It has been demonstrated that pollen grains contain NAD(P)H oxidases that induce oxidative stress in the airways, and this oxidative insult is critical for the development of allergic inflammation in sensitized mice. On the basis of this observation, we have examined whether pollen grain exposure triggers oxidative stress in dendritic cells (DCs), altering their functions. To test this hypothesis, human monocyte-derived DCs were treated with ragweed pollen grains. Our findings show that exposure to pollen grains induces an increase in the intracellular levels of reactive oxygen species in DCs. Our data also indicate that besides the NAD(P)H oxidases, other component(s) of pollen grains contributes to this phenomenon. Elevated levels of intracellular reactive oxygen species triggered the production of IL-8 as well as proinflammatory cytokines, such as TNF-α and IL-6. Treatment with pollen grains initiated the maturation of DCs, strongly upregulated the membrane expression of CD80, CD86, CD83, and HLA-DR, and caused only a slight increase in the expression of CD40. The pollen-treated DCs induced the development of naive T lymphocytes toward effector T cells with a mixed profile of cytokine production. Antioxidant inhibited both the phenotypic and functional changes of DCs, underlining the importance of oxidative stress in these processes. Collectively, these data show that pollen exposure-induced oxidative stress may contribute to local innate immunity and participate in the initiation of adaptive immune responses to pollen Ags.

Under normal conditions, plasmacytoid dendritic cells (pDCs) are located in peripheral lymphoid organs or circulate in the blood, from where they can migrate to sites of infection or inflammation. In inflamed tissues, pDCs can be exposed to elevated levels of reactive oxygen species produced by inflammatory cells and we presume that oxidative stress could affect the cellular responses of pDCs to microenvironmental stimuli. To explore this possibility, human pDCs isolated from peripheral blood of healthy donors were treated with H2O2 and R837 (a Toll-like receptor 7 ligand), separately and in combination. Our results demonstrate that treatment with a low concentration (0.01 µM) of H2O2 resulted in only slight changes in the expression of CD40, CD80, CD86, and CD83; however, low-dose H2O2 markedly decreased the expression of HLA-DQ on pDCs. Exposure to H2O2 did not trigger the release of IL-6, TNF-α, IL-8, or IFN-α from pDCs. Although addition of H2O2 did not modify the capacity of pDCs to activate allogeneic IL-17- or IFN-γ-producing T cells, it significantly increased the ability of pDCs to stimulate IL-4-secreting T cells. Exposure of pDCs to H2O2 before cocultivation with naïve autologous T cells significantly lowered IL-10 production by T cells, but did not affect IL-17 release. It was also observed that H2O2-exposed pDCs provided stronger stimuli for Th2 than for Th1 differentiation upon autologous activation, compared to untreated pDCs, possibly because of elevated surface expression of OX40-L. Most importantly, when pDCs were stimulated with R837 in the presence of H2O2, decreased phenotypic activation, decreased chemokine and cytokine release, and impaired allo- and autostimulatory functions of pDCs were detected, indicating that pDCs exposed to oxidative stress in vivo may have an anti-inflammatory or tolerogenic role in regulating adaptive immune responses.

Plasmacytoid pre-dendritic cells (pDCs) are able to prime and polarize naive T-cells, while also having an important effector function in antiviral immunity through the rapid and robust production of interferon-a. The main setback of pDCs investigation is the rarity and ex vivo fragility of these cells. Relative simple, reliable, and accurate methods for phenotypic analysis and functional studies of pDCs without isolation would be a great deal of interest. Fresh whole blood samples were analyzed by two-color and one-color flow cytometric pDC-identification assays. The changes in the surface expression of CD62L and HLA-DQ on pDCs in whole blood samples after 24-h treatment with imiquimod, a toll-like receptor 7 agonist, were analyzed. Our data demonstrate that the identification of pDCs in peripheral blood samples can be achieved by using only one fluorescent channel for blood dendritic cell antigen (BDCA)-4 staining combined with the light scatter parameters, thus leaving the other channels open for further phenotypic and/or functional analysis. Recently, several lines of evidence supported the involvement of pDCs in the development of several human diseases, so our new one-color identification approach may provide a useful tool for investigation of the pathomechanism of the relevant diseases by using common, 2-laser benchtop cytometers. ' 2008 International Society for Analytical Cytology Key terms plasmacytoid dendritic cells; flow cytometry; BDCA-4; phenotypic analysis PLASMACYTOID pre-dendritic cells (pDCs) represent a rare but unique cell population of innate immunity. Since their discovery in 1958 (1) the proper name of this special cell type has remained controversial due to the unclarified link between pDCs and the previously identified professional type I interferon-producing cells (IPCs) (2). IPCs have recently been described as a differentiation state of pDCs (3) that represent their resting form referred to as plasmacytoid dendritic cell precursors (4). The key function of pDCs is the recognition of viral nucleic acids, through a speciesand cell type-specific set of pattern recognition receptors such as toll-like receptors (TLR) that involve TLR7 and TLR9 as well as intracellular helicases (5). TLR7 recognizes viral single stranded RNA (ssRNA), but several synthetic compounds such as loxoribine, resiquimod (R848), and imiquimod (R837) are also able to bind to this receptor (6). TLR9 interacts with naturally occurring hypo-or unmethylated DNAsequences and with synthetic CpG-oligonucleotides (7), and upon ligation induces a type I interferon response in pDC. High expression of TLR7 and TLR9 is a hallmark of pDCs and B-cells, while monocytes and myeloid dendritic cells express TLR2 and TLR4, thus supporting the concept of independent differentiation pathways of these DC sublineages (8,9). Upon activation with specific TLR-ligands, pDCs differentiate to professional antigen-presenting cells and acquire a ''dendritic'' morphology, similar to conventional myeloid dendritic cells (cDCs).

Previous observations suggest that static magnetic field (SMF)-exposure acts on living organisms partly through reactive oxygen species (ROS) reactions. In this study, we aimed to define the impact of SMF-exposure on ragweed pollen extract (RWPE)-induced allergic inflammation closely associated with oxidative stress. Inhomogeneous SMF was generated with an apparatus validated previously providing a peak-to-peak magnetic induction of the dominant SMF component 389 mT by 39 T m 21 lateral gradient in the in vivo and in vitro experiments, and 192 mT by 19 T m 21 in the human study at the 3 mm target distance. Effects of SMF-exposure were studied in a murine model of allergic inflammation and also in human provoked skin allergy. We found that even a single 30-min exposure of mice to SMF immediately following intranasal RWPE challenge significantly lowered the increase in the total antioxidant capacity of the airways and decreased allergic inflammation. Repeated (on 3 consecutive days) or prolonged (60 min) exposure to SMF after RWPE challenge decreased the severity of allergic responses more efficiently than a single 30-min treatment. SMF-exposure did not alter ROS production by RWPE under cell-free conditions, while diminished RWPEinduced increase in the ROS levels in A549 epithelial cells. Results of the human skin prick tests indicated that SMF-exposure had no significant direct effect on provoked mast cell degranulation. The observed beneficial effects of SMF are likely owing to the mobilization of cellular ROS-eliminating mechanisms rather than direct modulation of ROS production by pollen NAD(P)H oxidases.

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