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PubMed1991

Mast cell numbers and staining characteristics in the normal and allergic human conjunctiva.

In this study we report the numbers and metachromatic dye-staining characteristics of mast cells (MCs) in the conjunctiva of normal subjects and patients with seasonal allergic conjunctivitis caused by pollenosis. In addition, we have used a monoclonal antibody to the MC-specific enzyme, tryptase, to enumerate tryptase-positive cells immunohistochemically. Tarsal conjunctival MCs were found to be present in increased numbers in the allergic compared to the nonallergic subjects. Most of the MCs exhibited staining that was formaldehyde resistant, compatible with their identification as connective tissue MCs or basophils. The high positive staining for tryptase confirmed that the metachromatic cells stained with toluidine blue were MCs and not basophils, both in normal and allergic subjects.

Adult↗
PubMed2005

Murine implantation serine proteinases 1 and 2: structure, function and evolution.

Implantation is a vital phase in pregnancy whereupon the hatched embryo invades into the uterine wall to establish intimate contacts with the mother for further development. Although it is generally believed that proteinases are major factors that confer the embryo its invasive character, the nature of proteinases involved in implantation remain mostly elusive. In this article, we review the organization, structure and postulated function of the implantation serine proteinase (ISP1 and 2) genes. The ISPs are embedded within a cluster of tryptase genes on mouse chromosome 17. They are most closely related to members of the mast cell tryptase family, indicating that they may possess some properties characteristic of tryptases including multimerization-dependent activation. The significant similarities found in regulatory regions of ISP genes, together with the observation that ISP proteins are co-expressed and heterodimerize in the embryo and uterus suggests that they are intimately co-regulated during implantation. Inhibition of ISP proteolytic function has implicated this enzyme in the processes of embryo hatching and implantation.

Amino Acid Sequence↗
PubMed2005

Protease-activated receptor 2 expression in trigeminal neurons innervating the rat nasal mucosa.

Protease-activated receptor 2 (PAR2) is activated by trypsin and mast cell tryptase to induce widespread inflammation by unknown mechanisms. Trypsin and tryptase were shown to activate sensory neurons to release substance-P and related peptides to mediate neurogenic inflammation. In the present study, the expression of PAR2 and tachykinins were investigated in rat trigeminal neurons that were identified by retrograde labeling with rhodamine dye from the nasal mucosa by using neuronal tracing in combination with immunohistochemistry. We found that large subpopulation of all trigeminal neurons (43.5+/-2.6%) identified by the pan-neuronal marker PGP 9.5 were stained with PAR2-immunoreactivity. Of all trigeminal neurons, 7.5+/-2.1% were immunoreactive for tachykinins and PAR2, and only 3.9+/-1.7% of all trigeminal neurons expressed tachykinins, but not PAR2-immunoreactivity. The present study also found that a large number trigeminal neurons innervating the nasal mucosa expressed PAR2-immunoreactivity. Of the rhodamine-labeled trigeminal neurons, 52.5+/-1.8% were immunoreactive for only PAR2 expression, 7.3+/-1.9% contained tachykinins and PAR2, and 3.1+/-0.4 of the rhodamine-labeled trigeminal neurons were non-immunoreactive PAR2, but were positive for tachykinins-immunoreactivity. In conclusion, based on the co-localization of PAR2 and tachykinins in trigeminal sensory neurons innervating the nasal mucosa, the present study suggests that, following an activation of PAR2 receptor in tachykinergic neurons by trypsin and mast cell tryptase, there may be a triggering of tachykinin-mediated phenomena such as neurogenic inflammation in allergic or non-allergic rhinitis.

Animals↗
PubMed2005

Benefit from anti-inflammatory treatment during clinical remission of atopic asthma.

