Development and field testing of a novel patient-reported outcome measure of dysphagia in patients with eosinophilic esophagitis.

BACKGROUND: Dysphagia is the hallmark of eosinophilic esophagitis (EoE), but no validated dysphagia instruments in this population exist. AIM: To develop and field test a patient-reported outcome (PRO) for dysphagia in subjects with EoE. METHODS: This was a multi-centre/multi-phase prospective study. The first phase developed a dysphagia questionnaire using qualitative methods. The second phase was a 30-day field trial to test the instrument and assess content validity. Adolescents and adults with EoE, active symptoms of dysphagia and oesophageal eosinophilia (≥15 eosinophils per high-power field) were enrolled. Solid-food-avoidance days, dysphagia days and actions taken to get relief were recorded. A dysphagia score was calculated and compared to the Straumann Dysphagia Instrument (SDI). RESULTS: Ten adolescents and 10 adults were included in the first phase and the Dysphagia Symptom Questionnaire (DSQ), a three-item daily electronic diary, was developed. In the second phase, 35 subjects finished the field trial (18 adults, 17 adolescents, mean age 24, 54% male, 95% white, 54% currently on topical corticosteroids). The median number of dysphagia days per week was 2 for adolescents vs. 4 for adults (P < 0.001), and 2 for those on topical steroids vs. 4 for those not on topical steroids (P < 0.001). The DSQ score strongly correlated with the number of dysphagia days (R = 0.96; P < 0.001) and the SDI (R = 0.77; P < 0.001). CONCLUSIONS: The DSQ, a three-question patient-reported outcome, was successfully developed and field tested. The DSQ had content validity and the score accurately measured dysphagia frequency and intensity. The Dysphagia Symptom Questionnaire is suitable for use in clinical trials of EoE patients with dysphagia.

Pharmacokinetics and safety of subcutaneous immune globulin (human), 10% caprylate/chromatography purified in patients with primary immunodeficiency disease.

Subcutaneous administration of intravenous immunoglobulin G (IgG) preparations provides an additional level of patient convenience and more options for patients with poor venous access or a history of intravenous IgG reactions. An open-label, pharmacokinetic trial (n = 32) determined the non-inferiority of the subcutaneous versus intravenous route of 10% caprylate/chromatography purified human immune globulin intravenous (IGIV-C; Gamunex®) administration by comparing the steady-state area under the concentration-versus-time curve (AUC) of total plasma IgG in patients with primary immunodeficiency disease. Patients on stable IGIV-C received two intravenous infusions (administered 3 or 4 weeks apart). Seven to 10 days after the second intravenous infusion, all patients switched to a weekly infusion of subcutaneous IGIV-C, with the dose equal to 137% of the previous weekly equivalent intravenous dose, for up to 24 weeks. Samples for pharmacokinetic analysis were collected during steady state for intravenous and subcutaneous IGIV-C treatments. The AUC(0-) τ geometric least-squares mean ratio was 0·89 (90% confidence interval, 0·86-0·92) and met the criteria for non-inferiority. The overall mean steady-state trough concentration of plasma total IgG with subcutaneous IGIV-C was 11·4 mg/ml, 18·8% higher than intravenous IGIV-C (9·6 mg/ml). Subcutaneous IGIV-C was safe and well tolerated. Subcutaneous IGIV-C infusion-site reactions were generally mild/moderate and the incidence decreased over time. No serious bacterial infections were reported. Weekly subcutaneous IGIV-C infusion using 137% of the weekly equivalent intravenous immunoglobulin dose provides an AUC comparable to intravenous administration, thus allowing patients to maintain the same IgG preparation/formulation if switching between intravenous and subcutaneous infusions.

Grass pollen immunotherapy inhibits seasonal increases in basophils and eosinophils in the nasal epithelium.

