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.

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.

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.

Mast cell tryptase stimulates the synthesis of type I collagen in human lung fibroblasts.

Mast cell activation is a characteristic feature of chronic inflammation, a condition that may lead to fibrosis as a result of increased collagen synthesis by fibroblasts. We have investigated the potential of tryptase, the major protease of human mast cells, to stimulate collagen synthesis in the human lung fibroblast cell line MRC-5. Tryptase was isolated from human lung tissue by ion-exchange and affinity chromatography. At concentrations of 18 and 36 mU/ml, tryptase stimulated both an increase in cell numbers, and a fivefold increase in DNA synthesis as determined by methyl-[3H]thymidine incorporation. Similar concentrations of tryptase resulted in a 2.5-fold increase in collagen synthesis as determined both by incorporation of [3H]proline into collagen, and by assay of hydroxyproline concentrations in the supernatants. There was also a twofold increase in collagenolytic activity in the culture medium after tryptase treatment, indicating that the increase in collagen synthesis was not a consequence of decreased collagenase production. All of these actions of tryptase were reduced in the presence of the protease inhibitors leupeptin and benzamidine hydrochloride, indicating a requirement for an active catalytic site. SDS-PAGE and autoradiographic analysis of the [3H]collagen produced by the cells revealed it to be predominantly type I collagen. Our findings suggest that the release of tryptase from activated mast cells may provide a signal for abnormal fibrosis in inflammatory disease.

Regulation of the activity of human chymase during storage and release from mast cells: the contributions of inorganic cations, pH, heparin and histamine.

Chymase, the major chymotryptic proteinase of human mast cells, can be released in substantial quantities following mast cell activation. As this enzyme is stored in the secretory granules in its fully active form, we have investigated various factors which might regulate its activity in storage and upon release. Chymase was purified from human skin by high salt extraction, cetylpyridinium chloride precipitation, heparin agarose affinity chromatography and gel filtration. Neither the addition of Mg2+ or Ca2+ (0.3-10 mM) nor their sequestration by EDTA had any effect on the rate of cleavage of the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Monovalent cations (Na+,K+) enhanced enzyme activity, but only at non-physiological concentrations (0.5-3.0 M), suggesting an ionic strength effect. At constant I = 0.15, enzyme activity was strongly pH-dependent: at pH 5.5 (the approximate pH of the mast cell granule) the activity was only 10% of that at pH 7.5 (the approximate pH of the extracellular space). Heparin, which is stored with chymase in the mast cell granule, accentuated this difference by enhancing activity at pH 7.5 by 33% and depressing it a pH 5.5 by 40%. Histamine at concentrations up to 50 mM (I = 0.15) had little effect on chymase activity at either pH, although high concentrations did attenuate the actions of heparin. It is concluded that pH and the interaction with heparin are central to the regulation of chymase activity within the granule and following release.

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.

Human mast cell chymase induces the accumulation of neutrophils, eosinophils and other inflammatory cells in vivo.

The roles of chymase in acute allergic responses are not clear, despite the relative abundance of this serine proteinase in the secretory granules of human mast cells. We have isolated chymase to high purity from human skin tissue by heparin-agarose affinity chromatography and Sephacryl S-200 gel filtration procedures, and have investigated the ability of human mast cell chymase to stimulate cell accumulation following injection into laboratory animals. Injection of chymase provoked marked neutrophilia and eosinophilia in the skin of Dunkin Hartley guinea-pigs. Compared with saline injected control animals, there were some 60 fold more neutrophils and 12 fold more eosinophils present at the injection site. Following injection of chymase into the peritoneum of BALB/c mice, there were up to 700 fold more neutrophils. 21 fold more eosinophils, 19 fold more lymphocytes and 7 fold more macrophages recovered than from saline injected controls at 16 h. Doses of chymase as low as 5 ng (1.7 x 10(-13) mole) stimulated an inflammatory infiltrate, and significant neutrophilia was elicited within 3 h. The chymase induced cell accumulation in both the guinea-pig and mouse models was dependent on an intact catalytic site, being reduced by co-injection of proteinase inhibitors or heat inactivation of the enzyme. Co-injection of histamine or heparin significantly reduced the chymase induced neutrophil accumulation, whereas neither histamine nor heparin by themselves had any effect on the accumulation of nucleated cells. No synergistic or antagonist interactions between chymase and tryptase were observed when these two major mast cell proteinases were co-injected into the mouse peritoneum. Our findings suggest that chymase may provide an potent stimulus for inflammatory cell recruitment following mast cell activation.

