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- Materials and methods
Background: The induction of an immune response to a biologically inert soluble protein requires an adjuvant. Here we have examined whether intact grains of pollen display such adjuvant effect, accounting for the immunogenic activity of pollen protein allergens that are devoid of intrinsic pro-inflammatory/adjuvant property.
Methods: Human monocyte-derived dendritic cells (DCs) were cultured with intact grains of grass or ragweed pollen for 48 h. The state of DCs maturation was analyzed by FACS and their cytokine production by ELISA. T cell priming activity of DCs was examined in co-cultures with naïve cord blood-derived CD4+ T cells.
Results: Contact with grains of pollen induced a distinct maturation program in immature DCs. Pollen up-regulated the expression of CD54, CD80, CD83, CD86, HLA-DR, CCR7, and CD40 on DCs. Moreover, CCR5 expression was up-regulated by pollen but suppressed by LPS. In sharp contrast to LPS-stimulated DCs, pollen-treated DCs did not produce cytokines [interleukin (IL)-10, IL-12, tumor necrosis factor (TNF)-α] but retained the ability to secrete high levels of these cytokines upon simulation with soluble CD40 ligand and interferon (IFN)-γ. Pollen-primed DCs strongly stimulated the proliferation of allogeneic naïve CD4+ T cells and promoted their development into effector cells producing high levels of IL-5 and IL-13 together with moderate levels of IFN-γ and IL-4.
Conclusion: Intact grains of pollen induce activation and maturation of DCs in vitro. Similar mechanisms may be effective in vivo, suggesting that pollen grain is not only an allergen carrier but also acts as an adjuvant in the induction phase of the allergic immune response.
Allergic diseases such as rhinitis or extrinsic asthma represent a major health problem of most modern societies, with significant economical consequences resulting from the reduced productivity of affected patients, as well as healthcare costs (1, 2). These diseases are caused by aberrant Th2 immune responses directed against protein Ags (also known as allergens) rapidly released by commonly inhaled small particles. Whereas the mechanisms whereby ongoing Th2 responses against these protein Ags lead to clinical asthma and rhinitis are understood in increasing detail, the early events involved in elicitation of the immune response or allergic sensitization are still unclear. Perhaps this is because the majority of studies conducted to date on the induction of immune or allergic responses to airborne allergens have relied on the utilization of purified allergen proteins (3, 4). However, we do not inhale pure protein Ags, but rather the small particles on which they are carried, such as pollen grains, their starch particles or other exogenous particulate materials (5, 6).
Pollen is one of the most frequent airborne allergens from the natural environment, leading to seasonal rhinitis in about 10–25% of the population (7, 8). However, healthy nonatopic individuals develop low grade and noninflammatory immune responses to pollen Ags following natural exposure. Pollen allergens are apparently very immunogenic as they induce in both healthy and allergic individuals the same number of pollen-specific CD4+ T cells. Although the latter recognize the same epitopes of the protein Ags, they differ by their cytokine production profile (9, 10).
The mechanisms leading to the induction of immune responses to innocuous antigens such as the proteins released by pollens are unknown. Studies in animal models on the immune response to biologically inert proteins such as ovalbumine (OVA), deposited on the airway mucosa have demonstrated the obligatory role of the adjuvants. No airway inflammatory response was generated in mice that had been sensitized with LPS-depleted OVA, whereas antigen-specific immune responses were induced in the presence of LPS with low and high doses inducing Th2 or Th1 responses, respectively (11). It has been suggested that the proteolytic activity of some purified allergens may account for their immunogenic activity by enhancing their trans-epithelial delivery and/or promoting local inflammation (12, 13). Der p1, the major allergen of house dust mite, is a cysteine protease that has been extensively investigated for its allergenic activity (12). No such proteolytic or intrinsic adjuvant activity has been described for pollen proteins. However, in vitro studies have shown that crude aqueous pollen extracts can directly recruit and activate polymorphonuclear neutrophils and eosinophils, inducing chemotaxis and cytokine secretion (14, 15).
Here we further explore the adjuvant activity of intact pollen grains and examine their ability to activate immature dendritic cells (DCs) and to influence their capacity to prime naïve T cells.
