Immunological profiling of key inflammatory drivers of nasal polyp formation and growth in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is a chronic inflammatory condition of the upper airways, often associated with the formation of nasal polyps (CRSwNP). It is well established that macroscopically normal (non-polypoidal) sinonasal mucosa in CRSwNP patients can undergo polypoidal change over time, turning into frank polyps. However, little is known about what drives this process. This study aimed to investigate potential drivers of nasal polyp formation or growth through comparison of the immunological profiles of nasal polyps with contiguous non-polypoidal sinonasal mucosa, from the same patients. METHODS: The immune profiles of three types of tissue were compared; nasal polyps and adjacent non-polypoidal sinonasal mucosa from 10 CRSwNP patients, and sinonasal mucosa from 10 control patients undergoing trans-sphenoidal pituitary surgery. Nasal polyp and control samples were also stimulated with Staphylococcus aureus enterotoxin B (SEB) using a nasal explant model, prior to cytokine analysis. Real time quantitative polymerase chain reaction (IL-5, T-bet, IL-17A, FoxP3, TLR-4, IL-8, IL-1beta and IL-6) and Luminex (IFNgamma, IL-5 and IL-17A) were used to quantify pro-inflammatory responses. RESULTS: Nasal polyps and contiguous non-polypoidal sinonasal mucosa from CRSwNP patients displayed a very similar pro-inflammatory profile. When stimulated with SEB, nasal polyps displayed a Th2/Th17 mediated response when compared to controls. CONCLUSIONS: In CRSwNP, nasal polyps and non-polypoidal sinonasal mucosa from the same patient displayed a similar pro-inflammatory profile skewed towards the Th2/Th17 pathway in nasal polyps following SEB stimulation, with evidence of disordered bacterial clearance. These factors may contribute to enhanced survival of bacteria and development of a chronic inflammatory milieu, potentially driving new polyp formation and recurrence following surgical removal.

Neutrophilia, gelatinase release and microvascular leakage induced by human mast cell tryptase in a mouse model: Lack of a role of protease-activated receptor 2 (PAR2).

BACKGROUND: Tryptase, the most abundant protease of the human mast cell, has been implicated as a key mediator of allergic inflammation that acts through activation of PAR2. OBJECTIVES: To investigate the contribution of PAR2 in the pro-inflammatory actions mediated by tryptase in a mice model. METHODS: We have injected recombinant human ßII-tryptase into the peritoneum of PAR2-deficient and wild-type C57BL/6 mice. After 6, 12 and 24 hours, mice were killed, peritoneal lavage performed and inflammatory changes investigated. RESULTS: Tryptase stimulated an increase in neutrophil numbers in the peritoneum, but responses did not differ between PAR2-deficient and wild-type mice. Heat inactivation of tryptase or pre-incubation with a selective tryptase inhibitor reduced neutrophilia, but neutrophil accumulation was not elicited with a peptide agonist of PAR2 (SLIGRL-NH2 ). Zymography indicated that tryptase stimulated the release of matrix metalloproteinases (MMP) 2 and 9 in the peritoneum of both mouse strains. Studies involving immunomagnetic isolation of neutrophils suggested that neutrophils represent the major cellular source of tryptase-induced MMP2 and MMP9. At 24 hours after tryptase injection, there was increased microvascular leakage as indicated by high levels of albumin in peritoneal lavage fluid, and this appeared to be partially abolished by heat-inactivating tryptase or addition of a protease inhibitor. There was no corresponding increase in levels of histamine or total protein. The extent of tryptase-induced microvascular leakage or gelatinase release into the peritoneum did not differ between PAR2-deficient and wild-type mice. CONCLUSIONS: Our findings indicate that tryptase is a potent stimulus for neutrophil accumulation, MMP release and microvascular leakage. Although these actions required an intact catalytic site, the primary mechanism of tryptase in vivo would appear to involve processes independent of PAR2.

