Mast cells are resident in tissues throughout the body, but are most common at sites that are exposed to the external environment, such as the skin, the airways, and the intestine. As such, mast cells are positioned to play a direct role in host defense against invading pathogens, in addition to their roles as modulators of other effector cell types [8
]. Recent studies have indicated that mast cells can function to kill bacteria or inhibit their growth by expression and secretion of the cathelicidin-related antimicrobial peptide CRAMP [9
]. As confirmed here, mast cell production of CRAMP is inducible by LPS [9
]. The aim of the present study was to define the signal transduction pathways regulating LPS-mediated induction in BMMCs. Since LPS-mediated induction of key mast-cell effector molecules is known to involve both MAPK and NF-κB signal transduction pathways [26
], components of these pathways were investigated.
It was found that BMMCs prepared from 2 strains of mice, C57BL/6 and BALB/c, contained easily detectable levels of CRAMP gene transcripts. Surprisingly, CRAMP expression was only inducible by LPS in the latter strain. It appeared that this lack of inducibility in C57BL/6-derived BMMCs was not due to a deficit of TLR4, the receptor through which LPS acts [32
]. The molecular basis for this differential inducibility in the 2 strains is currently under further investigation. In particular, it will be determined if the signal transduction pathways initiated by LPS binding differ in the 2 mouse strains. It will also be of interest to determine basal levels of CRAMP peptide in the medium of the 2 types of BMMCs, since some cathelicidins have been reported to bind to and hence neutralize LPS [13
]. It has also been proposed that LL-37 modulates the LPS/TLR4-mediated inflammatory response by inhibiting nuclear translocation of NF-κB, thus decreasing production of TNF-α and other cytokines [21
It was determined that CRAMP expression in BALB/c-derived mast cells was inducible by LPS, which also induces production of certain cytokines, including IL-13 [38
]. This is of interest since IL-13 (and IL-4) can reportedly suppress
induction of cathelicidin production by some cell types, such as antigen-exposed keratinocytes [40
]. In contrast, activation of mast cells with IL-4 appears to increase accumulation of cathelicidin protein [10
]. It was also reported that skin obtained from patients with atopic dermatitis have decreased cathelicidin LL-37 levels compared to normal skin and thus supports high levels of vaccinia virus replication, as is characteristic of eczema vaccinatum [40
]. Atopic dermatitis skin is characterized by overexpression of IL-4 and IL-13 [41
]. Thus, although mast cells may be a source of cathelicidins, as described above, their presence and activation in skin could in fact, through production of certain cytokines, result in suppression of production of the antimicrobial peptides by other cell types.
As noted above, LPS stimulation through TLR4 binding can activate both MAPKs and NF-κB, which then mediate specific gene expression [26
]. It was determined that LPS treatment (at 200 ng/ml) of BALB/c-derived BMMCs activated the 3 major MAPK modules, involving JNK1/2, ERK1/2 and p38 MAPK. LPS treatment did not appear to activate ERK5, a kinase which clearly plays an important role in mast cell activation through Fc
]. However, treatment with pharmacological inhibitors or specific siRNAs to decrease levels of the kinases indicated that JNK, ERK and p38 MAPK do not play significant roles in the LPS-mediated inducibility of CRAMP gene expression. The MEKK inhibitor PD98059, which prevents phosphorylation and activation of ERK, was used because specific siRNA constructs which specifically reduced ERK1/2 levels in BMMCs could not be identified. Finally, it was shown that overexpression of 2 upstream components of MAPK activation, MEKK2 and MEKK3, had no significant effect on CRAMP gene induction by LPS. The present study did not rule out the possibility that MAPKs might be involved in the translation, processing, or secretion of CRAMP peptides, all aspects of active CRAMP production which require further investigation.