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- Materials and methods
A number of cell types including epithelial cells and antigen-presenting cells such as DCs and macrophages, all of which are capable of interacting with inhaled allergens, are present in the lumen of the respiratory tract. Here we demonstrate for the first time the biological activity of intact grains of pollen in vitro on human dendritic cells. Our results suggest that pollen grains exert an adjuvant effect and, thereby, promote the induction of an immune response to a biologically inert protein carried by the pollen particle in allergic and nonallergic individuals. Although surprising, our results are in line with the recent observations on human neutrophils and eosinophils demonstrating that grains of pollen influence their effector functions by inducing their directed migration and secretion of granular proteins in an allergen-independent manner (14, 15).
In our hands, exposure to pollen grains mediated on immature DCs a distinct maturation program characterized by a strong up-regulation of CD54, CD80, CD83, CD86, HLA-DR and the chemokine receptor CCR7 contrasting with low levels of CD40. This functional modulation of DCs was distinct from the classical DC activation triggered by LPS and, most importantly, it was not mediated by purified allergen from pollen AgE, that did not affect the expression of co-stimulatory molecules on immature DCs and cytokine production by DCs (data not shown). Migration of CCR7-expressing DCs to the afferent lymphatics and into the T cell area is selectively promoted by the presence of PGE2 that facilitates the coupling of CCR7 to its signal transduction modules (25). Recently it has been shown that pollen grains release an exudate containing ‘LTB4-like’ and PGE2-like’ substances upon contact with the aqueous phases (14, 26). The recruitment of T cells into cell cycle and the subsequent rate of division have been reported to be enhanced by co-stimulatory molecules such as B7.1 and B7.2 (27). Likewise, our data have demonstrated that pollen-primed DCs acquired enhanced T-cell stimulatory capacity.
Skewing of naïve T cells toward a Th1 or Th2 response is a crucial process in determining the ultimate outcome of the immune response, and this is affected by the microenvironment of antigen-presenting DCs, as well as by the modulation of T-cell-receptor-mediated activation signals (28, 29). In our hands, T cells primed by DCs treated with grains of pollen showed increased Th2 cytokines production, such as IL-5 and IL-13. However, at the single-cell level the percentage of IFN-γ-producing cells was higher than that of IL-4-producing cells. Therefore, in our culture conditions pollen-primed DCs induced a mixed Th-cell polarization.
Our findings show that when DCs were exposed to grains of pollen, they did not produce or produce at very low levels cytokines IL-12, IL-10 and TNF-α. This is consistent with the profile of semi-mature DCs since the characteristics of these semi-mature but tolerogenic DCs are their expression of co-stimulatory molecules, but low or absent production of pro-inflammatory cytokines, in particular IL-12 (23). The finding that upon secondary stimulation pollen-primed DCs, in contrast to LPS-primed DCs, produced significant amounts of cytokines and were not ‘exhausted’ indicates their semi-mature state as well (24). The conditions of activation and degree of maturation of the immature DCs are crucial for the signals that are delivered to the T cells. Activation of DCs without concomitant full DC maturation most of time leads to the development of regulatory T cells (30). However, we found no evidence for such regulatory T cell induction by pollen-primed DCs in our culture conditions (data not shown).
A further question related to present data was how intact grains of pollen would have an access to DCs in physiological conditions. Several scenarios may be envisaged. Firstly, study showing an Ag-specific T cell activation and proliferation occurring after intra-tracheal instillation of Ag-pulsed DCs was taken to indicate that these DCs may cross the epithelial tight-junction barrier and go from the airways to the lymph nodes (31, 32). Secondly, DCs penetrate the tight junctions between epithelial cells and send dendrites outside the epithelium allowing them to directly sample the small fragments of particles of pollen as it has been described for some bacteria (33). Intact hydrated pollen grains generate reactive oxygen species (ROS) due to intrinsic NAD(P)H oxidase in them (34). Thus, it is tempting to speculate that these ROS could be implicated in the opening of the tight junction allowing the extrusion of dendrites. Finally, the particles (0.5–5 μm) released from grains of pollen were visualized inside of the cultured epithelial cells upon their exposure to pollen (34). This may represent another mechanism whereby pollen through the release of smaller particles gains an access to epithelial cells and DCs. This latter possibility may be attributed to the observation that the adjuvant effect of intact grains of pollen was contact-dependent (the particles did not pass through 0.45 μm Transwell system) and not related to the mediator release.
In summary, we provide evidence that pollen grains not only function as allergen carriers, but may have far more effects on human health by contributing to the manifestation or aggravation of allergic inflammation by virtue of their adjuvant effect on DCs.