Skeletal muscle weakness occurs during severe infections and may even result in the development of respiratory failure and death due to weakness of the diaphragm and other respiratory muscles (5
). The CC chemokines such as MCP-1 are classically considered to be chemotactic factors for mononuclear cells, whereas KC belongs to the ELR motif (glutamate-leucine-arginine)-positive group of CXC chemokines involved in neutrophil recruitment (34
). We have recently found that both CC and CXC chemokines are highly upregulated in the septic mouse diaphragm in vivo (12
), and infiltration of the diaphragm by macrophages and neutrophils during sepsis has been previously implicated in the pathogenesis of sepsis-induced diaphragmatic weakness (5
). Increased chemokine expression, leukocyte infiltration, and weakness are also found in the diaphragm and other muscles of mice suffering from muscular dystrophy (11
). Therefore, it is reasonable to speculate that chemokine expression by skeletal muscles is likely to play a significant role in the pathogenesis of these conditions.
In the present investigation, we observed that differentiated myotubes (from C2C12 cells or primary cells) and intact whole muscles all expressed TLR2, TLR4, TLR5, and TLR9. Our TLR expression pattern findings are generally consistent with those of other investigators, although there are some differences. Frost and colleagues have reported that TLRs 1 to 7 (but not TLR8 or TLR9) are expressed in C2C12 myotubes (16
). This differs from our own findings with respect to TLRs 3 and 7 (not detected in our study) as well as TLR9 (detected in our study). However, these authors also reported a failure to achieve functional responses (IL-6 expression) upon stimulation with TLR3 and TLR9 ligands (17
), which is in agreement with the results of our study. Two other groups have recently reported TLR9 mRNA expression in human skeletal muscle tissue (32
) and human myoblasts (38
). The latter study also found TLR3 expression in cultured human myoblasts and in diseased muscles from myopathic patients but not in normal healthy muscle fibers (38
). It is likely that the slightly divergent findings among these investigations is related at least in part to differences in tissue culture conditions (e.g., duration and stage of differentiation, etc.) as well as possible species differences.
To our knowledge, the present study is the first to systematically evaluate the ability of the different TLRs to mediate CC and CXC chemokine expression by skeletal muscle cells. We assessed a broad range of different TLR-ligand interactions and noted a complex pattern in which certain TLRs expressed by the cells responded to stimulation in a straightforward manner (TLRs 2 and 4), while others required immune modulation by another molecule to respond (IFN-γ for TLR5) or did not respond at all (TLR9). The ability of IFN-γ to potentiate TLR responses (as shown here for TLR5) is well described in other cell types (19
). Therefore, one possible reason for the lack of responsiveness to TLR9 ligand in our study is a similar dependence upon another as yet unidentified immunostimulatory molecule. In addition, while many cell types respond when CpG DNA is applied at the cell surface (22
), the subcellular location of TLR9 is largely endosomal (26
). It is thus possible that skeletal muscle cells require additional measures or a more prolonged period of exposure to achieve intracellular uptake of CpG DNA and effective TLR9 engagement.
Not surprisingly, we observed that the NF-κB pathway is activated after both TLR2 and TLR4 stimulation of skeletal muscle cells. However, a novel finding in our study was the documentation of major differences in the degree of NF-κB activation induced by TLR2 versus TLR4 stimulation of skeletal muscle cells. Hence, at TLR ligand doses which were supramaximal with respect to chemokine gene induction, TLR4 stimulation induced more rapid and complete IκB degradation than was observed after TLR2 stimulation. In keeping with these findings, TLR4 stimulation was also associated with dramatically higher NF-κB luciferase reporter activity. In addition, interference with NF-κB activation during TLR4 stimulation had greater inhibitory effects upon chemokine gene expression. The compound PDTC, which blocks the E3 ubiquitin ligase involved in IκBα degradation (20
), greatly reduced the levels of both KC and MCP-1 expression triggered by TLR4 stimulation but had no effect upon MCP-1 expression induced by TLR2 stimulation. Because PDTC also has antioxidant properties, and chemokine expression can be increased by mechanisms which are dependent upon reactive oxygen species (27
), we also evaluated the effects of free radical scavengers. We employed two potent antioxidants, NAC and catalase, with documented abilities to inhibit reactive oxygen species-mediated cytokine upregulation in muscle cells at the doses employed in this study (25
). In contrast to PDTC, these antioxidants had no significant effects upon TLR2- or TLR4-mediated chemokine gene expression, suggesting that free radicals were not responsible for chemokine induction in our experiments. Furthermore, as in the case of PDTC, dominant-negative IKKβ significantly inhibited KC and MCP-1 expression induced by TLR4 stimulation, whereas for TLR2 stimulation, it had substantially smaller effects upon KC and no impact at all upon MCP-1 expression. Therefore, it appears that NF-κB activation is greater in magnitude during TLR4 stimulation and also plays a more important role in TLR4- than in TLR2-mediated chemokine expression by skeletal muscle cells.
