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PubMed1995

Sarcoidosis patients have bronchial hyperreactivity and signs of mast cell activation in their bronchoalveolar lavage.

An increased (p < 0.001) frequency of bronchial hyperreactivity (BHR) was found in sarcoidosis patients as compared with healthy volunteers. The patients had more mast cells (p < 0.001) and tryptase (p < 0.001) in their bronchoalveolar lavage fluid, but there were no differences between BHR-positive and BHR-negative patients. Furthermore, the bronchoalveolar lavage fluid concentrations of macrophages, lymphocytes, and of the soluble components albumin, fibronectin, and vitronectin were also elevated in the sarcoidosis patients, indicating an ongoing inflammation in the airways and/or in the interstitium. We observed no significant differences in the parameters when the sarcoidosis patients were subdivided into BHR, clinical activity, or chest X-ray stages. Our findings may indicate a multifactorial background to the hyperreactivity.

Adolescent↗
PubMed1994

Mode of action of a topical steroid on immediate phase reaction after antigen challenge and nonspecific nasal hyperreactivity in nasal allergy.

This study compared the effects of 2-week administration of a topical steroid (fluticasone propionate [FP] 100 micrograms twice daily) with placebo in 28 patients with perennial nasal allergy who were allergic to house dust and mites in a double-blind randomized study. The number of inflammatory cells and decidual epithelial cells and concentrations of tryptase and eosinophil cationic protein in nasal lavages, and reactivity of the nasal mucosa to histamine and to antigen were investigated. The topical steroid, FP, significantly inhibited all of these assessments. The degree of improvement of nasal reactivity to histamine significantly correlated with the degree of decrease in eosinophil cationic protein levels.

Administration, Topical↗
PubMed1994

Histochemical and cytological characterizations of mucosal and connective tissue mast cells of Mongolian gerbils (Meriones unguiculatus).

Mast cells in jejunum, skin, and tongue of Mongolian gerbils (Meriones unguiculatus) were examined in terms of their histochemical, enzyme-histochemical, and ultrastructural properties. When glycosaminoglycans of mast cells in jejunum and tongue were characterized by measuring the critical electrolyte concentration, the salt concentration at which 50% of mast cells could be stained was > 1.0 M for those in the jejunum and also in the tongue, indicating that mast cells in both sites contained heparin. Enzyme-histochemical study revealed that mast cells in the jejunum of Mongolian gerbils were strongly positive for chymase and tryptase, whereas those in tongue and skin were essentially negative for both proteases. By electron microscopy, granular morphology and distribution of surface microfolds of mast cells in the jejunum were different from those in the ear skin. After in vivo stimulation with compound 48/80, however, mast cells in both sites showed remarkable degranulation. From these results, although Mongolian gerbils also have distinct mast cell subsets, their phenotypic properties are different from those of the previously known mast cell subsets of other animals.

Animals↗
PubMed1994

Inhibition of stem cell factor dependent formation of human mast cells by interleukin-3 and interleukin-4.

In murines, interleukins (IL) 3, 4, 9, and 10, nerve growth factor, and stem cell factor induce or promote growth and differentiation of mast cells (MC). Increased stimulation and synergy was observed when combinations of cytokines were used. In man, no growth factor for human MCs had been identified until recently, when SCF was found to induce in vitro growth and differentiation of human MCs. In the present study, the effects of recombinant human IL-3 and IL-4 on SCF-dependent differentiation of human MCs from their circulating progenitor cells in long-term culture were analyzed. Surprisingly, both IL-3 and IL-4 were found to inhibit SCF-dependent formation of human MCs (SCF, 100 ng/ml: 36.4 +/- 18.7 x 10(3)/ml; SCF + IL-3, 100 U/ml: 23.4 +/- 4.2 x 10(3)/ml; SCF + IL-4, 100 U/ml: 7.4 +/- 4.4 x 10(3)/ml) and synthesis of MC tryptase (SCF, 100 ng/ml: 73.2 +/- 17.6 ng/ml; SCF + IL-3, 100 U/ml: 10.8 +/- 3.1 ng/ml [p < 0.01]; SCF + IL-4, 100 U/ml: 8.1 +/- 1.5 ng/ml, [p = 0.02]). The inhibitory effects of these cytokines on SCF-dependent formation of human MCs were associated with an increase in the number of macrophages (IL-3) or lymphocytes (IL-4) in the same cultures and may be due to competitive recruitment of cells from a pool of multilineage hematopoietic progenitor cells.