BACKGROUND: Symptoms of allergic rhinitis are accompanied by infiltration of the nasal mucosa with inflammatory cells, predominantly eosinophils and metachromatic cells (basophils and mast cells). Specific immunotherapy (IT) reduces mucosal eosinophilia and numbers of metachromatic cells in the epithelium. A specific marker distinguishing basophils from mast cells was recently developed. OBJECTIVES: The basophil-specific monoclonal antibody 2D7 was used to determine the influence of subcutaneous IT on numbers of nasal mucosal basophils compared with the effects of IT on neutrophils, eosinophils and mast cells. METHOD: During a randomized, placebo-controlled trial of grass pollen IT in 44 adults with severe summer hay fever, nasal biopsies were taken at baseline, out of the pollen season, and at the peak of the pollen season following 2 years treatment. Biopsies were processed for immunohistochemistry for basophils (2D7+), mast cells (AA1+), eosinophils (MBP+) and neutrophils (neutrophil elastase+). RESULTS: In placebo-treated (PL) patients there were significant seasonal increases in basophils (P < 0.01), mast cells (P < 0.05) and eosinophils (P = 0.002) in the nasal submucosa. In IT-treated patients significant increases in 2D7+ cells (P < 0.01) and eosinophils (P = 0.01) but not AA1+ cells (P = 0.9) were observed. These differences were significant between groups for eosinophils (P < 0.05). In the epithelium there were seasonal increases in AA1+ cells and eosinophils in both groups (PL: P < 0.01, IT: P < 0.05 for both). The between-group difference was significant for eosinophils (P = 0.05). Basophils were observed in the epithelium of six out of 17 in the placebo group and one out of 20 in the IT group (P = 0.03). Neutrophil numbers remained constant in both epithelium and submucosa. CONCLUSION: Successful grass pollen immunotherapy was associated with inhibition of seasonal increases in basophils and eosinophils, but not mast cells or neutrophils within the nasal epithelium. Immunotherapy may act, at least in part, by reducing seasonal recruitment of basophils and eosinophils into the epithelium.

Basophil recruitment and IL-4 production during human allergen-induced late asthma.

BACKGROUND: Basophils represent an important source of inflammatory mediators and cytokines after IgE-dependent activation in human beings. OBJECTIVE: To assess the role of basophils in allergic asthma, we measured the number of basophils in the bronchial mucosa and their capacity to express IL-4 mRNA and protein during allergen-induced late asthmatic responses. METHODS: Fiberoptic bronchoscopic bronchial biopsies were obtained at 24 hours from sites of segmental bronchial allergen challenge and control sites in 19 patients with atopic asthma and 6 nonatopic healthy volunteers. Basophil numbers were assessed by immunohistochemistry through use of mAb 2D7. IL-4 mRNA--positive cells were detected through use of in situ hybridization and colocalized to basophils through use of sequential immunohistochemistry/in situ hybridization. IL-4 protein was detected and colocalized to basophils through use of dual immunohistochemistry. RESULTS: After allergen challenge, there was an increase in the median number of 2D7-positive basophils per square millimeter in the bronchial mucosa in patients with asthma (0.9 cells/mm(2) at baseline to 8.8 cells/mm(2) after challenge; P =.002), which also was significantly higher than what was seen in nonasthmatic controls (P =.01). Similarly, IL-4 mRNA--positive cells were increased at 24 hours in patients with asthma (1.4 to 14) in comparison with controls (0 to 0; P =.02). Colocalization studies revealed that 15% and 41% of the basophil population in patients with asthma after allergen-challenge expressed, respectively, IL-4 mRNA and protein. Conversely, 19% of IL-4 mRNA-positive cells and 72% of IL-4 protein--positive cells were accounted for by basophils. CONCLUSION: After allergen provocation in sensitive patients with atopic asthma, basophils are recruited to the bronchial mucosa and express IL-4 mRNA and protein, which might contribute to local IgE synthesis and/or tissue eosinophilia or other aspects of allergic inflammation during late responses and ongoing asthma.

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.

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.

Serum tryptase and the laboratory diagnosis of systemic mastocytosis.

Total tryptase levels of 20 ng/mL or higher in a baseline serum sample when the ratio of total to beta-tryptase is 20 or greater strongly suggest underlying systemic mastocytosis. Whether these criteria prove to be more sensitive than a bone marrow biopsy will require further study. Although the absolute level of total tryptase does not predict disease severity, it may provide a practical method for assessing the efficacy of therapeutic interventions designed to reduce the mast cell burden.

Increased numbers of both airway basophils and mast cells in sputum after allergen inhalation challenge of atopic asthmatics.

