Exposure to cadmium-contaminated soils increases allergenicity of Poa annua L. pollen.

BACKGROUND: Pollution is considered as one main cause for the increase of allergic diseases. Air pollutants may cause and worsen airway diseases and are probably able to make pollen allergens more aggressive. Previous studies looked at traffic-related air pollution, but no data about the effects of polluted soils on pollen allergens are available. We aimed to assess the effects of plant exposure to cadmium-contaminated soil on allergenicity of the annual blue grass, Poa annua L, pollen. METHODS: Poa plants were grown in soil contaminated or not contaminated (control) with cadmium. At flowering, mature pollen was analyzed by microscopy, to calculate the percentage of pollen grains releasing cytoplasmic granules, and by proteomic techniques to analyze allergen proteins. Allergens were identified by sera from grass pollen-allergic patients and by mass spectrometry. RESULTS: Pollen from Cd-exposed plants released a higher amount of allergenic proteins than control plants. Moreover, Cd-exposed pollen released allergens-containing cytoplasmic grains much more promptly than control pollen. Group 1 and 5 allergens, the major grass pollen allergens, were detected both in control and Cd-exposed extracts. These were the only allergens reacting with patient's sera in control pollen, whereas additional proteins strengthening the signal in the gel region reacting with patient's sera were present in Cd-exposed pollen. These included a pectinesterase, a lipase, a nuclease, and a secretory peroxydase. Moreover, a PR3 class I chitinase-like protein was also immunodetected in exposed plants. CONCLUSION: Pollen content of plants grown in Cd-contaminated soils is more easily released in the environment and also shows an increased propensity to bind specific IgE.

Differential activation of NF-kappa B subunits in dendritic cells in response to Gram-negative bacteria and to lipopolysaccharide.

Dendritic cell (DC) maturation is essential for the initiation of T-dependent immune responses. Nuclear factor kappa B/Rel (NF kappa B/Rel) transcription factors are ubiquitously expressed signalling molecules, known to regulate the transcription of a large number of genes involved in immune responses, including cytokines such as IL-1, IL-6, TNF-alpha and cell surface molecules (MHC class I and II, B7.2). In this study, we have compared the activation of five members of the NF-kappa B family, p65, c-Rel, p50, RelB and p52, during DC maturation in response to lipopolysaccharide (LPS) and to Salmonella typhimurium. We have shown that although the translocation of NF-kappa B occurred very early, 30 min after treatment with both S. typhimurium and LPS, bacteria-induced NF-kappa B activation was more pronounced. Four out of five members, i.e. p65, c-Rel, p50 and RelB, were similarly activated upon the two stimuli but with different kinetics. Indeed, we have observed that p65, c-Rel and p50 were translocated early, whereas RelB was translocated later in DC activation. This differential regulation suggests that the various members of NF-kappa B family can mediate distinct functions of DC physiology.

Physical contact between lipopolysaccharide and toll-like receptor 4 revealed by genetic complementation.

Some mammalian species show an ability to discriminate between different lipopolysaccharide (LPS) partial structures (for example, lipid A and its congener LA-14-PP, which lacks secondary acyl chains), whereas others do not. Using a novel genetic complementation system involving the transduction of immortalized macrophages from genetically unresponsive C3H/HeJ mice, we now have shown that the species-dependent discrimination between intact LPS and tetra-acyl LPS partial structures is fully attributable to the species origin of Toll-like receptor 4 (Tlr4), an essential membrane-spanning component of the mammalian LPS sensor. Because Tlr4 interprets the chemical structure of an LPS molecule, we conclude that LPS must achieve close physical proximity with Tlr4 in the course of signal transduction.

Microglia induce myelin basic protein-specific T cell anergy or T cell activation, according to their state of activation.