STUDY OBJECTIVES: Subjects with atopic asthma often experience a disappearance of symptoms around puberty. However, airway inflammation and remodeling may persist. It is unknown whether those findings warrant prolonged anti-inflammatory treatment despite the absence of symptoms. In this study, we investigated whether a short course of combined anti-inflammatory treatment would, also in this specific patient population, diminish airway inflammation and/or remodeling. DESIGN: A double-blind, randomized placebo-controlled trial was conducted in 28 asymptomatic subjects with a history of atopic asthma, with established bronchial hyperresponsiveness to methacholine (MCh) as non-invasive indicator of ongoing airway pathology. INTERVENTIONS: Intervention consisted of the salmeterol/fluticasone propionate combination (SFC) product (50/250 microg bid via the Diskus inhaler) or placebo for 3 months. MEASUREMENTS: The change in lung function (FEV1), bronchial response to MCh and adenosine monophosphate (AMP), the fraction of nitric oxide in exhaled air (FENO) and quality of life (QOL) scores were measured. Also, bronchial biopsies were taken and cryo sections immunostained for eosinophils (major basic protein, MBP) and mast cells (tryptase and chymase) before and after treatment. The change in reticular basement membrane (RBM) thickness, one of the parameters of airway remodeling, was also determined. RESULTS: SFC treatment improved hyperresponsiveness to MCh (P = 0.014) as well as AMP (P = 0.011), and reduced FENO (P < 0.001) significantly as compared with placebo. Lung function tended to improve (NS). Furthermore, SFC treatment reduced tryptase in the subepithelium of bronchial biopsy specimens (P = 0.01), and slightly reduced RBM thickness (P = 0.05). However, eosinophils in (sub)epithelium were not significantly affected; neither were chymase levels, blood eosinophils or QOL scores. CONCLUSIONS: We found that 3 months of treatment with fluticasone propionate and salmeterol reduced airway hyperresponsiveness, FENO and tryptase density in the airway mucosa as markers of airway inflammation. MBP density in the airway mucosa and QOL were, however, unchanged. The clinical relevance of these findings, especially with respect to the long-term outcome, has not been determined yet.

Adolescent↗
PubMed2005

Exposure to tobacco-derived materials induces overproduction of secreted proteinases in mast cells.

Mast cells reside at interfaces with the environment, including the mucosa of the respiratory and gastrointestinal tracts. This localization exposes mast cells to inhaled, or ingested, environmental challenges. In the airways of smokers, resident immune cells will be in contact with the condensed components of cigarette smoke. Mast cells are of particular interest due to their ability to promote airway remodeling and mucus hypersecretion. Clinical data show increased levels of mast cell-secreted tryptase and increased numbers of degranulated mast cells in the lavage and bronchial tissue of smokers. Since mast cell-secreted proteinases (MCPTs), including tryptases, contribute to pathological airway remodeling, we investigated the relationship between mast cell proteinases and smoke exposure. We exposed a mast cell line to cigarette smoke condensate (CSC). We show that CSC exposure increases MCPT levels in mast cells using an assay for tryptase-type MCPT activity. We hypothesized that this increase in MCPT activity reflects a CSC-induced increase in the cytosolic pool of proteinase molecules, via stimulation of MCPT transcription. Transcript array data suggested that mRNA changes in response to CSC were limited in number and peaked after 3 h of CSC exposure. However, we noted marked transcriptional regulation of several MCPT genes. CSC-induced changes in the mRNA levels for MCPTs were confirmed using quantitative RT-PCR. Taken together, our data suggest that chronic exposure to cigarette smoke up-regulates MCPT levels in mast cells at both the protein and the mRNA level. We suggest that the pathological airway remodeling that has been described in clinical studies of smoke inhalation may be attributable to MCPT overproduction in vivo.

Cell Line↗
PubMed2003

Expression of protease-activated receptor-1, -2, -3, and -4 in control and experimentally inflamed mouse bladder.

Inflammation underlines all major bladder pathologies and represents a defense reaction to injury involving a mandatory participation of mast cells and sensory nerves. Mast cells are particularly frequent in close proximity to epithelial surfaces where they are strategically located in the bladder and release their mediators in response to inflammation. Tryptase is specifically produced by mast cells and modulates inflammation by activating protease-activated receptors (PARs). We recently found that PAR-4 mRNA is up-regulated in experimental bladder inflammation regardless of the initiating stimulus. Because it has been reported that PAR-1, PAR-2, and PAR-3 may also be involved in the processes of inflammation, we used immunohistochemistry to characterize the expression of all known PARs in normal, acute, and chronic inflamed mouse bladder. We found that all four PARs are present in the control mouse bladder, and follow a unique distribution. All four PARs are co-expressed in the urothelium, whereas PAR-1 and PAR-2 are predominant in the detrusor muscle, and PAR-4 is expressed in peripheral nerves and plexus cell bodies. The strong expression of PARs in the detrusor muscle indicates the need for studies on the role of these receptors in motility whereas the presence of PAR-4 in nerves may indicate its participation in neurogenic inflammation. In addition, PARs are differentially modulated during inflammation. PAR-1 and PAR-2 are down-regulated in acute inflammation whereas PAR-3 and PAR-4 are up-regulated. Bladder fibroblasts were found to present a clear demarcation in PAR expression secondary to acute and chronic inflammation. Our findings provide evidence of participation of PARs in the urinary system, provide a working model for mast cell tryptase signaling in the mouse bladder, and evoke testable hypotheses regarding the roles of PARs in bladder inflammation. It is timely to understand the role of tryptase signaling and PARs in the context of bladder biology.

