In this study, we have shown that the following occur during infection of macrophages with L. monocytogenes: (1) an extracellular bacterial signal-dependent pathway is activated, leading to IL-1α gene expression and accumulation of pro-IL-1α in macrophages at an early stage of L. monocytogenes infection; (ii) secretion of the mature form of IL-1α at a late stage of L. monocytogenes infection is critically dependent on LLO-mediated bacterial escape from the phagosome; and (iii) entry of bacteria into the cytoplasm induces an increase in the intracellular calcium levels, resulting in the activation of a calpain protease that is necessary for the processing and secretion of mature IL-1α (Fig. ).
FIG. 8. Proposed model of IL-1α secretion in L. monocytogenes-infected macrophages. Upon L. monocytogenes infection, activation of the TLR/MyD88 signaling pathway induces IL-1α mRNA expression and pro-IL-1α synthesis independently of bacterial (more ...)
The TLR/MyD88-dependent signaling pathway is known to play a critical role in the induction of a variety of proinflammatory cytokines, including TNF-α, IL-12, IL-6, and IL-1β (18
). In the present study, we show that this extracellular pattern recognition receptor system can induce gene expression of IL-1α. However, this signal is unable to induce maturation of accumulated pro-IL-1α and secretion of mature IL-1α. Since MyD88 is an adaptor protein indispensable for signaling through TLRs, one or more of the TLRs may be involved in this process. Although TLR2 is known to be the major receptor recognizing Gram-positive L. monocytogenes
, we found that IL-1α secretion was not completely abolished in macrophages from TLR2−/−
mice (data not shown). It seemed that a number of TLRs, including TLR2, are engaged in the recognition of ligands of L. monocytogenes
, resulting in the induction of IL-1α mRNA expression.
The requirement for a further processing step has been well documented in the maturation and secretion of cytokines belonging to the IL-1 superfamily (3
). We have previously shown that LLO-dependent bacterial escape is critically important for the activation of caspase-1, required for the maturation and secretion of IL-18 (26
). The present study also indicated that LLO-dependent bacterial escape is essential for the maturation of IL-1α. On the other hand, Keller et al. showed that active caspase-1 contributes to secretion and processing of IL-1α in macrophages stimulated with LPS and ATP (31
). However, we did not detect any defect in the secretion of IL-1α in caspase-1−/−
macrophages after L. monocytogenes
infection. The discrepancy might be due to differences in the properties of stimuli. In their study, IL-1α was secreted from macrophages stimulated with LPS and ATP in a caspase-1-dependent manner, while to some extent, IL-1α was secreted caspase-1 independently. It appears that L. monocytogenes
possesses an ability to induce caspase-1-independent IL-1α secretion, although the molecular mechanism remains to be solved.
Compared to extensive studies on caspase-1 activation followed by processing and secretion of IL-18 or IL-1β in macrophages infected with L. monocytogenes
, the available information on IL-1α secretion has been limited. The results of our study have clearly indicated that IL-1α secretion in L. monocytogenes
-infected macrophages is not dependent on the formation of an ASC-containing inflammasome but is due to calcium-dependent protease activation, which was highly dependent on cytoplasmic entry of L. monocytogenes
. Calpain was described more than 40 years ago as a calcium-activated neutral protease originally detected in various tissues, including brain (24
) and skeletal muscles (35
). Now calpain is defined as an intracellular calcium-dependent cysteine protease, which is ubiquitously distributed, and more than 15 calpain proteins comprise the calpain family (23
). Calpain is reported to have two distinct domains that are similar to papain-like protease and calmodulin-like calcium-binding protein (39
). In experimental systems other than bacterial infection, there is increasing evidence that calpains participate in a variety of signal transduction pathways and function in important cellular processes (23
). Calpain gene disruption has revealed an essential role of calpain in embryonic development (4
). Mutation in a particular human calpain has been implicated in the progression of neuromuscular degeneration (40
). Thus, calpains appear to be involved in a variety of processes in mammalian development. So far, very few biochemical studies have proposed the involvement of a calcium-dependent calpain protease as the enzyme required for the processing of pro-IL-1α (32
). In this regard, our present finding may have added a novel insight into the process of L. monocytogenes
-induced maturation of IL-1α. There is no doubt that LLO-dependent cytoplasmic entry is critically involved in the elevation of the intracellular calcium level resulting in calpain activation. In addition, specific inhibitors for calpain I and calpain II exhibited only marginal effect, suggesting that a member of the calpain family other than calpain I or calpain II may be involved in this process. However, we cannot exclude the possibility that other calcium-dependent cysteine or cathepsin proteases may contribute to the processing of IL-1α either alone or in combination with calpain protease.