Cell Differentiation↗
PubMed1994

Fibroblasts determine the fate of Fc epsilon RI+ cell populations in vitro by selectively supporting the viability of mast cells while internalizing and degrading basophils.

To determine the fate of Fc epsilon RI+ cells on fibroblasts in vitro, human bone marrow derived CD34+ cells were cultured in the presence of recombinant human interleukin 3 and recombinant human hematopoietic stem cell factor for 3 weeks, and Fc epsilon RI+ cells were purified by immunomagnetic selection. This enriched Fc epsilon RI+ cell population consisted of 92-94% basophils and 3-5% mast cells as determined by morphologic, immunohistochemical, and ultrastructural criteria. The Fc epsilon RI+ cells were then cocultured with 3T3 fibroblasts. Basophils decreased markedly by 1 week and were absent from cocultures by 2-3 weeks, while the mast cell numbers on the fibroblast monolayers remained constant. Ultrastructural examination of cocultures at 2 days demonstrated phagocytosis of basophils by fibroblasts. By 1 week, phagocytosed basophil membranes and granules gave fibroblasts the superficial appearance of mast cells by toluidine blue staining. Mast cells surviving in cocultures could be distinguished from granule-containing fibroblasts by IgE surface labeling and by ultrastructural demonstration of tryptase-positive granules. Thus, while mast cells remain viable in coculture with 3T3 fibroblasts, basophils do not survive and are internalized and degraded by the fibroblast monolayer.

Basophils↗
PubMedDate not supplied

Nitrogen dioxide increases eosinophil activation in the early-phase response to nasal allergen provocation.

Recent studies have suggested that exposure to air pollutants may sensitise susceptible individuals to allergen. We have investigated the effect of exposure for 6 h to 400 ppb NO2 on nasal airways resistance (NAR) and changes in inflammatory mediators (IMs) in nasal lavage in subjects with a history of seasonal allergic rhinitis. In this single blind crossover study, 8 patients were randomised to exposure to either air or 400 ppb NO2 in air and evaluated for changes in NAR and IM, before and after exposure. Another 8 patients were further challenged with allergen after similar exposure regimes and then evaluated for changes in NAR and IMs. Exposure to air or NO2 did not alter either NAR or the levels of eosinophil cationic protein (ECP), mast cell tryptase (MCT), myeloperoxidase (MPO) or interleukin (IL)-8 in nasal lavage. MCT was significantly increased after allergen challenge following exposure to both air and NO2. In contrast, ECP was significantly increased by allergen challenge only after exposure to NO2. Neither MPO nor IL-8 were altered after allergen challenge. These results suggest that NO2 may increase eosinophil activation in the early-phase response to nasal allergen provocation in allergic rhinitis.

Adolescent↗
PubMedDate not supplied

Human keratinocytes release mast cell differentiation factors other than stem cell factor.

Besides stem cell factor (SCF), additional fibroblast-derived mast cell growth factors have previously been described. Since keratinocytes have also been shown to produce SCF, we have studied the ability of culture supernatants from the human HaCaT keratinocyte cell line to induce SCF-independent mast cell differentiation. The immature human mast cells of the HMC-1 line which express a mutant continuously activated SCF receptor were used as model target cells. Culture supernatants from differentiating keratinocytes (at day 11 of culture), and far less so those from proliferating keratinocytes (day 4 of culture), caused a marked, dose-dependent increase of histamine and tryptase in HMC-1 cells. This suggests that human HaCaT keratinocytes release mast cell differentiation factors other than SCF, to a degree related to their state of differentiation.

Biological Factors↗
PubMedDate not supplied

Phenotype of cells positive for interleukin-4 and interleukin-5 mRNA in allergic tissue reactions.

We have investigated the phenotype of cells positive for IL-4 and IL-5 mRNA in the nasal mucosa of subjects with allergic rhinitis and in bronchoalveolar lavage and bronchial biopsies from atopic asthmatic subjects. The method employed was immunochemistry followed by in situ hybridization using either 35S- or digoxigenin-labelled riboprobes. With nasal and bronchial tissue, this double ICC/ISH method revealed that more than 70% of IL-4 and IL-5 mRNA+ cells were T cells. The remaining IL-4 and IL-5 signals were co-localized to tryptase-positive mast cells and EG2+ eosinophils. Occasional IL-4 and IL-5 mRNA cells were observed in non-asthmatic control subjects, the large majority being CD3+ cells. These results indicate that CD3+ cells are the principal cellular source of IL-4 and IL-5 transcripts in atopic asthma and allergic rhinitis.