Animals↗
PubMed1999

Mast cells are augmented in deep vein thrombosis and express a profibrinolytic phenotype.

A number of recent data suggest that mast cells (MC) and their products are involved in the pathophysiology of thrombosis. In the current study, we have evaluated the number, distribution, and phenotype of MC in patients with deep vein thrombosis of the lower limb (DVT) (n = 15). Contralateral nonthrombosed limb veins served as control (CO). MC were examined by Giemsa staining and by immunohistochemistry using antibodies against tryptase, chymase, tissue-type plasminogen activator (tPA), urokinase (uPA), urokinase receptor (uPAR), and plasminogen activator inhibitors (PAI-1, PAI-2). We found an increase in the number of tryptase-positive MC in DVT compared with CO (DVT: 9.1+/-1.0 v CO: 4.7+/-0.6 MC/mm2, P < .05). Most of these MC appeared to accumulate in the adventitia of the thrombosed veins, in vicinity of the vasa vasorum. In both DVT and CO, MC reacted with monoclonal antibodies to c-kit, tryptase, and chymase. MC also stained positive for tPA and urokinase receptor, but did not express detectable PAI-1 or PAI-2. As compared with CO, a decreased proportion of MC in DVT was found to stain positive for chymase and tPA. Together, our results show that MC increase in number in DVT and express a profibrinolytic phenotype. We hypothesize that MC and MC-derived profibrinolytic molecules play a role in the pathophysiology of DVT.

Aged↗
PubMed1996

The presence of membrane-bound stem cell factor on highly immature nonmetachromatic mast cells in the peripheral blood of a patient with aggressive systemic mastocytosis.

BACKGROUND: Systemic mastocytosis is characterized by mast cell infiltration of bone marrow and tissues in the absence of identified circulating bone marrow-derived progenitors. A 58-year-old man was first seen with aggressive systemic mastocytosis manifested by urticaria pigmentosa, hepatosplenomegaly, generalized bone lesions, anemia, thrombocytopenia, monoclonal gammopathy, and increased urine histamine levels. OBJECTIVES AND METHODS: A rapidly progressive anemia and thrombocytopenia dictated a splenectomy. We sought to identify the mast cell progenitors in the peripheral blood and to provide evidence of their maturation in tissues with immunohistochemical and ultrastructural analyses. RESULTS: The peripheral blood contained 1% to 3% nonmetachromatic mononuclear cells with eccentric nuclei that expressed the mast cell proteases, tryptase and carboxypeptidase A, along with c-kit, stem cell factor (SCF), and high-affinity IgE receptor (Fc epsilon RI), but not chymase. Similar mononuclear cells colocalized in the spleen and lymph nodes with mature, metachromatic mast cells that expressed tryptase, chymase, carboxypeptidase A, c-kit, SCF, and Fc epsilon RI. Electron microscopy disclosed, at each site, a mature mast cell population with electron-dense, scroll-poor granules. CONCLUSIONS: The peripheral blood of a patient with aggressive systemic mastocytosis contained immature mononuclear cells of the mast cell lineage that express c-kit, SCF, tryptase, carboxypeptidase A, and Fc epsilon RI. These cells were also found in the skin, spleen, and lymph nodes where they presumably expand, differentiate, and mature, assuming the mast cell phenotype for those tissues characterized by metachromasia, expression of a full range of mast cell-related secretory granule proteases, and ultrastructural appearance. The presence of SCF on the surface membrane of the circulating, highly immature mast cells suggests an autocrine regulation of the c-kit-SCF interaction.

Cell Differentiation↗
PubMed1997

Eosinophils, mast cells, and basophils in induced sputum from patients with seasonal allergic rhinitis and perennial asthma: relationship to methacholine responsiveness.

