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Background: The basophil histamine release (HR) assay can be used for allergy diagnosis in addition to the conventional measurement of allergen-specific IgE (sIgE). Passive sensitization of basophils increases the versatility and allows testing the biological relevance of allergen-induced IgE cross-linking in any serum unbiased by the cellular component. However, not all the patient sera perform equally well and we hypothesized that the absolute level and fraction of sIgE affect the performance. Choosing birch pollen allergy as a model, we investigated the concentration of sIgE needed for successful passive sensitization using soluble- or matrix-fixed Bet v 1.Methods: Twenty-eight sera with Bet v 1 sIgE [7 sera within each allergy class (1: 0.1-0.70 kUA/L, 2: 0.71-3.50 kUA/L, 3: 3.51-17.50 kUA/L, and 4+: >17.50 kUA/L)] and a negative control serum pool were used to passively sensitize donor basophils, obtained from buffy coat blood ( n = 3). The cells were incubated (30 min) with a soluble allergen (rBet v 1 from 0.2 to 50 ng/ml), matrix-fixed allergen (ImmunoCAP™ containing recombinant Bet v 1), or phorbol 12-myristate 13-acetate (PMA)/ionomycin mixture (maximal HR) and released histamine was quantified fluorometrically. Results: The lowest level of Bet v 1 sIgE generating a detectable HR (HR > 10% of maximal release) in all the 3 runs was found to be 1.25 kUA/L (corresponding to allergy class 2, 0.71-3.50 kUA/L). Furthermore, sera from allergy classes 3 and 4+ ascertained a significant reproducible HR: 42/42 vs. 5/21 in allergy class 1 and 15/21 in allergy class 2. Using ImmunoCAP™s containing Bet v 1 as a matrix-fixed allergen system, similar results were obtained where the lowest sIgE concentration mediating an HR was 1.68 kUA/L and 7/7 for both allergy classes 3 and 4+.Conclusion: The results demonstrate that the IgE titer is strikingly robust in predicting the ability to sensitize basophils and produce a measurable HR.

Different protocols exist for in vitro development of HuMCs from hematopoietic stem cells, which results in distinct mast cells regarding molecular markers and activation patterns. Here, we introduce a SR profile using immunological, neurogenic, and pharmacological stimuli to characterize cellular functionality. Mast cells were obtained from three culture protocols using two types of PBdMCs (CD34(+) PBdMC or CD133(+) PBdMC) and one type of CBdMC (CD133(+) CBdMC). We analyzed resting cells for specific mast cell markers at protein and mRNA levels, thereby creating a molecular profile. To characterize the SR profile, we stimulated cells with anti-IgE, C3a, C5a, Substance P, or Compound 48/80 and measured the release of histamine and cytokines (IL-10, IL-13, GM-CSF, TNF-α). Molecular profiling revealed that CD133(+) CBdMC expressed less chymase, FcεRIα, and CD203c but more CD117 compared with CD34(+) and CD133(+) PBdMC. The SR profile for histamine release illustrated a functional heterogeneity between PBdMC and CBdMC. PBdMC released >10% histamine upon stimulation with anti-IgE, C3a, Substance P, and Compound 48/80, whereas CBdMC only reacted to C3a. Cytokine secretion was only detected after anti-IgE stimulation. Here, the SR profile identified the CD133(+) PBdMC as the most active cells regarding secretion of IL-10, IL-13, GM-CSF, and TNF-α. Cells from all three culture protocols, however, produced IL-10 spontaneously at comparable levels. We recommend validating mast cell cultures by means of molecular and SR profiles to characterize the mast cells and enhance consensus among studies.

BACKGROUND. IgE-mediated activation of mast cells has been reported to induce the release of tumour necrosis alpha (TNF-?), which may display autocrine effects on these cells by inducing the generation of the tissue remodelling protease matrix metalloproteinase-9 (MMP-9). While mast cells and basophils have been shown to express complementary and partially overlapping roles, it is not clear whether a similar IgE/TNF-?/MMP-9 axis exists in the human basophil. The purpose of this study was thus to investigate whether IgE-mediated activation of human basophils induces TNF-? and MMP-9 release. METHODS. Human peripheral blood mononuclear cells (PBMC), isolated basophils and monocytes were stimulated up to 21 h with anti-IgE. Mediator releases were assessed by ELISA, and surface expressions of mediators were detected by flow cytometry. Upregulation of cytokine production was detected by Western blot and polymerase chain reaction (PCR). RESULTS. IgE-mediated activation of basophils induced the synthesis and release of both TNF-? and MMP-9 from PBMC. In contrast, IgE-mediated activation of purified basophils induced the release and cellular expression of TNF-? but not MMP-9. Isolated monocytes did not release MMP-9 upon anti-IgE stimulation, but MMP-9 release was induced by stimulating monocytes with supernatants from activated basophils, and this release was inhibited by anti-TNF-? neutralizing antibodies. CONCLUSION. Our results strongly indicate that human basophils release TNF-? following IgE-dependent activation and that this cytokine subsequently stimulates MMP-9 release from monocytes. These findings support a direct involvement of basophils in inflammation as well as suggesting a role for the basophil in tissue remodelling.

