This study clearly shows that mast cells can function as effector cells in both host defense and pathogenesis of allergic disease through the direct activation of TLR4 and TLR2 on mast cells by microorganisms.
Mast cells represent a potential source of multifunctional cytokines that may participate in the recruitment and activation of other cells in the inflammatory microenvironment (27
). Mast cells responded to PGN or LPS via TLR2 or TLR4, respectively. Mast cell responses to LPS are completely impaired in the absence of the TLR4 but not the TLR2 molecule, suggesting that TLR2 is not involved in LPS response in mast cells and that the LPS-induced TLR4 responsiveness was not affected by the existence of the TLR2 molecule. A reciprocal result was obtained in PGN-TLR2 stimulation of mast cells.
Interestingly, upon PGN stimulation, mast cells released more IL-4 and IL-5, less TNF-α, and no IL-1β, even though TNF-α and IL-1β were predominant cytokines produced by mast cells upon LPS stimulation via TLR4 (18
). In addition, PGN could induce degranulation of mast cells via TLR2 accompanied by Ca2+
influx, whereas the activation of mast cells by LPS via TLR4 did not lead to degranulation (18
). So far, we still do not know how TLR2 is different from TLR4 in the signaling pathways of mast cell activation. To the extent that we analyzed the phosphorylation pattern of IκB-α and mitogen-activated protein kinases, we did not observe clear differences in TLR2 and TLR4 signaling pathways in mast cells (Figure and data not shown), even though the severely impaired proinflammatory cytokine production of TLR2- or TLR4-deficient mast cells was correlated with the loss of NF-κB activation. Although the intracellular domain of TLRs is highly conserved in each TLR (29
), recent findings suggest that downstream events are not always mediated by common components such as MyD88, an adaptor protein that links to the IL-1R–associated protein kinase and to the TNF receptor–activated factor 6 (30
). As TLR4 can activate both MyD88-dependent and -independent pathways that lead to different induction of genes in macrophages upon LPS stimulation (32
), it is possible that TLR2 and TLR4 use different pathways for activation of mast cells. While TLR4 recognizes relatively few ligands, TLR2 recognizes a wide variety of infectious pathogens (34
). Since it is reported that ligand specificity and signal-transducing ability of TLR2 are determined by heterodimeric interactions with other TLRs, such as TLR6 or TLR1 (35
), it would be useful to determine whether heterodimeric interaction of TLR2 and TLR6 is also required in mast cells. In particular, degranulation is a quite specific biological reaction confined to mast cells; it would be interesting to determine the TLR2-mediated specific signaling pathway that leads to this biological response. A recent report suggests that JAK3 is a key regulator of mast cell–mediated innate immunity, especially TNF-α production from mast cells after E. coli
stimulation and clearance of GNB (37
). Thus, it would also be helpful to know the relationship of JAK3 activation and TLR signaling in mast cells upon stimulation with GNB.
Mast cell–deficient WBB6F1
mice and the model of “mast cell knock-in” mice could be used to analyze the roles of mast cells in biological response in vivo. Consistent with previous work (18
mice were successfully reconstituted with BMMCs from genetically unmatched TLR4+/+
, or TLR2+/+
mice that are a mixture of C57BL/6 and 129SvJ strains. During the study period, the reconstituted mice did not show any abnormality. The mast cells in both peritoneal cavity and skin 5 weeks after reconstitution were no different in number, ability to develop, and ability to degranulate in vitro. Therefore, the differences in the mortality of mice after CLP and in the acute skin reaction after PGN injection were not due to the difference in development of BMMCs in the W/Wv
environments. To determine why mast cells can survive and differentiate in genetically unmatched environments, further investigations are required.
We have previously demonstrated that BMMCs from C3H/HeJ mice, which had constitutive mutation of the intracellular domain of TLR4, show impaired response to LPS by means of cytokine production and NF-κB activation. Our present study using TLR4–/–
mice, which lack whole molecules of TLR4, had results similar to those of C3H/HeJ mice. This again clearly indicates that TLR4 but not TLR2 is the critical receptor for activation of mast cells by LPS both in vitro and in vivo. Compared with in vitro results where BMMCs from TLR4–/–
mice completely lacked the LPS responses, determined by proinflammatory cytokine production and NF-κB activation, it seemed that W/Wv
mice showed a better survival rate than unreconstituted W/Wv
mice in the CLP model. These differences may reflect the involvement of other TLRs or other molecules, such as CD48 and C3 receptors, in activation of mast cells upon whole-microorganism stimulation in vivo (38
Since PGN caused degranulation and cytokine production of mast cells in vitro, the role of mast cells in PGN-induced cutaneous acute response was investigated. Wheal reaction was observed within 5 minutes after PGN challenge, but not after vehicle injection, and was gradually enhanced after 15 minutes. We used a mast cell knock-in system with W/Wv
mice to exclude the possibility that mediator released from other endogenous cells that express TLR2 can cause increased vascular permeability. The skin reactions were observed within 5 minutes and were not observed in TLR2–/–
mice, or W/Wv
mice reconstituted with TLR2–/–
BMMCs, strongly suggesting that degranulation of mast cells through TLR2 activation was responsible for these reactions. In fact, we could observe the highly degranulated mast cells 15 minutes after PGN application in the skin of wild-type, W/Wv
/WT, and W/Wv
mice, but not in the skin of TLR2–/–
mice, in the histological examination (Table ). Furthermore, TLR4–/–
mice and W/Wv
mice showed responses similar to those of wild-type mice, suggesting that TLR4 (on mast cells or other cells) in the skin is not involved in PGN-induced vasodilatation. In addition, application of LPS in the skin did not lead to degranulation of mast cells or vasodilatation similar to that induced by PGN, suggesting that response to LPS in the skin has a biologically different character from that of PGN-induced response. Although it has been reported that mast cells can be activated by GPB through IgE bound on cells (40
), this is, to our knowledge, the first evidence that shows the direct activation of mast cells via a specific pathogen recognition receptor, TLR2.
In summary, this study clearly shows that TLR4 but not TLR2 is the important molecule for mast cells in response to LPS challenge and that it functions in innate immunity against enterobacterial infection. In contrast, TLR2 but not TLR4 is responsible for activation of mast cells by S. aureus that might be involved in the pathogenesis of atopic dermatitis. Finding the microbial ligands and relevant receptors on mast cells, as well as signaling pathways that can activate mast cells, will elucidate the complexity of innate immunity and the roles of mast cells in conditions other than allergy. Also, the clarification of the mechanisms of mast cell activation by microorganisms through these TLRs may offer new insight into the treatment of allergic disease or inflammation in which bacterial infections and mast cell activations are common.