OBJECTIVES: We attempted to determine whether inflammation is present in induced sputum of patients with seasonal allergic rhinitis (AR) as compared with those with perennial asthma (AS) and examined its relationship with bronchial responsiveness to methacholine. METHODS: Sputum was induced in 30 patients with seasonal rhinitis in response to grass pollens only and in 15 patients with stable, asymptomatic asthma. The AR group was divided according to methacholine PD20 value: the AR- group (n = 15) had a methacholine PD20 greater than 24 micromol; the AR+ group (n = 15) had a methacholine PD20 ranging between 2.2 and 19.6 micromol. In the AS group, methacholine PD20 ranged between 0.42 and 2.6 micromol. The percentage of eosinophils and metachromatic cells (alcian blue-positive) was assessed in sputum by light microscopy. Tryptase-positive cells and EG2+ cells were identified by immunocytochemistry with the mouse anti-human mast cell-tryptase monoclonal antibody and the monoclonal anti-eosinophil cationic protein antibody. RESULTS: We found that the number of eosinophils in the AS group was greater than that in the AR+ group (p < 0.05) and in the AR- group (p < 0.01). Moreover, the eosinophil count was lower in the AR- group compared with the AR+ group (p < 0.05). Similarly, the number of EG2+ cells was greater in the AS group than in the AR group (p < 0.02) and the AR- group (p < 0.05). Moreover, the EG2+ cell count was lower in the AR- group than in the AR+ group (p < 0.05). The number of mast cells and basophils in the AS group was greater than that in the AR group (p < 0.05 and p < 0.01, respectively). Mast cells in sputum were tryptase-positive. Basophils were present in sputum from 23% of patients with AR and 53% of patients with asthma. There was a significant correlation between methacholine PD20 and eosinophils (p < 0.005) and mast cells (p < 0.02) but not with basophils in those patients showing a measurable methacholine PD20 (AR+ and AS groups). CONCLUSIONS: Inflammatory cells are present not only in the airways of patients with asthma but also in airways of patients with seasonal AR, even outside natural exposure. Moreover, we provide evidence for the presence of basophils in sputum of patients with asthma even during clinical remission. The presence of bronchial responsiveness is associated with an increase in the number of eosinophils and metachromatic cells. Our findings are consistent with the hypothesis that eosinophils, as well as mast cells, contribute to bronchial responsiveness not only in AS but also in seasonal AR.

Adult↗
PubMed1999

Upregulation of TNF-alpha and IL-3 expression in lesional and uninvolved skin in different types of urticaria.

BACKGROUND: Although mast cells are known to secrete a broad spectrum of proinflammatory and immunomodulatory cytokines, the role of these molecules in mast cell-dependent cutaneous inflammation is not clear. OBJECTIVE: We decided to study biopsy specimens from lesional and nonlesional skin of patients with acute, chronic recurrent, delayed pressure, and cold urticaria; from fleeting wheals of prick test reactions to allergens; and from normal skin of nonallergic subjects. METHODS: Cryostat sections were stained by immunohistochemistry with antibodies against IL-3, IL-8, TNF-alpha, and mast cell-specific tryptase. In serial sections with tryptase and each cytokine, reactivity of mast cells was studied as well. RESULTS: Compared with normal skin and prick test reactions, immunoreactivity for TNF-alpha and IL-3 was significantly increased on endothelial and perivascular cells of the upper dermis in all urticaria lesions. In nonlesional skin comparable upregulation was noted on endothelial cells and for TNF-alpha on perivascular cells of patients with delayed pressure urticaria. In addition, TNF-alpha was expressed throughout the epidermis in lesional and nonlesional skin of patients with all types of urticaria, but not in normal control subjects. Sequential biopsy specimens from patients with cold urticaria showed upregulation of TNF-alpha and IL-3 on endothelial cells 30 minutes after elicitation of lesions with an ice cube. In contrast to these findings, epidermal immunoreactivity, as well as endothelial and perivascular cell expression of IL-8, were only slightly altered in urticaria compared with normal skin. In sequentially stained sections, few tryptase-positive mast cells reacted to TNF-alpha, few reacted to IL-3 in pressure urticaria only, and practically none stained for IL-8. CONCLUSION: These findings suggest that the cytokines studied here are involved in the pathology of urticaria, possibly by inducing subthreshold inflammation in endothelial cells of uninvolved skin.

Cytokines↗
PubMed2004

Optimal skin prick wheal size for diagnosis of cat allergy.