Basophil infiltration in eosinophilic oesophagitis and proton pump inhibitor-responsive oesophageal eosinophilia.

BACKGROUND: The features of proton pump inhibitor-responsive oesophageal eosinophilia (PPI-REE) are similar to those of eosinophilic oesophagitis (EoE), but PPI-REE demonstrates symptomatic and histological responses to PPI therapy. Several studies have shown that basophils play a crucial role in the pathogenesis of allergic diseases. AIM: To identify and compare basophil infiltration in the oesophageal epithelium in patients with EoE, PPI-REE, gastroesophageal reflux disease (GERD) and normal oesophagus (controls). METHODS: Biopsy specimens from 43 patients, including 12 with EoE, 11 with PPI-REE, 10 with GERD and 10 normal oesophagus, were analysed. Immunohistochemistry was performed to quantify the number of basophils and mast cells in the oesophageal epithelium. Double immunofluorescence staining for thymic stromal lymphopoietin (TSLP) and basophils was performed. Patients with EoE were treated with swallowed fluticasone. RESULTS: There were no differences in clinical, endoscopic or histological features between patients with EoE and PPI-REE. There were more basophils and mast cells in patients with EoE and PPI-REE than in patients with GERD and control subjects. Basophil infiltration of the oesophageal epithelium in patients with EoE was higher than that in patients with PPI-REE (3.6 ± 2.8 per high power field vs. 1.2 ± 0.9 per high power field respectively; P = 0.02); however, there was no significant difference in mast cell infiltration between the two groups. TSLP was highly expressed in the oesophageal epithelium in areas infiltrated by basophils. Steroid therapy significantly decreased intraepithelial basophils in patients with EoE. CONCLUSION: Basophils may play an important role in the pathogenesis of eosinophilic oesophagitis.

Alpha-tryptase gene variation is associated with levels of circulating IgE and lung function in asthma.

BACKGROUND: Tryptase, a major secretory product of human mast cells has been implicated as a key mediator of allergic inflammation. Genetic variation in the tryptases is extensive, and α-tryptase, an allelic variant of the more extensively studied ß-tryptase, is absent in substantial numbers of the general population. The degree to which α-tryptase expression may be associated with asthma has not been studied. We have investigated the α-tryptase gene copy number variation and its potential associations with phenotypes of asthma. OBJECTIVES: Caucasian families (n = 341) with at least two asthmatic siblings (n = 1350) were genotyped for the α-tryptase alleles, using high-resolution melting assays. Standards for the possible α-/ß-tryptase ratios were constructed by cloning α-and ß-tryptase PCR products to generate artificial templates. Association analysis of asthma affection status and related phenotypes [total and allergen-specific serum IgE, bronchial hyperresponsiveness to methacholine, forced expiratory volume in 1s (FEV1 ) and atopy and asthma severity scores] was undertaken using family-based association tests (FBAT). RESULTS: Four consistent melting patterns for the α-tryptase genotype were identified with alleles carrying null, one or two copies of the α-tryptase allele. Possessing one copy of α-tryptase was significantly associated with lower serum levels of total and dust mite-specific IgE levels and higher FEV1 measurements, while two copies were related to higher serum concentrations of total and dust mite-specific IgE and greater atopy severity scores. CONCLUSIONS AND CLINICAL RELEVANCE: Associations of α-tryptase copy number with serum IgE levels, atopy scores and bronchial function may reflect roles for tryptases in regulating IgE production and other processes in asthma.

Basophil recruitment and activation in inflammatory skin diseases.

