Mast cells from patients with AE show an enhanced activation

Figure 9. A schematic model for activation of MCs by M. sympodialis extract. Non-sensitized MCs release cysteinyl leukotrienes after activation with M. sympodialis extract (A). IgE-sensitized MCs release cysteinyl leukotrienes, degranulate and produce MCP-1 but not IL-6 upon addition of M. sympodialis extract, as indicated in bold letters (B-C). M. sympodialis extract enhances the degranulation of IgE-receptor cross-linked MCs and by signaling through TLR-2 modifies their IL-6 production, as indicated in bold letters (D-F).

Taken together, our results demonstrate that M. sympodialis extract causes release of cysteinyl leukotrienes from non-sensitized MCs. In IgE-sensitized MCs, the extract induced release of cysteinyl leukotrienes, degranulation, MCP-1 production and activation of the MAPK ERK 1/2. Moreover, M. sympodialis extract enhanced IgE-dependent MC activation, inhibited activation of the MAPK ERK 1/2 and altered IL-6 production in a dose-dependent manner through the TLR-2/MyD88 pathway. Our findings (summarized in figure 9) imply that the effects of Malassezia on MCs might exacerbate the inflammation in AE.

4.4 MAST CELLS FROM PATIENTS WITH AE SHOW AN ENHANCED

MCs from healthy controls (HC). Studies on human MCs have mostly been conducted using cord derived MCs (CBMCs) (Saito et al., 1995), since peripheral blood-derived progenitors have a low proliferation rate, thereby limiting the possibilities of investigating adult human MCs. However, a protocol was recently established for generating high numbers of skin-like MCs from human peripheral blood (Lappalainen et al., 2007), facilitating more extensive studies of the role of MCs from adult individuals in different human diseases. We used this protocol in order to generate MCs from both AE patients and HC, and the obtained MCs were thereafter exposed to M.

sympodialis extract.

Both types of MCs generated showed homogenous cell growth, viability and morphology throughout the culture period of 9 to 11 weeks. Despite this, enhanced amounts of the granule mediators’ ȕ-hexosaminidase, histamine, and tryptase were found in MCs from AE patients as compared to HC. Following IgE-receptor cross-linking or culture with M. sympodialis extract, however, similar levels of the mediators were released from MCs of both HC and AE patients. One can speculate that upon a amplified activation, the MCs from AE patients may retain an augmented response to degranulate compared to MCs from HC. In vivo data support this, by augmented levels of histamine in skin and plasma of AE patients (Ring and Thomas, 1989). The enhanced levels of histamine and tryptase in AE patients’ MCs could have an impact on disease severity in the AE skin, since histamine recruits effector cells into tissues and affects their maturation, activation and polarization, thereby contributing to chronic inflammation (Jutel and Akdis, 2007). The observed enhanced granule content of tryptase in MCs derived from AE patients might also contribute to AE-related itch, since evidence has emerged for the role of tryptase in the itch mechanism of AE, through activation of the proteinase-activated receptor-2 (PAR-2) on nerve cells (Steinhoff et al., 2003).

To further investigate the reactivity of MCs derived from AE patients compared to HC we determined their ability to release inflammatory cytokines following stimulation with M. sympodialis extract. Interestingly, only MCs derived from AE patients responded to M. sympodialis extract by releasing the inflammatory cytokine IL-6. This cytokine has been demonstrated to modulate the Th1/Th2 differentiation of CD4⁺ T cells by promoting Th2 differentiation (Diehl and Rincon, 2002), an imbalance that has been associated with AE pathology (Akdis et al., 2006). In addition, IL-6 has been reported to support human MC growth (Kinoshita et al., 1999) and to increase MC histamine content (Kinoshita et al., 1999). Collectively, these data suggest that Malassezia compounds diffusing through the ruptured skin of AE patients, might, by inducing the release of IL-6 from activated MCs, augment the inflammatory response and may further enhance MC density as observed in the lesional skin of AE patients (Damsgaard et al., 1997).

In comparison to IL-6, M. sympodialis extract enhanced the IL-8 production of IgE-receptor cross-linked MCs from both AE patients and HC. These results indicate that M. sympodialis could aggravate an already ongoing allergic response in AE. Although the MCs from HC and AE patients responded with IL-8 release in a similar manner, one should keep in mind that elevated levels of IL-8 positive MCs have been noted in AE skin compared to in healthy skin (Fischer et al., 2006), which may result in an increased IL-8 release in AE compared to in HC.

Furthermore, IgE-receptor cross-linking is more likely to occur in patients with AE, who are sensitized to a variety of allergens (Akdis et al., 2006).

To elucidate the mechanism of Malassezia activation, we further investigated the presence of the fungal recognition receptors TLR-2 and Dectin-1 on MCs derived from HC and AE patients. These two receptors were chosen, since we in paper III demonstrated that M. sympodialis can activate murine BMMCs to produce IL-6 through the TLR-2/ MyD88 pathway, and it has previously been shown that the fungal product zymosan can induce MC activation through the Dectin-1 receptor (Olynych et al., 2006). No increase in TLR-2 mRNA expression was evident after MC activation, whereas, following IgE-receptor cross-linking the Dectin-1 receptor was exclusively up-regulated in MCs from HC. This finding could indicate that MCs from AE patients are unable to increase their Dectin-1 mRNA expression upon activation, and this would generate a defect response against fungal infection.

Dectin-1 has also been suggested to stimulate proliferation and activation of T-cells (Kimberg and Brown, 2008), which suggests a decreased ability of MCs in AE skin to activate T-cells. The role for the observed Dectin-1 mRNA up-regulation upon IgE-receptor cross-linking in MCs from HC could possibly function as an increased defense mechanism during a parasitic infection with the purpose of increasing T-cell recruitment.

In order to verify that Dectin-1 is also present on MCs in the skin, we stained skin sections from AE patients and HC. As previously shown by others (Damsgaard et al., 1997; Horsmanheimo et al., 1994), we could confirm an increase of tryptase-positive MCs in the AE lesional skin compared to HC skin. We also found that a majority of tryptase-positive MCs in both HC and AE patients’ skin expressed Dectin-1. To our knowledge, the expression of Dectin-1 has not been investigated in human skin prior to this study.

The observed differences in our study between MCs derived from AE patients and HC suggests that MCs from patients with AE may have a different gene expression profile compared to MCs from HC. This suggestion concord with the findings of Gabrielsson et al. who described a difference in gene expression between in vitro monocyte-derived dendritic cells from AE patients compared to HC (Gabrielsson et al., 2004).

In conclusion, the findings presented in this study suggest that MCs derived from AE patients and HC are different regarding the amount of preformed granule mediators, their IL-6 secretion in response to M. sympodialis, and in their ability to up-regulate the fungal recognition receptor Dectin-1 upon IgE-receptor cross-linking. These observed differences in MC activity provide new insights into the pathogenic mechanisms underlying AE and indicate an inherent difference between MCs from HC and AE patients.