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Airway diseases often feature persistent neutrophilic inflammation and infection. In cystic fibrosis bronchitis, for example, Pseudomonas aeruginosa is isolated frequently. Previously, this laboratory revealed that neutrophils become major sources of histamine in mice with tracheobronchitis caused by the wall-less bacterium Mycoplasma pulmonis. To test the hypothesis that more-broadly pathogenic P. aeruginosa (which expresses cell wall-associated LPS and novel toxins) has similar effects, we incubated naïve mouse neutrophils with two strains of P. aeruginosa. Strain PAO1 greatly increased neutrophil histamine content and secretion, whereas strain PA103 depressed histamine production by killing neutrophils. The histamine-stimulating capacity of PAO1, but not PA103-mediated toxicity, persisted in heat-killed organisms. In PAO1-infected mice, lung and neutrophil histamine content increased. However, PAO1 did not alter production by mast cells (classical histamine reservoirs), which also resisted PA103 toxicity. To explore mechanisms of neutrophil-selective induction, we measured changes in mRNA encoding histidine decarboxylase (rate-limiting for histamine synthesis), probed involvement of endotoxin-TLR pathways in Myd88-deficient neutrophils, and examined contributions of pyocyanin and exotoxins. Results revealed that PAO1 increased histamine production by up-regulating histidine decarboxylase mRNA via pathways largely independent of TLR, pyocyanin, and type III secretion system exotoxins. PAO1 also increased histidine decarboxylase mRNA in neutrophils purified from infected lung. Stimulation required direct contact with neutrophils and was blocked by phagocytosis inhibitor cytochalasin D. In summary, Pseudomonas-augmented histamine production by neutrophils is strain-dependent in vitro and likely mediated by up-regulation of histidine decarboxylase. These findings raise the possibility that Pseudomonas-stimulated neutrophils can enhance airway inflammation by producing histamine.

The immune system and the commensal bacteria in the intestine, which together form the intestinal symbiotic system, greatly contribute to regulation of allergy. Of the various types of cells constituting the intestinal immune system, this review focuses on epithelial cells and mast cells and the interaction of these cells with commensals. Mast cells express the high affinity IgE receptor FcepsilonRI which is essential to the induction of allergic inflammatory reactions. The molecular mechanisms of transcriptional regulation of genes encoding FcepsilonRI have been clarified. On the other hand, the expression of the molecules involved in microbe recognition is regulated in a specific manner in intestinal epithelial cells, which are continuously exposed to the commensals inhabiting the intestinal lumen, to prevent excessive inflammatory reactions. Microbial components directly regulate the functions of mast cells through Toll-like receptors. These aspects provide targets for the regulation of allergy based on the maintenance of the intestinal symbiotic system.

ANG is a plasma protein with angiogenic and ribonucleolytic activity implicated in tumor growth, heart failure, wound healing, asthma, and the composition of the adult gut microflora. Human mast cells (HuMC) are similarly associated with modulation of vascular permeability, angiogenic processes, wound healing, and asthma. We hypothesized that HuMC express and secrete ANG in response to divergent stimuli. ANG expression was evaluated in the LAD2 HMC, the HMC-1, and CD34+-derived HuMC, following exposure to live Escherichia coli, TLR ligands, or neuropeptides and following FcepsilonRI aggregation. Expression and production of ANG were determined by microarray analysis, qRT-PCR, confocal microscopy, and ELISA. Microarray analysis showed that ANG is up-regulated by LAD2 cells exposed to live E. coli. qRT-PCR analysis revealed that LAD2, HMC-1, and HuMC constitutively expressed ANG mRNA and that it was up-regulated by exposure to E. coli. Activation of HuMC by FcepsilonRI aggregation resulted in release of small amounts of ANG (<100 pg/mL), whereas compound 48/80, NGF, LPS, PGN, and flagellin activated HuMC to secrete >160 pg/mL ANG. These observations demonstrate that HuMC store and secrete ANG to a variety of stimuli and suggest that MC-derived ANG is available in the subsequent inflammatory response.

Background: Pulmonary colonization by Aspergillus fumigatus in chronic lung disease is associated with progressive decline in lung function even in the absence of specific allergic response. We hypothesized that A. fumigatus contributes to this decline by inducing pulmonary mast cell degranulation even in the absence of antigen-specific immunoglobulin E (IgE). Therefore, we investigated whether A. fumigatus can induce mast cell degranulation independently of IgE., Methods: We studied the interactions of Aspergillus species with mast cells in the absence of IgE in vitro with use of scanning electron microscopy. The extent of mast cell degranulation was quantified by measuring the release of beta-hexosaminidase., Results: Mature A. fumigatus hyphae induced mast cell degranulation in the absence of IgE. Hyphae of Aspergillus flavus, Aspergillus niger, and Aspergillus nidulans induced much less mast cell degranulation. Mast cell degranulation required direct contact with mature A. fumigatus hyphae, and was not induced by conidia or immature hyphae. Killed hyphae induced significant degranulation, whereas live hyphae from mutants deficient in the fungal development regulators StuA and MedA induced very little degranulation., Conclusions: Factors expressed on the surface of mature A. fumigatus hyphae that are controlled by StuA and MedA induce mast cell degranulation in the absence of IgE.

Stimulated mast cells release a variety of chemotactic factors such as tumor necrosis factor alpha (TNF-alpha) and leukotriene B4. Recent studies have shown that mast cell-derived TNF-alpha plays a critical role in host defense against Gram negative bacterial infections by the recruitment of neutrophils to the sites of infection. In the present study, we sought to investigate if mast cells release leukotriene (LT) B4 in response to bacteria and, if so, to establish its in vivo relevance. We show that mast cells release significant amounts of LTB4 and LTC4 in response to exposure to FimH-expressing type 1 fimbriated Escherichia coli in vitro. To test the functional significance of mast cell-derived LTs during an E. coli infection in vivo, we examined the effect of a LT-synthesis inhibitor, A-63162, on bacterial clearance and neutrophil influx in an infectious peritonitis model in mast cell-deficient mice (WBB6F1-W/WV) and their normal congenic control (WBB6F1-+/+) mice. Our results show that a treatment with A-63162 reduced neutrophil influx and bacterial clearance in the peritoneal cavities of mast cell-sufficient but not -deficient mice. Thus, mast cell-derived LTs contribute to host defense by mediating early neutrophil influx and bacterial clearance at sites of infection.

Rodent mast cells (MC) play critical roles in host defense against bacterial infection. However, bacteria-mediated signaling mechanisms in MC have not been studied. In addition, the response of human MC to bacteria is not fully investigated. This study examined the interaction between human MC and type 1 fimbriated Escherichia coli and the mechanisms involved using the human MC line HMC-1 5C6 and human cord blood-derived MC. These MC internalized significant numbers of FimH+ E. coli, but not its isogenic FimH- mutant. In HMC-1 cells, bacterial internalization was stimulated by protein kinase C (PKC) activation [short-term phorbol myristate acetate (PMA) treatment] and dramatically decreased by PKC inhibitors or PKC depletion (long-term PMA treatment). Moreover, bacterial internalization was accompanied by significant expression of PKCbeta1 and delta. Fluorescence microscopy demonstrated accumulation of PKCbeta1 on internalized bacteria. These data indicate that human MC has the capacity to internalize bacteria and PKC may be a critical intracellular mediator of this function.