The activation of DCs plays a critical role in the development of T cell–mediated immune responses. Our previous work has made it clear that low-molecular weight degradation products of HA (sHA) produced at sites of inflammation are key players in DC activation (5
). The exciting outcome of the study presented here is that the maturation and activation of human and murine DCs in response to sHA is dependent on a functional TLR-4 receptor complex. Thus, sHA was able to mediate activation of bone marrow–derived DCs from wild-type C3H/HeN and C57BL/10Sn mice but not of DCs from the respective closely related C3H/HeJ and C57BL/10ScCr mice, which on account of defects in TLR-4 are LPS nonresponder and exhibit no inflammatory response to endotoxin. In contrast, DCs from TLR-2–deficient mice retained the ability to respond to sHA. Furthermore, blockade of TLR-4 on human monocyte–derived DCs abrogated their ability to produce TNF-α in response to sHA. The stimulatory effects of sHA on DCs were mediated by components of the signaling pathway activated by TLR-4, including the phosphorylation of p38 and p42/p44 MAPK and the translocation of NF-κB to the nucleus.
It is becoming increasingly evident that subcomponents of the extracellular matrix, especially of those built of repeating disaccharide units, can have immunomodulatory functions. NF-κB and intracellular adhesion molecule 1 are induced in endothelial cells by dermatan sulfate, a substance with high structural homology to HA (34
), and low molecular weight heparan sulfate can modulate the migration of epidermal Langerhans cells, the immature precursors of skin DCs (35
). Nevertheless, it remains unclear whether these low molecular weight fragmentation products use the same or different receptors to exert their effects compared with the intact parental macromolecule. HMW-HA binds to CD44 and RHAMM (36
), but we and others could exclude binding of sHA fragments to these receptors (5
). Here we show for the first time that small fragmentation products of HA use receptors and signaling cascades which are different from those used by HMW-HA.
The concentrations of sHA needed to induce TLR-4–mediated signaling in DCs are several orders of magnitude higher than for LPS. This makes sense, as HA is a physiological constituent of the organism and minute amounts of degradation products enter into the bloodstream to be further degraded in the liver. Interestingly, higher levels of circulating HA are found during sepsis, correlating with disease severity and prognosis (38
). For these reasons, it seems most plausible that only high concentrations of sHA, occurring locally and exclusively at sites of inflammation, induce proinflammatory signals. Our preliminary data using suction blister fluid (24
) suggest that small glycosaminoglycan fragments of 4–6 oligosaccharides in size are indeed produced locally in inflamed skin (data not shown), consistent with this hypothesis.
There are many similarities between activation of DCs by LPS and by sHA. For example, DCs from TLR-4–deficient mice are nonresponsive to both LPS and sHA, but TLR-2–deficient DCs are still activated by both substances. Therefore, we performed extensive controls to exclude the possibility that our sHA preparations were contaminated with LPS. In this regard, it is also significant that while there are similarities between the signaling pathways induced by LPS and sHA, a critical difference is that LPS induces PI3-kinase (39
), whereas we show here that sHA does not. Moreover, ligation of the TLR-4 complex by LPS in mouse macrophages occurs very rapidly, with the receptor being downregulated within 1 h after stimulation (40
). In contrast, we found that both the intracellular signaling and TNF-α protein synthesis by sHA-stimulated DCs are notably delayed for ~5–6 h in comparison to LPS. Thus, although we cannot completely rule out the possibility that sHA might act synergistically with trace amounts of LPS present in our sHA preparations which are below the threshold of detection and which alone cannot mediate DC maturation, a much more likely interpretation of our results is that sHA is an independent inducer of DC activation which signals via TLR-4.
It remains to be shown how sHA induces TLR-4 signaling at the molecular level. Recent results demonstrate that TLR signaling is dependent on a complex series of protein interactions, which is well exemplified by LPS signaling via TLR-4 (13
). In addition to TLR-4, the serum protein LBP and the GPI-linked cell surface protein CD14 are required for the host response to LPS (16
). LPS binds to LBP, which in turn binds to CD14. The LPS-LBP-CD14 complex is then thought to interact with TLR-4, perhaps directly (15
), which in turn induces TLR-4 signaling via MyD88 and IRAK. TLR-4 also appears to contact LPS directly within the multiprotein complex. A further protein called MD-2 is also required for LPS-induced signaling (23
). This secreted extracellular protein binds to the extracellular portion of TLR-4 and possibly stabilizes TLR-4 dimers (23
). The complex molecular interactions involved in TLR signaling appears define the range of substances to which a given TLR can recognize, with the specificity determined at least in part by the TLR accessory molecules which bind to the ligand (42
In sHA-induced TLR-4 signaling, sHA might bind to known TLR-4 accessory molecules, but more likely to as-yet unidentified ones. This latter possibility is underscored by structural considerations: LPS and sHA share almost no homology, except for the highly diverse repeating sugar units at the end of the LPS molecule (43
). However, the binding of LPS to the TLR-4 complex and its activating effect on macrophages is mediated by the Lipid A moiety of the molecule (43
). Furthermore, other substances known to bind the TLR-4 complex show high diversity. Human heat shock protein 60 can induce a TLR-4 complex–dependent activation of macrophages by upregulation of adhesion molecules and the release of proinflammatory mediators (20
). Taxol, a dipertene purified from the bark of Taxus brevifolia
, also mediates a TLR-4– and MD-2–dependent activation of murine macrophages. Interestingly this effect could only be observed with murine cells, but not with human LPS responsive cells (21
). Therefore, it is significant that we found sHA to effectively activate both murine and human DCs which is suggestive of a highly conserved mechanism, since human TLR-4 has been described as being more effective in the discrimination of possible ligands (21
). Future work will focus on dissecting the way in which sHA interacts with components of the TLR-4 complex to induce the signal transduction which leads to DC maturation.
It is clear from the data presented here and from our previous studies (5
) that the sHA-induced DC activation is dependent on the induction of TNF-α secretion. Our data suggest that TLR-4 is required for this induction, as TLR-4–deficient DCs did not produce TNF-α in response to sHA, and an anti–TLR-4 antibody was able to block sHA-mediated TNF-α release by wild-type DCs. However, the anti–TLR-4 antibody had no effect on TNF-α production if given 7 h after sHA treatment ( A). This coincides with downregulation of TLR-4 expression in response to sHA, although there is clearly some TLR-4 on the surface of DCs 24 h after sHA treatment ( B and C). These data are consistent with the notion that TNF-α produced by DCs in response to sHA binds to the TNF receptor I on the same cells, which in turn delivers a signal that produces more TNF-α (45
The current understanding of TLRs and their associated molecules suggests that these multi-protein complexes developed in lower organisms such as Drosophila to provide innate immunity by recognizing conserved motifs present on pathogens. Therefore, it is fascinating that the data presented here suggest that the same molecular machinery involved in the innate immune response has perhaps been evolutionarily coopted to activate the immune response in response to degradation of endogenous extracellular matrix components during inflammation. Further study of the TLRs and the molecules they interact with will undoubtedly reveal many more new and exciting insights into the molecular mechanisms which regulate immunity.