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Heme Oxygenase

HO catalyzes the breakdown of heme to iron, biliverdin, and carbon monoxide.217 Three isoforms of HO

have been identified. HO-2 is constitutively expressed in many tissues, whereas HO-3 expression

appears to be limited to the brain. HO-1 is not expressed constitutively in most tissues, but is rapidly

upregulated by both heme and nonheme cellular stresses, including hypoxia, redox stress, and

inflammation. Additionally, NO is a potent inducer of HO-1. HO-1 has profound antiapoptotic and antiinflammatory effects. These cytoprotective effects have been attributed to the individual catalytic

products of heme metabolism. Biliverdin is converted to bilirubin by biliverdin reductase, and bilirubin

has been demonstrated to act as a potent intracellular antioxidant. Carbon monoxide alone can mimic

many of the actions of HO-1 and has been shown to protect in models of sepsis, hemorrhagic shock, and

ischemia/reperfusion when administered as an inhaled gas. The mechanisms of action of carbon

monoxide have both similarities and dissimilarities with NO, and is an area of active investigation.217

Hydrogen Sulfide

Hydrogen sulfide is a colorless, flammable gas, with the typically malodor of putrid eggs. It is highly

lipophilic and freely penetrates the cell wall, which greatly facilitates its biological activity.218 Recent

evidence highlights the widespread distribution of H2S in the plasma, brain, and other tissues. H2S is

formed in mammalian cells largely by the activity of two pyridoxal phosphate–dependent enzymes,

cystathione γ lyase (CSE), and cystathionine β synthetase (CBS) that utilize cysteine and homocysteine

to form H2S.219 Large amounts of these enzymes occur in the brain (CBS), liver (CSE), kidney (CSE) and

blood vessels (CSE). Interestingly, lipopolysaccharide exposure induces the expression of CSE,

suggesting that H2S may regulate inflammation.218 Both pro- and anti-inflammatory actions have been

described. H2S levels and CSE expression are increased in animal models of endotoxemia, sepsis, and

hemorrhagic shock.218,220,221 It has been shown to increase leukocyte attachment and rolling in jejunal

blood vessels and to increase ICAM-1 expression.222 In human monocytes, H2S donors induce the

formation of proinflammatory cytokines and chemokines via an NFκB mechanism.223 Inhibitors of CSE

reduce the inflammation in these animal models of sepsis and hemorrhage. By contrast, H2S decreases

LPS-induced upregulation of NFκB in RAW 264.7 macrophages, and H2S-releasing derivatives of

diclofenac exhibit greater anti-inflammatory activity in endotoxic shock.218,219,224

RECOGNITION AND ACTIVATION PROCESSES

Exogenous and Endogenous Danger Recognition

Multicellular organisms have evolved an essential mechanism of surveillance, defense, and repair of

injured cells. Implicit with this system is the ability to differentiate pathogens and damaged cells from

self. The initial response is orchestrated by the evolutionarily ancient and more universal innate

immune system, which employs monoclonal sets of recognition molecules called PRR.225,226 PRRs bind

conserved molecular structures found in large groups of pathogens, termed PAMPs. PAMPs are a

diverse set of microbial molecules, which share a number of different recognizable biochemical features

that alert the organism to intruding pathogens.225–227 These PAMPs are recognized by cells of innate and

acquired immunity, primarily through the Toll-like PRRs. Activation induces several signaling pathways,

most notably NFκB.225,227 Consequent to this activation, an immune response is triggered to destroy the

pathogen and/or pathogen-infected cells, and an adaptive response is initiated to select pathogenspecific T cells and antibodies for future occasions.

However, pathogens are not the only causative agents of tissue and cell damage. Cells, and thus

tissues, can be injured by various noxious insults: heat, cold, chemicals, radiation, ischemia, or direct

mechanical injuries. Evolution has enabled us to deal with these damages, which though not caused by

pathogens, still necessitate repair. It is becoming apparent that specific receptors exist by which to

recognize extrinsic threats (i.e., pathogen) and intrinsic altered self (cytokines, oxidized mitochondrial

DNA, HSPs, and uric acid).227

Danger-Associated Molecular Patterns

8 DAMPs, the endogenous equivalent of PAMPS, represent danger signals or “alarmins” and share many

characteristics similar to cytokines.227 DAMPs may be released following nonprogrammed cell death,

such as necrosis, and under such circumstances tend to elicit inflammation. By contrast, programmed

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cell death (i.e., apoptosis) incorporates mechanisms such as acetylation, to minimize the release of these

mediators and any subsequent inflammatory response. Cells of the immune system also can be induced

to secrete these mediators.227 This secretion may occur by specialized secretion systems or by the

classical ER–Golgi secretion pathway.227 Under these circumstances, DAMPs may facilitate the

inflammatory response by aiding the recruitment of innate immune cells, most notably DCs. In doing so,

they indirectly orchestrate the subsequent adaptive immune response and facilitate tissue repair.227 The

prototypical alarmin, HMGB1, has already been discussed.

