The effect of caffeic acid phenethyl ester on the functions of human monocyte-derived dendritic cells

Asthma is the leading chronic disease in children. Recent advance of asthma medication has decreased the mortality and morbidity of asthma. Among them, inhaled corticosteroid is the mainstay treatment. Other medications such as theophylline, long-acting beta₂-adrenergic agonist, leukotriene modifier and anti-IgE treatment, alone or in combination of inhaled corticosteroid, have increased the control of asthma. However, these drugs do have some side effects such as oral thrush, inhibition of growth[1] or increased risk of severe asthma exacerbation [2]. Difficult-to-treat asthma patients do still have persistent symptoms even with the best therapy [3]. Therefore, several other approaches to asthma treatment are extensively studied. The subcutaneous immunotherapy has some effect but the anaphylactic side effect and frequent injection limit the application [4]. Sublingual immunotherapy, which was safe in children, has only low to moderate clinical efficacy in mild to moderate persistent asthma [5, 6]. Other novel treatments in murine model of asthma, such as central deoxycytidyl-deoxyguanosine (CpG) dinucleotide inhalation, DNA vaccination and antisense oligonucleotide are not yet proved in human [7].

Propolis, the natural resinous products collected by honeybees from various plant sources, is well known for the management of respiratory problems in herbal medicine [8]. In clinical studies, Khayyal et al. administered the aqueous extract of propolis to patients with mild to moderate asthma daily for 2 months in combination with oral theophylline [9]. They found that the number of nocturnal asthma attacks decreased significantly in the propolis treatment group compared to the placebo group. In addition, the lung function of patients treated with propolis improved after 2 months while the placebo group did not. Finally, the sera of patients in the propolis group had significantly lower levels of tumor necrosis factor (TNF)-α, intercellular adhesion molecule (ICAM)-1, interleukin (IL)-6, IL-8, leukotrienes and prostaglandin (PG)E₂ after the 2 months treatment period, but these changes were not identified in the placebo group. In murine model of asthma, propolis extracts could suppress the serum levels of OVA-specific IgE and IgG₁, and airway hyperresponsiveness in OVA-sensitized mice. Besides, interferon (IFN)-γ, IL-6, and IL-10 secretion in OVA-stimulated splenocytes from the propolis groups was significantly lower than that of the control group [10].

The composition of propolis is highly variable, depending on the local plant, and is reported to contain approximately 50% resin and vegetable balsam, 30% wax, 10% essential and aromatic oils, 5% pollen, and 5% other substances (minerals) [8]. Further, propolis contains a mixture of biologically active chemicals including terpenes, cinnamic acid, caffeic acid and their esters, amino acids and flavonoids [11]. Caffeic acid phenethyl ester (CAPE), one of the most extensively studied components in propolis, is reported to have anti-tumor [12, 13], anti-inflammatory [14, 15] and antioxidant [16] properties. CAPE has also been found to suppress eicosanoid synthesis [14]. In immunological studies, CAPE is a potent inhibitor of mitogen-induced T cell proliferation, lymphokine production [17] and nuclear factor (NF)-κB activation [18‐20]. CAPE modulated nuclear binding of the NF-κB subunit p65/RelA, decreased expression of cytosolic IκBα [19] and inhibited NFAT dephosphorylation and transcriptional activity [20] in T cells.