Antigens, CD↗
PubMed1995

Effect of CC chemokines on mediator release from human skin mast cells and basophils.

Chemokines are considered important mediators of various inflammatory processes. In human basophils, different CC chemokines are known to stimulate release of histamine and generation of leukotriene (LT)C4. In the present study, we have evaluated the effect of RANTES (regulated upon activation, normal T cell expressed and secreted), monocyte chemotactic protein (MCP)-1, MCP-2, MCP-3, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta on mast cell activation. Whereas all these CC chemokines caused dose-dependent release of histamine from basophils in mixed human leukocyte suspensions, none of them was able to induce release of histamine as well as tryptase or prostaglandin (PG)D2 from human skin mast cells, nor did priming with these substances enhance IgE-mediated mediator release. In addition, all chemokines failed to promote changes in the cytosolic free calcium level in the human mast cell line HMC-1. These results add further evidence for the differences between human mast cells and basophils regarding cytokine-dependent activation.

Basophils↗
PubMed1996

Spontaneous and induced sputum to measure indices of airway inflammation in asthma.

Inhalation of hypertonic saline to induce sputum may alter cells and fluid-phase markers in sputum. We have compared indices of inflammation in sputum produced spontaneously with sputum induced by an aerosol of hypertonic saline. Twenty-three asthmatics produced spontaneous followed by induced sputum on the same day. The sputum specimen was separated from saliva within 2 h, dispersed with dithiothreitol (DTT) and processed to obtain cytospins and supernatant. The statistical power to detect a 20% difference in sputum parameters was > 90%. Results are expressed as median and interquartile range [IQR]. Induced sputum had a higher proportion of viable cells (77.0 [19.0] versus 47.0 [38.0]%, p < 0.001), less squamous cell contamination (1.0 [1.2] versus 1.8 [34.0]%, p < 0.001) and better quality cytospins (score of 8.0 [4.0] versus 4.0 [2.0], p < 0.001). It also had lower fluid-phase levels of eosinophil cationic protein (ECP) (1,358 [1,102] versus 1,574 [2,479] microg/L) and fibrinogen (1,560.0 [3,130.0] versus 4,350.0 [5,970.0] ng/ml) but only the latter was significantly different (p = 0.02). Induced sputum was similar to spontaneous sputum in weight (200.0 [219.0] versus 270.0 [227.0] mg), total cell count (3.3 [4.1] versus 3.5 [4.5] x 10(6)/ml), proportion of nonsquamous cells, and levels of tryptase. The agreement between induced and spontaneous measurements was good, but fluid-phase levels were affected by the low viability of some spontaneous samples. We conclude that for the indices measured in asthmatic subjects, induced sputum separated from saliva is similar to lower respiratory secretions expectorated spontaneously and has the advantage of better cell viability.

Adult↗
PubMed2000

Trypsin-induced, neurokinin-mediated contraction of guinea pig bronchus.

Proteases may act as cell signaling molecules via protease-activated receptors (PARs). PAR1, PAR3, and PAR4, but not PAR2, are activated by thrombin, whereas trypsin can activate PAR2 and PAR4. In this study, trypsin (3-100 nM) evoked concentration-dependent contractions of guinea pig isolated bronchus, however, thrombin (3-300 nM) was a weak spasmogen. Neither the PAR2-activating peptide SLIGRL (100 microM) nor mast cell tryptase (100 nM), a trypsin-like protease known to activate PAR2, evoked contraction. A role for neurokinins in trypsin-induced contraction is suggested by our observation that contractions to trypsin were markedly attenuated in the presence of neurokinin receptor antagonists. Depletion of neurokinins in sensory nerves with capsaicin also markedly reduced the ability of trypsin to evoke contraction. In electrophysiological studies, trypsin did not evoke action potentials in C-fiber afferents whose receptive fields were located in the trachea or main bronchi. The results from this study support the hypothesis that trypsin activates a mechanism allowing for local release of sensory neurokinins from afferent C-fibers and that this release occurs independently of the sensory function of these nerves.

Animals↗
PubMed2001

Safety and reproducibility of sputum induction in asthmatic subjects in a multicenter study.