S100 Proteins

The family of S100 proteins incorporates over 20 related calcium-binding proteins.227 S100A8 and

S100A9 form heterocomplexes in the cytosol of granulocytes, monocytes, and macrophages, whereas

S100A12 exists as homodimers in the cytoplasm of granulocytes. S100 proteins are actively secreted at

sites of inflammation via a nonclassical pathway. The receptors mediating their effects are still being

defined, though it appears that S100A12 and S100B interact with RAGE, whereas S100A8/9 may

interact with TLR receptors. S100 proteins have been shown to induce increased vascular permeability

and a prothrombotic effect. Recent studies implicate S100 proteins in the pathogenesis of autoimmune

arthritis and psoriasis.227

Uric Acid

Uric acid is released after cellular injury, and upon exposure to the extracellular environment,

precipitates to form monosodium urate (MSU).227 Uric acid stimulates dendritic maturation and, when

coinjected with antigen in vivo, significantly enhances the generation of responses from CD8+ T

cells.227 It has significant proinflammatory properties that are best evidenced in the disease gout, in

which uric acid accumulates in tissues and induces inflammation-dependent arthritis. MSU crystals

engage the inflammasome, resulting in the production of IL-1β and IL-18.227 Macrophages from mice

deficient in IL-1R or in various components of the inflammasome, such as caspase-1, ASC, and NALP3

are defective in MSU-induced cytokine secretion and have reduced inflammation.227 Extracellular uric

acid is eliminated by uricase.227

Receptors for Danger Recognition

9 Innate immunity is not antigen specific, but rather programmed to respond to groups of evolutionarily

conserved macromolecules that represent “patterns of danger” and signal a potential threat to the host.

PRRs identify these PAMPS and include the TLR and NOD-like receptors. Recent studies highlight the

promiscuity of these PRRs in also recognizing and mediating DAMP-dependent signaling. In fact,

multiple positive feedback loops between DAMPS and PAMPs and their overlapping receptors may

represent the molecular basis for the observation that infections, as well as nonspecific stress factors,

can trigger flares of autoimmune diseases (i.e., rheumatic).148 Additional novel receptors have

subsequently been identified and designated PRRs, including RAGE.226,227

CD14

The prototypical receptor identifying external infectious threat (i.e., gram-negative infection) is CD14.

CD14 was identified as the LPS receptor when transfection of CD14-negative CHO cells with CD14

conferred responsiveness to LPS.228 Its critical role in LPS recognition is underscored by the LPShyporesponsive phenotype of CD14–deficient mice. It has been identified on cells of the myeloid

lineage, B cells, liver parenchymal cells, and fibroblasts. Differential expression is observed; ranging

from high levels on peritoneal and pleural macrophages to lower levels on Kupffer cells, alveolar

macrophages, monocytes and PMN. Expression may be modified; human PMN express low levels of

CD14 that is upregulated by TNFα, G-CSF, GM-CSF, and fMLP. Prototypically, CD14 binds LPS bound to

LPS-binding protein (LBP) in combination with MD-2 and presents it to TLR4.228 MD-2 is required for

cellular responsiveness to LPS, as demonstrated by both transfection studies and an analysis of a CHO

cell line with mutated MD-2 gene. Most of the available evidence indicates that a complex of TLR4/MD2/CD14 directly binds LPS.228 CD14 is also important in TLR2 signaling, whereby it presents bacterial

products other than LPS.

Toll-Like Receptors

CD14 is a glycosylphosphatidylinositol-linked receptor devoid of any transmembrane domain. This, in

combination with the identification of a soluble form of CD14, necessitated identifying the manner by

which LPS induced activation.229 In 1996 the Toll protein in Drosophila was shown to be necessary for

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an effective immune response to the fungus Aspergillus fumigatus.225 In 1998, Poltorak et al.230

discovered that the lps gene in LPS-hyporesponsive C3H/HEJ mice encoded a murine member of the

TLR family. These data provided the initial evidence that mammalian TLRs function as PRRs.