BACKGROUND: The skin prick test is the diagnostic procedure of choice for determination of immediate hypersensitivity. A wheal diameter of 3 mm or larger is generally accepted as the cutoff for a positive test result, although the validity of this assumption has not been rigorously demonstrated. OBJECTIVE: To determine the skin prick wheal size that best identifies clinical allergy to cat. METHODS: Forty-five patients referred for evaluation of rhinoconjunctivitis underwent determination of atopic status by skin testing using the Greer Dermapik device and a combination of other modalities, including history, in vitro determination of specific IgE level, and nasal challenge with standardized cat pelt extract. Parameters evaluated before and after nasal challenge included symptom score and nasal lavage tryptase and prostaglandin D (PGD2) levels. RESULTS: The widely accepted 3-mm wheal for a positive skin test result to cat is highly sensitive but only moderately specific for diagnosis of cat allergy as evaluated by history, specific IgE level, postchallenge symptom score, and tryptase and PGD2 levels. Optimal cutoffs for a positive skin test result to cat based on receiver operating characteristic analysis and 95% positive predictive value were 5.5 mm or greater for each of these parameters. When a true-positive result for cat allergy was defined as a combination of positive history, specific IgE level, postchallenge symptom score, and tryptase and PGD2 levels and a true-negative result as all of these parameters being negative, a 6-mm cutoff was able to distinguish cat allergic from cat nonallergic individuals. CONCLUSION: In a potentially allergic population undergoing skin prick testing with the Greer Dermapik using standardized extracts, a 3-mm skin prick wheal will overestimate the presence of cat allergy. A 6-mm wheal appears to distinguish those individuals who are cat allergic from those who are not.

Adult↗
PubMed1998

Fluorescein-induced allergic reaction.

BACKGROUND: Adverse reactions following intravenous sodium fluorescein are very unusual and their mechanism is still uncertain. We report the case of a patient who suffered an adverse reaction during a fluorescein ocular angiography. Positive allergy tests to fluorescein suggest an IgE-mediated mechanism. OBJECTIVE: Report the allergy evaluation performed in a patient who suffered an adverse reaction during an intravenous fluorescein administration. METHODS: We selected the case of a patient who suffered dizziness, diaphoresis, generalized pallor, nausea, sphincter relaxation, hypotension, and intense malaise during a fluorescein ocular angiography and compared the results to other nonreactive subjects. Allergy evaluation: Prick and intradermal skin tests and serial determinations of serum tryptase were performed on the patient and four control subjects who underwent and tolerated the same procedure as well as on a patient who developed an intense vagal reaction during blood extraction. RESULTS: Positive skin tests and dramatic increase of serum tryptase (67U/I) were observed in our patient. The rest of the patients had negative skin tests and did not have any variation in their serum tryptase. CONCLUSIONS: An IgE-mediated mechanism is suggested as responsible for this adverse reaction. We recommend that a complete allergy evaluation should be performed in all patients who have adverse reactions to fluorescein in order to differentiate true allergic reactions from other types of reactions.

Aged↗
PubMed2000

Effect of current exposure to Der p 1 on asthma symptoms, airway inflammation, and bronchial hyperresponsiveness in mite-allergic asthmatics.

The existence of a dose-response relationship between indoor allergen exposure and sensitization has been widely described, but the effect of allergen exposure on asthma activity (symptoms, bronchial hyperresponsiveness [BHR], and inflammation) is not clear. Our aim was to determine the existence of an association among current exposure to mite allergens and symptoms, BHR, and airway inflammation assessed in blood and sputum from asthmatic patients sensitized to Dermatophagoides pteronyssinus. We selected 31 mild and recently diagnosed (12-24 months) asthma patients sensitized to D. pteronyssinus. Allergenic exposure (Der p 1, Der 2) was assessed by a commercial assay based on monoclonal antibodies (mAb), carried out on the dust samples collected from patients' beds in a standardized way. Patients completed an asthma symptom questionnaire and underwent skin tests, methacholine bronchial challenge, and sputum induction. Sputum cell profile was analyzed and eosinophil cationic protein (ECP), tryptase, albumin, and interleukin(IL)-5 levels were quantified in sputum supernatant. Total eosinophil numbers and ECP levels were measured in blood samples. Most patients were exposed to Der p 1 levels under 2 microg/g of dust. Der p 1 exposure was higher among the subjects with positive sputum tryptase detection (P = 0.020). Der p 1 levels showed a trend toward correlation with asthma symptoms (P = 0.066, r = 0.36) and correlated with sputum tryptase levels (P = 0.032, r = 0.42). No relationship between BHR, eosinophilic inflammation, and allergenic exposure was found. Our results suggest that asthma symptoms and lung mast-cell activation are at least partially dependent on current allergen exposure. The lack of correlation between mite exposure, eosinophilic inflammation, and BHR supports the role of other factors that enhance the immunologic response initiated by allergen, increasing the activity of asthma.