Mast cells and basophils are metachromatic cells that participate in allergic inflammation. Allergen challenge to the airways of atopic asthmatic individuals increases levels of metachromatic cells, which may reflect an increase in mast cells, basophils, or both. We conducted a study to characterize the kinetics of basophil and mast cell recruitment to the airways of atopic asthmatic subjects after allergen inhalation challenge, using monoclonal antibodies specific for each type of cell. Of 19 subjects, 14 developed both early- and late-phase asthmatic responses (dual responders [DRs]), whereas five developed only early asthmatic responses (early responders [ERs]) after allergen inhalation. There was a significant increase in the number of sputum eosinophils (p < 0.002) and basophils (p < 0.002) at 7 h and 24 h after challenge in both ERs and DRs. There was also a significant increase in the number of activated eosinophils (p = 0. 00002) and mast cells (p = 0.009) in sputum at 7 h and 24 h after challenge in DRs, but not in ERs (p > 0.4). DRs had a significantly higher number of allergen-induced sputum basophils than did ERs (p < 0.01), and sputum basophils correlated significantly with airway hyperresponsiveness (AHR) to methacholine at 24 h after challenge (r = 0.66, p = 0.002). DRs tended to have higher allergen-induced basophil levels than did ERs, which may contribute to the observed AHR.

The potential clinical utility of serum alpha-protryptase levels.

BACKGROUND: Because biopsy criteria for diagnosing systemic mastocytosis are not precise, the value of serum alpha-protryptase levels in the work-up of suspected systemic mastocytosis should be considered. OBJECTIVE: A retrospective analysis was performed on subjects with total tryptase serum levels that were high (>/=20 ng/mL), while beta-tryptase serum levels were normal (<1 ng/mL) or modestly elevated (1 to 5 ng/mL). METHODS: Over a 3.5-year period, 52 qualifying specimens were identified from 1369 consecutive samples. The corresponding subjects were divided into those with suspected mastocytosis and those with suspected anaphylaxis. Subjects with suspected mastocytosis were subdivided into 3 subgroups on the basis of biopsy results (positive, negative, or not available). Subjects with suspected anaphylaxis were subdivided into living and deceased subgroups. RESULTS: Among the 15 subjects who underwent biopsy, alpha-protryptase serum levels (the difference between directly-measured levels of serum total tryptase and beta-tryptase), when greater than 75 ng/mL (n = 9), were always associated with a positive biopsy result for systemic mastocytosis; levels from 20 to 75 ng/mL (n = 6) were associated with a positive biopsy result in 50% of subjects. alpha-Protryptase serum levels may be a more sensitive screening test than a bone marrow biopsy for this disorder. Also, elevated alpha-protryptase serum levels in some adult patients return to normal over time, suggesting that mast cell hyperplasia resolved in these patients. Finally, a high alpha-protryptase level may reveal anaphylaxis to be a presenting manifestation of systemic mastocytosis or mast cell hyperplasia. CONCLUSION: Levels of serum alpha-protryptase, relative to those of beta-tryptase, appear to be useful in the diagnostic work-up and follow-up of subjects with suspected systemic mastocytosis.

Immunohistochemical detection of human basophils in late-phase skin reactions.

BACKGROUND: Human basophils are difficult to detect with classic histochemical stains at sites of allergic inflammation. The 2D7 anti-basophil monoclonal antibody was used to identify basophils in skin during the late-phase response to a cutaneous allergen challenge. METHODS: The 2D7 monoclonal antibody was used on protease-digested sections of skin biopsy specimens obtained 6 and 24 hours after an allergen or buffer challenge. The skin chamber technique was used to compare buffer- and allergen-challenged sites at 6 hours, and intradermal injection of allergen was used to compare allergen-challenged sites at 6 and 24 hours. RESULTS: Dramatic increases in the numbers of 2D7+ cells and in tissue staining by 2D7 were observed 6 hours after allergen challenge compared with buffer challenge. Histamine levels in skin chamber fluid varied with 2D7+ cell concentrations. By 24 hours, 2D7+ cells and tissue staining appeared to diminish but were still detectable in the allergen-challenged sites. Basophils localized primarily in and around blood vessels, whereas mast cells remained mostly in the superficial dermis. Mast cells were 2D7- in both the allergen- and buffer-challenged skin. Metachromatic staining of 2D7+ basophils with toluidine blue was absent in these tissue sections. CONCLUSIONS: The 2D7 monoclonal antibody provides a more sensitive and precise marker than histochemical staining for human basophil involvement during the late-phase response to an allergen challenge. Basophil infiltration was observed at 6 hours only after allergen challenge and persisted at similar levels by 24 hours.