Subsequent studies have confirmed that TLR4 is the “LPS receptor” and that it is essential for the

defense against gram-negative microorganisms.228

Thirteen mammalian TLR receptors have been characterized. Each recognizes a specific set of

conserved microbial molecules, and as a family, they can detect most microbes.228 Interestingly, the

subcellular localization of different TLRs correlates to some extent with the molecular patterns of their

ligands and their function.231 TLR1, TLR2, and TLR4 are located on the cell surface and are recruited to

phagosomes after activation by their respective ligands. They are the receptors mediating the response

to exogenous insults including bacterial infection and trauma. By contrast, TLR3, TLR7, and TLR9, all of

which are involved in the recognition of nucleic-acid–like structures (i.e., viral DNA), are expressed

intracellularly.225,232

TLR4 is required for the innate response to gram-negative organisms and LPS, though other TLR, such

as TLR2, can also recognize and mediate this response.226 TLR4 has been shown to trigger the response

to additional ligands, including lipoteichoic acid (LTA) and peptidoglycans from gram-positive bacteria

and the fusion protein of the respiratory syncytial virus.233 Polymorphisms in the receptor confer

variability in function. Individuals with the D299G polymorphism in TLR4 demonstrate increased risk of

gram-negative infections, and other studies have linked this with an increased incidence of SIRS.234 In

addition, this polymorphism has been associated with alterations in the susceptibility to other

inflammatory and potentially infectious processes (carotid artery atherosclerosis, coronary artery

disease).234 HSP60 of Chlamydial origin has been found in atherosclerotic plaques and can bind TLR4.

Perhaps recognition of HSP60 by human TLR4 might exacerbate the inflammatory component of

atherosclerosis, whereas people with D299G polymorphism might be at least partly protected from this

exacerbation.234 TLR4 has been well-characterized as a PRR for DAMPS, including HSPs (HSP60, HSP70,

gp96), hyaluronate, heparan sulfate, and other matrix proteins. Because of its ability to recognize

endogenous proteins, TLR4 is implicated in a variety of diseases, including arthritis and

atherosclerosis.148,232

TLR2 recognizes lipoproteins derived from the cell wall of bacteria such as Treponema pallidum and

Mycoplasma fermentans, LTA from gram-positive bacteria (i.e., Streptococcal species), lipopeptides, LPS,

and lipid A.226,232,233 It also can recognize a host of DAMPs, including the HSPs, fibronectin, fibrinogen,

heparan sulfate and hyaluronate. Recent data suggest that TLR2 forms heterodimers with other TLR,

including TLR1 or TLR6 to recognize these DAMPs. A likely consequence of this cooperation is an

increased repertoire of ligand specificities. The R753Q polymorphism in the TLR2 is associated with

decreased response to these bacteria and may increase susceptibility to staphylococcal infections or

tuberculosis.232,234

Additional TLRs involved in inflammation include TLR9, which recognizes unmethylated CpG motifs

present in bacterial DNA. By contrast, most of the host mammalian genome is methylated. TLR9-ligand

engagement occurs intracellularly, in either endosomes or lysosomes, presumably following bacterial

lysis. Recent evidence suggests that TLR9 can identify host DNA released by dead or dying cells, and

hence, may be involved in the autoimmune diseases such as SLE.225 TLR5 recognizes flagellin of

bacterial flagella.235 TLR3 recognizes double-stranded RNA of both viral and endogenous sources.

Because of the later characteristic, TLR3 has been implicated in autoimmune arthritis.

TLRs induce signal transduction via their cytoplasmic Toll-interleukin-1 receptor (TIR) domains that

promotes the subsequent expression of a variety of host defense genes. These include inflammatory

cytokines and chemokines, antimicrobial peptides, costimulatory molecules, MHC molecules, and other

effectors. A considerable portion of the functional response is mediated by activating intracellular

signaling pathways that culminate in the induction of the transcription factor NFκB. Specifically, the

CD14/MD-2/TLR4 complex, upon engagement with LPS-LBP recruits the adapter protein MyD88, which

engages the serine/threonine kinase IRAK. IRAK undergoes autophosphorylation and recruits TRAF6.

Ultimately, activated IκK phosphorylates and targets for degradation the NFκB inhibitor IκB, which

enables the nuclear translocation of NFκB and transcription of inflammatory genes. AP-1 and members

of the MAPK transduction cascade are also activated by this mechanism. This pathway is critical to the

production of IL-12, TNFα, and IL-6. All TLRs signal through this conserved signaling cascade, and for

some MyD88 (TLRs 2, 6, 9) is their sole receptor-proximal adaptor. For instance, MyD88 is essential for

clearance of S. aureus, a gram-positive bacteria, which signals through TLRs 2, 6, 9.225,226,233

Subsequent studies utilizing MyD88-deficient mice suggested that alternate TLR pathways existed.

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