Allergens↗
PubMed1998

Ivermectin influence on the mast cell activity in nodules of onchocerciasis patients.

Onchocercal nodules were stained immunohistochemically using antibodies specific for human mast cells and IgE to elucidate the localization and frequency of mast cells after a single oral dose of 150 microg/kg ivermectin. Tryptase-and chymase-positive mast cells occurred predominantly in mixed inflammatory infiltrates and perivascularly, and never adhered to adult worms or microfilariae. Up to three days after ivermectin, mast cells and IgE-positive cells were markedly increased in the capsular area of nodules containing female worms with embryos and microfilariae compared to untreated nodules. In the centre of these nodules, around the adult Onchocerca volvulus, we found many tryptase-positive cells. More mast cells were IgE-positive than in untreated nodules, equalling the number of tryptase-positive mast cells. There was a clear correlation between the appearance of mast cells and the attacks on damaged microfilariae by eosinophils and macrophages and in the vicinity of adult worms by neutrophils that occur soon after ivermectin treatment. Onchocercomata harbouring female worms with oocytes only revealed, after all treatment intervals, the same mast cell numbers as untreated nodules. In conclusion, during the first three days after administration, ivermectin produces increased numbers of mast cells in nodules harbouring females with embryos and microfilariae, probably as part of an allergic reaction to the attacked microfilariae. Four to 19 days after ivermectin the number of mast cells in the entire nodule is no longer elevated.

Animals↗
PubMed2000

Effect of mast cell-derived mediators and mast cell-related neutral proteases on human dermal fibroblast proliferation and type I collagen production.

BACKGROUND: Possible involvement of mast cells in various fibrotic conditions has been suggested, but the relative contribution of each mast cell mediator and neutral protease to fibroproliferative activity remains to be elucidated. OBJECTIVE: We investigated the effect of mast cell-derived mediators and mast cell-related neutral proteases on type I collagen production and proliferation by human dermal fibroblasts. METHODS: Mast cell-derived mediators or neutral proteases were added to cultured fibroblasts from normal dermis, and cell proliferation and type I collagen synthesis were assayed. RESULTS: Fibroblast proliferation was increased by 2.8 x 10(-9) mol/L prostaglandin D(2) and 10 microgram/mL carboxypeptidase A, but not by leukotriene D(4) or cathepsin G at the concentrations studied. Proliferation was increased by tryptase in a concentration-dependent manner, and a significant increase was observed at concentrations of 1 and 10 microgram/mL. Production of type I collagen by fibroblasts was increased in the presence of 2.0 x 10(-9) mol/L leukotriene D(4) and 10 microgram/mL tryptase. CONCLUSION: Mast cell-derived mediators prostaglandin D(2) and leukotriene D(4) and mast cell-related neutral proteases carboxypeptidase A and tryptase increase the proliferation and type I collagen production of human dermal fibroblasts in various manners.

Cell Division↗
PubMed2000

Allergic fungal sinusitis: an immunohistologic analysis.