Dendritic cells (DCs), one of the most potent professional antigen-presenting cells (APCs), play an important role in the pathogenesis of asthma and allergic rhinitis [21]. DCs normally reside in the airway mucosa and interstitium in an immature state [22] and are specialized in capturing and processing antigens to form major histocompatibility complex (MHC) peptide complexes. Upon antigen or other stimulation, DCs mature with loss of endocytic/phagocytic receptors, upregulation of MHC molecules and costimulatory molecules, alterations in adhesion molecule and cytokine receptor expression and cytokine production, and changes in morphology [23‐27]. At the same time, the DCs migrate to the draining lymph nodes and present captured antigen to naïve T lymphocytes [23] by a stable, long-lasting immunologic synapse with T cells. MHC peptide interacts with the T cell receptors, costimulatory molecules interact with T cell-expressed coreceptors, and cytokines are released to polarize the T-cell response [28], thus results in the allergic airway inflammation. The role of DCs in the secondary immune response is further supported by the fact that their depletion at the time of allergen challenge abrogated all the features of asthma in murine model of asthma, including airway inflammation, goblet cell hyperplasia, and bronchial hyperresponsiveness [29].

Since Khayyal et al. demonstrated the beneficial effect of propolis in asthma patients [9] and Sy et al. reported the similar effect in murine model of asthma [10], we became interested in studying the impact of CAPE on dendritic cells in the pathogenesis of asthma. There was only one study that surveyed the immunosuppressive activity of CAPE on human DCs, which showed that CAPE blocked the L. major-induced IRF-1, IRF-8, IL-12 p35 and IL-12 p40 RNA expression in human MoDCs by quantitative real time RT-PCR as well as the IRF-8 nuclear translocation [30]. However, mite and pollens, instead of L. major, are the major allergens in allergic disease. In the present study, the authors investigated whether CAPE has regulatory effect on the cytokine and chemokine production and antigen presentation ability in mite stimulated human MoDCs. It is anticipated that this study would establish the role of CAPE on the function of DCs and might provide insight into the mechanisms of action and rationale of propolis administration in the management of asthma and other allergic disorders.

The main purpose of this study is to elucidate the regulatory effect of CAPE on human MoDCs. First, the cytokine (IL-12 p40, IL-12 p70, and IL-10) and chemokine (IP-10) production from MoDCs were evaluated. In our study, we used LPS as the stimulation other than crude mite extract because LPS is known to be ubiquitously present in the environment and induces Th1- and Th2-related cytokine and chemokine expression [34]. We found that LPS could induce MoDCs to secrete high levels of IL-12, IL-10 and IP-10 in healthy subjects and allergic patients (Figure 1) as previously stated [32, 35, 36]. Crude mite extract could also stimulate MoDCs to secrete high levels of IL-12 p40 and IP-10 in healthy subjects but high levels of IL-10 and IP-10 in allergic patients. The CAPE treatment could inhibit IL-12 and IL-10 secretion under LPS or crude mite extract stimulation in healthy subjects but only IL-10 secretion under crude mite extract stimulation in allergic patients. CAPE could only inhibit IP-10 secretion under crude mite extract stimulation but not LPS in both healthy subjects and allergic patients. Because the large variation was noted between individuals and the inhibitory effect of CAPE was dose-dependent (data not shown), we speculated that the higher concentration of CAPE may lead to significant IL-12 inhibition in allergic patients.