In contrast to these results, examination of the butyrate-induced activation of LL-37 expression in colon epithelial cells showed that the MEK inhibitor U0126 blocked LL-37 expression whereas the p38 inhibitor SB203580 did not [43
], indicating a stimulatory role for ERK, but not p38 MAPK, in cathelicidin gene expression. A more recent study [44
] suggests that butyrate-induced expression of CRAMP is dependent on both ERK and p38 MAPK activities. However, it has also been noted that proinflammatory mediators, including LPS, do not upregulate LL-37 production in colon epithelial cells [45
], suggesting that the pathway described here in LPS-treated mast cells may differ from the butyrate-induced pathway in colonic epithelial cells. Cell type-specific differences in the pathways leading to cathelicidin gene induction have also been suggested by experiments showing that the MEK-ERK inhibitor U0126 blocked the butyrate-induced expression of LL-37 in gastric and colon carcinoma cell lines, but not in hepatocellular carcinoma cells [46
]. Other studies indicate that the induction of LL-37 expression in epidermal keratinocytes by enforced expression of ASK1, a regulator of keratinocyte differentiation, is dependent on p38 activity [47
] and that the induction of LL-37 expression in an epithelial cell line by exposure to Mycobacterium bovis
BCG requires MEK1/2 and p38 activation [48
The involvement of NF-κB activation in LPS-mediated induction of CRAMP was also investigated. The change in abundance of IκBα and IκBβ after LPS treatment of BALB/c BMMCs indicated that NF-κB activation occurred in the system used here. Degradation of IκBs permits release and translocation of NF-κB to the nucleus, where it acts as a transcription factor [34
]. The essential role of NF-κB activation in LPS-mediated CRAMP induction was then demonstrated by 2 approaches: (1) decreased abundance of the p65 component of NF-κB through the use of a specific siRNA effectively abrogated CRAMP induction, and (2) overexpression of an IκBα construct resistant to phosphorylation-dependent degradation also effectively abrogated CRAMP induction. In this regard, it is of interest that butyrate, an inhibitor of histone deacetylases which upregulates LL-37 transcription in colonic epithelial cells [43
], reportedly prevents NF-κB activation by suppression of proteosome activity and stabilization of IκBα in these cells [49
]. However, it has been noted that protein acetylation is involved in NF-κB regulation at multiple levels, including through direct acetylation of NF-κB p65 and p50 subunits, indirect modulation of IKK activity, and regulation of NF-κB-dependent gene accessibility, through histone modifications [50
]. Thus, in some systems, deacetylase inhibitors, including butyrate, have been found to potentiate NF-κB activation [51
]. The effects of histone deacetylase inhibitors on CRAMP gene induction in mast cells is under investigation; preliminary results indicate that the potent histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) [52
] does not induce CRAMP gene expression in mast cells (data not shown).
It is also informative to compare the kinetics of LPS-mediated induction of CRAMP mRNA accumulation with the degradation of the IκBα and IκBβ inhibitors of NF-κB. As shown in figure , increased CRAMP mRNA levels are first seen at about 90 min after LPS addition, which follows the transient decrease in IκBα levels seen in figure and correlates with the major decrease observed in IκBβ levels beginning at about 90 min. Taken together with the observation that overexpression of the degradation-resistant form of IκBα abrogates the LPS-induced increase in CRAMP mRNA (fig. ), the results suggest that degradation of IκBα is necessary but not sufficient for CRAMP mRNA induction and that full induction requires the subsequent degradation of IκBβ.
As noted above, studies of the involvement of MAPKs in cathelicidin gene expression have yielded conflicting results, which may reflect different modes of regulation dependent on species, cell or tissue type and/or the method of cell stimulation. Differences in the involvement of NF-κB in cathelicidin gene expression may also exist. Thus, despite the demonstration here of a clear role for NF-κB in LPS-inducible CRAMP gene expression in BMMCs, Buchau et al. [53
] have recently suggested that NF-κB may play a negative regulatory role in cathelicidin gene expression. Their studies were performed with human keratinocytes treated with pimecrolimus and TLR2/6 ligand. A possible negative regulatory role for NF-κB in LL-37 expression would also be consistent with studies with corneal epithelial cells which were exposed to Pseudomonas
after induction of tolerance by low-dose treatment with flagellin, which resulted in impaired activation of NF-κB (and also of p38 and JNK) pathways yet augmented production of LL-37 and β-defensin-2 [54
]. In contrast, it has been reported that defensin expression can be induced by NF-κB in both intestinal epithelial cells and keratinocytes [55
]. Clearly, resolution of these different observations will require further comparative analyses.
The results presented here indicate that LPS-mediated CRAMP induction in mast cells is strongly dependent on NF-κB activity, but not on activation of MAPK modules involving JNK1/2, ERK1/2, ERK5 or p38 MAPK. This is of interest as most other major effector functions expressed by mast cells appear to be at least partially dependent on MAPK modules [1
]. The strong dependence on NF-κB activity which was observed is also noteworthy as defects in the NF-κB signal transduction pathway can lead to immunodeficiencies and increased susceptibility to infection [58
], the latter of which could be due, at least in part, to decreased CRAMP production.