Guinea pig lung tryptase. Localisation to mast cells and characterisation of the partially purified enzyme.

Tryptase (EC 3.4.21.59), the major secretory product of human mast cells, has become widely used as a biochemical marker for mast cells and mast cell activation, and is attracting attention as a mediator of allergic disease. However, there is little information available on the properties, or even the presence, of this protease in commonly used species of laboratory animals. We, here, report the demonstration and characterisation of this enzyme in the guinea pig lung. Tryptic activity resistant to alpha 1-proteinase inhibitor and soybean trypsin inhibitor was detected in sections of guinea pig lung tissue with the histochemical substrate Z-Gly-Pro-Arg-MNA. It was localised to mast cells and appeared to be present in all mast cells staining with Alcian Blue. A tryptic protease was purified 2400-fold from whole lung tissue by high salt extraction, cetylpyridinium chloride precipitation, heparin agarose chromatography, and gel filtration. This enzyme was found to be multimeric with a subunit of 38 kDa and a native molecular mass of 860 +/- 100 kDa. Inhibitor studies identified it as a serine protease. Like human tryptase, it was inhibited by leupeptin, benzamidine, and APC 366 (N-(1-hydroxy-2- naphthoyl)-L-arginyl(-L-prolinamide hydrochloride), but not by alpha 1-proteinase inhibitor, soybean trypsin inhibitor, or antithrombin III. Its response to changes in pH and ionic strength was similar to that of human tryptase. Differences between the guinea pig and human enzymes were seen in activity toward a panel fo 10 tryptic p_nitroanilide peptide substrates. Kinetic constants were determined for two of these: with L-Pyr-Pro-Arg-pNA the guinea pig tryptase had a similar Km but a 5-fold lower kcat than human tryptase, and with L-Pyr-Gly-Arg-pNA the guinea pig enzyme had a 10-fold lower Km and a 30% greater kcat than human counterpart. Heparin stabilised guinea pig tryptase, but did not alter its kinetic parameters as it did with human tryptase, decreasing the Km towards both substrates. The presence of a protease with similarities to human tryptase in the mast cells of guinea pigs suggests that this species may be an appropriate model to investigate the actions to tryptase in vivo, provided cognizance is taken of the differences that do exist.

Human mast cell tryptase attenuates the vasodilator activity of calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP) is localized in and released from sensory nerves. It is a potent and long acting vasodilator which has been suggested to play a role in the control of blood flow. Using HPLC and trichloroacetic acid precipitation techniques, we have examined the ability of human mast cell lysates and a purified preparation of mast cell tryptase to degrade CGRP. We found that CGRP is effectively cleaved by tryptase (Km = 6.8 x 10(-6) mol/L at 37 degrees). Enzymatic activity was inhibited by antipain, leupeptin, N-alpha-p-tosyl-L-lysine chloromethyl ketone, benzamidine or aprotinin, but not by soybean trypsin inhibitor or N-tosyl-L-phenylalanine chloromethyl ketone. The degradation of CGRP by lysates of purified skin mast cells showed a similar pattern of inhibition suggesting that tryptase may be the major enzyme involved. The activity of tryptase was not affected by the presence of heparin. Incubation of CGRP with tryptase resulted in a loss of its vasodilator activity as observed by intravital microscopy of the hamster cheek pouch microvasculature. CGRP preincubated with tryptase failed to relax arterioles when added topically. It is suggested that the catalysis of CGRP by tryptase could represent an important means by which the activity of this neuropeptide is regulated in vivo.

The origin and specificity of intrathecal IgG in chronic relapsing experimental allergic encephalomyelitis.

The source of IgG in the cerebrospinal fluid (CSF) in guinea pigs with chronic relapsing experimental allergic encephalomyelitis (CR-EAE) was investigated using quotient analysis of total IgG and albumin concentrations and by computing CSF-plasma ratios of specific IgG concentrations. Increased blood-CSF barrier (B-CSFB) permeability was shown by elevated albumin quotients in both relapse and remission phases of CR-EAE and intrathecal production of IgG was indicated by raised ratios of IgG to albumin in the CSF. Intrathecal IgG synthesis was greatest in guinea pigs which had little B-CSFB damage. When enzyme-linked immunosorbent assays (ELISA) for whole cord, myelin basic protein (MBP) or Mycobacterium tuberculosis were performed with CSF and plasma adjusted to the concentration of total IgG, the CSF/plasma ratios of ELISA results for specific antibodies were less then unity and ratios for whole cord and MBP were lower than those for M. tuberculosis. There was thus no evidence for a selective increase in the CSF of antibody specific either for the neuroantigens tested or for adjuvant components. The CSF-plasma ratios for each specific antibody were inversely correlated with the extent of total IgG intrathecal synthesis, suggesting that much of the antibody production within the CNS is the result of polyclonal B cell activation.

