Pollen grains are one of the most common inducers of allergic symptoms. Upon contact with mucosal surfaces of the upper respiratory tract, pollen grains rapidly release proteins/allergens into the aqueous phase. On the basis of a genetic susceptibility atopic individuals develop allergen-specific Th2-biased immune responses that ultimately lead to clinical manifestations of IgE-mediated hypersensitivity. Although the biology of Th2 cells in the effector phase of allergy is well understood, little is known about the mechanisms that control the initial Th2 polarization in response to exogenous allergens.
We recently demonstrated that pollen in addition to liberating protein allergens rapidly release various bioactive lipids into the aqueous phase (24
). These pollen-associated lipid mediators (PALMs) were shown to stimulate and attract cells of the innate immune system, such as neurophil and eosinophil granulocytes (25
). Here, we describe the effect of PALMs on the activation and functional maturation of human DCs. In addition, we demonstrate that Bet
.-APE and certain phytoprostanes identified in Bet
.-APE modulate the function of human DCs in a fashion that results in a preferential induction of Th2-dominated adaptive immune responses.
Activation of DCs with LPS depleted by Bet.-APE (LPS under the detection limit of the LAL test) resulted in moderate DC activation as documented by selective up-regulation of HLA-DR surface expression. When DCs were stimulated simultaneously with LPS plus Bet.-APE, the presence of Bet.-APE resulted in an additional up-regulation of CD80, CD86, and HLA-DR surface expression. At a functional level Bet.-APE–induced DC maturation resulted in an enhanced allostimmulatory activity as demonstrated by enhanced proliferative responses of naive allogeneic T cells. In addition, Bet.-APE treatment induced a dose-dependent inhibition of the LPS or CD40L induced IL-12 p70 production of DCs, whereas IL-6, IL-10, and TNFα production was not impaired. Thus, water-soluble factors released from pollen grains are capable to selectively modulate various DC functions, including the inhibition of activation-induced IL-12 release from human DCs. The reduced IL-12 production was confirmed at mRNA level, demonstrating that regulation occurred predominantly at the level of IL-12 p40 rather than IL-12 p35.
Maturation of DCs is stimulated by factors signaling tissue danger such as microorganisms, dying cells, or proinflammatory cytokines. Recently, a variety of factors has emerged that can limit DC maturation. For example intracellular cAMP-elevating agents, such as PGE2
, inhibit IL-12 and TNFα and enhance IL-10 expression by LPS-stimulated DCs (14
). In contrast, IL-10, glucocorticoids, and vitamin D3 interfere with DC maturation as a whole by blocking the up-regulation of presenting and costimulatory molecules (13
.-APE seemed to act independently of the above-cited mechanisms because its activity was not affected by indomethacin or neutralization of endogenous IL-10.
Recently a series of isoprostanes with the characteristic prostaglandin ring systems was discovered in plants and designated phytoprostanes (19
). Phytoprostanes are formed via autooxidation, which is initiated by free radical attack of α-linolenic acid yielding a linolenate radical that readily oxidizes and cyclizes to two regioisomeric, prostaglandin G–like compounds (34
). In vivo, PPG1
may be either reduced to PPF1
or converted to PPE1
, which itself may be dehydrated and isomerized to PPB1
In the present study we demonstrate for the first time that nonenzymatically formed phytoprostanes such as PPE1
, and PPB1
are present in aqueous pollen extracts in nanomolar concentrations as identified and quantified by NCI GC-MS (19
). Levels of PPF1
in organic extracts of birch pollen appear to be approximately 15 times more abundant in organic as compared with aqueous extracts (21
). These differences might reflect different extraction efficiencies as well as varying concentrations in pollen from different sources. A survey of PPF1
levels in fresh pollen from individual betula pendula
L. trees and different birch species extracted with organic solvents revealed that levels vary greatly and range from 2 to 33 μg/g pollen (unpublished data). A similar variance is expected for PPE1
levels. Phytoprostane levels in organic extracts reflect lipid peroxidation in pollen, and may only be of limited relevance for estimates of natural exposure levels on the mucus membranes. In contrast, analysis of phytoprostanes levels spontaneously released into the aqueous phase of the buffer used in this study, more closely mimics physiological exposure conditions.
PPE1, PPF1 and PPB1 were tested in their capacity to modulate the IL-12 production of human DCs. Interestingly, only PPE1 but not PPF1 or PPB1 inhibited the LPS or CD40-induced IL-12 production. Although Bet.-APE induced a functional and phenotypical maturation, none of the phytoprostanes tested had any significant effect on DC maturation (unpublished data). The modulatory effect of PPE1 on DC IL-12 production and the ensuing T cell response was dependent on the presence of a maturation signal such as LPS or CD40 ligation. The receptors and signal transduction pathways involved in these mechanisms are currently under investigation.
It is generally accepted that DCs instruct the immune system to initiate an Ag-specific response by providing naive Th cells with signal 1 (TRC triggering) and signal 2 (costimulation). In addition, it has recently been suggested that immature DCs in peripheral non lymphoid tissue can adopt different Th1- or Th2-promoting effector function, depending on the tissue- and/or pathogen-type context of their activation (14
). This DC-dependent component of the initial polarization of naive T cells (signal 3) was suggested to depend on pathogen-derived or -induced endogenous factors present in the local microenvironment at the time of antigen encounter. Our study demonstrates that this signal 3 can also be modulated by exogenous mediators such as phytoprostanes that are released from (under normal circumstances nonpathogenic) pollen grains upon contact with the airway mucosa.
Clearly, any extrapolation of these effects to the in vivo situation would partly depend on the expected concentration of pollen-derived lipids in the nasal or bronchial microenvironment. As demonstrated previously, concentrations of linolenic and linoleic acid in pollen are high (25
) and we assume that during pollen season the upper respiratory tract mucosa is exposed to biologically relevant concentration of various oxidized derivatives of these fatty acids. The effects of in vivo exposure to PALMs are currently under investigation.
Collectively, our data provide compelling evidence for the role of exogenous pollen-derived phytoprostanes in the decision-making process of DCs. We suggests that DCs that have been conditioned by PALMs, such as E1-phytoprostanes will provide one of the initial signals driving the development and perpetuation of Th2-dominated immune response in pollen allergy.