Microglial cells are non-professional antigen-presenting cells (APC) the function of which is still controversial. Here, we studied the function of microglia derived from H-2(u) mice. We show that these microglia express a low level of B7.2 and CD40 and, interestingly, lack surface expression of B7.1. Resting and IFN-gamma-activated microglia were unable to activate naive and primed myelin basic protein (MBP)-specific CD4(+) T cells in the presence of MBP and encephalomyelitic MBP Ac1-11 peptide. Furthermore, in the presence of Ac1-11 peptide, CD4(+) TCR-transgenic T cells became anergized. Microglia became professional APC only after a multistep activation process involving both stimulation through cytokines [granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-gamma] and cognate signaling (B7-CD28 and CD40-CD40 ligand interactions). As such they were able to present MBP to both unprimed and primed T cells. Co-culture of microglia with GM-CSF up-regulated co-stimulatory molecules, in particular B7.1. Additional activation with IFN-gamma induced MHC class II and CD40 up-regulation. CD40-CD40 ligand interaction significantly enhanced microglial ability to prime TCR-transgenic T cells and was essential for presentation of MBP to in vivo primed non-transgenic T cells. We propose that microglia may serve different functions under different inflammatory conditions, depending on the cytokine milieu and the type of cognate interaction they are involved in.

Dendritic cells as natural adjuvants.

Dendritic cells (DCs) are professional antigen presenting cells that hold the key to the induction of T-cell responses. Therefore, the use of DCs for immunotherapy to stimulate immune responses has recently raised a great deal of interest. Many clinical trials using DCs have been initiated to stimulate immune responses against tumors or infectious agents. Several issues need to be considered before DCs can be used successfully as natural adjuvants: DCs have to be generated in sufficient numbers; they should display morphological, phenotypical, and functional properties of DCs; and they should be able to present antigens. In the present review we focus on methods for the purification of DCs from human bone marrow and peripheral blood and for the optimization of in vitro cell culture systems. Methods to generate growth factor-dependent mouse DC lines are also described.

Retroviral gene transfer, rapid selection, and maintenance of the immature phenotype in mouse dendritic cells.

We used the retroviral vector PINCO [which expresses the green fluorescent protein (GFP) as a selectable marker], to infect growth factor-dependent immature D1 dendritic cells (DC). The efficiency of infection in different experiments was between 5 and 30%, but subsequent cell sorting led to a virtually homogeneous population of GFP-positive cells. Retroviral infection did not modify the immature DC phenotype, as shown by the low expression of major histocompatibility complex and co-stimulatory molecules. Furthermore, the GFP-positive D1 cells underwent full maturation after lipopolysaccharide treatment, as indicated by a high expression of cell-surface MHC and co-stimulatory molecules, and also by strong stimulatory activity in allogeneic mixed lymphocyte reaction. The high efficiency of this retroviral system, the rapidity of the technique, and the possibility to overcome in vitro selection make this method very attractive for the stable introduction of heterologous genes into proliferating immature mouse D1 cells. Furthermore, this approach is suitable for functional studies of new DC-specific genes involved in DC maturation and survival.

Bacteria and bacterial cell wall constituents induce the production of regulatory cytokines in dendritic cell clones.

The primary function of dendritic cells (DC) is the uptake, processing, and presentation of antigens to unprimed T cells, but the regulation of these functions is largely unknown. The study of the signals that maintain DC in a resting state or that drive their activation has been hampered by the difficulties in obtaining pure DC populations. The availability of immortalized DC clones from different tissues (spleen and skin) allowed us to investigate the regulation of cytokine production in response to physiological signals in the absence of contaminating cells. The DC clones exhibited the phenotypical and functional features of DC precursors and could phagocytose, albeit at a low rate, whole bacteria. Heat-inactivated bacteria and bacterial cell wall products were tested for cytokine induction. Lipopolysaccharide, lipoteichoic acid, and gram-negative bacteria were potent inducers of tumor necrosis factor alpha and interleukin 6 release, whereas gram-positive bacteria were less efficient. The results suggest that microbial infections can directly promote cytokine DC release of relevant inflammatory responses as well as in the autocrine activation of DC.