The safety of sputum induction and the reproducibility of measurements in induced sputum in multicenter studies is unknown. We examined the safety of sputum induction in a two-visit, six-center study in 79 subjects with moderate to severe asthma (mean +/- SD FEV(1) 71 +/- 12% predicted, 67% taking inhaled corticosteroids). In addition, we compared the reproducibility of markers of inflammation in induced sputum with the reproducibility of the FEV(1) and the methacholine PC(20). The FEV(1) decreased > or = 20% from the postbronchodilator baseline in 14% of all subjects and in 25% of subjects whose initial prebronchodilator baseline was 40 to 60% of predicted. All subjects responded promptly to additional albuterol treatment, and no subject developed refractory bronchoconstriction requiring treatment other than reversal of bronchospasm in the study laboratory. The reproducibility of measurements of the eosinophil percentage, eosinophil cationic protein, tryptase, and methacholine PC(20) were similar (concordance correlation coefficients of 0.74, 0.81, 0.79, and 0.74, respectively), without any significant among-center effect. We conclude that sputum induction can be performed safely in subjects with moderate to severe asthma in multicenter clinical trials when carried out under carefully monitored conditions. Importantly, we demonstrate that measurement of markers of inflammation in induced sputum is as reproducible as methacholine PC(20) and should prove useful in the assessment of airway inflammation in multicenter clinical trials.

Aged↗
PubMed1992

Bronchial exudation of bulk plasma at allergen challenge in allergic asthma.

This study examined plasma exudation into the bronchial lumen after allergen challenge. A novel low-trauma technique was developed to challenge and lavage a medium-sized lingular or middle lobe bronchus. Eleven subjects with challenge-assessed pollen-sensitive asthma were allocated to fiberbronchoscopy in the supine position. In the control bronchus 0.5 ml diluent was instilled. The bronchus was occluded proximally 3 min later by inflation of a balloon, and lavage was carried out twice with 25 ml saline. Incremental doses of allergen solution (0.5 ml) were then instilled in the contralateral lung. The challenge continued until a clearly visible bronchial reaction occurred and was immediately followed by the same lavage as on the control side. The lavage liquids were analyzed for the presence of plasma exudation and mast cell activation indices. On the allergen-challenged side, tryptase, reflecting mast cell activation, was increased by 150% (p < 0.01) compared with the control side. Fibrinogen (mol wt 340,000), reflecting large protein exudation, was increased by 840% (p < 0.05), and N-alpha-tosyl-L-arginine-methyl esterase activity, reflecting both large protein exudation and mast cell activation, increased by 480% (p < 0.01). The level of albumin (mol wt 69,000), the major luminal protein under baseline conditions, increased but not significantly. We conclude that activation of mast cells and luminal entry of little sieved plasma exudates occur early after endobronchial allergen provocation in human subjects with allergic asthma.

Albumins↗
PubMed1993

Cellular and biochemical analysis of induced sputum from asthmatic and from healthy subjects.

To determine the feasibility of cellular and biochemical analysis of sputum induced after inhalation of hypertonic (3%) saline, we analyzed sputum induced in 10 healthy and in 18 asthmatic subjects. We also analyzed saliva samples from all subjects. The entire sputum sample and the saliva sample were reduced using dithiothreitol, and cell counts and differentials were determined. Biochemical analysis was performed on sputum and saliva supernatants obtained after centrifugation. We found that induced sputum from asthmatic subjects had a higher percentage of eosinophils [8.1 +/- 3.43 (mean +/- SEM) versus 0.03 +/- 0.02%, p < 0.009] (after excluding squamous cells) and also had higher levels of albumin (232.3 +/- 54.8 versus 79.5 +/- 9.7 micrograms/ml, p < 0.02), fibrinogen (44.2 +/- 11.6 versus 11.9 +/- 2.5 micrograms/ml, p < 0.008) and eosinophil cationic protein (ECP) (142.6 +/- 34.2 versus 26.1 +/- 4.7 ng/ml, p < 0.006) but not of histamine or tryptase. In saliva, squamous cells made up more than 99% of the cells in both groups, and protein concentrations were not significantly different. We conclude that cellular and biochemical analysis of induced sputum is feasible in healthy and in asthmatic subjects and that it reveals differences similar to those reported from analyses of bronchial lavage fluid.

Adult↗
PubMed1997

Of mites and men: trypsin-like proteases in the lungs.