Effect of recombinant human IL-4 on tryptase, chymase, and Fc epsilon receptor type I expression in recombinant human stem cell factor-dependent fetal liver-derived human mast cells.

The effect of recombinant human IL-4 (rhIL-4) on the development of recombinant human stem cell factor-dependent fetal liver-derived mast cells was examined. RhIL-4 attenuates the number of mast cells that develop, preferentially affecting the MC(T) type of mast cell. Cellular levels of tryptase and chymase mRNA normalized to that of glyceraldehyde-3-phosphate dehydrogenase were not appreciably affected. Tryptase mRNA levels peaked at least 2 wk before tryptase protein and before chymase mRNA and protein, indicating that tryptase mRNA expression is an early marker of commitment to a mast cell lineage. In contrast, alpha-tryptase and beta-tryptase mRNA levels increased and decreased in parallel. The most dramatic effect of rhIL-4 was to induce expression of functional surface Fc epsilonRI. Expression was maximal by 21 days with 20 ng/ml of rhIL-4 and reached a plateau by 2 ng/ml of rhIL-4 at 4 wk. Fc epsilonRI+ cells increased modestly when myeloma IgE was added to the developing mast cells, but increased synergistically when both myeloma IgE and rhIL-4 were present together. Delayed addition of rhIL-4 progressively diminished Fc epsilonRI expression, as did withdrawal of rhIL-4 during the first 2 wk of culture. RhIL-4 selectively increased Fc epsilonRI alpha mRNA levels at least 10-fold. Mast cells developed in the presence of rhIL-4 released tryptase when exposed to anti-Fc epsilonRI alpha. In conclusion, induction of functional Fc epislonRI on recombinant human stem cell factor-dependent human fetal liver-derived mast cells by rhIL-4 harmonizes with the well-accepted ability of this cytokine to enhance IgE production by B cells.

Recombinant human granulocyte-macrophage colony-stimulating factor (CSF), but not recombinant human granulocyte CSF, down-regulates the recombinant human stem cell factor-dependent differentiation of human fetal liver-derived mast cells.

The effects of recombinant human granulocyte CSF (rhG-CSF) and recombinant human granulocyte-macrophage CSF (rhGM-CSF) on the recombinant human stem cell factor (rhSCF)-dependent development of human mast cells from fetal liver progenitors were examined. Mast cells were identified by immunohistochemical staining for tryptase and by flow cytometric analysis of surface Kit expression. Only rhGM-CSF affected mast cell development. When rhGM-CSF (1, 10, or 100 ng/ml) and rhSCF (50 ng/ml) were added to cell cultures from day 0, both the percentage and absolute numbers of mast cells were diminished after 4 wk compared with cultures exposed to rhSCF alone. Half of the maximal response was achieved at a dose of rhGM-CSF between 0.1 and 1 ng/ml. The Kit+ cells developing in the presence of rhGM-CSF and rhSCF exhibited an intensity of surface Kit expression comparable to that of cells exposed to rhSCF alone. Also, if the initial exposure to rhGM-CSF was delayed for 1 to 3 wk, attenuation of mast cell development waned. These findings are consistent with uncommitted progenitor cells being diverted to nonmast cell lineages by rhGM-CSF, while cells committed to a mast cell lineage, albeit immature, appear to be resistant to the lineage directives of rhGM-CSF. Exposure of fetal liver cells to rhGM-CSF for 1 to 3 days before addition of rhSCF further diminishes the number of mast cells that develop compared with the simultaneous addition of these growth factors on day 0. Whether administration of rhGM-CSF to humans before or together with rhSCF diminishes the mast cell hyperplasia that occurs with rhSCF alone remains to be determined.

Defective expression of early activation genes in cartilage-hair hypoplasia (CHH) with severe combined immunodeficiency (SCID).