A number of previous studies have documented the elevation of intracellular calcium levels, especially at the very early stage of infection with LLO-expressing L. monocytogenes
strains in mast cells, HEK cells, and J774 cells (16
). In all these reports, a quick and transient response in the increase in intracellular calcium was observed in a system employing in vitro
infection with an extremely high number of L. monocytogenes
cells (MOI = 100). Since pro-IL-1α has not yet accumulated immediately after the L. monocytogenes
infection, it is unlikely that this early calcium signaling actually contributes to the maturation of IL-1α that was observed in the later phase of infection. Under the present experimental condition using a MOI of 5, it was not possible to observe such a quick response as that reported, but instead, we have observed a significant response at the later stage. This long-lasting elevation of the intracellular calcium level at the later stage has not been described yet. After infection of macrophages in vitro
with L. monocytogenes
at MOI of 5, the entry of L. monocytogenes
into the cytosol was observed as early as 60 min when determined by comet tail staining under a fluorescence microscope. Thereafter, a gradual bacterial multiplication was noted (data not shown). Taking the into consideration that a long-lasting elevation of the intracellular calcium level started about 13 h after infection, it is likely that the accumulation of bacterial ligands inside the cytoplasmic space initiates the effective cellular response for calcium elevation. The absence of IL-1α processing in an experiment using bafilomycin A1 and the mutant Δhly
strain indicated that bacterial entry into the cytoplasm is a prerequisite for the increase in the intracellular calcium level at the later stage. We also found that IL-1α secretion was reduced when L. monocytogenes
infection was done in the presence of chloramphenicol (data not shown). This suggests that either intracellular bacterial growth or protein synthesis is essential for this process. It is not yet clear whether LLO itself is required or bacterial components other than the LLO molecule also contribute to the elevation of the calcium level, since bacteria never enter and multiply inside the cytoplasm in infection with the mutant strain and the wt in the presence of bafilomycin A1. Although our results demonstrated an LLO-dependent increase in the intracellular calcium level at the late stage, the precise mechanisms of the activation of this cytoplasmic entry-dependent calcium signaling remain to be analyzed. It has been reported that LLO forms a calcium-permeable pore, leading to intracellular calcium oscillations in mast cells and HEK cells (21
). We here showed that membrane perturbation induced by LLO initiated a calcium influx necessary for the processing and secretion of IL-1α. Recently Gekara et al. reported that LLO-expressing L. monocytogenes
infection induces calcium influx from the extracellular milieu and the release of calcium from intracellular stores by injury to the endoplasmic reticulum (21
). However, again these mechanisms were reported to be involved in an early increase in intracellular calcium levels. Since the LLO is known to be activated in an acidic environment in phagosomes but is not activated at a neutral or weakly alkaline pH in the cytosol of host macrophages (38
), other bacterial factors and processes after cytoplasmic entry may play a role in the elevation of calcium in this late stage of infection. Further studies are currently under way to elucidate the exact mechanisms involved. It will be intriguing to determine in the further study whether such a late-stage increase in intracellular calcium levels plays a role in other calcium-dependent cellular processes, which are reported to occur with cholesterol-dependent cytolysins/pore-forming toxins (8
There are several articles on the contribution of calpain to the host cell response to extracellular bacteria. Muller et al. reported that neisserial porin caused a rapid calcium influx and apoptosis in target cells through calpain activation (36
). A similar finding on the induction of calcium influx and apoptosis through calpain activation was found during group B Streptococcus
). Based on these reports, the possibility may be raised that calpain-dependent host cell death, if any, is involved in the secretion of mature IL-1α after processing in L. monocytogenes
-infected cells. Though we have not monitored the changes in membrane permeability or membrane damage in infected macrophages since the main focus of the present study was on calpain activation for IL-1α processing, such a possibility could be ruled out because we never detected the presence of the processed mature form of IL-1α in the cell lysate by Western blotting in macrophages which do not secrete mature IL-1α. Processing of pro-IL-1α into the mature form of IL-1α and the secretion of mature IL-1α seem to occur fundamentally as a single event governed by an activated calpain protease(s).
In conclusion, our study clearly indicated that L. monocytogenes-induced IL-1α secretion depends on two steps, LLO-independent expression/production of pro-IL-1α and processing/secretion of mature IL-1α that is induced by LLO-mediated cytoplasmic entry-dependent elevation of intracellular calcium, resulting in calpain activation. These findings may provide further insight into the interaction between the virulence factor of L. monocytogenes and the host cellular response. The overall effect of this mechanism observed in vitro in the context of the in vivo response needs to be addressed in a future study.