BACKGROUND: Basophils are blood leukocytes constituting less than 1% of leukocytes. They share morphological and functional similarities with mast cells, but recent studies indicate that basophils play non-redundant roles via the release of several cytokines and lipid mediators, as well as functioning as antigen presenting cells. However, basophil infiltration into the tissues in human skin diseases remains to be addressed. METHODS: The infiltration of basophils in 24 skin diseases (136 samples) was immunohistochemically analyzed using basophil-specific BB1 antibody. In addition, activation of blood basophils was examined by assessing CD203c expression with flow cytometry. RESULTS: Basophils were detected in skin lesions of atopic dermatitis, prurigo, urticaria, bullous pemphigoid, drug eruptions, eosinophilic pustular folliculitis, insect bites, scabies, Henoch-Schönlein purpura and dermatomyositis. While cell densities in urticaria, bullous pemphigoid and eosinophilic pustular folliculitis were prominent, much lower numbers of basophils were seen in lesional skin of atopic dermatitis. Basophils were entirely absent in psoriasis vulgaris, mastocytosis, tumoral lesions, systemic sclerosis, and systemic lupus erythematosus. Levels of CD203c expression on blood basophils from prurigo and urticaria patients were higher than those from healthy donors. CONCLUSIONS: Basophils infiltrate into skin lesions more commonly than previously thought, and thus they may play important roles in a variety of inflammatory skin diseases.

Basophils in the giant papillae of chronic allergic keratoconjunctivitis.

AIMS: The essential role of basophils as an initiator of chronic allergic reaction has been elucidated in mouse models. The aim of this present study was to analyse the in situ immunolocalisation of basophils and other relevant inflammatory cells in chronic allergic keratoconjunctivitis. METHODS: Transmission electron microscopic (TEM) analysis was carried out to examine the existence of basophils in the giant papillae obtained from atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC) patients. Cryostat sections of giant papillae were immunostained with basophil-specific antibody BB-1, and with anti-CD4, anti-CD8, anti-CD20, anti-major basic protein (MBP), anti-IgE and anti-FcepsilonRI-beta antibodies. RESULTS: TEM analysis confirmed the existence of basophils in the giant papillae. Small clusters of basophils were observed in the substantia propria of giant papillae, especially at the vicinity of vascular endothelium and subepithelial regions. BB-1-positive basophil clusters were surrounded by T cells, B cells, IgE-positive cells and MBP-positive eosinophils. No BB-1-positive basophils were observed in the control conjunctivae. CONCLUSION: Basophils may infiltrate from either vascular endothelium into the giant papillae. The existence of basophils at the centre of inflammatory cells suggests the role of basophils as an initiator of chronic allergic conjunctivitis.

Practical allergy (PRACTALL) report: risk assessment in anaphylaxis.

Effector mechanisms in anaphylaxis were reviewed. Current approaches to confirmation of the clinical diagnosis were discussed. Improved methods for distinguishing between allergen sensitization (which is common in the general population) and clinical risk of anaphylaxis (which is uncommon) were deliberated. Innovative techniques that will improve risk assessment in anaphylaxis in the future were described.

Polymorphism of the mast cell chymase gene (CMA1) promoter region: lack of association with asthma but association with serum total immunoglobulin E levels in adult atopic dermatitis.

BACKGROUND: Mast cell chymase has the potential to be an important mediator of inflammation and remodelling in the asthmatic lung. Previous studies have examined association between promoter polymorphism of the chymase gene (CMA1) and allergic phenotypes but the significance of this polymorphism is unclear. We have examined association of a CMA1 variant in relation to asthma in a large UK Caucasian family cohort. METHODS: A polymorphism of the CMA1 gene promoter (-1903G/A) was genotyped in 341 asthmatic families and in 184 non-asthmatic adults recruited from the UK PCR-RFLP based genotyping. Association with asthma diagnosis, atopy, specific and total IgE, and atopy and asthma severity was examined. RESULTS: Case-control studies did not reveal a significant difference in allele frequency between asthmatics and controls. A significant association was found between CMA1 genotypes and total IgE levels in subjects with self-reported eczema that remained significant after correction for multiple testing (median total serum IgE GG 297 kU/L, GA 144 kU/L, AA 48.4 kU/L, Pc=0.0032). CONCLUSION: These data suggest that CMA1 promoter polymorphism does not contribute to asthma susceptibility or severity but may be involved in regulating IgE levels in patients with eczema.