Cartilage-hair hypoplasia (CHH) is an autosomal recessive disease of unknown etiology characterized by metaphyseal dysostosis, unpigmented hair, and defective cellular immunity. We studied peripheral blood mononuclear cells (PBMC) of a boy with CHH and combined immunodeficiency in an attempt to characterize further the immune defect in this disease. Stimulation of his PBMC with mitogens was associated with severely depressed IL-2 and interferon-gamma (IFN-gamma) synthesis and IL-2 receptor alpha-chain (IL-2R alpha) expression and resulted in poor lymphocyte proliferation that was only modestly upregulated by the addition of recombinant IL-2 (rIL-2). The defective proliferation and lymphokine synthesis were not corrected by the addition of phorbol myristate acetate (PMA) and ionomycin, agents that bypass receptor-mediated signalling, indicative of a distal abnormality. Importantly, the levels of mRNA encoding c-myc, IL-2R alpha, IL-2 and IFN-gamma were markedly decreased in patient lymphocytes stimulated with PMA+ionomycin as compared to control lymphocytes. The defect in the expression of these early activation genes was selective in that induction by mitogens of mRNA encoding other early activation gene products such as c-fos and c-jun was not impaired. These results suggest that the underlying defect in this patient and perhaps others with CHH may be an abnormality in a component of intracellular signalling pathways or in a trans-acting factor which regulates the expression of a selected number of early activation genes.

Human mast cells derived from fetal liver cells cultured with stem cell factor express a functional CD51/CD61 (alpha v beta 3) integrin.

Human fetal livers contain progenitor cells that become mast cells after 4 weeks of culture with recombinant human stem cell factor. Expression of cell surface CD29 (beta 1), CD18 (beta 2), CD61 (beta 3), and beta 5 integrins was investigated on such cells by flow cytometry and adhesion measurements. High surface expression of CD49e, CD51, and CD61 along with kit was apparent by 4 weeks of culture, whereas expression of each at day 0 was low to undetectable. CD29 and CD49d were detected on cells from day 0 to 4 weeks of culture; CD49b, CD49c, CD49f, CD18, and CD54 expression was negligible. The fetal liver-derived mast cells spontaneously adhered to vitronectin. No evidence for degranulation was found during vitronectin-dependent adhesion. Adhesion occurred in part through the CD61/CD51 receptor. No evidence for adhesion to vitronectin through CD29 and beta 5 integrins was obtained. Almost all of the vitronectin-adherent cells expressed CD51, CD61, kit, and tryptase, and exhibited metachromasia with toluidine blue. Thus, among the fetal liver-derived cells, developing mast cells were selectively adherent to vitronectin. These mast cells and the other cell types present also adhere spontaneously to fibronectin and to laminin, this adhesion being partially inhibited by antibodies against CD61 and CD29 integrins. In conclusion, human mast cells acquire functional vitronectin receptors as they develop from fetal liver progenitors under the influence of rhSCF. This may be important for the recruitment, localization, and retention of developing mast cells.

Immunoelectron microscopic localization of galectin-3, an IgE binding protein, in human mast cells and basophils.

Galectin-3 is an endogenous soluble lectin within the family called galectins that bind beta-galactosides. Homologs of the protein isolated from different sources were previously designated as IgE-binding protein (epsilon BP), CBP35, CPB30, Mac-2, RL-29, RLL, L-29, and HL-29. All are now renamed galectin-3. This lectin is widely distributed in cells and tissues of mice, rats, dogs, hamsters, and humans. Light microscopic immunohistochemistry and ultrastructural immunogold labeling methods were used to determine the distribution of galectin-3 in human mast cells of several organs, in mast cells developed in vitro from human fetal liver cells, and in human peripheral blood basophils. Immunolabeling for the protein was observed in mast cells from all sources and in basophils. The lectin was detected in the nucleus and/or the cytoplasm. The nuclear labeling was over heterochromatin whereas euchromatin was unlabeled. Cytoplasmic labeling was concentrated over secretory granules. The intensity of staining generally was greater in mast cells of skin when compared with that of mast cells in other locations and with that of basophils. Studies have indicated that in mast cells galectin-3 may be involved in promoting their adhesion to basal laminae. In this study the localization of galectin-3 in the secretory granules of human mast cells and basophils suggests that these cells may release this lectin when activated to degranulate.

Quantitation of tryptase, chymase, Fc epsilon RI alpha, and Fc epsilon RI gamma mRNAs in human mast cells and basophils by competitive reverse transcription-polymerase chain reaction.