Udgivelsesdato: 2009-Mar-6, BACKGROUND: Animal models have shown histamine to be released from the skin during the acute phase of a burn injury. The role of histamine during the early phase of thermal injuries in humans remains unclear. PURPOSE: The objectives of this trial were to study histamine release in human skin during the acute phase of a standardized thermal injury in healthy volunteers. METHODS: Histamine concentrations in human skin were measured by skin microdialysis technique. Microdialysis fibers were inserted into the dermis in the lower leg in male healthy volunteers. A standardized superficial thermal injury was elicited by a heating thermode (49 degrees C) applied to the skin for 5 min. Histamine in dialysate was analyzed for up to 2 h after the injury using two different analytical methods. RESULTS: Spectrofluorometric assay of histamine showed no histamine release in separate studies using 2-min samples over 20 min (n = 6) and 5-10-min samples over 120 min (n = 8). The histamine values were at the limits of the quantification limit of the spectrofluorometric assay. Confirmatory studies using a sensitive radioimmunoassay confirmed no histamine release within the first hour of a thermal injury (baseline 11.6 +/- 1.8 nM vs. post-burn values of 14.8 +/- 1.8 nM, n = 8). CONCLUSIONS: Histamine is not released in human skin during the acute phase of a thermal injury.

Udgivelsesdato: 2003-Aug-15, We report that CCR3 is not expressed on freshly isolated peripheral and germinal B cells, but is up-regulated after stimulation with IL-2 and IL-4 (approximately 98% CCR3(+)). Ligation of CCR3 by eotaxin/chemokine ligand (CCL) 11 induces apoptosis in IL-2- and IL-4-stimulated primary CD19(+) (approximately 40% apoptotic cells) B cell cultures as well as B cell lines, but has no effect on chemotaxis or cell adhesion. Freshly isolated B cells express low levels of CD95 and CD95 ligand (CD95L) (19 and 21%, respectively). Expression is up-regulated on culture in the presence of a combination of IL-2, IL-4, and eotaxin/CCL11 (88% CD95 and 84% CD95L). We therefore propose that ligation of such newly induced CCR3 on peripheral and germinal B cells by eotaxin/CCL11 leads to the enhanced levels of CD95 and CD95L expression. Ligation of CD95 by its CD95L expressed on neigboring B cells triggers relevant death signaling pathways, which include an increase in levels of Bcl-2 expression, its functional activity, and the release of cytochrome c from the mitochondria into the cytosol. These events initiate a cascade of enzymatic processes of the caspase family, culminating in programmed cell death. Interaction between CCR3 and eotaxin/CCL11 may, besides promoting allergic reactions, drive activated B cells to apoptosis, thereby reducing levels of Ig production, including IgE, and consequently limit the development of the humoral immune response. The apoptotic action of eotaxin/CCL11 suggests a therapeutic modality in the treatment of B cell lymphoma.

Background: A growing body of evidence suggests that proinflammatory cytokines play a role in allergic inflammation by attracting and activating inflammatory cells. In this study, we have investigated the relationship between interleukin-8 (IL-8) in nasal lavage fluid and the local activation of eosinophils and neutrophils following nasal allergen challenge of allergic patients. Methods: Nasal challenges were performed with grass pollen extract in 14 allergic patients and 5 nonallergic controls. Nasal lavage fluid was collected repeatedly for 10 h, and the levels of eosinophil cationic protein (ECP) and myeloperoxidase (MPO) were used as markers of eosinophil and neutrophil activation, respectively. The levels of these molecules were compared with that of IL-8 in nasal lavage fluid. Results: Allergen challenge of allergic patients produced a significant late-phase increase in the levels of ECP and MPO. Furthermore, the level of MPO showed a highly significant correlation with the level of IL-8 in lavage fluid (r = 0.8, p < 0.0001), whereas there was no significant relationship between the levels of ECP and IL-8. Conclusion: Interestingly, our findings suggest that both eosinophils and neutrophils are activated following nasal allergen challenge. In addition, our results are consistent with the hypothesis that IL-8 acts as a chemoattractant/activator of neutrophils during the late phase of the allergic inflammation. In contrast, we were not able to demonstrate any significant relationship between the level of IL-8 in lavage fluid and the activation of eosinophils.

Human mast cells can be divided into two subtypes: MC(TC) cells, which contain tryptase and chymase, and MC(T) cells, which contain tryptase only. Herein we have used a combination of histamine, tryptase and chymase immunohistochemistry as a novel approach to the study of mast cells. Using this technique, we have discovered a new type of MC(TC) mast cell in biopsies of the nasal mucosa from healthy subjects and allergic patients. These mast cells have histamine-positive, dendrite-like cellular processes. Some cells have only one slender process, whereas other cells have several long processes extending from different parts of the cell body. Some of the cellular processes divide into two or three terminal branches, and histamine is sometimes found in small swellings along the course of the processes. Our findings contribute new aspects to the concept of mast cell heterogeneity. Thus, human mast cells may vary not only with respect to mediator content, but also with respect to gross morphologic features such as the presence of dendrite-like cellular processes. The recognition of this extreme heterogeneity may be an important step toward a better understanding of mast cell biology.