It is important to note that, despite a significantly lower level of NF-κB activation in the case of TLR2 stimulation, the magnitude of MCP-1 and KC mRNA expression was equally robust when cells were exposed to supramaximal doses of either PGN or LPS. This finding indicates that TLR2 and TLR4 must recruit different signaling pathways to regulate the same chemokine genes in skeletal muscle. A plausible candidate for another signaling molecule is the calcium-dependent serine/threonine phosphatase calcineurin, which is known to be expressed by skeletal muscle cells, where its actions in favoring the nuclear translocation of NFAT have been implicated in the regulation of skeletal muscle growth, differentiation, and specialization (9
). In activated T lymphocytes, calcineurin inhibition by FK506 has been found to have differential effects on chemokines, with several CC chemokines being repressed to variable degrees (MIP-1α, MIP-1β, RANTES), whereas a CXC chemokine (IP-10) actually demonstrated upregulation (41
). In addition, calcineurin has been reported to upregulate MCP-1 expression in vascular smooth muscle cells by augmenting mRNA stability (36
Here we show for the first time that calcineurin plays a major role in the regulation of chemokine expression by skeletal muscle cells. In this regard, we found that TLR2-mediated induction of both MCP-1 and KC was markedly attenuated by the calcineurin inhibitor FK506. On the other hand, during TLR4 stimulation, FK506 had a less pronounced inhibitory influence on KC expression, and no significant effect on MCP-1 expression could be demonstrated. Therefore, the impact of calcineurin inhibition on chemokine expression was substantially greater for TLR2- than TLR4-mediated responses, which is in direct contradistinction to the results obtained during NF-κB inhibition as discussed earlier. For MCP-1 in particular, there was a high degree of TLR-dependent differential regulation by NF-κB and calcineurin. Our findings are reminiscent of recent observations in human airway epithelial cells, in which it was reported that another calcineurin inhibitor, cyclosporine, achieved almost complete abrogation of TLR2-mediated induction of IL-8 (CXCL8) expression, whereas responses to heat-killed gram-negative bacteria (presumably TLR4 mediated to a large extent) could be inhibited by about 25% only (51
). This suggests that calcineurin may play a predominant role in the mediation of TLR2-stimulated chemokine responses not only within skeletal muscle but within other cell types as well.
It is of interest to consider the potential effects of calcineurin on several different transcription factors which could be involved in MCP-1 or KC gene regulation. One possibility would be an effect of calcineurin on NF-κB itself (46
), particularly since forced overexpression of activated calcineurin in C2C12 cells has been reported to increase NF-κB activity (2
), and mitochondrial stress can also lead to NF-κB activation in C2C12 cells through a calcineurin-dependent mechanism (4
). However, FK506 produced substantially less inhibition of chemokine expression during stimulation of TLR4 relative to TLR2, despite the fact that NF-κB activity during TLR4 stimulation was an order of magnitude higher than for TLR2. Therefore, it seems unlikely that the major effects of calcineurin inhibition observed during TLR2 stimulation were mediated through an effect on NF-κB. Calcineurin also has the potential to modulate several other proinflammatory signaling pathways which could be involved in TLR-mediated chemokine gene expression by skeletal muscle cells, such as NFAT, CREB, and C/EBP (18
). The latter, in particular, has been strongly implicated in transcriptional regulation of both MCP-1 (1
) and KC (7
) gene expression.
Recently, it has become apparent that chemokines have important biological functions in skeletal muscle which extend well beyond their classical roles as leukocyte chemoattractants. In particular, there is accumulating evidence for a significant role in muscle regeneration following injury (49
). In animal models of acute and subacute sepsis, skeletal muscle fiber injury has been demonstrated (14
). Recovery from injury involves myoblast precursors (called satellite cells) that are normally quiescent in adult skeletal muscle but which become activated to proliferate and migrate to form new muscle fibers (regeneration) when skeletal muscle is damaged (23
). It has recently been demonstrated that CCR2 (the major receptor for MCP-1) is expressed by skeletal muscle cells in vivo, and both CCR2 and MCP-1 are required for optimal functional recovery from injury (49
). In addition, RANTES (CCL5) has been shown to be a chemotactic factor for myoblasts (8
), and the CXC chemokine LIX (LPS-induced CXC chemokine) is expressed in satellite cells shortly after induced muscle injury (35
). Therefore, although the blocking of chemokine expression in muscle during sepsis could mitigate leukocyte-mediated adverse effects, there is also the potential for interference with muscle repair mechanisms. Accordingly, the rational design of therapeutic interventions in this area will require a detailed understanding of the roles played by specific chemokines in skeletal muscle during different stages of sepsis and recovery from injury. Finally, in view of the possible role of TLRs in sensing other types of cellular injury or stress beyond those associated with infectious insults (48
), it will be of considerable interest in future studies to determine whether TLR-mediated signaling plays a role in the augmented skeletal muscle chemokine gene expression found in noninfectious pathological conditions such as the muscular dystrophies and inflammatory myopathies.