Fibroblast-Derived Factors Induce Different Mast Cell Characteristics in Human Myeloid Cell Lines and Peripheral Monocytes.

Mast cell differentiation markers were studied by culturing monocytic (U937, THP1) a promyelotic (HL60) and a human mast cell line (HMC1) and peripheral human blood monocytes with fibroblast supernatants and feeder layers. Mast cell differentiation occurred on the basis of metachromasia, chloroacetate esterase, expression of mast cell-specific antigens (APAAP) and production of tryptase in all cell types, but to varying degrees and not at all with a FcεRI marker in monocytic lines. Direct cellular contact is thus not necessary for induction of mast cell differentiation by fibroblasts.

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.

Human mast cell tryptase: a stimulus of microvascular leakage and mast cell activation.

We have investigated the potential of tryptase to stimulate an increase in microvascular permeability following injection into the skin of guinea pigs. Tryptase was isolated from high salt extracts of human lung tissue by octyl-agarose and heparin-agarose chromatography. Injection of purified tryptase (2.5 ng-2.5 microg/site) into the skin of guinea pigs which had been injected intravenously with Evans blue dye provoked a dose-dependent increase in microvascular permeability. The skin reactions elicited by tryptase were apparent up to 80 min following injection, while histamine-induced microvascular leakage resolved completely by 40 min. Heat-inactivation of tryptase, or preincubating the proteinase with certain proteinase inhibitors, significantly reduced the extent of microvascular leakage, suggesting dependency on an intact catalytic site. No evidence was found for a synergistic or antagonistic interaction between tryptase (2.5 ng-2.5 microg/site) and histamine (1-10 microg/site) when these mast cell products were injected together. Addition of heparin to tryptase (10:1; w/w) prior to injection was without effect on tryptase-induced microvascular leakage. Pretreatment of guinea pigs with a combination of the histamine H1 receptor antagonist pyrilamine and the histamine H2 receptor antagonist cimetidine (both 10 mg/kg), partially abolished tryptase-induced microvascular leakage as well as attenuating the reaction to histamine. Reasoning that the microvascular leakage induced by tryptase is likely to involve the release of histamine, we investigated the ability of tryptase to stimulate histamine release from dispersed guinea-pig skin and lung cells in vitro. Tryptase was found to induce concentration-dependent histamine release from both sources of tissue. Mast cell activation stimulated by tryptase in vitro was inhibited by heat treating the enzyme or by addition of proteinase inhibitors, suggesting a requirement for an intact catalytic site. Histamine release was inhibited also by preincubating cells with the metabolic inhibitors antimycin A and 2-deoxy-D-glucose indicating that the mechanism was energy-requiring and non-cytotoxic. We conclude that human mast cell tryptase may be a potent stimulus of microvascular leakage. The activation of mast cells by this proteinase may represent an amplification process in allergic inflammation.

The induction of a prolonged increase in microvascular permeability by human mast cell chymase.

Chymase is a major constituent of the secretory granules of human mast cells, but little is known of the contribution of this serine proteinase in acute allergic reactions. We have purified chymase from human skin tissue, and have investigated its potential to induce microvascular leakage in vivo. Injection of chymase into the skin of guinea pigs provoked an increase in microvascular leakage within 20 min. Although skin reactions were smaller than those elicited with similar quantities of histamine at this time point, they were much longer-lived, and were still apparent 120 min following injection. Chymase induced microvascular leakage was reduced in the presence of soybean trypsin inhibitor, and abolished by heat inactivating the enzyme, indicating dependence on an intact catalytic site. Little evidence was found for synergistic interactions between chymase and either histamine or tryptase. Antihistamine pretreatment of animals did not reduce the magnitude of skin reactions to chymase suggesting that they were not mediated by histamine release. Chymase could contribute to increases in microvascular permeability following mast cell degranulation in allergic disease.

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.

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.