Endogenous TLR ligands and products of tissue destruction

It is now recognised that in addition to binding PAMPs, TLR's can also recognise endogenous signals released from injured tissue and cells undergoing necrotic cell death, such as HSP60. There are already examples of endogenous molecules signalling through TLR2, TLR3, TLR4 and TLR9. HSPs are recognised by TLR2 and TLR4, in particular HSP60 [69], HSP70 [70] and gp96 [71], which are released by cells undergoing necrosis. HSP-peptide complexes are able to elicit peptide specific CD8+ T cell responses without adjuvants [72-74] as well as delivering an endogenous maturation signal to antigen presenting dendritic cells (DC) [69-71].

DCs stimulated with heat shock protein gp96 produce similar levels of proin-flammatory cytokines, such as TNF-a, as DCs stimulated with bacterial LPS [71]. Similar results were obtained with macrophages treated with hsp60 and hsp70 [69, 70]. In murine DCs and macrophages HSPs signal in a MyD88 and TRAF6 dependent manner. Signalling through TLR2 is dependent on endocytosis of HSP60/70 and gp96 where as signalling via TLR4 is dependent on the co-receptor MD-2, similar to LPS signalling via TLR4. Studies showed that gp96 signals specifically through TLR2 and TLR4 while TLR3, TLR7, TLR8 and TLR9 showed no responsiveness implying specificity (in gain of function) [69-71]. However in studies of human macrophage-colony stimulating factor (M-CSF) derived macrophages, TLR4 signalling does not appear to employ MyD88 or Mal [75].

However, other studies have suggested that highly purified HSP70 does not induce TNF-a release from murine macrophages. The observed TNF-a inducing activity of HSP70 previously reported [70] has been suggested to be due to contamination of samples with LPS [76].

Another endogenous ligand that can activate TLR4 is cellular fibronectin, produced in response to tissue damage [77-80]. Okamura et al. were able to demonstrate that extra domain A (EDA)-fibronectin uses TLR4 as a receptor for the activation of a transfected TLR4 human embryonic kidney (HEK) 293 cell line [81]. Fibronectin contains alternatively spliced exons encoding type III repeat EDA and EDB. In response to exposure to EDA or EDB-containing fibronectin, synovial cells start producing proinflammatory cytokines and MMPs [79].

Infected cells are a source of endogenous TLR ligands. They release cytokines and chemokines as well as antimicrobial peptides, such as defensins [82-84]. One family member, P-defensin, functions as a chemoattractant of immature DCs through the CC chemokine receptor (CCR) 6 [85, 86]. In addition to this function it was shown that murine P-defensin2 acts directly on immature DCs through TLR4, inducing upregulation of co-stimulatory molecules and maturation of DCs [87]. As a consequence the immune response shifts to a Th1 dominated response, suggesting that P-defensin plays important role in the surveillance of pathogens. P-defensin may also act as a counter measure to suppressive microbial factors by generating a full scale Th1 immune response in the form of positive feedback.

Pre-packaged signals, which induce APC activation through TLR's, have been identified. These pre-packaged signals are released by cell necrosis, which could be induced by tissue damage, stress factors or infection, resulting in the release of cell components and the induction of an inflammatory response [88]. Apoptotic cells however retain their membrane integrity and are rapidly cleared by macrophages before lysis and therefore do not induce an inflammatory response [89]. Hence, DCs are activated by exposure to necrotic cells but not apoptotic cells [90, 91]. Recently the receptor and signalling molecules responsible were discovered. NF-kB activation in the RAW macrophage cell line through exposure to necrotic cells requires MyD88 and TRAF6 signalling from TLR2, while TLR4 and TLR6 were not required [92]. mRNA [93] and dsDNA [94] which can activate TLRs have been identified from necrotic cells. Additionally, IgG-chromatin complexes [95] were also shown to activate B cells via TLR's.

HEK293 cells stably expressing TLR3 can be stimulated by dsRNA leading to activation of NF-kB and the secretion of IL-12, IL-8 and IFN-a. DCs also respond to dsRNA by expressing activation markers, which can be inhibited, by a TLR3 specific antibody. DCs treated with endogenous dsRNA released from necrotic cells have been shown to produce IFN-a, this can be inhibited by pre-treating necrotic cells with RNase [93].

Similar results are obtained using dsDNA. Genomic dsDNA triggers the maturation of DCs and macrophages as measured by upregulation of MHC class I and II. Unmethylated CpG motifs do not appear to be responsible for the immune response induced by genomic dsDNA. The nature of this response also differs between bacterial CpG DNA and double-stranded murine DNA, the latter does not induce cytokine production. It has been hypothesised that genomic DNA may promote host survival by improving immune recognition of pathogens at sites of tissue damage or infection. However it is still unclear through which receptor this signal is transmitted. One candidate is TLR9, which recognises CpG DNA. It is a strong candidate as it is capable of recognising self-DNA (IgG-chromatin) complexes in B cells [95]. Endogenous DNA on its own is normally inert [96]. However, activation of the antigen receptor on B cells primes the cells so that TLR9 is able to be stimulated by endogenous DNA. The defining difference between bacterial and endogenous DNA is bacterial DNA is unmethylated whilst endogenous DNA has 70-80% of its CpGs methylated. Interestingly cells from autoimmune mice and humans show a decrease in this methylation [97] but elimination of methylation from murine DNA does not enable it to stimulate B cells [98]. So the mechanism by which bacterial and endogenous DNA activates TLR9 appears to be more complex than simply methylation.

Other endogenous TLR ligands can be generated from the remodelling or destruction of the extracellular space. Polysaccharide fragments of heparan sulfate as well as oligosaccharides of hyaluronic acid (HA) activate DCs as measured by co-stimulatory protein expression, morphology and T cell stimulation. Heparan sulfate is part of the cell membrane as well as of the extracellular matrix (ECM) [99]. While soluble heparan sulfate is not found in healthy tissues in significant amounts, its level rises in the extracellular fluid around wounds [100], the synovium of arthritic joints [101] and the urine of patients with infections [102]. HA is the major component of the ECM, at sites of inflammation it is rapidly degraded [103, 104]. Inhibition of TLR4 or use of mutated TLR4 in DCs inhibited this response to soluble heparan sulfate [105] as well as to HA [106] indicating a role for TLR4 in the recognition of these molecules. The molecular mechanism of this recognition is not known.

The clotting factor fibrinogen is another example of an endogenous TLR4 lig-and. Fibrinogen is usually found in the vasculature, however due to inflammation and the resulting increased permeability of endothelial cells, fibrinogen diffuses into the extravascular space [107]. Macrophage cell lines treated with fibrinogen secrete chemokines such as macrophage inflammatory protein 1a (MIP1a), MIP1P and MIP2, which requires functional TLR4 [108]. Fibrinogen can therefore enhance an already ongoing immune response by activating macrophages, and the recruitment of further leucocytes (Fig. 3).

TLRs can bind many endogenous ligands in addition to their better characterised pathogenic ligands (see Tab. 1). Most have been identified for TLR4 which binds chemically unrelated and structurally different endogenous ligands. Heparan sulfate, HA, HSP60, 70 and gp 96, fibrinogen and fibronectin share no major homology with LPS. Added to this list are also saturated fatty acids, but not unsaturated acids. These were shown to induce NF-kB in a macrophage cell line via TLR4 [109]. It is likely that the list of endogenous TLR ligands is far from complete and will grow in the years to come.

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