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These mice, in response to many TLR ligands, including peptidoglycan and unmethylated CpG motifs,

did not activate NFκB or MAPK. Surprisingly, however, LPS could still activate NFκB and MAPK, though

in a delayed fashion. Subsequently, a MyD88-independent pathway has been characterized that is

utilized by TLR4 and utilizes a distinct adaptor protein called TIR domain–containing adapter protein

TIRAP or Mal. A dominant negative mutant of TIRAP specifically impairs TLR4- and TLR2- but not IL1R or TLR9-induced NFκB, indicating a specificity of TIRAP for the TLR4 pathway. A third TIRcontaining adaptor molecule, TIR-domain containing adaptor inducing IFN-β (TRIF) also appears to

mediate a MyD88-independent pathway.225,226,233

Most of this research has occurred in the realm of sepsis and provided insight into the manner by

which organisms identify infectious threats and activate the inflammatory pathway to preserve the host.

However, several circumstances of sterile inflammation suggest a role of the TLRs in mediating

inflammation in response to endogenous danger signals (uric acid, mitochondrial DNA, HMGB1).228

Hepatocellular injury consequent to ischemia/reperfusion has been shown to be dependent on TLR4.32

RAGE

RAGE is an immunoglobulin superfamily molecule that belongs to the multiligand receptors that

recognize families of ligands rather than a single polypeptide.97,227,236,237 It has a single transmembrane

spanning domain and a highly charged cytoplasmic tail that though lacking known signaling motifs, is

critical for cellular activation. Though RAGE knockout mice are viable and fertile, they display a wide

range of defects. Most of these defects are subtler than expected, leading to the suggestion that other

receptors with overlapping function might exist. Signaling appears to necessitate clustering into a

particular orientation, which facilitates binding of cytosolic signaling complexes.97,236 Its ligands include

products of nonenzymatic glycosidation (e.g., advanced glycation endproducts or AGEs), the amyloid-β

precursor protein, the S100/calgranulin family of proinflammatory cytokine-like mediators, and the

high mobility group 1 DNA binding proteins (HMGB-1). Its expression is upregulated at sites of diverse

pathologies including atherosclerosis and Alzheimer’s disease. In fact RAGE-mediated cellular

stimulation is thought to increase expression of the receptor itself, thereby generating a positive

feedback mechanism and perpetuating the proinflammatory state.97,236,237

Recent studies demonstrate the activation of multiple signal transduction cascades subsequent to

ligand binding, including the family of MAPKs (p38, ERK 1/2, and JNK) and the rho gtpases (cdc42 and

rac).237 Like TLRs and IL-1R, RAGE engagement leads to NFκB activation, suggesting that both receptor

usage and signaling pathways evoke similar responses when cells are activated by PAMPs and DAMPs.

It is currently hypothesized that RAGE–ligand interaction induces a new heightened basal state of

activation. With a superimposed stimulus, cellular perturbation is magnified. Rather than returning to

homeostasis, cellular signal transduction mechanisms favor augmented dysfunction.97,236

Studies have implicated RAGE to be an important receptor during various acute and chronic

inflammatory processes, including tumor biology. RAGE expression and function directly correlated

with tumor growth in RAGE-transfected C6 glioma cells. In a murine tumor model, mice treated with

soluble RAGE, which functions as a decoy, exhibited a reduction in lung metastases, cellular invasion,

and expression of matrix metalloproteinases.237

RAGE has also been identified as a receptor for S100A12, a member of the S100/calgranulin family of

proinflammatory mediators. Endothelial cells cultured with S100A12 displayed RAGE-dependent

expression of VCAM-1 and tissue factor. Mononuclear phagocytes displayed S100A12-RAGE chemotaxis,

expression of tissue factor, and elaborated IL-1 and TNFα. In vivo studies of delayed-type

hypersensitivity demonstrated reduced inflammatory response when mice were treated with anti-RAGE

F(ab′)2, anti-S100 F(ab′)2, or soluble RAGE. Treated animals also displayed reduced NFκB activation

and IL-1 and TNFα expression.237

The angiopathy of diabetes is thought to be consequent to inflammatory processes generated by

elevated glucose concentrations. Hyperglycemia has been demonstrated to activate numerous signaling

cascades. One process that might mediate these effects is through the nonenzymatic formation of

AGEs.97,236,237 AGE-RAGE interactions on endothelium lead to the expression of procoagulant tissue

factor and VCAM-1 and macrophages-released tissue factor. Animals treated with soluble RAGE showed

decreases in atherosclerotic lesion area and number and a marked reduction in lesion complexity. Also,

treated animals had reduced expression of adhesion molecules, tissue factor, MCP-1, and matrix

metalloproteinases.97,236,237

CD91

The immunological properties of HSPs were discovered by their ability to elicit antitumor immunity. It

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was later discovered that this antigenic specificity was determined by the peptides they chaperoned.