Mucosal T-cell phenotypes in persistent atopic and nonatopic rhinitis show an association with mast cells.

BACKGROUND: Allergic rhinitis is characterized by selective expansion of T cell subsets with a CD4+ phenotype. Recently, we identified a subpopulation of nonallergic rhinitis subjects with increased epithelial mast cell and eosinophil populations, suggestive of local mucosal allergy. Previously, T cell subsets have not been characterized in this subselection of nonallergic subjects and furthermore, their relationship to mast cell and basophil effector cells remain unidentified. OBJECTIVE: To determine if a subpopulation of nonallergic subjects with idiopathic rhinitis (IR) have localized allergy confined to their nasal mucosa by comparing the T cell subsets and major histocompatibility complex (MHC) II expressing cells to persistent allergic rhinitis (PAR). Furthermore, the relationship between T cell subsets and mast cells/basophils was investigated. METHODS: None of the symptomatic patients in this study were clinically allergen-challenged. Nasal turbinate mucosa was removed from patients with PAR, IR and normal controls. Morphometry was performed on immunostained sections for T cell subset populations including CD3+, CD4+, CD8+, CD25+, CD45RA+, CD45RO+, human leucocyte antigen (HLA)-DRalpha (MHC class II), mast cell tryptase and for basophils. RESULTS: Subjects with persistent allergic rhinitis differed to normal controls in showing significantly increased numbers of total (CD3+), activated (CD25+) and allergen-naïve (CD45RA+) T lymphocytes in their nasal mucosa (P < 0.025). The naïve CD45RA+ memory T cells correlated to mucosal mast cells in PAR (P = 0.03). IR patients differ to allergic subjects in showing significantly reduced numbers of epithelial HLA-DRalpha+ cells (P = 0.007), but increased numbers of CD8+ lymphocytes (P = 0.02). The CD8+ T cells correlated with mucosal mast cell numbers (P = 0.02). In both rhinitis groups, basophils were present in very low numbers obviating the need for statistical analysis. CONCLUSION: PAR is characterized by increased numbers of CD3+, CD25+ and CD45RA+ T lymphocytes compared with normal mucosa. Allergic and nonallergic rhinitis groups can be separated by significant differences in the number of epithelial antigen presenting cells (APCs) (HLA-DRalpha+) and sub-epithelial activated (CD25+) T cells. Moreover, IR patients do not significantly differ to their allergic counterparts with respect to total (CD3+) and naïve (CD45RA+) T cell numbers, or numbers of epithelial activated (CD25+) lymphocytes. IR subjects show significantly increased numbers of CD8+ lymphocytes compared with control mucosa and although our findings suggest that the initiating inflammatory events may differ, both rhinitis groups show a similarity in pathology involving mucosal mast cells with an association to infiltrating T cells.

Evidence of mast-cell activation in a subset of patients with eosinophilic chronic obstructive pulmonary disease.

Although asthma has been viewed mainly as an eosinophilic disease, and chronic obstructive pulmonary disease (COPD) as a neutrophilic disease, recent studies have shown increased neutrophil counts in severe asthma and sputum eosinophilia in some COPD patients. In an attempt to further characterise these two syndromes according to pathology, the current authors have conducted a study of induced sputum in 15 subjects with COPD, 17 asthmatics, and 17 nonatopic healthy individuals. Sputum was analysed for cytology and levels of eosinophil cationic protein (ECP), albumin, tryptase and soluble intercellular adhesion molecule-1. The COPD subjects differed from the asthmatics as they had higher sputum neutrophil and lower columnar epithelial cell counts, but there were no differences in any soluble marker studied. When compared to control subjects, both the asthmatic and COPD subjects had raised eosinophil counts and ECP levels. In a subset of COPD subjects with sputum eosinophilia (>3% of total cells), significantly increased levels of tryptase were detected. In conclusion, although chronic obstructive pulmonary disease is a more neutrophilic disease than asthma, the two diseases are difficult to distinguish on the basis of sputum levels of the soluble markers traditionally associated with asthma. However, a subset of patients with chronic obstructive pulmonary disease with airway eosinophilia and mast-cell activation might represent a distinct pathological phenotype.

