Histamine is an important mediator in the development of allergic reactions. Only a small subset of human cell types is able to produce histamine. No previous studies have shown that human neutrophils are among them. The present work was undertaken to analyze whether human neutrophils produce histamine, and to determine what agonists are involved in histamine production by human neutrophils. The expression of histidine decarboxylase in human neutrophils was established by quantitative PCR, Western blotting, and flow cytometry analysis. The activity of the enzyme was determined by ELISA, which measured histamine in the culture supernatant of neutrophils stimulated with a set of classical agonists. Human neutrophils are bona fide histamine-producing cells. Neutrophils store ∼0.29 pg/cell and release ∼50% of the histamine content in an antigen-dependent manner and on stimulation with other neutrophil agonists. Basal expression of histidine decarboxylase, the rate-limiting enzyme in histamine production, is higher in neutrophils from patients with allergies than from healthy donors. Our results cannot be ascribed to cell contamination for several reasons. LPS failed to induce histamine release by basophils, whereas it induced histamine release by neutrophils; and we did not detect basophils, monocytes, or lymphocytes in our neutrophil preparations. Eosinophils, albeit detected, were only 0.001-0.004% of the final cell population, and they did not store or release histamine on antigen or LPS stimulation. Antigens to which patients with allergies were sensitized stimulated release of histamine from neutrophils. These observations represent a novel view of neutrophils as possible source of histamine in the allergic diseases.

These data demonstrate that the exposure of neutrophils to allergens leads to generation of MMP-9, which may then lead to remodelling in asthma.

Introduction Neutrophils have been involved in sepsis-induced organ damage. Neutrophils could be directly activated by TLR binding ligands including LPS. IRAK-1 is one of many intracellular proteins that are activated upon stimulation of TL receptors. This triggers a series of events that results in the migration of NF-κB into the nucleus and the activation NF-κB-dependent genes. Objectives To identify a single nucleotide polymorphism at position 532 (coding SNP) in volunteers and patients with sepsis. To determine whether IRAK-1 SNP532 results in a decrease in neutrophil NF-κB activation in volunteers and patients with sepsis. To evaluate neutrophil gene expression patterns in IRAK-1 SNP532 and wildtype patients with sepsis. Methods Thirty severe sepsis patients and 34 healthy volunteers were enrolled in this study. Peripheral blood was obtained and neutrophils were isolated by plasma-percoll gradients after dextran sedimentation of erythrocytes. Neutrophils from volunteers were resuspended in RPMI and cultured with or without 100 ng/ml LPS for 60 min. The electrophoretic mobility shift assay technique was used to measure the NF-κB activation. Real-time PCR allelic discrimination assays were developed by the assay-by-design service offered by Applied Biosystems (Foster City, CA, USA). Probe and primer combinations were designed at the single nucleotide polymorphism 532. PCR reactions were performed according to the manufacturer's manual using the Applied Biosystems 7500 Real-Time PCR system. Microarray analysis was used to evaluate the neutrophil gene expression in unstimulated neutrophils and after LPS stimulus. Results The median AUC for NF-κB activation was higher in wildtype genotyped neutrophils as compared with IRAK-1 SNP532 genotyped neutrophils (85.2 vs 100.5, P = 0.05) (Fig. 1). In terms of kinetics pattern, we found some differences on nuclear levels of NF-κB in neutrophils from volunteers cultured with LPS. At 30 min after LPS, the culture nuclear translocation of NK-κB was significantly greater in wildtype genotyped neutrophils than in IRAK-1 SNP532 genotyped neutrophils. Even after 60 min, the NF-κB translocation remained high in wildtype genotyped neutrophils, while in IRAK-1 SNP532 genotyped neutrophils the NF-κB translocation was similar to baseline (Fig. 2). In unstimulated neutrophils from septic patients, the NF-κB translocation was significantly lower in IRAK-1 SNP532 genotyped neutrophils than in wildtype genotyped neutrophils (1.20 vs 2.10, P = 0.05) (Fig. 3). Finally, the expression of some inflammatory related genes (IL-8, IL1β, MIP-2, COX-2, and SOD2) was decreased in IRAK-1 SNP532 genotyped neutrophils. Conclusion IRAK-1 SNP532 genotyped neutrophils from volunteers (after LPS ex vivo challenge) and from septic patients are associated with lower NF-κB activation and lower expression of some IRAK1-related genes. These results demonstrate that IRAK1 Figure 2 Mut 0 WT 0 Mut 30 WT 30 Mut 60 WT 60 0.0 2.5 5.0

CD66b is a member of the carcinoembryonic antigen family, which mediates the adhesion between neutrophils and to endothelial cells. Allergen-specific immunotherapy is widely used to treat allergic diseases, and the molecular mechanisms underlying this therapy are poorly understood. The present work was undertaken to analyze A) the in vitro effect of allergens and immunotherapy on cell-surface CD66b expression of neutrophils from patients with allergic asthma and rhinitis and B) the in vivo effect of immunotherapy on cell-surface CD66b expression of neutrophils from nasal lavage fluid during the spring season. Myeloperoxidase expression and activity was also analyzed in nasal lavage fluid as a general marker of neutrophil activation.ResultsCD66b cell-surface expression is upregulated in vitro in response to allergens, and significantly reduced by immunotherapy (p<0.001). Myeloperoxidase activity in nasal lavage fluid was also significantly reduced by immunotherapy, as were the neutrophil cell-surface expression of CD66b and myeloperoxidase (p<0.001). Interestingly, CD66b expression was higher in neutrophils from nasal lavage fluid than those from peripheral blood, and immunotherapy reduced the number of CD66+MPO+ cells in nasal lavage fluid. Thus, immunotherapy positive effects might, at least in part, be mediated by the negative regulation of the CD66b and myeloperoxidase activity in human neutrophils.

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