanced, as demonstrated by decreased 7-hour FEV1 and increased eosinophilia and percentage of metachromatic cells in the 7-hour sputum sample. Increased allergen-induced mast cell degranulation could, in part, explain the increased asthmatic responses to allergen after β2-agonist treatment and could contribute to the deterioration of asthma control that is associated with regular use of β2-agonist by potentiating allergic inflammation. (J Allergy Clin Immunol 2000;106:57-64.)

Release of mast cell tryptase from human colorectal mucosa in inflammatory bowel disease

Release of mast cell tryptase from human colorectal mucosa in inflammatory bowel disease

Immunofluorescent staining for mast cells in idiopathic pulmonary fibrosis: quantification and evidence for extracellular release of mast cell tryptase.

In many diseases, retrospective analysis for determining the presence of mast cells has been difficult because of their loss of metachromatic staining properties once tissue has undergone formalin fixation. We quantified mast cells in peribronchiolar tissue of idiopathic pulmonary fibrosis (IPF) and in normal human lung by using rabbit antiserum to human mast cell tryptase. In lung biopsy specimens from 15 patients with IPF, the mean number of mast cells per high-power field in connective tissue directly adjacent to the lumen of small airways (0.5 to 2 mm in diameter) and other fibrotic foci was 29.9 +/- 10.8 in comparison with 13.7 +/- 3.5 in 16 normal controls (P < 0.001). In addition, mast cells in cases of IPF had an altered appearance--irregularity of the plasma membrane and release of extracellular tryptase. We conclude that the number of mast cells is increased in IPF and that the altered appearance of the mast cells suggests that they are activated and undergoing degranulation.