IL-12, produced by mature DCs, has a central role in initiating a specific T cell-mediated immune response [37, 38], driving Th1 cell activation and differentiation [39‐42], and inducing production of IFN-γ and lytic activity [43, 44] IL-10, the well known regulatory cytokine, can have different functions in immune differentiation and regulation. On one hand, IL-10 and TGF-β have been shown to induce regulatory T cells. These two cytokines, produced early in the infection could activate T cell subsets such as IL-10 and IFN-γ producing T cells [45]. Neutralization of IL-12 inhibited the generation of the double producing T cell lines, indicating the role of IL-12 in inducing IL-10 and IFN-γ producing T cells. Therefore, the principle source of IL-10 in persistent infection may be T cells that also produce IFN-γ, which was important in the modulation of the inflammatory response [45]. On the other hand, IL-10 produced by DCs and macrophages promotes Th2 responses by abrogation of IL-12 generation [46]. DCs from IL-10 treated cultures can induce naïve T cells to differentiate into IL-4-secreting T cells [47], while DCs treated with anti-IL-10 Ab have an increased capacity to activate allogeneic T cells and prime naïve T cells to a more prominent Th1 polarization [48]. Further, the IL-10-induced anergic state in human CD4⁺ T cells can be reversed by mature DCs [49]. Therefore, the IL-10 produced by DCs may promote Th2 responses but not T cell anergy when co-cultured with mature DCs. In the present study, it appeared that CAPE inhibited cytokine production by MoDCs without preference of Th1 or Th2 prone cytokines in both healthy subjects and allergic patients. IP-10 is a potent chemokine for activated T cells, natural killer cells, and mast cells [50]. In individuals with established allergic inflammation, IP-10 is capable of worsening preexisting asthmatic airway inflammation [51]. CAPE inhibited IP-10 production by crude mite extract-stimulated MoDCs, which could prevent the IP-10 induced airway inflammation in asthmatic patients under mite exposure. Therefore, CAPE inhibited cytokine and chemokine production by MoDCs, and the inhibition was extensive and may have influenced the T cell priming, activation, inflammatory cells recruitment and the further airway inflammation.

NF-κB activation may be responsible, in part, for increased expression of many inflammatory genes in asthma [52]. NF-κB normally binds to IκBα to inhibit NF-κB nuclear translocation from the cytosol to the nucleus for further enhancement of inflammatory and associated gene transcription. Once cells are exposed to inflammatory stimuli such as LPS, IκBα is phosphorylated by IKK complex and degraded, leading to the nuclear translocation of activated NF-κB [53‐56]. Different inhibitory results of CAPE on NF-κB signaling pathway were noted in the literatures. In T cells, the direct inhibition of NF-κB nuclear translocation [18, 20], and the indirect inhibition of IκBα phosphorylation [20] were noted. In these two articles, CAPE could inhibit IκBα resynthesis instead of cytosolic IκBα degradation [18, 20]. However, the inhibitory effect of IκBα degradation of CAPE was observed in human middle ear epithelial cells [57] and gastric epithelial cells [58]. CAPE inhibited IκB degradation in monocytic cells but not astroglial cells, in which the suppression of activated IKK was shown [59]. It seemed that in different cells and possible different concentrations of CAPE (10 μM ~100 μM) would result in different inhibitory mechanisms. In the present study, we found that CAPE inhibited IκBα phosphorylation (Figure 5A) and NF-κB activation (Figure 5B) in human MoDCs in a dose-dependent manner. CAPE could also inhibit IκBα degradation (see Additional file 1 Figure S1B). We speculate that the inhibition of IκBα phosphorylation leads to the inhibition of IκBα degradation, so the nuclear translocation of NF-κB is inhibited for further enhancement of inflammatory and associated gene transcription. Jayakumar et al. also reported that pretreatment with CAPE blocked NF-κB p50, NF-κB p65 nuclear translocation in human MoDCs [30]. Since the promoters of hIL-12 p35 and hIL-12 p40 gene contain κB binding sites [60], and previous studies implicates the specific role of NF-κB factors in IL-12 transcription [30, 61, 62], we also suggested that the inhibition of NF-κB signaling by CAPE might result in the decreased production of IL-12 p40 and p70. The MAPK signaling in the MoDCs maturation may also induce the inflammatory cytokine production [63]. Here, we found that CAPE did not appear to alter the MAPK signaling pathway in human MoDCs, while p38 MAPK and ERK were activated in C6 glioma cells by the treatment of CAPE [64]. Therefore, new experiments addressing the detailed mechanism of inhibition effect of CAPE in cytokine and chemokine production are required.