It is no surprise to anyone who has tried to memorize clotting and complement cascades that there are a great many trypsin-like proteases-too many, it may seem. This fecund enzyme family encompasses such a range of biological roles that other important members and functions are sure to await discovery. Of known trypsin-like proteases, few, if any, are lung-specific. Nonetheless, many contribute in ways critical to lung function, such as fighting microbial invaders, regulating the formation and removal of polymerized fibrin, and rejecting tumors or transplanted tissues. Some of these enzymes, such as plasminogen activators and mast cell tryptases, are native to the lung and live where they work; identical enzymes live and work in other tissues. Other enzymes, such as most complement and hemostatic proteases, are migrants, typically born elsewhere (e.g., the liver). They pass through the lung, looking for something to do; if nothing is found, they pass on, circulating, perhaps to return later. Still others are unwanted, uninvited intruders, such as dust mite proteases. This minireview provides a selective glimpse of the lives of some of the trypsin-like enzymes at work in the lung and airways.

Animals↗
PubMed1997

IL-13 mRNA and immunoreactivity in allergen-induced rhinitis: comparison with IL-4 expression and modulation by topical glucocorticoid therapy.

The allergen-induced late nasal response (LNR) is associated with high expression of interleukin-4 (IL-4) and IL-5 messenger RNA (mRNA) in the nasal mucosa, suggesting a role for Th2-type cytokines in the development of the LNR. Moreover, topical corticosteroid-mediated inhibition of the LNR is accompanied by inhibition of IL-4, but not IL-5, mRNA expression, IL-13 shares a number of functions with IL-4, including IgE switching and vascular cell adhesion molecule-1 (VCAM-1) upregulation. We investigated the expression of IL-13 mRNA and immunoreactivity in nasal biopsies from 10 normal subjects and 20 subjects with allergic rhinitis. IL-4 mRNA expression was examined in the same subjects. The allergic rhinitis patients were randomized to receive a 6-wk treatment with either topical fluticasone propionate (n = 10) or placebo (n = 10) nasal spray twice daily. A nasal biopsy was taken before treatment and 24 h after local nasal allergen provocation with a grass-pollen extract. Before treatment, there was no significant difference between the allergic rhinitis patients and controls in the expression of IL-13 mRNA and immunoreactivity. After allergen provocation, we observed a significant increase in IL-13 mRNA-positive and immunoreactive cells at 24 h only in subjects given placebo (P < 0.001). Inhibition of the LNR after corticosteroid treatment was associated with a marked decrease in allergen-induced IL-13 mRNA-positive (P < 0.001) and immunoreactive cells (P < 0.001). In subjects given placebo, 76.9 +/- 5.5% of IL-13 mRNA-positive cells observed after allergen were CD3+, whereas 11.2 +/- 2.7% coexpressed immunoreactivity for mast-cell tryptase. In these subjects, increases in cells expressing IL-13 mRNA were greater than for IL-4 mRNA (P = 0.001), and double in situ hybridization studies revealed that 100% of the IL-4 mRNA-positive cells coexpressed IL-13 mRNA, whereas 66.6 +/- 10.5% of IL-13 mRNA-positive cells coexpressed IL-4 transcripts after allergen challenge. The results of this study suggest that IL-13 expression is a prominent feature of the LNR, and that inhibition of the LNR following steroid therapy may be partly attributable to inhibition of IL-13 expression.

Administration, Intranasal↗
PubMed1998

Assessment by nasal biopsy of long-term use of mometasone furoate aqueous nasal spray (Nasonex) in the treatment of perennial rhinitis.

Allergic rhinitis is associated with specific histopathologic changes in the nasal mucosa including squamous metaplasia and local eosinophilia. Previous studies have shown that mometasone furoate aqueous nasal spray is effective and well tolerated in reducing perennial rhinitis and seasonal allergic rhinitis symptoms. We undertook a multicenter, open-label study to evaluate, by nasal biopsy, the tissue changes associated with mometasone furoate use (200 microg/day) during a 12-month treatment period in patients with perennial rhinitis. Of the 69 patients enrolled in the study, 52 completed all 12 months of treatment. Nasal biopsy specimens obtained from patients at baseline and after treatment were evaluated in a blinded fashion by computerized image analysis, qualitative histologic examination, and immunocytochemistry. Morphologic examination of nasal biopsy specimens showed a decrease in focal metaplasia, no change in epithelial thickness, and no sign of atrophy after treatment with mometasone furoate. Immunocytochemical analyses of nasal biopsy specimens obtained before and after treatment revealed a significant decrease in major basic protein-positive eosinophils and tryptase-positive mast cells in the epithelium and lamina propria after treatment. Mometasone furoate appeared to attenuate the inflammatory process by reducing the extent of inflammatory cell infiltration, particularly of eosinophils. This study demonstrated that long-term administration of mometasone furoate is not associated with adverse tissue changes in the nasal mucosa of patients with perennial rhinitis.

Administration, Intranasal↗