Elevated serum concentrations of beta-tryptase, but not alpha-tryptase, in Sudden Infant Death Syndrome (SIDS). An investigation of anaphylactic mechanisms.

BACKGROUND: Sudden Infant Death Syndrome, (SIDS) or cot death, remains the most common category of post-perinatal death in the UK. By definition, the cause of death is unknown, but a long-standing theory is that some of these deaths could be the result of anaphylaxis. OBJECTIVE: To investigate the potential contribution of anaphylactic mechanisms to deaths in infancy by determining relative levels of alpha- and beta-tryptases and both total and allergen-specific IgE in sera from groups of infants whose deaths were attributed to SIDS or to other causes. METHODS: Serum samples were collected at the time of post-mortem examination from infants whose death was classed as SIDS (n = 40) and from a comparison group in which cause of death had been established (n = 32). Serum tryptase concentrations were measured with a radioimmunoassay with monoclonal antibody G5 which detects primarily beta-tryptase or an ELISA with antibody AA5 which has equal sensitivity for alpha- and beta-tryptases. Levels of total IgE and IgE specific for casein, beta-lactoglobulin, house dust mite and moulds were determined. RESULTS: Analysis of the results of the two assays for tryptase indicated that levels of the beta-like tryptase (the form secreted on anaphylactic degranulation) were significantly higher in serum from infants with SIDS compared with those whose death was explained. There was no evidence for an increase in serum levels of alpha-tryptase (the variant secreted constitutively from mast cells). Total levels of serum IgE did not differ between the two groups and, reflecting the low circulating IgE concentrations in infancy, an elevation in IgE specific for the panel of allergens was not detected. CONCLUSIONS: In a proportion of SIDS victims there may be increased serum levels of beta-like tryptase, a marker for anaphylaxis. The failure to detect an increase in alpha-tryptase would suggest that mast cell hyperplasia is not a feature of cot death. The nature of the inciting agents remains unclear, but anaphylaxis deserves serious consideration as a possible cause of sudden death in infancy.

Segmental bronchoprovocation in allergic rhinitis patients affects mast cell and basophil numbers in nasal and bronchial mucosa.

Mast cells and basophils are cells that play an important role in the initiation and control of allergic inflammation in asthma and rhinitis. This study was undertaken to determine the presence and dynamics of mast cells and basophils in the nasal and bronchial mucosa of allergic rhinitis patients after segmental bronchial provocation (SBP). Eight nonasthmatic, grass pollen-allergic rhinitis patients and eight healthy controls were included. Bronchial and nasal biopsies, as well as blood samples, were taken before (T(0)) and 24 h (T(24)) after SBP. Immunohistochemical staining was performed for mast cells (tryptase and chymase; phenotypes MC(T), MC(TC), MC(C)) and basophils (BB1). In the bronchial mucosa, the number of BB1(+) cells increased significantly (p < 0.05) in allergic rhinitis patients after SBP. In the nasal mucosa, the numbers of MC(C) and MC(TC) cells decreased significantly, whereas the numbers of [BB1(+)] cells increased significantly in allergic rhinitis patients after SBP (p < 0.05). In blood, the number of basophils decreased (p < 0.05) and the level of interleukin (IL)-5 increased (p < 0.05) in atopic patients after SBP. No significant changes could be observed in healthy controls. This study shows that SBP in nonasthmatic allergic rhinitis patients reduces numbers of mast cells in the nose as a result of enhanced degranulation. At the same time, there is evidence for an influx of basophils from the blood into the nasal and bronchial mucosae.