Upon maturation, DCs can present captured antigens to T cells to induce proliferation and cytokine production in T cells [27]. To mimic in vivo system, we chose "autologous" naïve CD4⁺ T cells for coculture to evaluate the ability of antigen presentation and T cell priming of MoDCs. Surprisingly, CAPE did not inhibit the cytokine production of T cells (Figure 4A) as seen in MoDCs. The lymphoproliferation assay (a marker of T cell activation triggered by APCs) [65] was not inhibited in both healthy subjects and allergic patients when co-cultured with CAPE treated DCs either (Figure 4B). This result might be explained by the fact that the coculture system was in a "renewed" culture medium (2% human AB serum of RPMI). Therefore, the effect of decreased cytokine and chemokine production by MoDCs on T cell priming would be abolished in the experiment. As CAPE did not inhibit the upregulation of MHC molecules and costimulatory molecules in mature MoDCs, the antigen presenting ability of MoDCs in the present study were not expected to differ.

It is interesting about the polarization of crude mite extract. It induced MoDCs to produce large amount of IL-12 p40 but little IL-10 in healthy subjects, and lower IL-12 p40 but higher IL-10 in allergic patients, which indicated that crude mite extract was Th2-prone in allergic patients but not in non-atopic healthy subjects. The phenomenon was also proved in cocultured naïve CD4⁺ T cells. The lower IFN-γ/IL-5 in allergic patients indicated a Th2 polarization while the higher IFN-γ/IL-5 in healthy subjects favored a Th1 polarization. The different polarizations by Der p-pulsed MoDCs due to the allergic status of the donors were also reported previously [35, 36]. These studies used purified Der p 1 for stimulation. They all found that IL-4 or IL-5 was increased in co-cultured autologous CD4⁺ T cells from mite-sensitized allergic patients while IFN-γ was increased in those from healthy subjects.

The major limitation of our present study is failure of mimicking microenvironment of DC-T cell interaction in vivo with cytokine and chemokine in which immunity can develop [66]. Therefore, the effect of the decreased IL-12, IL-10 and IP-10 production by MoDCs treated with CAPE in priming T cells cannot be surveyed in the present study. IL-12 is related to the T cell polarizing and T cell survival [67], thus the decreased IL-12 in microenvironment may influence the T cell activation and proliferation. To determine whether the decreased cytokine secretion of MoDCs would influence the T cells activation, we collected the supernatant of pulsed MoDCs mixed with serum free RPMI 1640 (1:1) to culture autologous naïve CD4⁺ T cells. The cytokine production and lymphoproliferation were inhibited in CAPE treated group (data not shown). However, the CAPE was in the supernatant and could directly inhibit the T cells activation through NF-κB and NFAT pathway [19, 20]. The decreased activation of T cells may be due to both the decreased cytokine and chemokine secretion from MoDCs and the CAPE direct effect. The other limitation is that the in vitro human MoDCs cannot represent the in vivo tissue DCs. To study the effect of CAPE on the function of circulating DCs will be the future work.

Our results showed that CAPE significantly inhibited IL-12 p40, IL-12 p70, IL-10 protein expression in mature healthy human MoDCs stimulated by lipopolysaccharides (LPS) and IL-12 p40, IL-10, IP-10 stimulated by crude mite extract. CAPE significantly inhibited IL-10 and IP-10 but not IL-12 expression in allergic patients' MoDCs stimulated by crude mite extract. In contrast, the upregulation of costimulatory molecules in mature MoDCs was not suppressed by CAPE. Therefore, the antigen presentation ability of MoDCs through MHC peptide complex and costimulatory molecules was not changed in this study after evaluating T cell lymphoproliferation and cytokine production. The mechanism of the inhibition of cytokine and chemokine production in MoDCs was thought to be related to NF-κB signaling, but not the MAPK pathway. The findings of this study provide new information and evidence for regulatory effect of CAPE on MoDCs relevant to the pathogenesis of allergic airway disease, such as asthma and allergic rhinitis. Further, it is possible to suggest that CAPE and propolis might be useful in the management of allergic disorders since CAPE inhibited cytokine and chemokine production in MoDCs and thus resulted in a decrease in downstream inflammatory process.