APCs can bind and internalize HSP–peptide complexes derived from virus-infected cells or tumors and

represent them on major histocompatibility MHC class I molecules. In addition, APC engagement of

HSP–peptide complexes induces maturation, the expression of costimulatory molecules, and the

induction and release of cytokines (TNFα, IL-1, IL-12, IL-6, GM-CSF, MIP-1, RANTES, and NO).238,239

CD91 had been identified as a receptor for the serum protein α2 macroglobulin, a natural protease

inhibitor that like HSPs is found across many species. In fact α2 macroglobulin is the evolutionary

precursor of the C3 complement component. By binding pathogen proteases utilized during invasion and

shuttling them for endocytosis, α2 macroglobulin hinders pathogen invasion.238,240

However, such a method of host defense would be ineffective for intracellular pathogens that gain

access by alternative means, such as mimicking host proteins and developing ligands for host receptors.

Hence, another method for signaling danger is necessary. It is currently thought that upon cell death,

HSPs contained within the cell transfer information regarding the infected intracellular environment to

CD91. This large cell surface receptor, complexed with the HSPs, is internalized and delivers antigen to

the classical MHC class I pathway. The MHCs bind and present them to CD8+ T cells. On recognizing

nonself, T cells are induced to proliferate and mediate killing. This mechanism could be generalized to

implicate CD91 and HSP interactions in the recognition of all cellular stress that culminates in injury

and death.240

CD91 was first identified by Binder et al. as the receptor for gp96 and then by Basu et al. as a

common receptor for other HSPs (HSP70, HSP90, and calreticulin).241,242 This large multidomain 600-

kD protein possesses multiple binding sites for at least 32 ligands. The binding of this receptor to many

members of the HSP family has been corroborated by several independent functional and structural

studies.240

Several in vivo studies suggest a physiological relevance for HSP-CD91 interaction. Mice immunized

with tumor-derived gp96–peptide complexes reject a subsequent tumor challenge. If CD91 antiserum is

mixed with the HSP–peptide inoculum, the mice fail to reject the tumors. Srivastava et al. noted that

blocking of CD91 completely inhibits the phenomenon of representation of peptides that are carried or

chaperoned by HSPs, suggesting that not only is CD91 a receptor for HSPs, but it may also be the sole

receptor involved in antigen representing.239,242

Figure 7-12. Autophagy/mitophagy. Autophagy is inhibited by the metabolic growth rheostat mTOR that inhibits autophagy when

ATP and nutrients are plentiful. Sepsis and metabolic stress lead to inhibition of mTOR and initiation of the isolation membrane.

Atg12-ATG5-Atg16L and LC3-II localize to the phagophore to capture selected targets for degradation. Upon completion of

autophagosome formation, the Atg12-Atg5-Atg16L complex dissociates while LC3-II remains on it. The autophagosome ultimately

fuses with the lysosome to form an autolysosome, where its contents are degraded.

HSPs have been shown to chaperone antigenic peptides (tumor, viral, minor histocompatibility

antigens) from all cellular compartments. The family of HSPs thus appear to be a universal mechanism

for antigen capture, and they permit a high-efficiency antigen uptake through a receptor-mediated

mechanism.

Evidence that HSP–peptide–CD91 interactions serve to signal cellular stress is beginning to culminate.

Basu et al. demonstrate that Hsp70, Hsp90, gp96, and calreticulin are released from cells as a result of

necrotic cell death, but not apoptotic cell death.241 Similarly, Melcher reported that tumor cells

undergoing necrotic death are highly immunogenic as compared to those undergoing apoptotic death.243

Actual necrosis may be unnecessary as stressed cells and cancer cells have been reported to express cell

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