Tryptase and agonists of PAR-2 induce the proliferation of human airway smooth muscle cells.

Airway remodeling with smooth muscle cell (SMC) hyperplasia is a feature of chronic asthma. We investigated the potential for tryptase, the major secretory product of human mast cells, to act as a growth factor for human airway SMCs. Because this serine protease can activate proteinase-activated receptor-2 (PAR-2), we also examined the actions of SLIGKV, a peptide agonist of PAR-2. Incubation with lung tryptase provoked a twofold increase in [(3)H]thymidine incorporation; a similar increase in cell numbers was found when we used the MTS assay. The effect was catalytic site dependent, being abolished by the protease inhibitors leupeptin and benzamidine and by heat inactivation of the enzyme. Tryptase-induced DNA synthesis was inhibited by preincubation of the cells with pertussis toxin, calphostin C, or genistein. Transduction mechanisms are thus likely to involve a pertussis toxin-sensitive G protein, protein kinase C, and tyrosine kinase. SLIGKV elicited a response on SMCs similar to that of tryptase. Tryptase could provide an important stimulus for SMC proliferation in asthmatic airways, by acting on PAR-2.

Mass, charge, and subcellular localization of a unique secretory product identified by the basophil-specific antibody BB1.

BACKGROUND: BB1 is a basophil-specific mAb (Lab Invest 1999;79:27-38). The identity of the corresponding antigen has not been determined, but it gives a granular appearance on staining and is secreted on activation of basophils. OBJECTIVE: We sought to further characterize the basophilspecific antigen identified by BB1. METHODS: Intracellular localization was determined by flow cytometry and by immunogold labeling and electron microscopy. Physical chemical properties were investigated by gel filtration chromatography and preparative isoelectric focusing. RESULTS: In flow cytometry, permeabilization of cells increased immunofluorescence 100-fold, confirming the predominantly intracellular localization of the antigen. It was further localized to the secretory granules by immunoelectron microscopy. Double labeling with a CD63-specific antibody demonstrated selective binding of BB1 to the granule matrix. Gel filtration chromatography indicated that the antigen is secreted as a complex of approximately 5 x 10(6) d, which was well resolved from the 210-kd supramolecular complex containing tryptase. The antigen was degraded by pronase. Isoelectric focusing indicated a highly basic protein with an isoelectric point of 9.6. CONCLUSION: With its granule localization, release on cell activation, and unique properties, the antigen identified by BB1 could be a novel mediator of allergic disease. We propose the name basogranulin for this novel basophil-specific protein.

Chemokine-induced cutaneous inflammatory cell infiltration in a model of Hu-PBMC-SCID mice grafted with human skin.

Recently, certain chemokines and chemokine receptors have been preferentially associated with the selective recruitment in vitro of type 1 T cells, such as IP-10 and its receptor CXCR3, or type 2 T cells such as monocyte-derived chemokine (MDC) and eotaxin and their receptors CCR4 and CCR3. Very few models have provided confirmation of these findings in vivo. Taking advantage of the humanized SCID mouse model grafted with autologous human skin, the ability of the chemokines IP-10, MDC, eotaxin, and RANTES to stimulate cell recruitment was investigated. Intradermal IP-10 injection resulted in an influx of CD4+ T lymphocytes but also surprisingly in the recruitment of dendritic cells. MDC recruited mainly CD8+ T lymphocytes, and had little effect on eosinophils. As predicted, eotaxin was a potent inducer of eosinophil and basophil migration, also recruiting CD4+ T cells. RANTES, a ubiquitous chemokine associated with both type 1 and type 2 profiles, was able to recruit all cell types. CXCR3-positive cells were preferentially recruited by IP-10, whereas CCR3- and CCR4-positive cells were predominantly found after injection of eotaxin and MDC. Thus, in a human environment in vivo, some chemokines have the ability to recruit cells expressing chemokine receptors preferentially expressed on type 1 or type 2 cells. Further investigations revealed that MDC and eotaxin induced the recruitment of type 2, but not type 1, cytokine-producing cells. RANTES, on the other hand, induced the migration of both type 1 and type 2 cytokine-secreting cells, whereas IP-10 did not induce the recruitment of either subtype. These studies provide detailed information on the properties of MDC, eotaxin, IP-10, and RANTES as chemotactic molecules in skin in vivo. The use of the humanized SCID mouse model grafted with human skin is validated as a useful model for the evaluation of chemokine function in the inflammatory reaction, and suggests that therapeutic targeting of certain chemokines might be of interest in diseases associated preferentially with a type 1 or type 2 profile.