BACKGROUND: Allergic fungal sinusitis is a noninvasive form of fungal sinusitis that has recently been delineated as a distinct clinicopathologic entity. It is increasingly recognized as a cause of chronic sinusitis, with the primary causative agents being members of the Dematiaceae fungus family. Although its immunopathogenesis has not been elucidated, the eosinophil is a prominent inflammatory cell on histologic examination. OBJECTIVE: We sought to characterize the involvement of eosinophils in sinus tissue and accompanying mucin from patients with allergic fungal sinusitis. As a comparison, neutrophil and mast cell involvement was also evaluated in the same group of patients. METHODS: Tissue specimens from 8 patients with allergic fungal sinusitis, along with 8 nasal polyp specimens from patients without allergic fungal sinusitis, were stained by using indirect immunofluorescence for eosinophil granule major basic protein (MBP). Neutrophil elastase and mast cell tryptase staining was also performed on the same allergic fungal sinusitis and nasal polyp tissues. RESULTS: MBP was diffusely localized within the mucin, showing intense staining at the periphery and variable staining of degenerated cell clusters throughout. Extracellular MBP in the mucin was strikingly greater than intact eosinophil staining. Diffuse extracellular neutrophil elastase was also present in the mucin. Mucinous areas showed no tryptase localization. Adjacent nonmucinous areas of respiratory mucosa showed predominantly cellular staining with eosinophil MBP, neutrophil elastase, and mast cell tryptase. MBP staining of nasal polyps showed a predominantly cellular pattern with focal areas of extracellular deposition. CONCLUSIONS: Given the known toxicities of eosinophil granule MBP and neutrophil elastase, their extracellular presence supports the contribution of these proteins in the pathogenesis of allergic fungal sinusitis and further indicates that eosinophil and neutrophil activation occurs in the disease.

Adult↗
PubMed2000

Tear and conjunctival changes during the allergen-induced early- and late-phase responses.

BACKGROUND: Allergic eye disease is common, but little is known about the underlying disease mechanisms. Conjunctival allergen challenge causes symptoms similar to those of seasonal allergic conjunctivitis and is a useful model to study. OBJECTIVE: We have used allergen challenge to investigate the course of the ocular response, tear inflammatory mediators, tissue adhesion protein expression, and cellular infiltration. METHODS: Eighteen atopic patients and 4 nonatopic control subjects were challenged with extracted mixed grass or Dermatophagoides pteronyssinus in one eye and control vehicle in the other. The clinical response was recorded, and tears were collected over a 6-hour period. Conjunctival biopsy specimens were taken from the challenged eye at 6 or 24 hours. RESULTS: An early-phase response (maximal at 20 minutes) showed a significant increase in tear histamine and tryptase levels, reducing to control levels again by 40 minutes. At 6 hours, a late-phase response occurred with increased symptoms, a second peak of tear histamine and eosinophil cationic protein but not tryptase, upregulation of the adhesion molecules E-selectin and intercellular adhesion molecule, and a cellular infiltrate of mast cells, neutrophils, eosinophils, macrophages, and basophils, with T cells increased only in bulbar biopsy specimens. CONCLUSIONS: The early peaks of tear histamine plus tryptase indicate that the mast cell is responsible for the early-phase response, but basophils may be involved in the late-phase response. Both tear and biopsy findings underline the significance of the late-phase response as the transition between a type I response and clinical disease.

Adolescent↗
PubMed2001

Disialoganglioside GD3 is selectively expressed by developing and mature human mast cells.

BACKGROUND: Disialoganglioside GD3 is expressed on the surface of selected cell types. Anti-GD3 mAb administered to human subjects with malignant melanoma produces signs and symptoms of immediate hypersensitivity reactions. OBJECTIVE: The expression of GD3 by human mast cells was assessed during mast cell development in vitro and in samples of lung and skin. METHODS: GD3 on tissue- and in vitro-derived mast cells was analyzed after double labeling of cells for tryptase (G3 mAb) or Kit (YB5.B8 mAb) and GD3 (R24 mAb). Glycolipids in extracts of fetal liver-derived mast cells were examined by using high-performance thin-layer chromatography. RESULTS: Flow cytometry showed that the percentage of GD3+ cells increased in parallel to Kit+ cells during the recombinant human stem cell factor-dependent development of fetal liver-derived mast cells. Double-labeling experiments showed that GD3+ cells were also surface Kit+ and granule tryptase positive, identifying them as mast cells in preparations of lung-, skin-, fetal liver-, and cord blood-derived cells. The major acidic glycolipid detected was NeuAcalpha2-8NeuAcalpha2-3Galbeta1-4Glcbeta1-1'Cer (GD3). Among peripheral blood leukocytes, only basophils and about 10% of the T cells were labeled with anti-GD3 mAb. Anti-GD3 mAb-conjugated magnetic beads were used to purify mast cells to greater than 90% purity from dispersed skin cells enriched to approximately 12% purity by means of density-dependent sedimentation but were less proficient for dispersed human lung mast cells, most likely because of other cell types that express GD3. CONCLUSION: GD3 is expressed on the surface of developing human mast cells in parallel to tryptase in secretory granules and, like Kit, can serve as a target for their enrichment by immunoaffinity techniques.

Cells, Cultured↗