Competitive reverse transcription-PCR assays developed for human tryptase, chymase, Fc epsilon RI alpha, and Fc epsilon RI gamma mRNA molecules were applied to the HMC-1 leukemic mast cell line, the KU812 leukemic basophil cell line, mast cells dispersed from lung and skin, and peripheral blood basophils. Relative amounts of alpha-tryptase and beta-tryptase mRNA were determined by analysis of BseAI digests of PCR products. Tryptase expression was highest in tissue-derived mast cells, lowest in basophils and KU812 cells, and intermediate in HMC-1 cells. beta-Tryptase mRNA predominated in HMC-1 and KU812 cells; mixtures of alpha- and beta-tryptase were found in tissue mast cells; and alpha-tryptase predominated in basophils. Chymase mRNA was more abundant in skin-derived (nearly all of the MCTC type) than lung-derived (variable amounts of MCTC and MCT cells) mast cells. Small amounts of chymase mRNA were detected in HMC-1 cells; none was found in basophils, in KU812 cells, or in the one preparation of 100% MCT cells derived from lung. Comparable amounts of Fc epsilon RI alpha and Fc epsilon RI gamma mRNA molecules were measured in basophils and tissue-derived mast cells, lesser amounts were detected in KU812 cells, and almost none was detected in HMC-1 cells. Thus, steady state levels of the granule and membrane resident molecules examined in our study are transcriptionally regulated in mast cells and basophils.

Quantitation of inflammatory cells in the nasal mucosa of patients with allergic rhinitis and normal subjects.

BACKGROUND: The role of inflammatory cells at the local site of allergic inflammation in the nose is unclear. METHODS: Nasal biopsy specimens were obtained from 10 patients with symptomatic seasonal allergic rhinitis and 10 normal subjects. Freeze-dried paraffin-embedded sections were stained for mononuclear cells and eosinophils. Tissues in Carnoy's fixative were stained for mast cells. RESULTS: T cells were much more plentiful than B cells or macrophages, and no significant differences were found between the two groups in the number of T cells, T-cell subsets, B cells, and macrophages. However, the number of CD25+ cells (lymphocyte activation markers) and the number of eosinophils were significantly higher in the allergic group than in the control group. There were no significant differences between the two groups in the total mast cell number. However, mucosal type mast cells were slightly increased, and a higher ratio of mast cells were costained for IgE in the allergic group. IgE+ cells mostly constained for mast cell tryptase and did not costain for J chain. CONCLUSIONS: These data suggest that unlike granulocytes, in some mononuclear cells qualitative, not quantitative, changes may be important in allergic rhinitis and that IgE may not be locally produced in the nasal mucosa.

Human mast cell heterogeneity.

Mast cell neutral proteases are the most precise markers of heterogeneity among human mast cells. Two types of human mast cells have been recognized. MCTC cells contain tryptase together with chymase, cathepsin-G like protease, and mast cell carboxypeptidase; MCT cells contain tryptase, but lack the other neutral proteases present in MCTC cells. All mast cells develop from hemopoietic stem cells. In vitro procedures for studying mast cell growth have been developed, using the major human mast cell growth factor, stem cell factor (SCF, also called Kit-ligand). Cultures of hemopoietic progenitor cells in the presence of SCF alone result in selective differentiation to mast cells. The same progenitor cells can be induced to differentiate into other lineages when SCF is used with various lineage-specific colony-stimulating factors such as erythropoietin for erythrocytes. Mast cell development from hematopoietic progenitors may represent a "default pathway," occurring optimally in a permissive microenvironment such as skin, bowel, and lung. The presence or absence of certain cytokines in blood and bone marrow may create a non-permissive environment, thus the absence of granulated mast cells in such locations.

Interleukin-4 inhibits the expression of Kit and tryptase during stem cell factor-dependent development of human mast cells from fetal liver cells.

Although interleukin-4 (IL-4) in mice is known to augment the proliferation of mast cells and to modulate the expression of certain mast cell protease transcripts, its effect on human mast cells is less well understood. The current study examined the effects of recombinant human IL-4 (rhuIL-4) on stem cell factor (SCF)-dependent fetal liver-derived human mast cells in liquid culture. In no case did rhuIL-4 augment proliferation of mast cells. rhuIL-4 selectively inhibited certain aspects of the development of mast cells in cultures of fetal liver cells with rhuSCF. These include lower numbers and percentages of cells expressing tryptase and surface Kit, smaller cells, and lower contents of cells for tryptase, histamine, and Kit. Development of metachromasia was not attenuated. The downregulation of Kit, the surface receptor for SCF, is probably a critical factor, because cells lacking this molecule would not be able to respond to SCF. In contrast to mast cell progenitors, mast cells already developed in vitro from fetal liver cells are relatively resistant to rhuIL-4, but are still dependent for survival on the presence of rhuSCF.