The activation of synovial mast cells: modulation of histamine release by tryptase and chymase and their inhibitors.

Mast cells have been implicated as having pivotal roles in arthritis, but little is known of the processes leading to the activation of synovial mast cells or their potential for pharmacological control. We have investigated the ability of tryptase and chymase, and inhibitors of these major mast cell proteases to modulate the activation of mast cells from human synovial tissue. The tryptase inhibitor drug N-(1-hydroxy-2-naphthoyl)-L-arginyl-L-prolinamide hydrochloride (APC366) inhibited immunoglobulin E (IgE)-dependent histamine release in a dose-dependent manner, with about 70% inhibition being achieved at a dose of 300 microM. Histamine release stimulated by calcium ionophore A23187 was also inhibited by this compound. The chymase inhibitor chymostatin inhibited IgE-dependent histamine release by approximately 60% at 1 microg/ml. Tryptase at concentrations of 3.0 microg/ml and greater stimulated histamine release from synovial cells, which was dependent on catalytic activity, whereas chymase had little effect on these cells. The activation of mast cells by tryptase may represent an amplification process in the synovium. The mast cell stabilising properties of inhibitors of tryptase and chymase could be of therapeutic value in arthritis.

Basophil and eosinophil accumulation and mast cell degranulation in the nasal mucosa of patients with hay fever after local allergen provocation.

BACKGROUND: Basophils and mast cells have certain similarities and are believed to be important in upper and lower respiratory allergy. OBJECTIVE: We sought to apply immunohistology to investigate the distribution and numbers of mast cells and basophils in the nasal mucosa after allergen provocation. METHODS: Allergen challenge with grass pollen was performed in 9 patients with seasonal allergic rhinitis out of the hay fever season. Nasal biopsy specimens were taken before and approximately 1 hour, 24 hours, and 1 week after intranasal allergen provocation. We determined relative numbers and their phenotypic characteristics by using mAbs specific for tryptase, chymase, IgE, eosinophils (BMK-13), and a new mAb against basophils (BB1) by using immunohistochemistry in frozen sections. RESULTS: In the nasal mucosa at baseline, practically no basophils were found in the epithelium. A significant increase in numbers was found in the epithelium and lamina propria of the nasal mucosa in the early phase as early as 1 hour after allergen provocation. At 24 hours and 1 week after allergen provocation, a significant increase in basophil numbers was found in the lamina propria only. The proportion of mast cells staining for chymase in the lamina propria decreased from a median of 38% (range, 0%-82%) to 14% (range, 0%-78%) within 1 hour of allergen provocation. The proportion of mast cells staining for chymase increased from 1% (range, 0%-86%) at baseline to 21% (range, 3%-85%) within 1 hour of allergen provocation. One week after provocation, mast cells returned to baseline numbers. A definite tissue eosinophilia was observed after allergen provocation. CONCLUSION: Basophil numbers are increased in the epithelium and lamina propria of the nasal mucosa of subjects with rhinitis after allergen challenge, with influx already apparent at 1 hour. Moreover, changes in mast cell percentages and numbers were observed within 1 hour of allergen provocation.